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Introduction
Bioactive peptides produced from natural sources have been widely investigated. The studies presented the possible roles of food-derived bioactive peptides in decreasing the risk of different kinds of diseases, especially neurodegenerative disease.1 Most of the protein isolates and hydrolysates are commonly being prepared from protein-rich sources like soy and whey.2,3 There have been relatively few studies that have focused on examining the bioactive peptide properties of rice. Rice (Oryza sativa L.) is the main food of Thai population and a cheap product having nutrients including vitamins, minerals and fiber. Pigmented rice is a type of rice grown in Thailand recently utilized as a healthy food. An outer layer of pigmented rice is a good source of fat, protein and antioxidants. Serveral studies reported that peptide-derived from pigmented rice bran contains large amounts of fiber and bioactive phytochemicals such as, tocopherols, tocotrienols, oryzanols, vitamin B complex and phenolic compounds.4-6 A study showed the pigmented rice could significantly prevent memory impairment and hippocampal neurodegeneration in hippocampus.7 The objective of this study was to investigate the effects of protein hydrolysate type A-2 from pigmented rice on learning and memory in b-amyloid injected rats.
Materials and Methods
Plant materials and reagents
Hydrolysate peptide type A-2 from pigmented rice was studied and prepared by Prof. Dr. Bungorn Sripanidkulchai and the Center for Research and Development of Herbal Health Products (CRD-HHP) at Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand.
Animals and treatments
All experiments were conducted under the National-Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Khon Kaen University (Approval No. 0514.1.12.2/76). Male Wistar rats weighing 180 220 grams were used in this study. Rats were maintained on a 12-h light/dark cycle with free access to commercial food pellets and drinking water. All rats were trained with the novel object recognition (NOR) test for five days to assess their learning ability and were divided into six groups (n = 9): Sham vehicle, vehicle plus Ab, donepezil plus Ab, HPR10 plus Ab, HPR20 plus Ab and HPR40 plus Ab. All treatments were performed by gastric gavage with biomedical needles at 8.00 to 9.00 a.m. for 56 consecutive days. At day 56, the rats in groups 2-6 were injected with Ab25-35 into both sides of the lateral ventricle, whereas the vehicle rats in the first group received the sham injections. Ten days after Ab25-35 injection, the rats were tested for the behavioral performance using NOR tasks (Figure 1).
Figure 1 Timeline of drug administration and behavioral tests. (Aβ: β‐amyloid (25‐35), D: donepezil, HPR: protein hydrolysate type A-2 from pigmented rice, NOR: novel object recognition, V: vehicle)
Ab25-35 Treatment
All surgical procedures were conducted under aseptic conditions and sodium pentobarbital (35 mg/kg b.w., i.p., Sigma-Aldrich, Germany) anesthesia. Rats were maintained in a stereotaxic holder. A midline sagittal incision was made in the scalp and two holes were drilled in the skull over the lateral ventricles using the following coordinates: AP = −0.8 mm, L = ±1.5 from the bregma.8-10 The dura was perforated with the needle of the microsyringe, which was inserted 3.8 mm beneath the dura mater. Two microliters of either aggregated Ab25-35 were injected into both sides of lateral ventricles. After surgery, the animals were returned to their home cages.
Novel object recognition (NOR)
The NOR test was used to examine hippocampus-dependent memory.11 Briefly, rats were habituated in an open-field arena for 5 minutes (min) before training. During the training phase, two identical objects were placed in two locations and each rat was allowed to explore the objects for 5 min. In the testing phase, one of the objects was replaced with a novel object. Rats were placed again in the apparatus to explore the objects for 5 min after a retention interval of 5 min and a 24 h delay (Figure 1). Each groups ability to recognize the novel object was determined using a discrimination index (DI) calculated for each animal using the formula12: N - F/N + F, which corresponds to the difference between the time exploring the novel (N) and the familiar object (F), corrected for total time exploring both objects. The result can vary between +1 and -1, where a positive score indicates more time spent with the novel object, a negative score indicates more time spent with the familiar object, and a zero score indicates a null preference.
Statistical analysis
All data were expressed as mean standard error of the mean (S.E.M.). Statistical analysis of the experimental data was carried out using GraphPad Prism (version 5). Significance of differences among groups were analyzed using a one-way ANOVA and a Newman-Keuls post hoc test. The criterion for statistical significance was p< 0.05.
Results
Effect of hydrolysate peptide type A-2 on recognition memory
During training session, the results showed no significant difference in the time spent exploring the two identical objects (Figure 2). In the first and second period (delay 5 min, 24 h), Ab25-35 exhibited cognitive deficits by decreasing the discriminative index (Figure 3, 4). When compare to the V+Ab group, HPR20-, HPR40-treated rats showed a significantly higher discriminative index during 5 min and 24 hours delay testing phase.
Figure 2 Effects of HPR type A-2 on memory performance in Ab-induced rats as measured by the discrimination index (DI) in the training phase of NOR task. Data are presented as mean ± S.E.M. (n = 9/group).
Figure 3 Effects of HPR type A-2 on memory performance in Ab-induced rats as measured by the discrimination index (DI) after a five-minutes delay of NOR task. Data are presented as mean ± S.E.M. (n = 9/group), * = significant differences from vehicle+Ab group at p < 0.05.
Figure 4 Effects of HPR type A-2 on memory performance in Ab-induced rats as measured by the discrimination index (DI) after 24-hours delay of NOR task. Data are presented as mean ± S.E.M. (n = 9/group), * = significant differences from vehicle+Ab group at p < 0.05.
Discussion
Previous studies have shown that pigmented rice could enhance the cognitive performance as well as reverse the deleterious effects of brain ageing.7 The present study used the novelty of exploration to investigate the effect of HPR type A-2 on recognition based on the amount of time that rats spend exploring the presented two objects.13 Recognition memory is the ability of an individual to recognize the previous events or objects. The novel environment is matched with the previously stored memory for the identification.14 In line with previous studies, we observed that Ab caused deterioration in the NOR test both in short- and long-term memory in rat.15,16 When HPR type A-2 plus Aβ was administered, it took longer for the rats to recognize the novel object compared to the familiar object indicating that HPR type A-2 had a neuroprotective effect on the Aβ25-35-injected rats. However, this neuroprotective effect was not detected at low doses (10 mg/kg BW). This is probably due to this dose of HPR type A-2 extract is not sufficiency to search the therapeutic level. During training periods, rats in all groups did not show significant difference among the two identical objects. An alternative possibility is that since both objects were new objects, rats can not separate the objects (Figure 2).
Conclusion
These findings do provide initial evidence that HPR type A-2 may be benefit to be used for neuroprotective effect.
Acknowledgement
This study was supported by Agricultural Research Development Agency (Public Organization), Thailand and the Center for Research and Development of Herbal Health Products, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, Thailand.
References
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