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In vivo investigation on the potential of galangin, kaempferol and myricetin for protection of d -galactose-induced cognitive impairment
Food Chemistry 135 (2012) 2702–2707
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
In vivo investigation on the potential of galangin, kaempferol and myricetin for protection of D-galactose-induced cognitive impairment Yongfang Lei a, Jinglou Chen b, Wenting Zhang a, Wei Fu a, Guanghua Wu a, Han Wei b, Qing Wang c, Jinlan Ruan b,⇑ a
Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Key Laboratory of Natural Medicinal Chemistry and Resources Evaluation of Hubei Province, College of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China c Laboratory of Wuhan Botanical Garden, The Chinese Academy of Sciences, Wuhan, China b
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Article history: Received 7 September 2011 Received in revised form 8 June 2012 Accepted 5 July 2012 Available online 15 July 2012 Keywords: Neuroprotective nature Flavonols Extracellular signal-regulated kinases (ERK) Cyclic AMP response element binding protein (CREB) Oxidative stress
a b s t r a c t The potential of three natural flavonols (galangin, kaempferol and myricetin) to protect against D-galactose-induced cognitive impairment in mice was investigated. After 8 weeks treatment, the mice were assessed by behavioural tests. The levels of oxidative stress, the amount of Na+,K+-ATPase and extracellular signal-regulated kinases (ERK)-cyclic AMP response element binding protein (CREB) signaling pathway in hippocampus were also analysed. It was found that all the three dietary flavonols could ameliorate the oxidative stress, enhance the activity of Na+,K+-ATPase and regulate the expression of ERK-CREB pathway in mice. However, only kaempferol and myricetin could significantly improve the learning and memory capability when compared with D-galactose model. Our results suggest that the presence of hydroxyl groups in the B ring of flavonols may have contribution to the neuroprotective activity. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In nowadays, increased numbers of people are obsessed by ageing and neurological deficits all over the world. The cognitive damage often hampers the patients’ routine duties and daily lives. The free radical damage and the resultant cellular dysfunctions are believed to be major causes of neurodegenerative disorders such as Alzheimer’s disease (AD) (Jeong et al., 2011; Zurich & MonnetTschudi, 2009). It has been approved that the hippocampus is responsible for the regulation of learning and memory (El-Omri et al., 2010). Unfortunately, the hippocampus is considered as the most vulnerable brain region to oxidative stress (Yargicoglu, Sahin, Gümüsßlü, & Ag˘ar, 2006). Under physiological condition, the reactive oxygen species (ROS) are scavenged by antioxidant enzymes in vivo. However, when the antioxidant defence is reduced, the neuronal cell will be easily attacked by excess levels of ROS (Huang et al., 2007). Furthermore, oxidative stress modulates a number of signaling targets in nervous system including the extracellular signal-regulated kinases 1/2 (ERK1/2) and the cyclic AMP response ⇑ Corresponding author. Address: Key Laboratory of Natural Medicinal Chemistry and Resources Evaluation of Hubei Province, College of Pharmacy, Huazhong University of Science and Technology, No. 13 Hangkong Rd, Wuhan 430030, Hubei Province, China. Fax: +86 27 83692762. E-mail address: [email protected] (J. Ruan). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.07.043
element binding protein (CREB) Zhang and Jope (1999). Both of them belong to the mitogen-activated protein kinase (MAPK) signaling system, which is highly expressed in postmitotic neurons of the adult nervous system (Haddad, 2005). Flavonols, which belong to the large natural antioxidants group of flavonoids, are widely presented in habitual food such as onions, leeks and broccoli (Spencer, Vauzour, & Rendeiro, 2009). Previous studies indicate that flavonols have great antioxidant activity and can prevent the decrease in the activities of antioxidant enzymes in vivo (Jeong et al., 2011; Škerget et al., 2005). It has also been demonstrated that flavonols can inhibit the age-related cognition declines through stimulating the expression of ERK1/2 (Spencer et al., 2009). Therefore, the natural flavonols represent a huge source of potential agents with neuroprotective properties. Galangin (3,5,7-trihydroxyflavone), kaempferol (3,5,7,40 -tetrahydroxyflavone), and myricetin (3,5,7,30 ,40 ,50 -hexahydroxyflavone) are widely distributed in the human daily diet such as fruits, beverages, tea and vegetables (Aherne & O’Brien, 2002). Kaempferol protects PC12 cells against the oxidative stress induced by H2O2 (Hong et al., 2009). Plants rich in myricetin also show potential neuroprotective effects (Yao & Vieira, 2007). Furthermore, it was reported that the intake of flavonols including quercetin, kaempferol and myricetin has favourable effect on the cognitive performance (Spencer et al., 2009).
Y. Lei et al. / Food Chemistry 135 (2012) 2702–2707
The chemical structures of galangin, kaempferol and myricetin are shown in Fig. 1. It is obvious to notice that the three molecules have similar structures with two aromatic rings (A and B). The only difference, as shown in Fig. 1, exists in the numbers of hydroxyl group in B ring, which structure is believed to be closely related with the biological activities of flavonols (Karawajczyk et al., 2006). Although quite a lot of research has been carried out on galangin, kaempferol and myricetin, however, seldom work has been carried out on the neuroprotective effects of the three above-mentioned flavonols or the possible relationship between the structure and bioactivity, for instance, the effect of the number of hydroxyl groups in the B ring on the neuroprotective function. Therefore, in the present study, the potential of the three dietary flavonols was systematically studied in vivo. As high levels of D-galactose (D-gal) can be oxidised into free radicals and induce impairment of neurogenesis in the hippocampus, D-gal induced senescent mice, which considered as a good model for studying neurodegenerative diseases, were applied in the present study (Sun et al., 2007; Zhong, Ge, Qu, Li, & Ma, 2009). The aim of this work was to investigate the protective effect of the three dietary flavonols on D-galinduced cognitive impairment in mice. This work also made preliminary exploration of whether the number of hydroxyl groups in the B ring of flavonols would influence their neuroprotective nature.
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lege, Huazhong University of Science and Technology, Wuhan, China. The animals were housed in a controlled room (temperature 22 ± 3 °C and humidity 50 ± 10%) and were kept under 12 h light:12 h dark cycle. The mice were fed with standard diet and water ad libitum and acclimated 7 days before they were used for the study. After acclimatisation to the laboratory conditions, the mice were randomly divided into five groups (n = 10): the vehicle control group (C), the D-gal model group (DM), the galangin treated group (GT), the kaempferol treated group (KT), and the myricetin treated group (MT). D-gal at the dose of 150 mg/kg once a day for 8 weeks was subcutaneously injected to the mice in DM, GT, KT, and MT groups. PBS buffer with the same volume was given to those of vehicle control group. Mice of GT, KT, and MT groups were orally administered with galangin, kaempferol, and myricetin respectively, at 100 mg/kg/d 1 h after the injection of D-gal. At the same time, the mice of the vehicle control group and the D-gal model group were treated with same volume of physiological saline (with 0.1% Tween 80) for 8 weeks. At the end of the treatment period, behavioural tests and biochemical measurements were performed as follows. All experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the Huazhong University of Science and Technology Committee on Animal Care and Use. 2.3. Behavioural tests
2. Materials and methods 2.1. Reagents Galangin, kaempferol, and myricetin (purity > 98%) were purchased from Baoji Biological Development Co., Ltd., (Baoji, China). D-gal was purchased from Sigma–Aldrich (St. Louis, MO, USA). The commercial kits used for the determination of thiobarbituric acid reactive substances (TBA-RS), superoxide dismutase (SOD), and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) were purchased from Jiancheng Institute of Biotechnology (Nanjing, China). The total protein extraction kit was purchased from ProMab (Richmond, CA, USA). The BCA kit used for the determination of protein content was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The antibodies used for western blot were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other solvents and chemicals used in the study were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Animals and administration The Kunming mice, weighing 18–22 g and 5 weeks old, were obtained from the Experimental Animal center, Tongji Medical Col-
All behavioural tests were conducted at the same time of the day (9:00 am to 6:00 pm) for 7 days. Morris water maze (MWM) test, which performed as previously described was applied to evaluate the learning and memory capability of mice (Sun et al., 2007). The experimental apparatus consisting of a circular water tank (120 cm in diameter, 50 cm in height) was filled with water (22 ± 1 °C) to a depth of 30 cm. The water was rendered opaque by adding black nontoxic carbon ink. The water maze was conceptually divided into four equal quadrants (east, south, west, and north). A black platform (10 cm in diameter, 29 cm in height) was submerged 1 cm below the water surface and placed at the midpoint of east quadrant. The mice were placed in the water facing the pool wall at one of the three starting quadrant points (south, west, and north). They were allowed to swim for 60 s to find the hidden platform and rest on it for 15 s. Mice that did not reach the platform within the given time were gently guided to the platform and also allowed to rest for 15 s. The time of the latency to reach the platform was recorded. The mean data from daily test were used for statistical analysis. To determine whether the animal would take a spatial learning strategy to locate the platform, a single spatial probe trial was assessed on the 7th day. The platform was removed from the water tank, and the mice were allowed to swim freely from different start quadrant points (south, west, and north), each for 60 s. The time spent in the target quadrant (east quadrant), the distance moved in the target quadrant, the crossing number of the platform (the place where the platform should be) and the time of first crossing the platform were measured. The records were analysed by a computerised video imaging analysis system (EthoVision, Noldus Information Technology BV, Wageningen, The Netherlands). 2.4. Preparation of tissue samples
Fig. 1. The structures of galangin, kaempferol and myricetin.
The animals were deeply anaesthetised and sacrificed by decapitation 60 min after behavioural testing. Brains were quickly removed and dissected on ice to obtain the hippocampus. One half of the hippocampus tissues were homogenised in ice-cold physiological saline. The homogenate (10%) was centrifuged at 4000g at 4 °C for 10 min, and the supernatant was used for biochemical
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assay. The other half of hippocampus tissues were stored at 80 °C until the western blot analysis. 2.5. Biochemical analysis Protein concentration of each sample was determined based on bicichoninic acid (BCA) method where bovine serum albumin was used as the standard (Jeong et al., 2005). The levels of TBARS and SOD were measured as previous reported (Zhong et al., 2009). Briefly, in the hippocampus tissues, the lipid peroxidation process may form malondiadehyde and other TBARS, and these TBARS can be measured at the wavelength of 532 nm by reacting with TBA to form a stable chromophoric production. Tetraethoxypropane (10 nmol/mL) was used as the standard. The assay for total SOD was based on its ability to inhibit the oxidation of oxymine. The absorbance was measured at 550 nm. The activity of Na+,K+-ATPase was measured based on the recommended method and the absorbance was measured at 660 nm (Sun et al., 2007). All the manipulations followed the directions of the commercially available kits. The activities of SOD and Na+,K+-ATPase were expressed as U/mg protein, and the concentration of TBARS was expressed as nmol malondialdehyde/mg protein. 2.6. Western blot analysis The hippocampal expressions of ERK1/2, phopho-ERK1/2, CREB and phospho-CREB were evaluated by western blot analysis (Lei et al., 2011). The tissue samples were triturated in liquid nitrogen and the total protein was extracted using a protein extraction kit. Protein concentrations were determined using the BCA protein assay kit. The two kits were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Protein samples (50 lg) were separated by 12% SDS–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane (Roche Diagnostics Corporation, Indianapolis, IN, USA) by electrophoretic transfer (Bio-Rad Laboratories, Inc., Benicia, CA, USA). Transferred membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), and then incubated overnight at 4 °C with different primary antibodies as follows: anti-ERK1/2 (1:500), anti-CREB (1:500), anti-phopho-ERK1/2 (1:400), or anti-phospho-CREB (1:400). After washing by TBST for three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies in TBST with 3% nonfat milk for 1 h at room temperature. Immunoblots were developed on films using the enhanced chemiluminescence technique (Super Signal West Pico; Pierce Biotechnology, Rockford, IL, USA). Quantification of bands was determined by densitometric analysis using Quantity One imaging software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The data were normalised using GAPDH (1:1000) as an internal control. 2.7. Statistical analysis The values were presented as mean ± S.D. Results were analysed statistically by one-way ANOVA followed by Tukey’s multiple comparison using SPSS 11.5 Software for Windows. Differences were considered as significant at p < 0.05. Figures were made using Origin 6.0 Software for Windows. 3. Results 3.1. Morris water maze test Fig. 2A indicates the latencies for mice in different groups to reach the hidden platform: day, F (5, 280) = 21.000, p < 0.01; group,
F (4, 280) = 18.724, p < 0.01; day by group interaction, F (20, 280) = 0.934, p = 0.544; 2-way ANOVA. During the training period, the time that mice spent to find the hidden platform from 0th to 6th day was decreased. However, the vehicle control showed continuous decrease while D-gal model only slightly reduced the time for finding the platform which indicating that treatment with D-gal for 8 weeks could induce noticeable neurologic impairment and cognitive impairment. Our results also demonstrated that both kaempferol and myricetin (100 mg/kg/d) could prevent the D-gal induced platform finding impairment as mice from KT and MT groups took less time to find the platform in all the 6 training days as compared with D-gal model. The platform crossing number, the time of first crossing the platform, the time spent in the target quadrant and the distance moved in the target quadrant after the 6 training days were recorded to evaluate the spatial learning and memory ability of mice on the 7th day, as shown in Fig. 2B–E. It can be seen that while the moving time and swimming distance of the D-gal model mice were remarkably decrease as compared to those of the vehicle control (p < 0.05), the time of first crossing was significantly (p < 0.05) longer with fewer platform crossings numbers in the target quadrant. The impairment in the spatial learning and memory ability by D-gal was attenuated by the dietary flavonols. Treatment with 100 mg/kg/d kaempferol or myricetin for 8 weeks could obviously (p < 0.05) increase the number of platform crossings, the time and distance spent in the target quadrant, while also reduced the time of first crossing. However, there was no significant difference between the mice of KT and MT. On the other hand, although galangin could slightly ameliorate the spatial learning and memory ability of mice, it failed to show a significant effect when compared with the D-gal model (p > 0.05). 3.2. Biochemical analysis Effects of galangin, kaempferol and myricetin on the levels of TBARS, and the activities of SOD and Na+,K+-ATPase in hippocampus of senescent mice induced by D-gal are shown in Table 1. DGal resulted in a significant decrease in the activities of Na+,K+ATPase and SOD and increase in the levels of TBARS as compared with vehicle control. All the three flavonols could produce significant (p < 0.05) effects on elevating the activities of SOD and Na+,K+ATPase, as well as on reducing the TBARS levels in mice. However, there were no significant difference (p > 0.05) among galangin, kaempferol and myricetin. 3.3. Western blot analysis Fig. 3 shows the effects of galangin, kaempferol and myricetin on the regulation of the expression for ERK1/2-CREB pathway. Dgal inhibited the expressions of phospho-ERK1/2 and phosphoCREB when compared with the vehicle control. Galangin, kaempferol and myricetin significantly (p < 0.05) activated the phosphorylations of ERK1/2 and CREB. It is also worth noting that the more number of hydroxyl group is within B ring of the three flavonol molecules, the more noticeable level of the ERK1/2-CREB expression was noticed (p < 0.05). 4. Discussion D-gal-induced neurodegeneration model was applied in this study to investigate the neuroprotective activities of three natural dietary flavonols (galangin, kaempferol and myricetin). In general, the results showed that 8 weeks administration of kaempferol or myricetin can obviously attenuate the D-gal-induced cognitive impairment in mice.
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Fig. 2. Effects of galangin, kaempferol and myricetin on the spatial learning and memory ability of mice. Values are expressed as means ± S.D. from each group (n = 8–10). Values sharing the same letter (a, b, c) in each row do not differ significantly at p < 0.05 (SPSS). (A) The time mice spent to find the hidden platform from 0th to 6th day. (B) The platform crossing number of mice on the 7th day. (C) The time of mice spent in the target quadrant on the 7th day. (D) The time of mice first crossing the platform on the 7th day. (E) The distance of mice moved in the target quadrant on the 7th day.
Neurodegenerative disease is a complex pathophysiological process. It is generally suggested that the oxidative stress plays a key role in the nerve damage (Jeong et al., 2011). Previous studies show that ROS participates in neurodegenerative diseases and the oxidative metabolism is very active in brain (Yao & Vieira, 2007; Zhong et al., 2009). In normal cases, the generation and elimination
of ROS is in a dynamic balance. It was reported that superoxide anion, a major free radical, is mainly catalysed into H2O2 by SOD in vivo (Palsamy, Sivakumar, & Subramanian, 2010). However, when the balances were disrupted, the activity of antioxidase will decrease and the levels of ROS will increase. The excessive production of ROS will impair brain functions, especially the hippocampus
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Table 1 Effects of three dietary flavonols on the levels of SOD, TBARS and Na+,K+-ATPase in hippocampus of senescent mice induced by D-gal. Vehicle control SOD (U/mg protein) TBARS (nmol/mg protein) Na+,K+-ATPase (U/mg protien)
a
75.97 ± 4.73 4.30 ± 0.32a 2.60 ± 0.43a
D-gal
Galangin
model b
40.69 ± 6.38 7.00 ± 1.68b 1.32 ± 0.28b
Kaempferol c
54.50 ± 4.50 5.63 ± 0.55c 1.90 ± 0.59c
Myricetin d
62.41 ± 6.02 5.22 ± 0.55c 2.14 ± 0.59c
60.00 ± 6.02cd 5.05 ± 0.43c 1.80 ± 0.35c
Values are given as means ± SD from 8 mice in each group. a,b,c,dValues sharing a common superscript (a, b, c, d) in a row do not differ significantly at p < 0.05 (SPSS). Both c and d do not differ significantly with cd.
Fig. 3. Effects of galangin, kaempferol and myricetin on the expressions of ERK1/2, phospho-ERK1/2, CREB and phospho-CREB in hippocampus. Values are expressed as means ± S.D. from each group (n = 8). Values sharing the same letter (a, b, c, d, e) in each row do not differ significantly at p < 0.05 (SPSS). (A) Representative western blot images. (B) The p-ERK/ERK ratios in mice hippocampus. (C) The p-CREB/CREB ratios in mice hippocampus.
which is mainly responsible for learning and memory (Yargicoglu et al., 2006). Furthermore, it is necessary for the brain to maintain relevant cellular Na+/K+ gradients for normal nerve impulse propagation, neurotransmitter release and cation homeostasis, as Na+,K+-ATPase is an imperative enzyme for sustaining the Na+/K+ gradients (Sun et al., 2007). Unfortunately, as a consequence of oxidative damage, the activity of Na+,K+-ATPase decreases and finally leads to the impairment in learning and memory (Zhong et al., 2009). In this study, daily treatment of galangin, kaempferol or myricetin for 8 weeks can obviously increase the activities of SOD and Na+,K+-ATPase, and decreased the levels of TBARS in hippocampus when compared with D-gal model. In addition, evidence shows that MAPKs are highly expressed in postmitotic neurons of the adult nervous system and are necessary for maintaining normal nervous functions (Hong et al., 2009). MAPKs are activated in response to growth factor stimulation of cells in culture, and fall into four different subgroups: the ERK1/2 MAPK, the Jun N-terminal kinases (JNKs) MAPK, the p38 MAPK and the ERK5 MAPK (Plotnikov, Zehorai, Procaccia, & Seger, 2011). The ERK1/2 is one of the most-studied members and mainly found in the hippocampus (Qi et al., 2008). It is documented that phosphorylation of ERK1/2 influences the survival, differentiation and cycle regulation of many nerve cells (El-Omri et al., 2010; Hong et al., 2009). As a result, the activation ERK1/2 MAPK pathway is necessary for nervous system functions. CREB is a major downstream target of ERK1/2 MAPK pathway and also critical for a lot of cellular processes including proliferation and adaptive responses (Zhou, Yao, Zhang, & Chang, 2002). Recent studies have shown that the phosphorylation of CREB is necessary for the induction of the ERK-dependent plasticity (Qi et al., 2008). The activation of ERK1/2-CREB pathway is a necessary step for the learning and memory (Atkins, Falo, Alonso, Bramlett, & Dietrich, 2009; Haddad, 2005). Our western blotting observations show that galangin, kaempferol and myricetin can treat D-gal-induced impairment of ERK1/2 and CREB activations in mice hippocampus, suggesting that the three natural flavonols were able to exert neuroprotective effects via activating the phosphorylations of ERK1/2 and CREB. The current investigations indicated that all of the three natural flavonols could reduce the level of oxidative stress and enhance the
Na+,K+-ATPase activity in vivo. On the other hand, galangin, kaempferol and myricetin could activate the phosphorylations of ERK1/2 and CREB. It was worthy of notice that, when the number of hydroxyl group in the B ring (galangin, kaempferol and myricetin) increased, the flavonols up-regulated the expression of ERK1/ 2-CREB pathway in an obviously incremental manner (p < 0.05). These findings suggested that the hydroxyl in the B ring played an important role in up-regulating the hippocampus expression of ERK1/2-CREB pathway. In the Morris water maze test, although all the three flavonols exerted positive effects to protect mice from D-gal-induced neurologic impairment, galangin failed to show a significant difference (p > 0.05) on attenuating the cognitive impairment when compared with the D-gal model group. However, kaempferol and myricetin did obviously (p < 0.05) enhance the spatial learning and memory ability of mice and there was no significant difference (p > 0.05) between kaempferol and myricetin treated mice. In summary, kaempferol and myricetin are able to significantly attenuate D-gal-induced cognitive impairment in mice via up-regulating the expression of ERK1/2-CREB pathway, enhancing the activities of Na+,K+-ATPase and reducing the level of oxidative stress in hippocampus. The presence of hydroxyls in B ring of flavonols is necessary for their neuroprotective activity and the increase in the number of hydroxyl group also contributed to up-regulation of the hippocampus expression of ERK1/2-CREB pathway in the Dgal-induced cognitive impairment mice. Acknowledgements This study was financially supported by The state natural science fund (NO30973864). References Aherne, S. A., & O’Brien, N. M. (2002). Dietary flavonols: chemistry, food content, and metabolism. Nutrition, 18, 75–81. Atkins, C. M., Falo, M. C., Alonso, O. F., Bramlett, H. M., & Dietrich, W. D. (2009). Deficits in ERK and CREB activation in the hippocampus after traumatic brain injury. Neuroscience Letters, 459, 52–56. El-Omri, A., Han, J., Yamada, P., Kawada, K., Ben-Abdrabbah, M., & Isoda, H. (2010). Rosmarinus officinalis polyphenols activate cholinergic activities in PC12 cells
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
In vivo investigation on the potential of galangin, kaempferol and myricetin for protection of D-galactose-induced cognitive impairment Yongfang Lei a, Jinglou Chen b, Wenting Zhang a, Wei Fu a, Guanghua Wu a, Han Wei b, Qing Wang c, Jinlan Ruan b,⇑ a
Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Key Laboratory of Natural Medicinal Chemistry and Resources Evaluation of Hubei Province, College of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China c Laboratory of Wuhan Botanical Garden, The Chinese Academy of Sciences, Wuhan, China b
a r t i c l e
i n f o
Article history: Received 7 September 2011 Received in revised form 8 June 2012 Accepted 5 July 2012 Available online 15 July 2012 Keywords: Neuroprotective nature Flavonols Extracellular signal-regulated kinases (ERK) Cyclic AMP response element binding protein (CREB) Oxidative stress
a b s t r a c t The potential of three natural flavonols (galangin, kaempferol and myricetin) to protect against D-galactose-induced cognitive impairment in mice was investigated. After 8 weeks treatment, the mice were assessed by behavioural tests. The levels of oxidative stress, the amount of Na+,K+-ATPase and extracellular signal-regulated kinases (ERK)-cyclic AMP response element binding protein (CREB) signaling pathway in hippocampus were also analysed. It was found that all the three dietary flavonols could ameliorate the oxidative stress, enhance the activity of Na+,K+-ATPase and regulate the expression of ERK-CREB pathway in mice. However, only kaempferol and myricetin could significantly improve the learning and memory capability when compared with D-galactose model. Our results suggest that the presence of hydroxyl groups in the B ring of flavonols may have contribution to the neuroprotective activity. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction In nowadays, increased numbers of people are obsessed by ageing and neurological deficits all over the world. The cognitive damage often hampers the patients’ routine duties and daily lives. The free radical damage and the resultant cellular dysfunctions are believed to be major causes of neurodegenerative disorders such as Alzheimer’s disease (AD) (Jeong et al., 2011; Zurich & MonnetTschudi, 2009). It has been approved that the hippocampus is responsible for the regulation of learning and memory (El-Omri et al., 2010). Unfortunately, the hippocampus is considered as the most vulnerable brain region to oxidative stress (Yargicoglu, Sahin, Gümüsßlü, & Ag˘ar, 2006). Under physiological condition, the reactive oxygen species (ROS) are scavenged by antioxidant enzymes in vivo. However, when the antioxidant defence is reduced, the neuronal cell will be easily attacked by excess levels of ROS (Huang et al., 2007). Furthermore, oxidative stress modulates a number of signaling targets in nervous system including the extracellular signal-regulated kinases 1/2 (ERK1/2) and the cyclic AMP response ⇑ Corresponding author. Address: Key Laboratory of Natural Medicinal Chemistry and Resources Evaluation of Hubei Province, College of Pharmacy, Huazhong University of Science and Technology, No. 13 Hangkong Rd, Wuhan 430030, Hubei Province, China. Fax: +86 27 83692762. E-mail address: [email protected] (J. Ruan). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.07.043
element binding protein (CREB) Zhang and Jope (1999). Both of them belong to the mitogen-activated protein kinase (MAPK) signaling system, which is highly expressed in postmitotic neurons of the adult nervous system (Haddad, 2005). Flavonols, which belong to the large natural antioxidants group of flavonoids, are widely presented in habitual food such as onions, leeks and broccoli (Spencer, Vauzour, & Rendeiro, 2009). Previous studies indicate that flavonols have great antioxidant activity and can prevent the decrease in the activities of antioxidant enzymes in vivo (Jeong et al., 2011; Škerget et al., 2005). It has also been demonstrated that flavonols can inhibit the age-related cognition declines through stimulating the expression of ERK1/2 (Spencer et al., 2009). Therefore, the natural flavonols represent a huge source of potential agents with neuroprotective properties. Galangin (3,5,7-trihydroxyflavone), kaempferol (3,5,7,40 -tetrahydroxyflavone), and myricetin (3,5,7,30 ,40 ,50 -hexahydroxyflavone) are widely distributed in the human daily diet such as fruits, beverages, tea and vegetables (Aherne & O’Brien, 2002). Kaempferol protects PC12 cells against the oxidative stress induced by H2O2 (Hong et al., 2009). Plants rich in myricetin also show potential neuroprotective effects (Yao & Vieira, 2007). Furthermore, it was reported that the intake of flavonols including quercetin, kaempferol and myricetin has favourable effect on the cognitive performance (Spencer et al., 2009).
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The chemical structures of galangin, kaempferol and myricetin are shown in Fig. 1. It is obvious to notice that the three molecules have similar structures with two aromatic rings (A and B). The only difference, as shown in Fig. 1, exists in the numbers of hydroxyl group in B ring, which structure is believed to be closely related with the biological activities of flavonols (Karawajczyk et al., 2006). Although quite a lot of research has been carried out on galangin, kaempferol and myricetin, however, seldom work has been carried out on the neuroprotective effects of the three above-mentioned flavonols or the possible relationship between the structure and bioactivity, for instance, the effect of the number of hydroxyl groups in the B ring on the neuroprotective function. Therefore, in the present study, the potential of the three dietary flavonols was systematically studied in vivo. As high levels of D-galactose (D-gal) can be oxidised into free radicals and induce impairment of neurogenesis in the hippocampus, D-gal induced senescent mice, which considered as a good model for studying neurodegenerative diseases, were applied in the present study (Sun et al., 2007; Zhong, Ge, Qu, Li, & Ma, 2009). The aim of this work was to investigate the protective effect of the three dietary flavonols on D-galinduced cognitive impairment in mice. This work also made preliminary exploration of whether the number of hydroxyl groups in the B ring of flavonols would influence their neuroprotective nature.
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lege, Huazhong University of Science and Technology, Wuhan, China. The animals were housed in a controlled room (temperature 22 ± 3 °C and humidity 50 ± 10%) and were kept under 12 h light:12 h dark cycle. The mice were fed with standard diet and water ad libitum and acclimated 7 days before they were used for the study. After acclimatisation to the laboratory conditions, the mice were randomly divided into five groups (n = 10): the vehicle control group (C), the D-gal model group (DM), the galangin treated group (GT), the kaempferol treated group (KT), and the myricetin treated group (MT). D-gal at the dose of 150 mg/kg once a day for 8 weeks was subcutaneously injected to the mice in DM, GT, KT, and MT groups. PBS buffer with the same volume was given to those of vehicle control group. Mice of GT, KT, and MT groups were orally administered with galangin, kaempferol, and myricetin respectively, at 100 mg/kg/d 1 h after the injection of D-gal. At the same time, the mice of the vehicle control group and the D-gal model group were treated with same volume of physiological saline (with 0.1% Tween 80) for 8 weeks. At the end of the treatment period, behavioural tests and biochemical measurements were performed as follows. All experiments were performed in compliance with the Chinese legislation on the use and care of laboratory animals and were approved by the Huazhong University of Science and Technology Committee on Animal Care and Use. 2.3. Behavioural tests
2. Materials and methods 2.1. Reagents Galangin, kaempferol, and myricetin (purity > 98%) were purchased from Baoji Biological Development Co., Ltd., (Baoji, China). D-gal was purchased from Sigma–Aldrich (St. Louis, MO, USA). The commercial kits used for the determination of thiobarbituric acid reactive substances (TBA-RS), superoxide dismutase (SOD), and Na+,K+-adenosine triphosphatase (Na+,K+-ATPase) were purchased from Jiancheng Institute of Biotechnology (Nanjing, China). The total protein extraction kit was purchased from ProMab (Richmond, CA, USA). The BCA kit used for the determination of protein content was purchased from Beyotime Institute of Biotechnology (Shanghai, China). The antibodies used for western blot were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other solvents and chemicals used in the study were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 2.2. Animals and administration The Kunming mice, weighing 18–22 g and 5 weeks old, were obtained from the Experimental Animal center, Tongji Medical Col-
All behavioural tests were conducted at the same time of the day (9:00 am to 6:00 pm) for 7 days. Morris water maze (MWM) test, which performed as previously described was applied to evaluate the learning and memory capability of mice (Sun et al., 2007). The experimental apparatus consisting of a circular water tank (120 cm in diameter, 50 cm in height) was filled with water (22 ± 1 °C) to a depth of 30 cm. The water was rendered opaque by adding black nontoxic carbon ink. The water maze was conceptually divided into four equal quadrants (east, south, west, and north). A black platform (10 cm in diameter, 29 cm in height) was submerged 1 cm below the water surface and placed at the midpoint of east quadrant. The mice were placed in the water facing the pool wall at one of the three starting quadrant points (south, west, and north). They were allowed to swim for 60 s to find the hidden platform and rest on it for 15 s. Mice that did not reach the platform within the given time were gently guided to the platform and also allowed to rest for 15 s. The time of the latency to reach the platform was recorded. The mean data from daily test were used for statistical analysis. To determine whether the animal would take a spatial learning strategy to locate the platform, a single spatial probe trial was assessed on the 7th day. The platform was removed from the water tank, and the mice were allowed to swim freely from different start quadrant points (south, west, and north), each for 60 s. The time spent in the target quadrant (east quadrant), the distance moved in the target quadrant, the crossing number of the platform (the place where the platform should be) and the time of first crossing the platform were measured. The records were analysed by a computerised video imaging analysis system (EthoVision, Noldus Information Technology BV, Wageningen, The Netherlands). 2.4. Preparation of tissue samples
Fig. 1. The structures of galangin, kaempferol and myricetin.
The animals were deeply anaesthetised and sacrificed by decapitation 60 min after behavioural testing. Brains were quickly removed and dissected on ice to obtain the hippocampus. One half of the hippocampus tissues were homogenised in ice-cold physiological saline. The homogenate (10%) was centrifuged at 4000g at 4 °C for 10 min, and the supernatant was used for biochemical
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assay. The other half of hippocampus tissues were stored at 80 °C until the western blot analysis. 2.5. Biochemical analysis Protein concentration of each sample was determined based on bicichoninic acid (BCA) method where bovine serum albumin was used as the standard (Jeong et al., 2005). The levels of TBARS and SOD were measured as previous reported (Zhong et al., 2009). Briefly, in the hippocampus tissues, the lipid peroxidation process may form malondiadehyde and other TBARS, and these TBARS can be measured at the wavelength of 532 nm by reacting with TBA to form a stable chromophoric production. Tetraethoxypropane (10 nmol/mL) was used as the standard. The assay for total SOD was based on its ability to inhibit the oxidation of oxymine. The absorbance was measured at 550 nm. The activity of Na+,K+-ATPase was measured based on the recommended method and the absorbance was measured at 660 nm (Sun et al., 2007). All the manipulations followed the directions of the commercially available kits. The activities of SOD and Na+,K+-ATPase were expressed as U/mg protein, and the concentration of TBARS was expressed as nmol malondialdehyde/mg protein. 2.6. Western blot analysis The hippocampal expressions of ERK1/2, phopho-ERK1/2, CREB and phospho-CREB were evaluated by western blot analysis (Lei et al., 2011). The tissue samples were triturated in liquid nitrogen and the total protein was extracted using a protein extraction kit. Protein concentrations were determined using the BCA protein assay kit. The two kits were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Protein samples (50 lg) were separated by 12% SDS–polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane (Roche Diagnostics Corporation, Indianapolis, IN, USA) by electrophoretic transfer (Bio-Rad Laboratories, Inc., Benicia, CA, USA). Transferred membranes were blocked for 1 h at room temperature with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), and then incubated overnight at 4 °C with different primary antibodies as follows: anti-ERK1/2 (1:500), anti-CREB (1:500), anti-phopho-ERK1/2 (1:400), or anti-phospho-CREB (1:400). After washing by TBST for three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies in TBST with 3% nonfat milk for 1 h at room temperature. Immunoblots were developed on films using the enhanced chemiluminescence technique (Super Signal West Pico; Pierce Biotechnology, Rockford, IL, USA). Quantification of bands was determined by densitometric analysis using Quantity One imaging software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The data were normalised using GAPDH (1:1000) as an internal control. 2.7. Statistical analysis The values were presented as mean ± S.D. Results were analysed statistically by one-way ANOVA followed by Tukey’s multiple comparison using SPSS 11.5 Software for Windows. Differences were considered as significant at p < 0.05. Figures were made using Origin 6.0 Software for Windows. 3. Results 3.1. Morris water maze test Fig. 2A indicates the latencies for mice in different groups to reach the hidden platform: day, F (5, 280) = 21.000, p < 0.01; group,
F (4, 280) = 18.724, p < 0.01; day by group interaction, F (20, 280) = 0.934, p = 0.544; 2-way ANOVA. During the training period, the time that mice spent to find the hidden platform from 0th to 6th day was decreased. However, the vehicle control showed continuous decrease while D-gal model only slightly reduced the time for finding the platform which indicating that treatment with D-gal for 8 weeks could induce noticeable neurologic impairment and cognitive impairment. Our results also demonstrated that both kaempferol and myricetin (100 mg/kg/d) could prevent the D-gal induced platform finding impairment as mice from KT and MT groups took less time to find the platform in all the 6 training days as compared with D-gal model. The platform crossing number, the time of first crossing the platform, the time spent in the target quadrant and the distance moved in the target quadrant after the 6 training days were recorded to evaluate the spatial learning and memory ability of mice on the 7th day, as shown in Fig. 2B–E. It can be seen that while the moving time and swimming distance of the D-gal model mice were remarkably decrease as compared to those of the vehicle control (p < 0.05), the time of first crossing was significantly (p < 0.05) longer with fewer platform crossings numbers in the target quadrant. The impairment in the spatial learning and memory ability by D-gal was attenuated by the dietary flavonols. Treatment with 100 mg/kg/d kaempferol or myricetin for 8 weeks could obviously (p < 0.05) increase the number of platform crossings, the time and distance spent in the target quadrant, while also reduced the time of first crossing. However, there was no significant difference between the mice of KT and MT. On the other hand, although galangin could slightly ameliorate the spatial learning and memory ability of mice, it failed to show a significant effect when compared with the D-gal model (p > 0.05). 3.2. Biochemical analysis Effects of galangin, kaempferol and myricetin on the levels of TBARS, and the activities of SOD and Na+,K+-ATPase in hippocampus of senescent mice induced by D-gal are shown in Table 1. DGal resulted in a significant decrease in the activities of Na+,K+ATPase and SOD and increase in the levels of TBARS as compared with vehicle control. All the three flavonols could produce significant (p < 0.05) effects on elevating the activities of SOD and Na+,K+ATPase, as well as on reducing the TBARS levels in mice. However, there were no significant difference (p > 0.05) among galangin, kaempferol and myricetin. 3.3. Western blot analysis Fig. 3 shows the effects of galangin, kaempferol and myricetin on the regulation of the expression for ERK1/2-CREB pathway. Dgal inhibited the expressions of phospho-ERK1/2 and phosphoCREB when compared with the vehicle control. Galangin, kaempferol and myricetin significantly (p < 0.05) activated the phosphorylations of ERK1/2 and CREB. It is also worth noting that the more number of hydroxyl group is within B ring of the three flavonol molecules, the more noticeable level of the ERK1/2-CREB expression was noticed (p < 0.05). 4. Discussion D-gal-induced neurodegeneration model was applied in this study to investigate the neuroprotective activities of three natural dietary flavonols (galangin, kaempferol and myricetin). In general, the results showed that 8 weeks administration of kaempferol or myricetin can obviously attenuate the D-gal-induced cognitive impairment in mice.
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Fig. 2. Effects of galangin, kaempferol and myricetin on the spatial learning and memory ability of mice. Values are expressed as means ± S.D. from each group (n = 8–10). Values sharing the same letter (a, b, c) in each row do not differ significantly at p < 0.05 (SPSS). (A) The time mice spent to find the hidden platform from 0th to 6th day. (B) The platform crossing number of mice on the 7th day. (C) The time of mice spent in the target quadrant on the 7th day. (D) The time of mice first crossing the platform on the 7th day. (E) The distance of mice moved in the target quadrant on the 7th day.
Neurodegenerative disease is a complex pathophysiological process. It is generally suggested that the oxidative stress plays a key role in the nerve damage (Jeong et al., 2011). Previous studies show that ROS participates in neurodegenerative diseases and the oxidative metabolism is very active in brain (Yao & Vieira, 2007; Zhong et al., 2009). In normal cases, the generation and elimination
of ROS is in a dynamic balance. It was reported that superoxide anion, a major free radical, is mainly catalysed into H2O2 by SOD in vivo (Palsamy, Sivakumar, & Subramanian, 2010). However, when the balances were disrupted, the activity of antioxidase will decrease and the levels of ROS will increase. The excessive production of ROS will impair brain functions, especially the hippocampus
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Table 1 Effects of three dietary flavonols on the levels of SOD, TBARS and Na+,K+-ATPase in hippocampus of senescent mice induced by D-gal. Vehicle control SOD (U/mg protein) TBARS (nmol/mg protein) Na+,K+-ATPase (U/mg protien)
a
75.97 ± 4.73 4.30 ± 0.32a 2.60 ± 0.43a
D-gal
Galangin
model b
40.69 ± 6.38 7.00 ± 1.68b 1.32 ± 0.28b
Kaempferol c
54.50 ± 4.50 5.63 ± 0.55c 1.90 ± 0.59c
Myricetin d
62.41 ± 6.02 5.22 ± 0.55c 2.14 ± 0.59c
60.00 ± 6.02cd 5.05 ± 0.43c 1.80 ± 0.35c
Values are given as means ± SD from 8 mice in each group. a,b,c,dValues sharing a common superscript (a, b, c, d) in a row do not differ significantly at p < 0.05 (SPSS). Both c and d do not differ significantly with cd.
Fig. 3. Effects of galangin, kaempferol and myricetin on the expressions of ERK1/2, phospho-ERK1/2, CREB and phospho-CREB in hippocampus. Values are expressed as means ± S.D. from each group (n = 8). Values sharing the same letter (a, b, c, d, e) in each row do not differ significantly at p < 0.05 (SPSS). (A) Representative western blot images. (B) The p-ERK/ERK ratios in mice hippocampus. (C) The p-CREB/CREB ratios in mice hippocampus.
which is mainly responsible for learning and memory (Yargicoglu et al., 2006). Furthermore, it is necessary for the brain to maintain relevant cellular Na+/K+ gradients for normal nerve impulse propagation, neurotransmitter release and cation homeostasis, as Na+,K+-ATPase is an imperative enzyme for sustaining the Na+/K+ gradients (Sun et al., 2007). Unfortunately, as a consequence of oxidative damage, the activity of Na+,K+-ATPase decreases and finally leads to the impairment in learning and memory (Zhong et al., 2009). In this study, daily treatment of galangin, kaempferol or myricetin for 8 weeks can obviously increase the activities of SOD and Na+,K+-ATPase, and decreased the levels of TBARS in hippocampus when compared with D-gal model. In addition, evidence shows that MAPKs are highly expressed in postmitotic neurons of the adult nervous system and are necessary for maintaining normal nervous functions (Hong et al., 2009). MAPKs are activated in response to growth factor stimulation of cells in culture, and fall into four different subgroups: the ERK1/2 MAPK, the Jun N-terminal kinases (JNKs) MAPK, the p38 MAPK and the ERK5 MAPK (Plotnikov, Zehorai, Procaccia, & Seger, 2011). The ERK1/2 is one of the most-studied members and mainly found in the hippocampus (Qi et al., 2008). It is documented that phosphorylation of ERK1/2 influences the survival, differentiation and cycle regulation of many nerve cells (El-Omri et al., 2010; Hong et al., 2009). As a result, the activation ERK1/2 MAPK pathway is necessary for nervous system functions. CREB is a major downstream target of ERK1/2 MAPK pathway and also critical for a lot of cellular processes including proliferation and adaptive responses (Zhou, Yao, Zhang, & Chang, 2002). Recent studies have shown that the phosphorylation of CREB is necessary for the induction of the ERK-dependent plasticity (Qi et al., 2008). The activation of ERK1/2-CREB pathway is a necessary step for the learning and memory (Atkins, Falo, Alonso, Bramlett, & Dietrich, 2009; Haddad, 2005). Our western blotting observations show that galangin, kaempferol and myricetin can treat D-gal-induced impairment of ERK1/2 and CREB activations in mice hippocampus, suggesting that the three natural flavonols were able to exert neuroprotective effects via activating the phosphorylations of ERK1/2 and CREB. The current investigations indicated that all of the three natural flavonols could reduce the level of oxidative stress and enhance the
Na+,K+-ATPase activity in vivo. On the other hand, galangin, kaempferol and myricetin could activate the phosphorylations of ERK1/2 and CREB. It was worthy of notice that, when the number of hydroxyl group in the B ring (galangin, kaempferol and myricetin) increased, the flavonols up-regulated the expression of ERK1/ 2-CREB pathway in an obviously incremental manner (p < 0.05). These findings suggested that the hydroxyl in the B ring played an important role in up-regulating the hippocampus expression of ERK1/2-CREB pathway. In the Morris water maze test, although all the three flavonols exerted positive effects to protect mice from D-gal-induced neurologic impairment, galangin failed to show a significant difference (p > 0.05) on attenuating the cognitive impairment when compared with the D-gal model group. However, kaempferol and myricetin did obviously (p < 0.05) enhance the spatial learning and memory ability of mice and there was no significant difference (p > 0.05) between kaempferol and myricetin treated mice. In summary, kaempferol and myricetin are able to significantly attenuate D-gal-induced cognitive impairment in mice via up-regulating the expression of ERK1/2-CREB pathway, enhancing the activities of Na+,K+-ATPase and reducing the level of oxidative stress in hippocampus. The presence of hydroxyls in B ring of flavonols is necessary for their neuroprotective activity and the increase in the number of hydroxyl group also contributed to up-regulation of the hippocampus expression of ERK1/2-CREB pathway in the Dgal-induced cognitive impairment mice. Acknowledgements This study was financially supported by The state natural science fund (NO30973864). References Aherne, S. A., & O’Brien, N. M. (2002). Dietary flavonols: chemistry, food content, and metabolism. Nutrition, 18, 75–81. Atkins, C. M., Falo, M. C., Alonso, O. F., Bramlett, H. M., & Dietrich, W. D. (2009). Deficits in ERK and CREB activation in the hippocampus after traumatic brain injury. Neuroscience Letters, 459, 52–56. El-Omri, A., Han, J., Yamada, P., Kawada, K., Ben-Abdrabbah, M., & Isoda, H. (2010). Rosmarinus officinalis polyphenols activate cholinergic activities in PC12 cells
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