Germinated Brown Rice Attenuates Cell Death in Vascular Cognitive Impaired Mice and Glutamate-Induced Toxicity In HT22 Cells

Subscriber access provided by JAMES COOK UNIVERSITY LIBRARY Bioactive Constituents, Metabolites, and Functions Germinated brown rice attenuate cell death in vascular cognitive impaired mice and glutamate-induced toxicity in HT22 cells Eve Mon Oo, Katesirin Ruamyod, Ladawan Khowawisetsut, Chairat Turbpaiboon, Vipavadee Chaisuksunt, Panapat Uawithya, Nanthanit Pholphana, Nuchanart Rangkadilok, and Supin Chompoopong J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b07957 • Publication Date (Web): 10 Apr 2020 Downloaded from pubs.acs.org on April 23, 2020 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. 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However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties. Page 1 of 49 Journal of Agricultural and Food Chemistry 1 TITLE AND AUTHORSHIP 2 Germinated brown rice attenuate cell death in vascular cognitive impaired mice and 3 glutamate-induced toxicity in HT22 cells 4 Eve Mon Oo†, Katesirin Ruamyod±, Ladawan Khowawisetsut#, Chairat Turbpaiboon†, Vipavadee Chaisuksunt§, 5 Panapat Uawithya±, Nanthanit Pholphana+, Nuchanart Rangkadilok+, Supin Chompoopong†* 6 †Department of Anatomy, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. 7 ±Department 8 #Department of Parasitology, Faculty of Medicine Siriraj Hopsital, Mahidol Univeristy, Bangkok 10700, Thailand. 9 §Department 10 +Laboratory 11 Thailand of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. of Anatomy, Faculty of Medicine, Chaing Mai University, Chiang Mai 50200, Thailand. of Pharmacology, Chulabhorn Research Institute (CRI), Kamphaeng Phet 6, Laksi, Bangkok 10210, 12 13 *Corresponding author, E-mail: supin.cho@mahidol.ac.th Tel: +66-2-4197035, Mobile +66-818242606, Fax +66-2- 14 4198523 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 15 ABSTRACT 16 Germinated brown rice (GBR) with un-polishing, soaking and germinating processes can improve the texture, 17 flavor and nutritional values including GABA and phenolic contents. The effect of GBR was firstly investigated 18 in vascular cognitive impaired mice and glutamate-induced toxicity in HT22 cells with respect to standard pure 19 GABA. Feeding mice with GBR for 5 weeks showed the neuroprotection. In this study, the modified bilateral 20 common carotid artery occlusion mice model was mild but the significant difference in cognitive impairment was 21 still shown. Like pure GABA, GBR decreased cognitive deficits in memory behavioral tests and significantly 22 attenuated hippocampal neuronal cell death at P<0.001. Similar to GABA (0.125 µM), 100 µg/ml of GBR increased 23 HT22 cell viability after glutamate toxicity. GBR affected less apoptotic cell death and less blocking by GABAA 24 antangonist, bicuculline than GABA. Taken together, the underlying mechanism of GBR protection may mediate 25 though GABAA receptor and its phenolic contents. 26 27 KEYWORDS: germinated brown rice, gamma-amino butyric acid (GABA), ischemic-reperfusion, cognitive 28 impairment, HT22 cells ACS Paragon Plus Environment Page 2 of 49 Page 3 of 49 Journal of Agricultural and Food Chemistry 29 INTRODUCTION 30 Vascular cognitive impairment (VCI), the second leading form of dementia after Alzheimer’s disease 31 (AD) is a progressive disease caused by reduced blood flow to the brain or defined as cerebral hypoperfusion, and 32 affects cognitive abilities especially executive function 1. In acute insults like ischemic stroke, excess extracellular 33 glutamate may lead to excitotoxicity via the over activation of ionotropic glutamate receptors, oxidative stress 34 inside the cells and eventually cause cell death. Clinical imaging studies in the aging patients suggest that the 35 reduction of blood flow appears early and even precedes the onset of AD pathology and the degree correlated with 36 the severity of dementia 2. Recently, the contributions of cerebral hypoperfusion to AD have been investigated to 37 elucidate the pathogenesis of AD 2. 38 It is widely acceptable that brown rice (BR) is a healthier food than white rice since it contains higher 39 nutritional and biofunctional or bioactive components. However, it has been consumed less than white rice since 40 its texture is not as fine as white rice while eating. When BR undergoes germination process by soaking in water 41 for a considerable period of time to produce sprout, not only does it improve its eating texture and flavor but also 42 its nutrient levels increase significantly including gamma-amino butyric acid (GABA) and other antioxidants such 43 as phenolic substances 3-4. This brown rice is termed as germinated brown rice (GBR). GBR has been proved to 44 have beneficial effects on various organs 5. Increasing reported evidence suggest the nutraceutical property of GBR 45 which prevents the apoptosis, decreases reactive oxygen species formation in SH-SY5Y cells 6-8, reduces TNF- 46 alpha both in serum and brain and attenuates dopaminergic cell loss in rotenone-induced Parkinson-like disease 47 rat models4 may help to protect neurodegenerative diseases. 48 As a non-clinical practice, GABA has been recently used as food supplements and reported to help 49 alleviate anxiety and/or improve sleep quality 9. The major composition in 100 g of GBR compared to BR has 50 been previously 10 reported. There were no difference in energy (389.2 Kcal), protein (6.9 g), fat (2.6 g), 51 carbohydrate (84.4 g), total vitamin E (1.19 mg) and dietary fiber (3.5 g), but a significantly higher level of γ- 52 oryzanol (13.3/ 11.4 mg), total phenolic content (69.6/ 37.1 mg) and GABA (11.9/2.6 mg) was found in GBR 53 than BR. The promising GABA contents in GBR have been reported to be 10 times higher than those in white 54 rice 10. In addition, the review of the mechanism by which GABA, an inhibitory neurotransmitter, is sensitive to 55 cerebral ischemia was reported concerning how neuronal death can be prevented by GABAergic drugs 11. 56 GABAergic effects have been used as an established clinical practice, for examples, using as sedative, 57 anxiolytic, and anti-epileptics. Moreover, GABA agonists increased surviving of pyramidal cells in CA1 58 hippocampal region by decreasing the phosphorylated c-Jun, thus preventing the apoptotic cell death 12-14. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 59 Nevertheless, studies on the direct effects of GBR on vascular cognitive impairment and glutamate- 60 induced toxicity are limited, and since neurodegeneration due to glutamate toxicity is believed to underlie the 61 neurodegenerative diseases, it may be hypothesized that GBR has some alleviate effects. The present study was 62 aimed to examine the effects of GBR in comparison to GABA on memory deficits in mice with mild cerebral 63 hypoperfusion using mouse model of modified bilateral common carotid artery occlusion (BCCAo) and 64 glutamate-induced HT22 cell death. Pure GABA was used as a standard compound. ACS Paragon Plus Environment Page 4 of 49 Page 5 of 49 Journal of Agricultural and Food Chemistry 65 66 MATERIALS AND METHODS Preparation of GBR 67 GBR was prepared from paddy rice, not milled rice, (Oryza saliva L. cultivor), Hom Mali 105 or 68 KhaoDawk Mali 105 (KDML 105) which was grown and provided from Ban-Srangming community enterprise, 69 Muang Sam Sip district, Ubon Ratchathani, the northeastern part of Thailand followed Thai agriculture standard 70 TAS 4003-2012, National Bueau of Agricultural Commodity and Food Standards. The germination process was 71 prepared accordingly to the method developed by Dr. Kasinee Chantharasophon, Faculty of Science, Ubon- 72 Ratchathani-Rajabhat University. Five hundred grams of paddy rice were rinsed 2 times with clean tap water, then 73 soaked in 2.5 L of water in a container for 24 hours at room temperature and the water was changed every 8 hours. 74 After washing 2 times with clean tap water, followed by filtering, the paddy rice was placed on the double layers 75 of cotton cloth for germination in a closed and dark container at 35°C with water spray every 6 hours for 20- 36 76 hours. According to Cho et al 3, the presence of moisture is essential for the germination. The higher degree of 77 germination has been done by simple soaking followed by O2 free incubation. The moisture content was less than 78 13% by rice grain weight. The moisture content was calculated by (initial weigh-final weight) /initial weight x 79 100. It resulted in germination of greater than 80% of 1 mm budding. Then the germinated paddy rice was washed 80 twice with clean tap water, followed by air drying and parboiling of GBR with steam performed in a close 81 container for 7 minutes. GBR was transferred to the temperature bin for drying with hot air temperature at 50°C, 82 tempered for 15 hours before milling. After milling, GBR was freeze-dried, ground into fine powder, packed in 83 aluminum foil vacuo bags and stored at -20°C till further study. 84 According to the previous report 10 that GBR from the similar source, Khao Dawk Mali 105 and the same 85 procedure of germination process has GABA content about 11.9 µg /100 mg of GBR. Therefore, in this study, 86 pure GABA compound (Sigma-Aldrich, A5835, BioXtra, 99%, USA) was used as a standard compound for the 87 comparison both in vivo study (11.9 µg of GABA/100 mg of GBR) and in vitro study (0.125 µM of GABA and 88 100 µg/ml of GBR). 89 For 5 week-GBR treatment, Zhang et al 15 previously proposed an alternative to gavage for long term 90 oral administration by using gelatin blocks containing GBR. In this study, 2400 mg of standard mice food and 91 GBR was trapped in 10 ml of boiled water containing 500 mg of gelatin (as shown in Fig 2B). 92 For in vitro study, aqueous extract of GBR was used and prepared by dissolving the GBR powder in 93 sterile distilled water. The GBR solution was well mixed, warmed in water bath at 70°C for 30 minutes, and 94 centrifuged at 5,000 rpm, 4°C for 30 minutes. The supernatant was collected and passed through 0.45 µm filter. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 95 Page 6 of 49 Chemicals 96 Standards -Aminobutyric acid (GABA) and amino acids (Sigma-Aldrich, St. Louis, MO, USA), HPLC 97 grade methanol and acetonitrile (Merck, Darmstadt, FR, Germany), and all chemicals (Sigma-Aldrich St. Louis, 98 MO, USA) were analytical grade. Ultrapure water obtained through a Milli-Q water purification system 99 (Millipore, Bedford, MA) was used throughout this experiment. 100 Determination of GABA and Amino acids Contents 101 Sample was analyzed using modified protocol from Thai Agricultural Standard methods, National 31 and Long, 2017 32. 102 Bureau of Agricultutal Commodity and Food Standards Rice samples were accurately 103 weighed as 0.10 g, and then mixed with 1.8 mL of ultrapure water and 0.2 mL of 5-sulfosalicylic acid (5% w/v). 104 After vortex mixing, the mixture was agitated by tube rotator for 10 min and centrifuged at 1876×g for 20 minutes. 105 The extracted sample was filtered through 0.2 µm PVDF filter membrane (Chrom Tech, MN, USA) and further 106 derivatized using an automated online derivatization program as followed, sample (0.50 µL) was added to the 1.30 107 µL of borate buffer (0.4 M, pH 10.2), and then 0.50 µL of o-phthalaldehyde reagent (OPA), followed by 0.20 µL 108 of 9-fluorenylmethyl chloroformate reagent (FMOC-Cl), and finally 7.40 µL of injection diluent. After each 109 reagent addition, five mixing cycles were performed. 110 The derivatized sample (9.9 µL) was analyzed using HPLC (Agilent Technologies, Baudrats, Germany) 111 equipped with Infinity Lab Poroshell HPH-C18 column (4.6 x 100 mm, 2.7 µm) fitted with guard column (Agilent 112 Technologies, CA, USA). A mobile phase consisting of 10 mM Na2HPO4, 10 mM Na2B4O7, and 5 mM NaN3 (pH 113 8.2) (solvent A) and acetonitrile : methanol : water (45:45:10, v/v/v) (solvent B) was running with a gradient 114 system: 0-0.35 min, 2% B; 0.35-10 min, 2-48% B; 10-14.5 min, 48% B; 14.5-14.6 min, 48-100% B; 14.6-17 min, 100% 115 B; 17-17.1 min, 100-2%B, and equilibrated at this condition (2% B) for 3min with a flow rate of 1.5 mL/min. The 116 column compartment was controlled at 40oC. The sample was detected using a fluorescence detector (FLD) set at 117 excitation and emission wavelengths of 340 nm and 450 nm, respectively. The recoveries of GABA and amino 118 acids were in a range of 90.26-97.57% (GABA was at 96.81%). 119 Animals 120 Five-week old ICR male mice weighing between 25-32 g were purchased from Nomura Siam International 121 Co. Ltd. (Bangkok, Thailand). Animals were housed as one per cage under a 12/12h light-dark cycle, and were 122 ad libitum accessible to food and water. All experimental procedures were performed according to the protocols ACS Paragon Plus Environment Page 7 of 49 Journal of Agricultural and Food Chemistry 123 approved by the Animal Ethics Committee, Faculty of Medicine Siriraj Hospital, Mahidol University (SI-ACUP 124 008/2559). 125 After 1 week of habituation, mice were randomly divided into sham-operated groups and carotid- 126 occluded groups, each of which was divided into three subgroups of treatments according to feeding food; 1) 127 standard mice food, 2) GBR-containing gelatin food and 3) GABA-containing gelatin food. Therefore, 128 Therefore, six groups (n=12 each) included Sh, Sh-GBR, Sh-GABA, CO, CO-GBR and CO-GABA groups. 129 It has been reported 16 that mice at the age of 3-4 weeks (weaning time) approximately on 28th day after 130 birth can feed themselves and drink on their own. In this study, GBR treatment as gelatin block feeding was 131 started 1 week after habituation. The pilot study was performed for being sure that mice can have GBR 132 containing gelatin block. It was found that maximal five gelatin blocks/day/mouse were eaten. Therefore, six 133 gelatin blocks were supplied daily to ensure the ad libitum food access. The weight of gelatin blocks was 134 measured, the daily consumption of gelatin block intake and GBR intake was calculated and compared between 135 pre-fed and post-fed weights of gelatin blocks. 136 To mimic the effect of GBR consuming in human, the timeline of this study (Fig 1) indicated that five 137 week-GBR feeding was performed before and after BCCAo according to the previous study of GBR treatment 138 in β amyloid induced mice model 17. 139 Carotid Occlusion for Cerebral Hypoperfusion 140 Two vessel occlusion surgery was used to induce cerebral hypoperfusion and mimic the VCI models as 141 described previously by Song et al, 2017 18. Mice were subjected randomly to either sham operation or mild 142 cerebral hypoperfusion induction with BCCAo. Under intraperitoneal injection of ketamine (80 mg/kg) and 143 xylazine (8 mg/kg) mixture, a small ventral incision was made on the midline of the neck, the common carotid 144 artery was exposed and carefully separated from the carotid sheath, the cervical sympathetic and vagus nerves. 145 Carotid-occluded mice underwent permanent ligation of right common carotid artery occlusion (RCCAo) on day 146 0, followed by temporary ischemic-reperfusion with transient ligation of left common carotid artery occlusion 147 (LCCAo) for 20 minutes and then released for reperfusion on day 7. Sham-operated mice underwent the identical 148 surgical procedure without artery occlusion. During recovery from anesthesia, mice were placed on a heating 149 pad or supplemental heat sources to maintain the body temperature at 37.5 ± 0.5°C and prevent hypothermia 19. 150 The duration of 20 minutes ischemia was sufficient to ascertain the ischemic injury and did not show un- 151 necessary protective effect from other sources 20-21. For the subgroup of treatments, GBR or GABA was ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 152 continually fed for completing five week-feeding time. All mice were sacrificed on day 21 after behavior tests as 153 shown in figure 1. 154 Rapid Murine Coma and Behavioral Scale (RMCBS) 155 Previous report described by Carroll et al 22 showed that the level of illness, as objectively determined by 156 RMCBS was corroborated by inter-operator validation. It was significantly correlated with intracerebral pathology. 157 In the present study, The neurological score, RMCBS was determined for neurological symptom or sensori-motor 158 functioning at 4 access points of RCCAo (pre-RCCAo, 3 or 24 or 72 hours after RCCAo) and 4 access points of 159 LCCAo (pre-LCCAo, 3 or 24 or 72 hours after LCCAo). 160 Ten parameters were observed within three minutes including motor performances, hygiene-related 161 behavior (grooming), gait, balance, body position, touch escape, pinna reflex, toe pinch reflex, limb strength, and 162 aggression. Each parameter was scored 0 to 2, with a 0 score correlating with the lowest function and a 2 score 163 correlating with the highest. The RMCBS observation was recorded by video camera and blindly analyzed by 164 two investigators. Mean total score of 18-20 indicated no sensori-motor deficits. 165 Morris Water Maze (MWM) Test 166 The MWM test was conducted to evaluate the performance of spatial learning and memory as described 167 in previous study 23-24. One week after BCCAo (Figure 1) when the surgical wounds were completely healed, the 168 circular pool with a diameter of 55 cm and height of 25 cm filled with opacified water (20 cm depth) at 25 ± 1 °C 169 and contained a hidden escape platform (10 cm) which was submerged 1.5 cm below the surface of the water. The 170 MWM test is a vision dominant, all spatial signs around the maze were fixed at same location. The pool was 171 divided into four quadrants, the platform was placed in the center of one fixed quadrant for all trials. An overhead 172 video camera was used to track mice while in the pool. Firstly, all mice have learned to experience with the visible 173 platform, mice were gently guided to the platform if they failed to find it within 60 s. During learning period, any 174 differences in latency time existed among each mouse, it was likely a problem with vision or motor deficit rather 175 than learning and memory 25. Therefore, such a mouse was excluded from experiment. Training period (4 trials per 176 day) was continued for 4 days and the escape latency time was recorded. Finally, the retention of spatial memory 177 was assessed using a probe trial for 60-second, the hidden platform was removed and starting from the same ACS Paragon Plus Environment Page 8 of 49 Page 9 of 49 Journal of Agricultural and Food Chemistry 178 location. Using a video analysis and modeling tool, Tracker V.4.11.0 program, the percentages of swimming path 179 (distance in the target zone), the quadrant occupancy time (time in the target quadrant) and the number of platform 180 crossing (the frequency of crossings over the original platform location), were analyzed. 181 Trace Fear Conditioning (TFC) Test 182 As previously described 26-28, the trace fear conditioning (TFC) task is a non-vision dominant behavioral 183 test performed in the chamber (25 cm x 31 cm x 25 cm) with tone and shock stimulation sources. The TFC includes 184 3 sessions: pre-training, training and cued test sessions. As shown in Figure 1, on the first day of TFC test, the 185 pre-training session, mice were allowed to freely explore the chamber for 10 minutes to get familiar context. On 186 next two days of training session, after 3 minutes habituation to chamber, mice were trained by six cycles of 187 conditioned-stimulus (CS) and unconditioned-stimulus (US) with an inter-stimulus trace interval of 20 seconds. 188 The CS was 20 seconds duration of 80 decibels monotone sound with an intensity of 3000 Hz. The US was one 189 second of foot shock with 0.3 mA applied to the metal grid floor of chamber. During 120 seconds of inter-trial 190 interval, this shock frightened mice to stop moving and stay immobile, termed as “freezing”. Two minutes after 191 the last shock, mice were returned to their home cages. 192 The cue test session was performed for next 4 days. To create a distinct context in cue test session, the 193 sidewalls of chamber were changed by adding a white plastic board and metal grids for shock stimulation were 194 removed. After 3 minutes habituation, six times of CS were introduced without receiving any foot shock. One 195 minute after last tone sound, mice were brought to their home cages. After each trial, the chamber surface was 196 cleaned with 70% ethanol to eliminate a smell. 197 Freezing behavior was recorded and analyzed by ImageFZ software 27. Freezing after the tone in cued 198 test indicated that the mice had a good cognitive ability since the mice could recognize that there would be the 199 foot shock after tone sound. Fear memory was assessed as the total amount of freezing behavior with complete 200 lack of movement, except for respiration during memory testing. Values were reported as percentage of time spent 201 freezing. 202 Histopathological Examination 203 At the end of experiment, mice were deeply anesthetized with ketamine (80 mg/kg) and xylazine (8 204 mg/kg) mixture, intraperitoneally and perfused transcardially with 0.9% saline followed by 4%paraformaldehyde 205 (PF) in 0.1 M phosphate buffer (pH 7.4). Brains were immediately collected and placed in 4% PF for post 206 fixation overnight at 4°C. With a standard paraffin processing, block of brain were cut at bregma -2.92 to -3.72 207 mm into 5 µm thickness. To confirm the survival of pyramidal cells, the brain sections were stained with 1.5% ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 208 cresyl violet for 30 minutes as described previously 29 that has high affinity to Nissl body. In the hippocampal 209 CA1 and CA3 area, surviving neurons were defined as a light stained cells with vesicular nucleus and prominent 210 1-2 nucleoli. Ischemic dead neurons exhibit features including dark stained cells with pyknotic nucleus and 211 shrunken cytoplasm. 212 For quantitative analysis of cresyl violet staining, three non-overlapping fields within hippocampal 213 CA1 and CA3 of each section were taken by Olympus microscope digital camera, DP73. Using ImageJ software 214 version 1.52a, the percentage of surviving and dead neurons were analyzed per area (135 mm2) and expressed as 215 mean ± SEM. 216 HT22 Cell Culture 217 The immortalized mouse hippocampal HT22 cell line, HT22 cells (SCC129, Sigma-Aldrich) were 218 grown as monolayers in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine 219 serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin (P4458, Sigma-Aldrich). HT22 cells were 220 maintained at 37˚C in a humidified incubator containing 5% CO2 30. When cell growth reached 90-95% 221 confluency, cells were split and subcultured by 0.05% trypsin/EDTA followed by DMEM containing FBS every 222 2 days at a 1 to 3-8 ratio in order to maintain exponential growth. 223 Cell Viability Assay 224 Cultured HT22 cells were seeded in 96-well plates and grown to a confluence of 60-70% at the density 225 of 0.5 x 104 cells/ml. At first, cell viability assay has been performed for testing the concentration effects of 226 glutamate (2-10 mM, Sigma-Aldrich, G5889), GBR (10-4000 µg/ml), GABA (0.0125-5 µM) and bicuculline 227 (GABAA receptor antagonist, 1.25-125 nM) after 24 hours of incubation at 37°C under a 5% CO2 humidified 228 atmosphere. After finding the appropriate concentration of each compound, the effect of GBR against glutamate 229 toxicity was determined on HT22 cells. Preliminary in vitro study has shown that pre-treated with GBR for 24 230 hours before glutamate induction could not alleviate the cell death. Therefore, in this study pre-treated with GBR 231 for 24 hours and then exposed to glutamate in the presence of GBR (co-treated) for another 24 hours were used 232 and could gain more cells for the evaluation of the cell viability. Bicuculline incubation was performed for a few 233 minutes before pre-treatment of GBR. 234 At the end of treatments, the culture medium was replaced by a solution of 3- (4, 5-dimethylthiazol-2- 235 yl-2,5-diphenyltetrazolium bromide (MTT) at 0.5 mg/ml in fresh medium. After incubation at 37°Cfor 3 hours, ACS Paragon Plus Environment Page 10 of 49 Page 11 of 49 Journal of Agricultural and Food Chemistry 236 the supernatants were discarded and 100 µl of DMSO was added and mixed thoroughly to dissolve the resultant 237 dark blue crystal. The absorbance was determined with an automatic plate reader at 540 nm by spectrophotometry 238 machine (Biotek, USA, Synergy H1). The corrected absorbance of each sample was calculated by subtracting the 239 culture medium background (blank control) from the assay readings. Percentage of cell viability was calculated 240 using corrected absorbance of samples, when compared with cell control. All data have done in a triplicate manner. 241 Since GABA is previously known as GBR content and shown the protective effect, it has also been used in vitro 242 study as a positive standard for comparison. 243 Quantitative Analysis of Apoptotic Cells by Flow Cytometry 244 Regarding Fluorescence activated cell sorting (FACS), HT22 cells were seeded at the density of 105 245 cells/ml in each well of 6-well plates. The glutamate, GBR, GABA and bicuculline were incubated as mentioned 246 above. After incubation time, the cells were collected by trypsinization. The aspirated medium and washing PBS 247 were also collected as the possibility of containing the dead cells. After trypsinized, the cells were centrifuged at 248 2500 rpm for 5 minutes at 4°C. Then, the cell pellet was washed in PBS. After that, the binding buffer, 249 propidium iodide (PI) staining and annexin V-FITC from the detection kit (Sigma-Aldrich, APOAF) were added 250 and incubated in dark for 1 hour. The cells were analyzed by flow cytometry instrument BD FACSCalibur 4C 251 (Becton Dickinson, SN: E5809). 252 Statistical Analysis 253 Quantitative data is represented as mean ±standard error of mean (SEM). Statistical analyses were 254 completed using the Statistical Package for Social Science (SPSS) program version 18 (SPSS Inc., Chicago). 255 Comparison among groups was analyzed using One-way ANOVA test with Tukey post-hoc multiple 256 comparison. The changes of body weight in week by week were analyzed by paired samples t-test. Results were 257 considered statistically significant when P < 0.05, 0.01 and 0.001. Data were tabulated and plotted using 258 SigmaPlot/SigmaStat 12.5 (Systat Software GmbH, Erkrath, Germany). ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 259 260 RESULTS Food Intake 261 As shown in Figure 2A, the total food intake (gelatin block, Figure 2B) was not significantly different 262 among groups with average, about 1236±50 gram/kg/day. To report consumed amount of GBR after calculation, 263 when compared between sham- operated and carotid occluded groups, the average consumed GBR (Figure 2C) and 264 GABA (Figure 2D) showed no significant difference, with average, about 11.25±0.25 gram/kg/day and 265 1360.7±0.02 µg/kg/day, respectively. Therefore, in this study, mice with a significantly different food intake were 266 excluded. 267 Body Weight 268 During 5 weeks of food treatments, the average body weight of various groups as compared in Figure 269 3A, showed no significant differences. However, the weight differences after operation was shown in Figure 3B. 270 There was a significant increase in weight loss after first operation or RCCAo in carotid occluded group under 271 GBR treatment (CO-GBR) at both compared to sham-operated group, P<0.05 and compared to second operation 272 or LCCAo, P<0.01. Some weight loss found in sham-operated groups might be resulted from aneasthesia and 273 operation. While that in carotid-occluded groups may cause by the sudden reduction of blood flow to the brain and 274 transient disturbances in normal physiological homeostasis. 275 Sensorimotor Deficits on Rapid Murine Coma and Behavioral Scale (RMCBS) Test 276 To consider sensorimotor deficits after operation as shown in Table 1, the mean total score of RMCBS 277 in the carotid occluded group (8.3 ± 0.6 and 12.2 ± 0.8) markedly decreased when compared with that in the 278 sham-opereated group (15.3 ± 0.7 and 18.7 ± 0.3) at 3 and 24 hours, respectively (P<0.001) following the 279 induction of cerebral hypoperfusion after RCCAo. GBR treatment could not ameliorate the injury and the 280 RMCBS was still significantly decreased at 8.4 ± 0.5 and 13.1 ± 1.2, when compared with that in the sham- 281 opereated group (P<0.001). The similar results were also shown after LCCAo. Interestingly, the higher mean 282 total score of RMCBS (17.1 ± 0.7) returned at 72 hours after arterial occlusion with no significant difference to 283 the sham-opereated group (18.6 ± 0.4). 284 Cognitive Deficits on Morris Water Maze (MWM) and Trace Fear Conditioning (TFC) Tests ACS Paragon Plus Environment Page 12 of 49 Page 13 of 49 Journal of Agricultural and Food Chemistry 285 As shown in Figure 4, cognitive deficits caused by BCCAo were assessed in the MWM test based on 286 visual cues for spatial learning and memory and TFC test for non-vision dominant memory deficits. In the MWM 287 test, the typical swimming path (Figure 4A) of the last training day with hidden platform test, the significant 288 decrease in path length is indicative of successful learning of the platform position. Longer path length in the 289 carotid occluded group indicates that mice may use an inappropriate searching sign to locate the hidden platform 290 which also resulted in longer escape latency time. During 4 days of training, the carotid occluded group exhibited 291 longer escape latency. Comparisons of individual day values (Figure 4B and 4C), escape latency on the following 292 day gradually decreased in all mice group. Furthermore, the carotid occluded mice began to have notably longer 293 escape latencies than sham-operated mice at the second and third training day (P< 0.05), which lasted for 4 days, 294 (P< 0.001 at the fourth day), and was also plotted for comparison only trial of the fourth day in Figure 4D. In the 295 probe trial test, swimming time in the target quadrant was used to evaluate the retention performance. The sham- 296 operated groups swam longer in the target quadrant than the carotid occluded group (P< 0.001, Figure 4E). In 297 agreement, frequency of platform crossing was decreased in the carotid occluded group (P< 0.01, Figure 4F). 298 However, five weeks of GBR treatment, the longer path length and longer escape latency time due to 299 BCCAo were attenuated significantly (P< 0.001 at the fourth day, Figure 4B, 4C and 4D). The result of probe trial 300 test indicated that mice in GBR treated group spent significantly more time in the target quadrant (P< 0.001, Figure 301 4E), and higher frequency of platform crossing (P< 0.001, Figure 4F) when compared to carotid occluded group. 302 In addition, GBR treated mice could show more improvement of these indicated parameters of memory deficits 303 than GABA control treated group (Figure 4C, 4D, 4E and 4F). 304 The TFC test, a non-vision dominant trace fear conditioning test was suggested as the better evaluation for 305 the cognitive performance. As shown in Figure 4G using TFC instrument, after experienced six pairs of tone-shock, 306 mice exhibited freezing behavior even when only tone was present in the cued test, which means they have 307 remembered the association between tone and shock. In the cued test for 4 day evaluation, freezing percentage of 308 the carotid occluded mice gradually decreased in the second day and were significantly lower than that of sham- 309 operated group since the first day (P< 0.01, Figure 4H and 4I). This decrease of freezing time was reversed 310 obviously in the GBR treated group (P< 0.01, Figure 4H). GBR treatment showed similarly ameliorated cognitive 311 deficits to pure GABA treatment (Figure 4I) but affected higher freezing percentage than GABA since the first ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 312 day. This result indicated the protective effects of GBR on cognitive impairment after cerebral hypoperfusion 313 resulted from BCCAo. 314 CA1 and CA3 Hippocampal Cell Death 315 Nissl staining for the dorsal hippocampus were carried out to investigate the mechanism underlying the 316 degradation of the neurons related to the cognitive deficits. In Figure 5, the histopathological examination showed 317 that there were pathological changes occurred in CA1 (Figure 5B) and CA3 (Figure 5D) subfield. Dead neurons with 318 irregular-shaped, shrunken cytoplasm and extensively dark pyknotic nucleus and indistinct nuclear border were 319 observed in the carotid occluded group. Histopathological analysis showed that BCCAo caused marked CA1 320 (Figure 5A) and CA3 (Figure 5C) pyramidal cell loss in carotid occluded group versus sham operated group. GBR 321 treatment significantly attenuated the percentage of pyramidal cell death of hippocampal CA1 (less than GABA 322 treatment) and CA3 (more than GABA treatment) subfield in the cerebral hypoperfusion mice after BCCAo 323 (P<0.001, Figure 5A and 5C). 324 HT22 Cell Viability 325 .The protective activity of GBR against glutamate-induced cell death was investigated by the MTT assay. 326 Pure GABA has been used as a standard compound because it has been previously shown to have 327 neuroprotective activity and reported as the composition in GBR. The viability of HT22 cells exposed to 4mM 328 of glutamate for 24 hours decreased to 57% significantly when compared to the untreated control cells (Figure 329 6A). The decrease of HT22 cell viability in a dose dependent manner by various concentration of GBR and 330 GABA was shown in Figure 6B. GBR prevented glutamate (4mM)-induced HT22 cell death, and the highest 331 protective effect was achieved with 100 µg/ml of GBR (118.6 ± 1.7% cell viability, P< 0.001, Figure 6D). GBR 332 affected more viability of HT22 cells than 0.125 µM of pure GABA (96.9± 9.9% cell viability) at P< 0.05. Upon 333 comparing the cell viabilities in the cases of 12.5nM of bicuculline which is the GABAA receptor (Figure 6E), it 334 has been found that the protective effect of GBR showed less viability of HT22 cells (55.3±9.6%) as the same 335 manner as the effect of GABA (49.7±2.4%). The HT22 cells treated with 100 µg/ml of GBR shared a similar 336 morphology with the normal control cells when compared with the glutamate-treated cells (Figure 6C). Most of 337 HT22 cells undergo degenerate containing short processes and reduce in number of cells following glutamate 338 toxicity. GBR showed similar effect to GABA on the ameliorated glutamate toxicity. Some HT22 cells showed ACS Paragon Plus Environment Page 14 of 49 Page 15 of 49 Journal of Agricultural and Food Chemistry 339 the differentiation and containing long processes. The protective effect of GBR or GABA against glutamate 340 toxicity was abolished by bicuculline at 1.25, 12.5 and 125 nM with showing the decrease in the number of 341 surviving HT22 cells. 342 Apoptotic Cell Death Related to GBR and GABA Effects on Glutamate Toxicity 343 Glutamate induces neuronal cell death which was mediated through necrosis and apoptotic process. To 344 observe the protective effects of GBR on glutamate-induced apoptosis, annexin V/propidium iodide double 345 staining was performed and analyzed by flow cytometry. In Figure 7A, the percentage of dead or late apoptotic 346 HT22 cells in the glutamate treated group decreased (50.6±1.9%) when compared with the case of the cell control 347 group (4.5±0.6%). However, when the glutamate-treated cells were pre- and co-treatment with GBR, cell death was 348 attenuated by GBR (6.1±0.9%). This improvement of apoptotic cell death was shown in the similar manner to 349 GABA (5.6±1.3%). In addition, the incubation with bicuculline, a GABAA receptor antagonist (1.25 nM) showed 350 less abolished effects in GBR treated group (27.9±4.1%, P< 0.01) than GABA treated group (44.5±3.4%, P< 351 0.001). These results suggested that GBR prevented glutamate-induced HT22 apoptosis. The attenuation effects 352 may be mediated via GABAA receptor. However, the result was not only from the GABA content itself but also 353 from the other constituents of GBR like antioxidants. 354 Chemical Analyses of Germinated Brown Rice (GBR) 355 The health-promoting potential of GBR may be due, in part, to the bioactive compounds present in the 356 GBR, as shown in Figure 8, the determination of GABA and amino acids contents using HPLC analysis. The 357 combination of extraction and HPLC methods was consistent for the separation of these compounds of GBR 358 (GBR sample, PH0001). According to the standard GABA 1.00 µg/mL (Figure 8A), the HPLC fingerprint of GBR 359 (Figure 8B) showed that GBR contained small amounts of GABA (peak at 5.2 min, 2.84±0.02 µg/100 mg dry 360 weight) and amino acids including Asp, Glu, Ser, Gln, Gly and Tyr (Table 2). ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 361 DISCUSSION 362 The present study demonstrated that cerebral hypoperfusion produced neuronal cell damage in the 363 hippocampus as well as vascular cognitive impairment in performance on the MWM and TFC tests. Treatment 364 with GBR protected against cell damage in the brain and attenuated cerebral ischemia-induced learning and 365 memory deficits. Neuronal apoptosis might cause this memory impairment. Moreover, the protective capability of 366 GBR was compared to pure GABA on glutamate-mediated neuronal toxicity in HT22 murine hippocampal 367 neuronal cells. The 4mM of glutamate has reduced the cell viability around 57% after 24 hour treatment. 368 Interestingly, the decrease in cell viability by glutamate was recovered by pre and co-treatment with the GBR 369 which increased cell viability to more than 80% and better than GABA. Specificity, the protective effect of GBR 370 has not been previously reported in cerebral ischemia and HT22 cells against glutamate toxicity, the protection 371 was more pronounced in GBR than that of GABA. Therefore, the underlying molecular mechanism associated 372 with the GBR-mediated neuroprotection was investigated. Flow cytometry, annexin V/propidium iodide double 373 staining was used to discriminate between apoptotic and necrotic cells and found that pre and co-treatment of 374 GBR with 4mM glutamate treatment affected less percentage of apoptotic cell death (6.1±0.9%) and less blocking 375 by bicuculline (27.9±4.1%,) than GABA (5.6±1.3% and 44.5±3.4%, , respectively). Hence, this study showed that 376 bicuculline, GABAA receptor antagonist inhibited GBR attenuation of apoptotic HT22 cell death against 377 glutamate-induced neurotoxicity less than GABA. 378 Moreover, the HPLC fingerprint of GBR showed less amount of GABA content than that of previous 379 reports. GBR has been previously reported that its bioactive contents vary widely. As previously described by 380 Lone and Thuy (2019) 33 that the optimum conditions, major factors such as soaking time, pH, germination time 381 and temperature play important roles for obtaining the equal bioactive contents of GBR. Therefore, the delayed 382 analyzing process and the freezing storage of crude GBR may affect the amount of GABA available in different 383 batches. 384 However, GBR still show the similar effect to pure GABA with previously reported dose. This GBR 385 attenuation may include the activity of other compounds in GBR contents too. The other compounds in GBR 386 (PH0001) was also analyzed (not shown in the result session) and compared to two colored rice line (RR365, 387 RR368) including total phenolic content (mg/g); 0.1193±0.0020 compared to 0.167±0.0320, 0.1701±0.465, 388 respectively and proanthocyanidine (mg/g); 0.7728±0.0240 compared to 1.6408±0.0643, 2.0037±0.0559, 389 respectively. Therefore, these total phenolic content and proanthocyanidine in GBR which have been previously ACS Paragon Plus Environment Page 16 of 49 Page 17 of 49 Journal of Agricultural and Food Chemistry 390 shown the anti-oxidant activity and its GABA contents may mediate the neuroprotection by attenuated apoptotic 391 HT22 cell death against glutamate-induced neurotoxicity. 392 Cerebrovascular diseases including ischemic and hemorrhagic conditions are leading causes of death and 393 disability worldwide. Ischemic stroke or cerebral hypoperfusion is induced by a transient or permanent occlusion 394 in the cerebral vessel, resulting in neuronal death and associated behavioral deficits, including sensorimotor 395 dysfunction, spatial orientation disorder, and learning and memory impairment 396 therapeutic agents are still required to assist the patients with these diseases. GBR, the natural product was 397 proposed to show the nutraceutical activity as neuroprotection. In this study, the GBR gelatin block was used 398 instead of gavage in long term oral feeding, the amount of food intake can confirm that all mice groups could 399 receive equal amount of GBR treatment. GBR was fed pre-and co-treatments with BCCAo for five weeks (as 400 shown in Figure 1) for studying the protective effect mimic continuous consuming rice daily in human. 34-36. Up till now, the effective 401 In preclinical studies, a suitable animal model with long-lasting and/or progressive cognitive deficits and 402 neuronal damage is very important for the efficacy of potential neuroprotective drugs. To investigate the 403 neuroprotection effect of GBR, this study is the first report for using the modified BCCAo mice model with 67.5% 404 of survival rate, compared with the study of Cechetti (90%) 37. The cerebral ischemia was mild enough and did 405 not disturb the ability of swimming in MWM test because the RMCBS showed recovery of sensori-motor function 406 within 72 hours. This study is agree with previous reports by Combs and D'Alecy 38 that motor deficits showed at 407 24 hours following ischemia and by Capdeville et al 39 that a 50% reduction of sensorimotor functioning in global 408 ischemia rodent models showed at 3 hours after reperfusion. 409 BCCAo is the most common model for understanding the pathophysiology of vascular dementia and 40-41. 410 evaluating the therapeutic potential of drugs The Morris water maze was used to test cognitive function, 411 where the hidden platform trial measured acquisition and the probe trial measured memory retention 24. Five week 412 feeding with 100 mg/kg per day of GBR feeding significantly decreased the latency of carotid occluded mice to 413 reach the platform compared with vehicle control or sham-operated mice and increased the time to spend 414 swimming in the platform quadrant, which suggested that GBR treatment could improve cognitive dysfunction 415 caused by cerebral hypoperfusion. A non-vision dominant TFC test is more applicable since the carotid occlusion 416 may impair retina due to anatomical implication from the ophthalmic branch of internal carotid artery 28. In TFC 417 test, the fear that develops to the cues relates to hippocampal function and the designed trace interval can alter the ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Page 18 of 49 418 circuitry involved in memory formation. A more complex circuitry involves both hippocampus and prefrontal 419 cortex 42. 420 After ischemic insults, the most vulnerable regions for cell death is the hippocampus, which plays a 421 major role in learning and memory 43. Previous studies reported that histological finding of hippocampal CA1 422 pyramidal cells, which are Glutamatergic or aspartatergic, show marked necrosis after transient ischemia 423 Reducing neuronal damage in the hippocampus can improve memory performance in the Morris water maze 45 . 424 In this study, neuropathological alterations in the hippocampus of mice after BCCAo was shown using Nissl 425 staining, but GBR treatment reduced this damage. GBR treatment also attenuated the decrease in the percentage 426 of survival pyramidal cells induced by BCCAo. The pathogenesis of vascular dementia or VCI is still not clear. 427 Klejman et al (2005) 46 reported that the symptoms of VCI may correlate with hippocampal injury induced by the 428 increase of free radicals 44. 429 After the onset of cerebral hypoperfusion could lead to energy depletion in the ischemic-vulnerable 430 neurons, subsequent spontaneous depolarization, release of excitatory neurotransmitter glutamate, triggering 431 glutamate excitotoxicity, subsequently leading to oxidative stress and apoptotic death in post-synaptic neurons. 432 The neuronal death in turn stimulated the astrocytic and microglial activation that can induce the 433 neuroinflammation and more pronounced neuronal death 47 434 Chronic cerebral hypoperfusion caused the damages of membranes and induced lipid peroxidation from 435 free-radical generation 48. In addition, oxidative stress caused the increase in intracellular reactive oxygen species 436 (ROS) contributes to neurodegenerative disorders, including Alzheimer’s disease, and vascular dementia. 437 Disrupted protein synthesis can lead to obvious cognitive deficits 49 438 The suitable GBR intake dose for human can be calculated from the present data of GBR intake in mice 439 (11gram/kg/day of GBR intake). According to the previous reports 50, human equivalent dose was calculated not 440 only from the body weight but also from other factors that account for the difference in metabolic rate: the body 441 surface area, pharmacokinetics and physiological times. Using the equation of the previous reports 50, the 60kg 442 weight for adult human should consume approximately 53-55 gram of GBR per day. This would help conduct a 443 similar human study in a well-designed method thereby providing hope for human health promotion. 444 Highly sensitive to glutamate, an immortalized mouse hippocampal cell line, HT22 cells, has been studied 445 in the neurotoxicity and oxidative stress induced neurodegenerative diseases. In HT22 cells, an increased 446 extracellular glutamate concentration leads to reverse action of cystein-glutamate antiporter, mediating entry of ACS Paragon Plus Environment Page 19 of 49 Journal of Agricultural and Food Chemistry 447 glutamate into the cells and release of cysteine from the cells, thereby depleting the antioxidant gluthatione, 448 causing the oxidative stress, the release of apoptosis inducing factors and eventually the neuronal death. Oxidative 449 stress and the significant drop in glutathione levels can also be found during cerebral ischemia 51. 450 The present data in Figure 5D and 7A pointed that the protective effect of GBR is caused by the activities 451 of not only GABA but also other contents, probably γ –oryzanol and phenolic compounds. Although there is a 452 controversy whether GABA can cross the blood brain barrier , the HPLC analysis of GBR in Zhang et al 52 showed 453 that GBR feeding for 40 days increased GABA level in hippocampus. In the present study, long term feeding GBR 454 for 36 days may increase GABA level in hippocampus. GABA has an inhibitory action on post-synaptic potential, 455 thereby counteracting the glutamate excitotoxicity through chloride influx, preventing cell death in ischemic mice. 456 The phenolic compound and γ-oryzanol found in GBR are potent antioxidant, anti-inflammatory, anti-apoptotic 458 compounds. Both of them can cross the blood brain barrier Like previous reports 53-54, γ-oryzanol might regulate 459 enhance glutathione level. Phenolic compounds might increase in erythropoietin expression in the hippocampus 460 through MEK/ERK/p90RSK signaling pathway 461 receptor 462 compounds might provide some neurotrophic factors that is necessary to protect pyramidal cell death in CA1 and 463 CA3, attenuate the memory deficits in MWM and TFC tests and reduce apoptotic cell death in vitro. 457 quantitative changes on hippocampal proteome to enhance cognition and activate Akt/Nrf2/GCLC pathways to 56, 55 and mimic the actin of BDNF through the tyrosine kinase likewise in previous reports. Hence, the combination effects of GABA, γ-oryzanol and phenolic 464 GABAA receptors are widely available in the hippocampus in vivo since GABAergic inhibitory 465 interneurons are present among pyramidal cells. In vitro, despite there was no previous study that proved GABAA 466 receptors are expressed in HT22 cells, it is possible that pre-treatment of GBR and GABA acts like a 467 differentiating factor to HT22 cells and probably induced the expression of membranous GABA receptors on 468 HT22 cells since GABA has trophic effects on the developing neurons 469 GABA with glutamate, the HT22 cells undergoes glutamate toxicity which is counteracted by GABA in GBR 470 through GABAA receptors because these activities can be blocked by GABAA antagonist, bicuculline. 57-58. With the co-treatments of GBR or 471 In CNS, GABA shows two opposing action on the CNS, depending on mature or immature brain. In 472 mature brain, activation of GABAA hyperpolarizes the post synaptic membrane, generating an inhibitory synaptic 473 response, thereby opposing the action of glutamatergic synapses. On the contrary, in immature brain, activation of 474 GABAA depolarizes the neuron membrane This depolarization regulates the proliferation, migration and 475 differentiation of neural progenitor cells, the elongation of neurites, the formation of synapses, the growth of 476 embryonic and neural stem cells and potentiate the action of glutamatergic synapses 57. Previously reported that ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 477 the switch of GABA function from depolarization to hyperpolarization occurs usually in postnatal day 14 in the 478 mice 59 and GABA level in the hippocampus did not change at 3 weeks and 16 weeks of mice age. For the five- 479 weeks old mice used in the present study, the GABA action is hyperpolarization and oppose the action of 480 glutamatergic synapses. The decrease of GABA caused by injury at this age, GABA supplement might promote 481 health benefits. 482 The current study suggests that GBR showed the neuroprotective effects and this might provide some 483 benefits of GBR consuming instead of white rice daily. Moreover, the implications of this study is the advantage 484 of rice agriculture that can provide better human health by the developed methods for improving the nutritional 485 values that support health benefits whenever, if the similar study can be further conducted and applied in human. 486 The limitation of this study were that the GABA content was not measured in serum and brain to prove the exact 487 amount of GABA in GBR treatment. The molecular mechanism still need further studies for clearly describing 488 how its GABA rescues the hippocampal neuronal death. 489 In conclusion, these findings suggested that GBR provided significant neuroprotection in mice subjected 490 to cerebral hypoperfusion by attenuating vascular cognitive impairments and in glutamate-induced toxicity HT22 491 cells by inhibiting neuronal apoptosis, which is associated with the GABAA receptor. Although the details of how 492 GBR improves cognitive function are not yet clear, this study may provide a new strategy to counter neuronal cell 493 death caused by cerebral hypoperfusion. ACS Paragon Plus Environment Page 20 of 49 Page 21 of 49 Journal of Agricultural and Food Chemistry 494 ABBREVIATIONS 495 GBR, Germinated Brown Rice; GABA, γ-amino-butyric acid; Sh, Sham; CO, Carotid Occlusion; LCCAo, Left 496 common carotid artery occlusion; RCCAo, Right common carotid artery occlusion; RMCBS, Rapid Murine Coma 497 and Behavioral Score; MWM, Morris Water Maze; TFC, Trace fear conditioning. 498 DECLARATIONS 499 Conflicts of interest: The authors declare that they have no conflict of interests. 500 Ethic approval: All experimental procedures were performed according to the protocols approved by 501 the Animal Ethics Committee, Faculty of Medicine Siriraj Hospital, Mahidol University (SI-ACUP 008/2559) 502 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry 503 REFERENCES 504 1. Gurol, M. E., Cerebral hypoperfusion and white matter disease in healthy elderly and patients with Alzheimer's 505 506 disease. Eur J Neurol 2013, 20 (2), 214-5. 2. 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Koh, P.-O., Ferulic acid attenuates the down-regulation of MEK/ERK/p90RSK signaling pathway in focal cerebral ischemic injury. Neuroscience Letters 2015, 588, 18-23. 645 56. Stagni, F.; Giacomini, A.; Guidi, S.; Emili, M.; Uguagliati, B.; Salvalai, M. E.; Bortolotto, V.; Grilli, M.; 646 Rimondini, R.; Bartesaghi, R., A flavonoid agonist of the TrkB receptor for BDNF improves 647 hippocampal neurogenesis and hippocampus-dependent memory in the Ts65Dn mouse model of DS. 648 Experimental neurology 2017, 298 (Pt A), 79-96. ACS Paragon Plus Environment Page 26 of 49 Page 27 of 49 649 650 Journal of Agricultural and Food Chemistry 57. Ganguly, K.; Schinder, A. F.; Wong, S. T.; Poo, M., GABA itself promotes the developmental switch of neuronal GABAergic responses from excitation to inhibition. Cell 2001, 105 (4), 521-32. 651 58. Obrietan, K.; Gao, X. B.; Van Den Pol, A. N., Excitatory actions of GABA increase BDNF expression via a 652 MAPK-CREB-dependent mechanism--a positive feedback circuit in developing neurons. J Neurophysiol 653 2002, 88 (2), 1005-15. 654 59. Levav-Rabkin, T.; Melamed, O.; Clarke, G.; Farber, M.; Cryan, J. F.; Dinan, T. G.; Grossman, Y.; Golan, H. 655 M., A Sensitive Period of Mice Inhibitory System to Neonatal GABA Enhancement by Vigabatrin is 656 Brain Region Dependent. Neuropsychopharmacology 2010, 35 (5), 1138-1154. 657 658 ACKNOWLEDGEMENTS 659 The authors would like to thanks Professor Dr. Prasert Sobhon, Faculty of Science, Mahidol University 660 and Associate Professor Dr. Jutamaad Satayavivad, Chulabhorn Research Institute (CRI) for their important 661 guidance and helpful discussion. For contributing in animal surgery, behavioral tests experiment and 662 reagent/material/analysis tools, special thanks present to Ms. Farhana Padungraksart, Dr. NutthaponYookong and 663 Dr. Tanapol Limboonreun. This work was also supported by the PhD Thesis research fund form MY-NORTH 664 project and Mahidol University. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figures Captions Figure 1. Shown is the timeline of experimental studies. RCCAo, right common carotid artery occlusion; LCCAo, left common carotid artery occlusion; RMCBS, rapid murine coma and behavioral scales; MWM, Morris water maze; TFC, trace fear conditioning test. Figure 2. The total food intake expressed as gram/kg/day (A) was not significantly different among groups. Food containing gelatin blocks has been shown in (B). The average consumed GBR expressed as gram/kg/day (C) and GABA expressed as µg/kg/day (D) were calculated and shown no significant difference between sham-operated and carotid-occluded groups. Figure 3. Body weight gain. In (A), no significant difference of average body weight among groups was shown throughout the experiment. In (B), the significant increase of weight difference only in CO-GBR was found between first and second operation when compared to Sh. Significant difference when compared to Sh; * P<0.05. Significant difference when compared to first operation; ##P<0.01. Figure 4. Memory deficit was evaluated by Morris water maze (A-F) and trace fear conditioning (TFC) test (GI). The swimming path (A) on the last day of hidden platform test was tracked by “Tracker" program. The significant difference were shown in escape latency time of hidden platform tests (day 1 to 4) between Sh and CO groups after GBR (B) and GABA (C) treatments. The comparison among groups were also shown including escape latency time for trial at day 4 (D), quadrant occupancy time (E) for the probe trial test and number of platform crossing (F). Using TFC instrument (G), the significant increase in the percentage of freezing in cue tests after GBR (H) and GABA (I) treatments was shown. Significant difference when compared to Sh; *P <0.05, **P <0.01, ***P<0.001. Significant difference when compared to CO; #P<0.05, ##P<0.01, ###P<0.001. Figure 5. In CA1 and CA3 of dorsal hippocampus, significant increase in percentage of surviving pyramidal neurons (compared with dead neurons) were shown per 135 mm2 (A and C, respectively). The histomorphology of the representative photomicrographs of CA1 (Panel B) and CA3 (Panel D) were shown; surviving neurons with circular nucleus and prominent 1-3 nucleoli, and dead neurons with irregular-shaped, shrunken cytoplasm and extensively dark pyknotic nucleus. Significant difference at ***P<0.001 when compared to Sh, ###P<0.001 when compared to CO. Scale bar is 50µm. Figure 6. Concentration effects of Glutamate (A), GBR and GABA (B) on HT22 cell viability were analyzed by using MTT assay, the obtained optical density was converted into % of cell viability. The standard pure GABA concentration was calculated according to the previous report of GABA content in GBR. Significant difference when compared to 0 mM of glutamate. **P<0.01, ***P<0.001. The protective effect of GBR was compared to ACS Paragon Plus Environment Page 28 of 49 Page 29 of 49 Journal of Agricultural and Food Chemistry GABA against glutamate toxicity (Panel C) on the morphological changes of HT22 cells with/without Bi at 1.25, 12.5 and 125nM. Using MTT assay (D), pre-treatments of GBR for 24 hours followed by another 24 hours of cotreatment with glutamate toxicity were performed on HT22 cell viability. For the study in (E), cells was incubated first with Bi for a few minutes followed by pre- and co-treatment of GBR. **P<0.01 when compared to cell control and #P<0.05, ##P<0.01, ###P<0.001 when compared to glutamate toxicity. Figure 7. Comparison of the percentage of late apoptotic HT22 cells (A) compared among groups were plotted and shown as FACS analysis (panel B). Flow cytometric analysis expressed as 4 quadrants; the percentage of early necrotic (upper left), dead or late apoptotic (upper right), viable cells (lower left) and early apoptotic (lower right) cells, ***P<0.001 when compared to cell control, $$P<0.01 ###P<0.001 when compared to glutamate toxicity, when compared to GBR+Glu, and +++P<0.001 when compared to GABA+Glu. Figure 8. HPLC chromatograms of standard GABA 1.00 µg/mL (A) and GBR sample, PH0001 (B). ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Page 30 of 49 Tables Table 1. Mean Total Score of RMCBS was Recorded at Each Access Point and Expressed as mean±SEM. Sh 19.0 ± 0.3 15.3 ± 0.7 18.7 ± 0.3 Sh-GBR 19.0 ± 0.2 13.3 ± 0.3 18.6 ± 0.8 Sh-GABA 18.7 ± 0.4 12.5 ± 1.5 18.7 ± 0.3 CO 18.6 ± 0.3 8.3 ± 0.6*** 12.2 ±***0.8*** CO-GBR 18.0 ± 0.4 8.4 ± 0.5*** 13.1 ± 1.2*** 13.6 ± 1.2*** 72 hours 18.6 ± 0.4 18.2 ± 1.3 19.0 ± 0.3 17.1 ± 0.7 17.7 ± 1.0 16.6 ± 1.0 Pre-LCCAo 3 hours 24 hours 19.0 ± 0.3 15.5 ± 0.8 18.7 ± 0.4 18.9 ± 0.2 13.5 ± 0.2 18.4 ± 0.6 18.8 ± 0.3 13.1 ± 1.2 18.8 ± 0.3 15.8 ± 2.5 7.7 ± 1.1*** 11.6 ± 0.7*** 18.3 ± 1.2 9.3 ± 1.1*** 12.0 ± 3.2*** 19.2 ± 0.5 8.3 ± 1.0*** 15.9 ± 0.7*** 72 hours 18.7 ± 0.3 18.4 ± 0.9 19.3 ± 0.3 16.6 ± 0.7 16.5 ± 1.5 17.3 ± 1.0 After LCCAo after RCCAo Access points Pre-RCCAo 3 hours 24 hours *** P<0.001 when compared to Sh within the specified access point. ACS Paragon Plus Environment CO-GABA 18.5 ± 0.2 7.2 ± 0.7*** Page 31 of 49 Journal of Agricultural and Food Chemistry Table 2. Amino Acids and GABA Contents in GBR Samples Amino acids and GABA contents (µg/100 mg dry weight) Sample GBR Asp Glu Ser Gln Gly GABA Tyr 4.16 ± 0.03 11.29 ± 0.11 3.47 ± 0.07 0.61 ± 0.04 1.69 ± 0.01 2.84 ± 0.02 2.72 ± 0.04 Limit of detection (LOD) of amino acids and GABA are 0.02 µg/100 mg dry weight. Limit of quantitation (LOQ) of amino acids are 0.10 µg/100 mg dry weight and GABA is 0.05 µg/100 mg dry weight. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figures Graphics Figure 1. ACS Paragon Plus Environment Page 32 of 49 Page 33 of 49 Journal of Agricultural and Food Chemistry Figure 2. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figure 3. ACS Paragon Plus Environment Page 34 of 49 Page 35 of 49 Journal of Agricultural and Food Chemistry Figure 4. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figure 5. ACS Paragon Plus Environment Page 36 of 49 Page 37 of 49 Journal of Agricultural and Food Chemistry Figure 6. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figure 7. ACS Paragon Plus Environment Page 38 of 49 Page 39 of 49 Journal of Agricultural and Food Chemistry Figure 8. ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry TOC (Graphic abstract) ACS Paragon Plus Environment Page 40 of 49 Page 41 of 49 Journal of Agricultural and Food Chemistry TOC (Graphic abstract) 209x147mm (300 x 300 DPI) ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figure 1. Shown is the timeline of experimental studies. RCCAo, right common carotid artery occlusion; LCCAo, left common carotid artery occlusion; RMCBS, rapid murine coma and behavioral scales; MWM, Morris water maze; TFC, trace fear conditioning test. 163x31mm (300 x 300 DPI) ACS Paragon Plus Environment Page 42 of 49 Page 43 of 49 Journal of Agricultural and Food Chemistry Figure 2. The total food intake expressed as gram/kg/day (A) was not significantly different among groups. Food containing gelatin blocks has been shown in (B). The average consumed GBR expressed as gram/kg/day (C) and GABA expressed as µg/kg/day (D) were calculated and shown no significant difference between sham-operated and carotid-occluded groups. 85x230mm (300 x 300 DPI) ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figure 3. Body weight gain. In (A), no significant difference of average body weight among groups was shown throughout the experiment. In (B), the significant increase of weight difference only in CO-GBR was found between first and second operation when compared to Sh. Significant difference when compared to Sh; * P<0.05. Significant difference when compared to first operation; ##P<0.01. 85x152mm (300 x 300 DPI) ACS Paragon Plus Environment Page 44 of 49 Page 45 of 49 Journal of Agricultural and Food Chemistry Figure 4. Memory deficit was evaluated by Morris water maze (A-F) and trace fear conditioning (TFC) test (G-I). The swimming path (A) on the last day of hidden platform test was tracked by “Tracker" program. The significant difference were shown in escape latency time of hidden platform tests (day 1 to 4) between Sh and CO groups after GBR (B) and GABA (C) treatments. The comparison among groups were also shown including escape latency time for trial at day 4 (D), quadrant occupancy time (E) for the probe trial test and number of platform crossing (F). Using TFC instrument (G), the significant increase in the percentage of freezing in cue tests after GBR (H) and GABA (I) treatments was shown. Significant difference when compared to Sh; *P <0.05, **P <0.01, ***P<0.001. Significant difference when compared to CO; #P<0.05, ##P<0.01, ###P<0.001. 175x187mm (300 x 300 DPI) ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figure 5. In CA1 and CA3 of dorsal hippocampus, significant increase in percentage of surviving pyramidal neurons (compared with dead neurons) were shown per 135 mm2 (A and C, respectively). The histomorphology of the representative photomicrographs of CA1 (Panel B) and CA3 (Panel D) were shown; surviving neurons with circular nucleus and prominent 1-3 nucleoli, and dead neurons with irregular-shaped, shrunken cytoplasm and extensively dark pyknotic nucleus. Significant difference at ***P<0.001 when compared to Sh, ###P<0.001 when compared to CO. Scale bar is 50µm. 177x194mm (300 x 300 DPI) ACS Paragon Plus Environment Page 46 of 49 Page 47 of 49 Journal of Agricultural and Food Chemistry Figure 6. Concentration effects of Glutamate (A), GBR and GABA (B) on HT22 cell viability were analyzed by using MTT assay, the obtained optical density was converted into % of cell viability. The standard pure GABA concentration was calculated according to the previous report of GABA content in GBR. Significant difference when compared to 0 mM of glutamate. **P<0.01, ***P<0.001. The protective effect of GBR was compared to GABA against glutamate toxicity (Panel C) on the morphological changes of HT22 cells with/without Bi at 1.25, 12.5 and 125nM. Using MTT assay (D), pre-treatments of GBR for 24 hours followed by another 24 hours of co-treatment with glutamate toxicity were performed on HT22 cell viability. For the study in (E), cells was incubated first with Bi for a few minutes followed by pre- and co-treatment of GBR. **P<0.01 when compared to cell control and #P<0.05, ##P<0.01, ###P<0.001 when compared to glutamate toxicity. 209x297mm (300 x 300 DPI) ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry ACS Paragon Plus Environment Page 48 of 49 Page 49 of 49 Journal of Agricultural and Food Chemistry Figure 7. Comparison of the percentage of late apoptotic HT22 cells (A) compared among groups were plotted and shown as FACS analysis (panel B). Flow cytometric analysis expressed as 4 quadrants; the percentage of early necrotic (upper left), dead or late apoptotic (upper right), viable cells (lower left) and early apoptotic (lower right) cells, ***P<0.001 when compared to cell control, ###P<0.001 when compared to glutamate toxicity, $$P<0.01 when compared to GBR+Glu, and +++P<0.001 when compared to GABA+Glu. 210x203mm (300 x 300 DPI) ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Figure 8. HPLC chromatograms of standard GABA 1.00 µg/mL (A) and GBR¬¬ sample, PH0001 (B). 175x156mm (300 x 300 DPI) ACS Paragon Plus Environment Page 50 of 49