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Protective effect of Polygonum multiflorum Thunb on amyloid β-peptide 25-35 induced cognitive deficits in mice
Journal of Ethnopharmacology 104 (2006) 144–148
Protective effect of Polygonum multiflorum Thunb on amyloid -peptide 25-35 induced cognitive deficits in mice Min-Young Um, Won-Hee Choi, Ji-Yun Aan, Sung-Ran Kim, Tae-Youl Ha ∗ Food Function Research Division, Korea Food Research Institute, Seongnam 463-746, Republic of Korea Received 9 January 2005; received in revised form 5 August 2005; accepted 27 August 2005 Available online 10 October 2005
Abstract Amyloid  protein (A) may be neurotoxic during the progression of Alzheimer’s disease by eliciting oxidative stress. This study was designed to determine the effect of Polygonum multiflorum Thunb water extract (PWE) on A25-35-induced cognitive deficits and oxidative stress in mice. Mice were fed experimental diets comprising either 0.5 or 1% PWE for 4 weeks, and then received a single intracerebroventricular (i.c.v.) injection of A25-35 (10 g/mouse). Behavioral changes in the mice were evaluated using passive avoidance and water-maze tests. The consumption of PWE significantly ameliorated the cognitive deficits caused by i.c.v. injection of A25-35. The A25-35 treatment accelerated the lipid peroxidation, and PWE attenuated the A-induced increase in brain levels of thiobarbituric acid reactive substances. There was an increase in glutathione peroxidase activity in PWE-treated groups. The acetylcholinesterase activity in the brain and serum was lower in PWE supplemented groups than in the only A-injected group. These findings suggest that PWE exerts a preventive effect against cognitive deficits induced by A25-35 accumulation in Alzheimer’s disease, and that this effect is mediated by the antioxidant properties of PWE. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Amyloid  protein; Antioxidative capacity; Cognitive deficits; Polygonum multiflorum Thunb
1. Introduction Alzheimer’s disease (AD) is characterized by progressive cognitive function deficits due to the presence of numerous senile plagues and neurofibrillary tangles in brain regions (Yankner, 1996). Amyloid  (A) peptide is a major component of these plagues (Golde et al., 1992; Yan et al., 2004). The accumulating evidences suggest that oxidative stress is involved in the mechanism of A-induced neurotoxicity (Behl et al., 1992; Yamada et al., 1999; Zhu et al., 2004). The levels of lipid peroxidation, protein carbonyl, and 8-hydroxyl-2-deoxyguanosine are higher in the brains of AD patients than in aged-matched control brains (Sayre et al., 1997; Smith et al., 1997; Morishima et al., 2001). Antioxidants, such as Vitamin E, Ginkgo biloba, and fer-
Abbreviations: A, amyloid  protein; AChE, acetylcholinesterase; AD, Alzheimer’s disease; GPx, glutathione peroxidase; i.c.v., intracerebroventricular; MDA, malondialdehyde; PBS, phosphate buffered saline; PM, Polygonum multiflorum Thunb; PWE, Polygonum multiflorum Thunb water extract; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances ∗ Corresponding author. Tel.: +82 31 780 9054; fax: +82 31 780 9225. E-mail address: [email protected] (T.-Y. Ha). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.08.054
ulic acid have been used in attempts to treat/prevent AD (Sano et al., 1997; Christen, 2000; Yan et al., 2001). The mechanisms underlying the preventive effect of antioxidants against AD are unclear, but there is accumulating evidence that oxidative stress contributes to neurotoxicity caused by A in vitro and in vivo (Stackman et al., 2003; Tamagno et al., 2003). Furthermore, it was reported that A deposition causes selective neuronal loss and is related to dysfunction and degeneration of basal forebrain cholinergic neurons. The activity of acetylcholinesterase (AChE), responsible for acetylcholine hydrolysis, has been shown to be increased within and around amyloid plagues (Atack et al., 1983). An increase in the AChE activity promotes the assembly of A into fibrils, and it has been suggested that AChE plays a pathogenic role in AD by influencing the process leading to A toxicity (Melo et al., 2003). Polygonum multiflorum Thunb (PM), the root of a Korean medicinal herb, has been used for a long time as an antiaging agent. Recent studies have demonstrated that PM exerts hypocholesterolemic, antitumor, and vasorelaxant effects (Zhang et al., 1983; Xiao et al., 1993). The efficacy of PM in treating chronic disorders may be mediated by its antioxidative properties. Chen et al. (1999) identified that gallic acid, catechin,
M.-Y. Um et al. / Journal of Ethnopharmacology 104 (2006) 144–148
and 2,3,5,4 -tetrahydroxystilbene-2-O--d-glucoside in the ethyl acetate fraction of PM extracts showed strong antioxidant activities. Chan et al. (2002) showed that PM ethanol extract supplemented groups had a lower percentage of lipofuscin and a lower malondialdehyde (MDA) concentration in the brain. In screening study of AChE inhibitory activity and neuroprotective effect from some medicinal plants used in oriental medicines, we found out that PM water extract (PWE) showed a potent protective effect against A-induced neuronal cell death and AChE inhibitory activity in vitro. This result in the preliminary study prompts us to investigate whether PWE can exhibit protective effect on cognitive deficits in mice. We examined the behavioral changes, AChE activity, level of lipid peroxidation, and antioxidant enzyme activities to evaluate effect of PWE on cognitive deficits and oxidative stress induced in A-treated mice. 2. Materials and methods 2.1. Preparation of PWE The roots of Polygonum multiflorum Thunb, family Polygonacease, originated from Korea were purchased from Kyungdong Oriental medicine market (Seoul, Korea), and identified by Professor Y.M. Park, Department of Life Science, Cheongju University. Voucher specimens (KFRI-PM03002) were preserved in Korea Food Research Institute. Dried PM roots were cut into small pieces and extracted three times with 10 volumes of distilled water at 100 ◦ C for 3 h. The water extract was filtered with filter paper (Whatman No. 2, USA). The supernatants were concentrated under reduced pressure with a vacuum rotary evaporator. The concentrated extracts were freeze-dried. Finally, 11.1 g of the dried extract was obtained from 100 g of the roots of PM and stored at −20 ◦ C until use. 2.2. Animals and intracerebroventricular (i.c.v.) injection of Aβ25-35 Male ICR mice (5-week-old; Bio Genomics, Korea) were used in the experiments. The mice were housed in a room maintained at 23 ± 1 ◦ C with a 12-h light/12-h dark cycle and fed for 4 weeks ad libitum. The experimental diet was based on the AIN76 formula, and comprised either 0.5 or 1% PWE. The A25-35 peptide was dissolved in PBS. An i.c.v. injection of A25-35 (10 g/mouse) was performed using the procedure established by Laursen and Belknap (1986). In brief, each mouse was injected at the bregma with a 50 l Hamilton microsyringe fitted with a 26-gauge needle, the tip of which was adjusted to be inserted by 2.4 mm. The i.c.v. injection volume was 10 l. Control animals were injected with PBS. Fifty mice were randomly divided into five groups. All animal procedures were approved by Institutional Animal Care and Use Committee of Korea Food Research Institute.
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illuminated and one dark, equipped with a grid floor and shock generator. The day after A25-35 injection, mice were trained in the passive avoidance task. During the training trial, each mouse was placed in the lighted compartment, and as soon as it entered the dark compartment the door was closed, and it received an inescapable shock (0.3 mA, 3 s). The next day, the mouse was again placed in the lighted compartment, and the time until it returned to the dark compartment was measured as the step-through latency (with a maximum of 300 s). 2.4. Water-maze test The water maze was slightly modified from the Morris water task. The experimental apparatus consisted of a circular water tank (diameter 100 cm; height 35 cm) containing water at 23 ◦ C to a depth of 15 cm and rendered opaque by the addition of powdered milk. A platform was positioned inside the tank with its top submerged 2 cm below the water surface in the target quadrant of the maze. After several trials, the test was conducted on the day of injection of A peptide. In each training trial, the time required to escape onto the hidden platform was recorded. The number of times the platform was not found was also recorded. 2.5. Measurement of AChE activity AChE activity in the brain was measured using the method of Ellman et al. (1961). Acetylthiocholine iodide was used as a substrate. The hydrolysis of acetylthiocholine was determined by monitoring the formation of the yellow 5-thio-2-nitrobenzoic acid at a wavelength of 412 nm. Protein concentration was determined by the method of Lowry et al. (1951). Serum AChE activity was assayed with cholinesterase kit from Sigma Chemical (St. Louis, MO, USA) following a modified version of the method of Rappaport et al. (1959). 2.6. Measurement of lipid peroxide levels and antioxidant enzyme activity Lipid peroxidation in the brain was determined by measuring the formation of thiobarbituric acid reactive substances (TBARS) according to the method of Ohkawa et al. (1979). A standard curve was obtained using 1,1,3,3-tetramethoxypropane. Catalase activity was measured using the method of Aebi (1974). Brain homogenate was reacted with hydrogen peroxide, and the decomposition of hydrogen peroxide was determined spectrophotometrically at 240 nm. Superoxide dismutase (SOD) activities were assayed according to Marklund and Marklund (1974). Glutathione peroxidase (GPx) activity was measured according to Lawrence and Burk (1976). The decrease in NADPH was recorded at 340 nm and is expressed here using a molar extinction coefficient of NADPH of 6.22 mM−1 cm−1 . 2.7. Statistics
2.3. Passive avoidance test Passive avoidance was tested using a two-compartment shuttle chamber (256000 series, TSE Systems, Germany), one
Statistical analysis was performed using one-way analysis of variance, with Duncan’s multiple test used for group comparison with P < 0.05.
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3. Result 3.1. PWE improves cognitive deficits in Aβ25-35-treated mice It was suggested previously that the i.c.v. injection of A2535 causes memory deficits and lowers choline transferase in hippocampus (Tang et al., 2002; Stepanichev et al., 2004). In the present study, an i.c.v. injection of 10 g of A25-35-induced cognitive dysfunction as assessed by passive avoidance and Morris water-maze tests. Changes in the step-through latency in passive avoidance are shown in Fig. 1. There was no significant difference between the normal and normal PBS groups (i.c.v. injection of PBS), but significantly reduced step-through latency in the A25-35-injected control group (25.3% reduction compared to normal, P < 0.05). This result confirmed that cognitive deficits induced by A i.c.v. were not attributable to the i.c.v. injection itself. As shown in Fig. 1, step-through latency was increased up to 2.5-fold by consumption of the 1% PWE diet (P < 0.05). As shown in Fig. 2, injection of A25-35 increased the escape latency and error frequency in the Morris water-maze test. Treatment of mice with PWE for 4 weeks attenuated the increase in the escape latency almost to that in the normal group, but the change was not dose dependent. Changes in error frequency in each group showed a similar pattern to the escape latency.
Fig. 2. Effect of PWE treatment on performance in the water-maze task by A25-35-treated mice. Escape latency (A) and error frequency (B) were determined. Values are means ± S.E.M. (n = 10; * P < 0.05 compared to controls; # P < 0.05 compared to normals).
3.2. Effect of PWE on AChE activity in the brain and serum 3.3. Effect of PWE on lipid peroxide levels in the brain To determine the effect of PWE on AChE, we evaluated AChE activity in the brain and serum (Table 1). Exposure to A25-35 had no significant effect on AChE activity in brain, but 0.5% PWE resulted in the reduction of AChE activity. In the group consuming 1% PWE, the AChE activity was significantly decreased compared to the control group. The activity of AChE in serum was significantly higher in the control group than in the normal group (21.0%, P < 0.05). In the group consuming 1% PWE, the activity of AChE in serum was significantly lower than that in the control group (25.3% reduction compared to control, P < 0.05).
Lipid peroxide levels in the brain of A25-35-treated mice are shown in Fig. 3. Injection of A25-35 into the cerebral ventricle increased lipid peroxide levels in the brain. PWE slightly reduced the lipid peroxide (TBARS) levels in the brain of A2535 injected mice. 3.4. Effect of PWE on antioxidant enzyme activity in the brain The effect of PWE on specific activities of catalase, SOD, and GPx in the brain is shown in Table 2. Treatment with A25-35 did not alter the activity of catalase in the brain. However, the activity of catalase was decreased in the PWE-treated Table 1 Effect of PWE treatment on acetylcholinesterase activity in the serum and brain
Fig. 1. Protective effect of PWE on A25-35-induced cognitive deficits in mice. The learning and memory performance was assessed by the passive avoidance test. Values are means ± S.E.M. (n = 10; * P < 0.05 compared to controls; # P < 0.05 compared to normal).
Group
Brain (M/(min mg protein))
Serum (Rappaport units/ml)
N NP C 0.5% PW 1% PW
17.0 ± 2.2 ab 17.9 ± 0.9 a 18.4 ± 0.4 a 15.0 ± 0.5 b 14.8 ± 0.3 b
179.9 177.2 219.3 177.6 163.6
± ± ± ± ±
27.8 b 8.6 b 11.7 a 8.3 b 2.2 b
(1) Values are means ± S.E.M. (n = 10). (2) One Rappaport unit is that amount of cholinesterase that will liberate 1 mol of acetic acid from acetylcholine in 30 min at 25 ◦ C and pH 7.8 under the conditions of this test. (3) AChE activities are expressed as the amount of 5-thio-2-nitro-benzoic acid produced by the hydrolysis of substrate. (4) Values with different letters within a row are significantly different at α = 0.05 by Duncan’s multiple-range test.
M.-Y. Um et al. / Journal of Ethnopharmacology 104 (2006) 144–148
Fig. 3. Effect of PWE on TBARS levels in brain tissue of mice treated with A25-35 (* P < 0.05 compared to controls; # P < 0.05 compared to normals). Table 2 The effect of PWE on specific activities of catalase, SOD, and GPx in brain homogenates Group
Catalase (units/ (min mg protein))
SOD (units/ (min mg protein))
GPx (nmol/ (min mg protein))
N NP C 0.5% PW 1% PW
2.96 ± 0.42 n.s. 3.31 ± 0.28 3.37 ± 0.37 2.34 ± 0.16 2.27 ± 0.20
8.94 ± 1.48 n.s. 9.33 ± 0.47 8.88 ± 0.97 6.67 ± 0.16 8.01 ± 0.37
34.81 ± 7.67 ab 35.87 ± 3.43 a 26.97 ± 1.34 ab 24.68 ± 1.10 ab 34.16 ± 5.78 b
(1) Values are mean ± S.E.M. (n = 10). (2) Values with different letters within a column are significantly different at α = 0.05 by Duncan’s multiple-range test. (3) n.s., not significant.
group, with the lowest activity exhibited in mice consuming 1% PWE. The SOD activities did not differ significantly between the groups. The activity of GPx in brain tissue was lower in A2535-treated mice than in normal mice. However, the activity of GPx in brains of mice consuming 1% PWE was increased up to the normal level. 4. Discussion The present study has revealed a neuroprotective effect of PM on A-induced cognitive deficits in mice. Memory associated behavior did not differ between the PBS-injected and normal groups. Wang et al. (2001) similarly demonstrated that i.c.v. injection of A25-35-induced impairment of memory as assessed by passive avoidance and Morris water-maze tests. A has the potential to induce oxidative stress in the brain (Behl and Sagara, 1997). Moreover, it has been reported that A induces the production of hydrogen peroxide and lipid peroxide in hippocampal neurons of the rat brain (Yatin et al., 2000). Jhoo et al. (2000) showed the induction of 4-hydroxy-2-nonenal and 8hydroxy-2 -deoxyguanosine (a marker of oxidative damage to DNA) immunoreactivities following infusion of A1-42 in rat brain. In the present study, we found a significantly increased level of TBARS in mice brain after a single injection with A2535. Furthermore, imbalances in each antioxidant enzyme were also observed. Kim et al. (2003) demonstrated that continuous i.c.v. infusion of A1-42 in rat resulted in a significant decrease in protein expression of SOD, GPx, and glutathioneS-transferase- in rat brain, from which they suggested that
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A1-42 impairs antioxidant capacity. We found that consumption of a diet containing PWE ameliorated cognitive deficits in A25-35-injected mice. Especially, the data show that the step-through latency in passive avoidance increased in a dosedependent manner, but not in the water-maze test. Also, consumption of PWE decreased the escape latency almost to normal levels. It is possible that neuroprotection plays a role in the favorable effect of PWE on A25-35-induced cognitive deficits. Antioxidants, such as ␣-tocopherol and ferulic acid protect against learning and memory deficits induced by A (Yamada et al., 1999; Yan et al., 2001). The AChE activity has been shown to be increased within and around amyloid plaques in Alzheimer’s brain (Ulrich et al., 1990). The enhancement of AChE activity induced by A25-35 is mediated by oxidative stress (Melo et al., 2003). The AChE activity in the brain and serum was increased in mice treated with A25-35 when compared with the normal in our experiment. In addition, the A25-35-induced increase in AChE was attenuated by PWE consumption. PM is a medicinal plant that has antioxidant properties in vitro (Ryu et al., 2002) and delays aging responses in vivo (Chiu et al., 2002) involving oxidative stress. Also, previous studies have shown that PM ethanol extract suppressed lipid peroxidation in the mitochondria of rat heart (Chen et al., 1999). Chan et al. (2002) demonstrated that PM ethanol extract significantly improved learning and memory deficits in SAMP8 (a murine AD model), and lowered lipofuscin percentages and MDA concentrations in hippocampus, and increased total thiol concentrations. Catalase, SOD, and GPx are involved in the reduction in reactive oxygen species and peroxides produced in living organisms as well as in the detoxification of certain compounds of exogenous origin, and thus play a primary role in the maintenance of a balanced redox status (Kweon et al., 2003). Treatment of mice with PWE for 4 weeks decreased TBARS level and increased GPx activity in the brain, while having no significant effect on catalase and SOD activity. These findings can be attributed to GPx exhibiting a higher sensitivity to lipid peroxidation than catalase or SOD. Therefore, we suggest that accumulation of lipid peroxides by A was reduced by PWE via antioxidative mechanisms. In conclusion, we suggest that PWE markedly improves cognitive deficits induced by A25-35, and that this effect is mediated by the antioxidant properties of PWE. Future studies should determine the specific components in PWE responsible for preventing cognitive impairment. References Aebi, H., 1974. In: Bergmeyer, H.U. (Ed.), Catalase. Methods of Enzymatic Analysis. Academic Press, New York and London, pp. 637–684. Atack, J.R., Perry, E.K., Bonham, J.R., Perry, R.H., Tomlinson, B.E., Blessed, G., Fairbairn, A., 1983. Molecular forms of acetylcholinesterase in senile dementia of Alzheimer type: selective loss of the intermediate (10S) form. Neuroscience Letters 40, 199–204. Behl, C., Davis, J., Cole, G.M., Schubert, D., 1992. Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochemical and Biophysical Research Communications 186, 944–950. Behl, C., Sagara, Y., 1997. Mechanism of amyloid beta protein induced neuronal cell death: current concepts and future perspectives. Journal of Neural Transmission. 49, 125–134.
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Protective effect of Polygonum multiflorum Thunb on amyloid -peptide 25-35 induced cognitive deficits in mice Min-Young Um, Won-Hee Choi, Ji-Yun Aan, Sung-Ran Kim, Tae-Youl Ha ∗ Food Function Research Division, Korea Food Research Institute, Seongnam 463-746, Republic of Korea Received 9 January 2005; received in revised form 5 August 2005; accepted 27 August 2005 Available online 10 October 2005
Abstract Amyloid  protein (A) may be neurotoxic during the progression of Alzheimer’s disease by eliciting oxidative stress. This study was designed to determine the effect of Polygonum multiflorum Thunb water extract (PWE) on A25-35-induced cognitive deficits and oxidative stress in mice. Mice were fed experimental diets comprising either 0.5 or 1% PWE for 4 weeks, and then received a single intracerebroventricular (i.c.v.) injection of A25-35 (10 g/mouse). Behavioral changes in the mice were evaluated using passive avoidance and water-maze tests. The consumption of PWE significantly ameliorated the cognitive deficits caused by i.c.v. injection of A25-35. The A25-35 treatment accelerated the lipid peroxidation, and PWE attenuated the A-induced increase in brain levels of thiobarbituric acid reactive substances. There was an increase in glutathione peroxidase activity in PWE-treated groups. The acetylcholinesterase activity in the brain and serum was lower in PWE supplemented groups than in the only A-injected group. These findings suggest that PWE exerts a preventive effect against cognitive deficits induced by A25-35 accumulation in Alzheimer’s disease, and that this effect is mediated by the antioxidant properties of PWE. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Amyloid  protein; Antioxidative capacity; Cognitive deficits; Polygonum multiflorum Thunb
1. Introduction Alzheimer’s disease (AD) is characterized by progressive cognitive function deficits due to the presence of numerous senile plagues and neurofibrillary tangles in brain regions (Yankner, 1996). Amyloid  (A) peptide is a major component of these plagues (Golde et al., 1992; Yan et al., 2004). The accumulating evidences suggest that oxidative stress is involved in the mechanism of A-induced neurotoxicity (Behl et al., 1992; Yamada et al., 1999; Zhu et al., 2004). The levels of lipid peroxidation, protein carbonyl, and 8-hydroxyl-2-deoxyguanosine are higher in the brains of AD patients than in aged-matched control brains (Sayre et al., 1997; Smith et al., 1997; Morishima et al., 2001). Antioxidants, such as Vitamin E, Ginkgo biloba, and fer-
Abbreviations: A, amyloid  protein; AChE, acetylcholinesterase; AD, Alzheimer’s disease; GPx, glutathione peroxidase; i.c.v., intracerebroventricular; MDA, malondialdehyde; PBS, phosphate buffered saline; PM, Polygonum multiflorum Thunb; PWE, Polygonum multiflorum Thunb water extract; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances ∗ Corresponding author. Tel.: +82 31 780 9054; fax: +82 31 780 9225. E-mail address: [email protected] (T.-Y. Ha). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.08.054
ulic acid have been used in attempts to treat/prevent AD (Sano et al., 1997; Christen, 2000; Yan et al., 2001). The mechanisms underlying the preventive effect of antioxidants against AD are unclear, but there is accumulating evidence that oxidative stress contributes to neurotoxicity caused by A in vitro and in vivo (Stackman et al., 2003; Tamagno et al., 2003). Furthermore, it was reported that A deposition causes selective neuronal loss and is related to dysfunction and degeneration of basal forebrain cholinergic neurons. The activity of acetylcholinesterase (AChE), responsible for acetylcholine hydrolysis, has been shown to be increased within and around amyloid plagues (Atack et al., 1983). An increase in the AChE activity promotes the assembly of A into fibrils, and it has been suggested that AChE plays a pathogenic role in AD by influencing the process leading to A toxicity (Melo et al., 2003). Polygonum multiflorum Thunb (PM), the root of a Korean medicinal herb, has been used for a long time as an antiaging agent. Recent studies have demonstrated that PM exerts hypocholesterolemic, antitumor, and vasorelaxant effects (Zhang et al., 1983; Xiao et al., 1993). The efficacy of PM in treating chronic disorders may be mediated by its antioxidative properties. Chen et al. (1999) identified that gallic acid, catechin,
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and 2,3,5,4 -tetrahydroxystilbene-2-O--d-glucoside in the ethyl acetate fraction of PM extracts showed strong antioxidant activities. Chan et al. (2002) showed that PM ethanol extract supplemented groups had a lower percentage of lipofuscin and a lower malondialdehyde (MDA) concentration in the brain. In screening study of AChE inhibitory activity and neuroprotective effect from some medicinal plants used in oriental medicines, we found out that PM water extract (PWE) showed a potent protective effect against A-induced neuronal cell death and AChE inhibitory activity in vitro. This result in the preliminary study prompts us to investigate whether PWE can exhibit protective effect on cognitive deficits in mice. We examined the behavioral changes, AChE activity, level of lipid peroxidation, and antioxidant enzyme activities to evaluate effect of PWE on cognitive deficits and oxidative stress induced in A-treated mice. 2. Materials and methods 2.1. Preparation of PWE The roots of Polygonum multiflorum Thunb, family Polygonacease, originated from Korea were purchased from Kyungdong Oriental medicine market (Seoul, Korea), and identified by Professor Y.M. Park, Department of Life Science, Cheongju University. Voucher specimens (KFRI-PM03002) were preserved in Korea Food Research Institute. Dried PM roots were cut into small pieces and extracted three times with 10 volumes of distilled water at 100 ◦ C for 3 h. The water extract was filtered with filter paper (Whatman No. 2, USA). The supernatants were concentrated under reduced pressure with a vacuum rotary evaporator. The concentrated extracts were freeze-dried. Finally, 11.1 g of the dried extract was obtained from 100 g of the roots of PM and stored at −20 ◦ C until use. 2.2. Animals and intracerebroventricular (i.c.v.) injection of Aβ25-35 Male ICR mice (5-week-old; Bio Genomics, Korea) were used in the experiments. The mice were housed in a room maintained at 23 ± 1 ◦ C with a 12-h light/12-h dark cycle and fed for 4 weeks ad libitum. The experimental diet was based on the AIN76 formula, and comprised either 0.5 or 1% PWE. The A25-35 peptide was dissolved in PBS. An i.c.v. injection of A25-35 (10 g/mouse) was performed using the procedure established by Laursen and Belknap (1986). In brief, each mouse was injected at the bregma with a 50 l Hamilton microsyringe fitted with a 26-gauge needle, the tip of which was adjusted to be inserted by 2.4 mm. The i.c.v. injection volume was 10 l. Control animals were injected with PBS. Fifty mice were randomly divided into five groups. All animal procedures were approved by Institutional Animal Care and Use Committee of Korea Food Research Institute.
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illuminated and one dark, equipped with a grid floor and shock generator. The day after A25-35 injection, mice were trained in the passive avoidance task. During the training trial, each mouse was placed in the lighted compartment, and as soon as it entered the dark compartment the door was closed, and it received an inescapable shock (0.3 mA, 3 s). The next day, the mouse was again placed in the lighted compartment, and the time until it returned to the dark compartment was measured as the step-through latency (with a maximum of 300 s). 2.4. Water-maze test The water maze was slightly modified from the Morris water task. The experimental apparatus consisted of a circular water tank (diameter 100 cm; height 35 cm) containing water at 23 ◦ C to a depth of 15 cm and rendered opaque by the addition of powdered milk. A platform was positioned inside the tank with its top submerged 2 cm below the water surface in the target quadrant of the maze. After several trials, the test was conducted on the day of injection of A peptide. In each training trial, the time required to escape onto the hidden platform was recorded. The number of times the platform was not found was also recorded. 2.5. Measurement of AChE activity AChE activity in the brain was measured using the method of Ellman et al. (1961). Acetylthiocholine iodide was used as a substrate. The hydrolysis of acetylthiocholine was determined by monitoring the formation of the yellow 5-thio-2-nitrobenzoic acid at a wavelength of 412 nm. Protein concentration was determined by the method of Lowry et al. (1951). Serum AChE activity was assayed with cholinesterase kit from Sigma Chemical (St. Louis, MO, USA) following a modified version of the method of Rappaport et al. (1959). 2.6. Measurement of lipid peroxide levels and antioxidant enzyme activity Lipid peroxidation in the brain was determined by measuring the formation of thiobarbituric acid reactive substances (TBARS) according to the method of Ohkawa et al. (1979). A standard curve was obtained using 1,1,3,3-tetramethoxypropane. Catalase activity was measured using the method of Aebi (1974). Brain homogenate was reacted with hydrogen peroxide, and the decomposition of hydrogen peroxide was determined spectrophotometrically at 240 nm. Superoxide dismutase (SOD) activities were assayed according to Marklund and Marklund (1974). Glutathione peroxidase (GPx) activity was measured according to Lawrence and Burk (1976). The decrease in NADPH was recorded at 340 nm and is expressed here using a molar extinction coefficient of NADPH of 6.22 mM−1 cm−1 . 2.7. Statistics
2.3. Passive avoidance test Passive avoidance was tested using a two-compartment shuttle chamber (256000 series, TSE Systems, Germany), one
Statistical analysis was performed using one-way analysis of variance, with Duncan’s multiple test used for group comparison with P < 0.05.
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3. Result 3.1. PWE improves cognitive deficits in Aβ25-35-treated mice It was suggested previously that the i.c.v. injection of A2535 causes memory deficits and lowers choline transferase in hippocampus (Tang et al., 2002; Stepanichev et al., 2004). In the present study, an i.c.v. injection of 10 g of A25-35-induced cognitive dysfunction as assessed by passive avoidance and Morris water-maze tests. Changes in the step-through latency in passive avoidance are shown in Fig. 1. There was no significant difference between the normal and normal PBS groups (i.c.v. injection of PBS), but significantly reduced step-through latency in the A25-35-injected control group (25.3% reduction compared to normal, P < 0.05). This result confirmed that cognitive deficits induced by A i.c.v. were not attributable to the i.c.v. injection itself. As shown in Fig. 1, step-through latency was increased up to 2.5-fold by consumption of the 1% PWE diet (P < 0.05). As shown in Fig. 2, injection of A25-35 increased the escape latency and error frequency in the Morris water-maze test. Treatment of mice with PWE for 4 weeks attenuated the increase in the escape latency almost to that in the normal group, but the change was not dose dependent. Changes in error frequency in each group showed a similar pattern to the escape latency.
Fig. 2. Effect of PWE treatment on performance in the water-maze task by A25-35-treated mice. Escape latency (A) and error frequency (B) were determined. Values are means ± S.E.M. (n = 10; * P < 0.05 compared to controls; # P < 0.05 compared to normals).
3.2. Effect of PWE on AChE activity in the brain and serum 3.3. Effect of PWE on lipid peroxide levels in the brain To determine the effect of PWE on AChE, we evaluated AChE activity in the brain and serum (Table 1). Exposure to A25-35 had no significant effect on AChE activity in brain, but 0.5% PWE resulted in the reduction of AChE activity. In the group consuming 1% PWE, the AChE activity was significantly decreased compared to the control group. The activity of AChE in serum was significantly higher in the control group than in the normal group (21.0%, P < 0.05). In the group consuming 1% PWE, the activity of AChE in serum was significantly lower than that in the control group (25.3% reduction compared to control, P < 0.05).
Lipid peroxide levels in the brain of A25-35-treated mice are shown in Fig. 3. Injection of A25-35 into the cerebral ventricle increased lipid peroxide levels in the brain. PWE slightly reduced the lipid peroxide (TBARS) levels in the brain of A2535 injected mice. 3.4. Effect of PWE on antioxidant enzyme activity in the brain The effect of PWE on specific activities of catalase, SOD, and GPx in the brain is shown in Table 2. Treatment with A25-35 did not alter the activity of catalase in the brain. However, the activity of catalase was decreased in the PWE-treated Table 1 Effect of PWE treatment on acetylcholinesterase activity in the serum and brain
Fig. 1. Protective effect of PWE on A25-35-induced cognitive deficits in mice. The learning and memory performance was assessed by the passive avoidance test. Values are means ± S.E.M. (n = 10; * P < 0.05 compared to controls; # P < 0.05 compared to normal).
Group
Brain (M/(min mg protein))
Serum (Rappaport units/ml)
N NP C 0.5% PW 1% PW
17.0 ± 2.2 ab 17.9 ± 0.9 a 18.4 ± 0.4 a 15.0 ± 0.5 b 14.8 ± 0.3 b
179.9 177.2 219.3 177.6 163.6
± ± ± ± ±
27.8 b 8.6 b 11.7 a 8.3 b 2.2 b
(1) Values are means ± S.E.M. (n = 10). (2) One Rappaport unit is that amount of cholinesterase that will liberate 1 mol of acetic acid from acetylcholine in 30 min at 25 ◦ C and pH 7.8 under the conditions of this test. (3) AChE activities are expressed as the amount of 5-thio-2-nitro-benzoic acid produced by the hydrolysis of substrate. (4) Values with different letters within a row are significantly different at α = 0.05 by Duncan’s multiple-range test.
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Fig. 3. Effect of PWE on TBARS levels in brain tissue of mice treated with A25-35 (* P < 0.05 compared to controls; # P < 0.05 compared to normals). Table 2 The effect of PWE on specific activities of catalase, SOD, and GPx in brain homogenates Group
Catalase (units/ (min mg protein))
SOD (units/ (min mg protein))
GPx (nmol/ (min mg protein))
N NP C 0.5% PW 1% PW
2.96 ± 0.42 n.s. 3.31 ± 0.28 3.37 ± 0.37 2.34 ± 0.16 2.27 ± 0.20
8.94 ± 1.48 n.s. 9.33 ± 0.47 8.88 ± 0.97 6.67 ± 0.16 8.01 ± 0.37
34.81 ± 7.67 ab 35.87 ± 3.43 a 26.97 ± 1.34 ab 24.68 ± 1.10 ab 34.16 ± 5.78 b
(1) Values are mean ± S.E.M. (n = 10). (2) Values with different letters within a column are significantly different at α = 0.05 by Duncan’s multiple-range test. (3) n.s., not significant.
group, with the lowest activity exhibited in mice consuming 1% PWE. The SOD activities did not differ significantly between the groups. The activity of GPx in brain tissue was lower in A2535-treated mice than in normal mice. However, the activity of GPx in brains of mice consuming 1% PWE was increased up to the normal level. 4. Discussion The present study has revealed a neuroprotective effect of PM on A-induced cognitive deficits in mice. Memory associated behavior did not differ between the PBS-injected and normal groups. Wang et al. (2001) similarly demonstrated that i.c.v. injection of A25-35-induced impairment of memory as assessed by passive avoidance and Morris water-maze tests. A has the potential to induce oxidative stress in the brain (Behl and Sagara, 1997). Moreover, it has been reported that A induces the production of hydrogen peroxide and lipid peroxide in hippocampal neurons of the rat brain (Yatin et al., 2000). Jhoo et al. (2000) showed the induction of 4-hydroxy-2-nonenal and 8hydroxy-2 -deoxyguanosine (a marker of oxidative damage to DNA) immunoreactivities following infusion of A1-42 in rat brain. In the present study, we found a significantly increased level of TBARS in mice brain after a single injection with A2535. Furthermore, imbalances in each antioxidant enzyme were also observed. Kim et al. (2003) demonstrated that continuous i.c.v. infusion of A1-42 in rat resulted in a significant decrease in protein expression of SOD, GPx, and glutathioneS-transferase- in rat brain, from which they suggested that
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A1-42 impairs antioxidant capacity. We found that consumption of a diet containing PWE ameliorated cognitive deficits in A25-35-injected mice. Especially, the data show that the step-through latency in passive avoidance increased in a dosedependent manner, but not in the water-maze test. Also, consumption of PWE decreased the escape latency almost to normal levels. It is possible that neuroprotection plays a role in the favorable effect of PWE on A25-35-induced cognitive deficits. Antioxidants, such as ␣-tocopherol and ferulic acid protect against learning and memory deficits induced by A (Yamada et al., 1999; Yan et al., 2001). The AChE activity has been shown to be increased within and around amyloid plaques in Alzheimer’s brain (Ulrich et al., 1990). The enhancement of AChE activity induced by A25-35 is mediated by oxidative stress (Melo et al., 2003). The AChE activity in the brain and serum was increased in mice treated with A25-35 when compared with the normal in our experiment. In addition, the A25-35-induced increase in AChE was attenuated by PWE consumption. PM is a medicinal plant that has antioxidant properties in vitro (Ryu et al., 2002) and delays aging responses in vivo (Chiu et al., 2002) involving oxidative stress. Also, previous studies have shown that PM ethanol extract suppressed lipid peroxidation in the mitochondria of rat heart (Chen et al., 1999). Chan et al. (2002) demonstrated that PM ethanol extract significantly improved learning and memory deficits in SAMP8 (a murine AD model), and lowered lipofuscin percentages and MDA concentrations in hippocampus, and increased total thiol concentrations. Catalase, SOD, and GPx are involved in the reduction in reactive oxygen species and peroxides produced in living organisms as well as in the detoxification of certain compounds of exogenous origin, and thus play a primary role in the maintenance of a balanced redox status (Kweon et al., 2003). Treatment of mice with PWE for 4 weeks decreased TBARS level and increased GPx activity in the brain, while having no significant effect on catalase and SOD activity. These findings can be attributed to GPx exhibiting a higher sensitivity to lipid peroxidation than catalase or SOD. Therefore, we suggest that accumulation of lipid peroxides by A was reduced by PWE via antioxidative mechanisms. In conclusion, we suggest that PWE markedly improves cognitive deficits induced by A25-35, and that this effect is mediated by the antioxidant properties of PWE. Future studies should determine the specific components in PWE responsible for preventing cognitive impairment. References Aebi, H., 1974. In: Bergmeyer, H.U. (Ed.), Catalase. Methods of Enzymatic Analysis. Academic Press, New York and London, pp. 637–684. Atack, J.R., Perry, E.K., Bonham, J.R., Perry, R.H., Tomlinson, B.E., Blessed, G., Fairbairn, A., 1983. Molecular forms of acetylcholinesterase in senile dementia of Alzheimer type: selective loss of the intermediate (10S) form. Neuroscience Letters 40, 199–204. Behl, C., Davis, J., Cole, G.M., Schubert, D., 1992. Vitamin E protects nerve cells from amyloid beta protein toxicity. Biochemical and Biophysical Research Communications 186, 944–950. Behl, C., Sagara, Y., 1997. Mechanism of amyloid beta protein induced neuronal cell death: current concepts and future perspectives. Journal of Neural Transmission. 49, 125–134.
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