Uncaria rhynchophylla Ameliorates Cognitive Deficits Induced by D-galactose in Mice

• Abstract
• Introduction
• Materials and Methods
• Reagents and chemicals
• Plant material and extraction
• Animals
• Groups and drug treatment
• Open-field test
• Morris water maze test
• Preparation of the brain tissue samples
• Assay of the MDA level
• Assay of the GSH level
• Assay of the Ach level
• Measurement of the AChE activity
• Protein assay
• Statistical analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

The stem with hooks of Uncaria rhynchophylla is a component herb of many traditional formulae for the treatment of neurodegenerative diseases. However, scientific evidence of the efficacy of Uncaria rhynchophylla in the treatment of Alzheimer's disease (AD) in animal models is lacking. Thus, in the present study, we investigated whether the 70 % aqueous ethanol extract of Uncaria rhynchophylla (EUR) could protect against D-galactose (D-gal)-induced cognitive deficits in mice. Mice were given a subcutaneous injection of D-gal (50 mg/kg) and orally administered EUR (100, 200, or 400 mg/kg) daily for 8 weeks. The effect of EUR on D-gal-induced cognitive deficits was evaluated by measuring behavioral and neurochemical parameters of AD and the antioxidant status of brain tissue. The results showed that EUR (200 or 400 mg/kg) significantly increased exploratory behavior (assessed by an open-field test) and improved spatial learning and memory function (assessed by the Morris water maze test) in D-gal-treated mice. In addition, EUR (200 or 400 mg/kg) significantly increased the levels of acetylcholine and glutathione and decreased the activity of acetylcholinesterase and the level of malondialdehyde in the brains of D-gal-treated mice. These results indicate that EUR ameliorates cognitive deficits induced by D-gal in mice, and that this action may be mediated, at least in part, by the inhibition of acetylcholinesterase activity and the enhancement of the antioxidant status of brain tissue.

Introduction

Alzheimer's disease (AD) is a multifaceted neurodegenerative disorder characterized by the progressive deterioration of cognitive function and memory, which is associated with widespread neuronal loss and the deposition of senile plaques [1]. Epidemiological studies show that the number of AD patients worldwide reached 25 million in 2000, and predict that this number will exceed 114 million by 2050 if there is no new preventive or therapeutic approaches to be established [2]. Current clinical strategies for AD treatment aim at delaying the progression of deterioration in AD patients by using acetylcholinesterase inhibitors (AChEIs) such as tacrine, donepezil, rivastigmine, and galantamine. These drugs have been used successfully for treating cognitive deficits; however, they have undesirable side effects in AD patients, including hypertensive crisis, nausea, diarrhea, and vomiting [3]. Thus, there is an unmet need for safe, better tolerated, and powerful drugs for the treatment of AD.

Recently, herbal remedies have attracted a great deal of attention as alternative and supplemental medicines. Herbal extracts have been proven to be useful in the treatment of age-associated diseases [4]. Uncaria rhynchophylla (Miq.) Miq. ex Havil. (Rubiaceae) is one of the best-known herbs in China, Korea, and Japan. It has been used for thousands of years in Chinese medicine to relieve headache, dizziness, tremors, and convulsions resulting from hypertension [5], [6]. It is a component herb of many popular herbal formulae, such as Chotosan (Gouteng-San in Chinese) and Yokukansan (Yigan-San in Chinese), prescribed for the treatment of AD [7], [8]. Previous studies have shown that Uncaria rhynchophylla exhibits various kinds of pharmacological properties, including antihypertensive [5], anti-inflammatory [9], anticonvulsant [6], anxiolytic [10], neuroprotectant [11], and antioxidant activities [6]. It has also been shown to inhibit beta-amyloid (Aβ) fibril formation and disassemble preformed Aβ fibrils [12]. Excessive accumulation of Aβ plays a pivotal role in the pathogenesis of AD as a neurotoxic agent [13]. As we have known, no previous work has been done to investigate whether Uncaria rhynchophylla has a cognition-improving effect in animal models of AD.

D-galactose (D-gal) is found in lactose (milk sugar). It can be metabolized to glucose 6-phosphate in the liver at a normal concentration. However, at high concentration levels, it can turn into aldose and hydroperoxide under catalysis by galactose oxidase, leading to the excessive production of reactive oxygen species [14]. Recent studies have demonstrated that rodents receiving continuous subcutaneous injections of D-gal show progressive deterioration in learning and memory capacity [14], [15], [16], pathological changes in the brain including a decrease of antioxidant enzyme activity and increase of the production of free radicals [17], [18], and impairment of neurogenesis [15], [19] and cholinergic neurons [20], which resemble brain aging processes. Therefore, long-term injection of D-gal in rodents has been widely used for inducing brain aging in the animals [15], [16], [17], [18], [19], which serve as a feasible model for investigating the mechanism of oxidative stress-induced AD.

In the present study, we tried to examine whether Uncaria rhynchophylla can protect against D-gal-induced cognitive deficits in mice. To investigate the biochemical mechanism involved in such protection, we measured the levels of glutathione (GSH), malondialdehyde (MDA), acetylcholine (Ach), and activity of acetylcholinesterase (AChE) in the brain of D-gal-treated mice.

Materials and Methods

Reagents and chemicals

D-galactose (D-gal), thiobarbituric acid, sodium dodecyl sulfate, tetramethoxypropane, trichloroacetic acid, 5,5′-dithiobis-2-nitrobenzoic acid, calabarine sulfate, trichloracetic acid, acetylthiocholine iodide, hydroxylamine hydrochloride, ferric chloride, and dithiobisnitrobenzoic acid (DTNB) were purchased from Sigma-Aldrich. Calabarine sulfate was purchased from Tianjin Yifang Technology Co. Ltd. Vitamin E (98 % pure) was purchased from Hangzhou Huadong Medicine Group Wufeng Pharmaceutical Co. Ltd. Standard substances (rhynchophylline, isorhynchophylline, corynoxeine, and isocorynoxeine, 98 % pure) were purchased from Chengdu Mansite Pharmacetical Co. Ltd. All other reagents and chemicals used in the study were of analytical grade.

Plant material and extraction

The dried hook-bearing stem and branch of Uncaria rhynchophylla was purchased from Zhixin Pharmaceutical Co. It was authenticated to be the dried rhizome of Uncaria rhynchophylla (Miq.) Jacks. (Gou-teng in Chinese), according to the guidelines of the Chinese Pharmacopoeia (2010), by Ms. Y. Y. Zong, School of Chinese Medicine, the Chinese University of Hong Kong, Hong Kong, where a voucher specimen (No. 091220) has been deposited. Uncaria rhynchophylla (400 g) was macerated in 4 L of 70 % aqueous ethanol for 24 h at room temperature and then refluxed for 30 min. Extraction was repeated twice. The pooled extract was combined and concentrated under reduced pressure, followed by freeze drying. The yield of the extract (EUR) was approximately 14 % (w/w). The extract was analyzed by high-performance liquid chromatography (HPLC) as follows. A Nucleosil 100 C18 HPLC column (4.6 mm × 250 mm) was used for separation. The mobile phase consisted of 0.01 mmol/L triethylamine in water (solvent A) and methanol (solvent B). Separation was achieved by a linear gradient elution from 60 % to 85 % solvent B over 40 min at a flow rate of 1.0 mL/min. The eluate was monitored by a diode array detector at a wavelength of 245 nm. The extract was found to contain 0.40 % rhynchophylline, 0.20 % isorhynchophylline, 0.26 % corynoxeine, and 0.55 % isocorynoxeine ([Fig. 1]).

Animals

Ten-week-old male ICR mice were obtained from the Laboratory Animal Services Center, the Chinese University of Hong Kong. The animals were maintained on a 12-h light/dark cycle under controlled temperature (22 °C ± 2 °C) and humidity (50 % ± 10 %) levels, and given standard diet and water ad libitum. They were allowed to acclimatize for 7 days before use. The experiments were approved by the Animal Experimentation Ethics Committee of the Chinese University of Hong Kong on April 30, 2009 (Ref. No. 09/010/MIS), and conformed to the guidelines of the Principles of Laboratory Animal Care (NIH publication No. 80-23, revised 1996).

Groups and drug treatment

Mice were randomly assigned to six groups of ten individuals each: control, D-gal plus vehicle (D-gal-treated control), D-gal plus EUR (100 mg/kg), D-gal plus EUR (200 mg/kg), D-gal plus EUR (400 mg/kg), and D-gal plus vitamin E (100 mg/kg). In the clinical practice of Chinese herbal medicine, the prescribed daily dose of Uncaria rhynchophylla is usually 9 g of plant material which was equivalent to the middle dose (200 mg extract/kg) used in this study. The dose of the positive control (reference compound), vitamin E, was taken from a previous study [18]. D-gal was dissolved in physiological saline. Except for controls, the mice received a daily subcutaneous injection of D-gal at a dose of 50 mg/kg, while those in the control group received an injection of the same volume of physiological saline for 8 weeks. Both EUR and vitamin E were dissolved in 0.1 % Tween 80 and given intragastrically 30 min before D-gal injection. All drugs were given daily between 9:00 a. m. and 11:00 a. m. for 8 weeks. Twenty-four hours after the last EUR or vitamin E administration, the behavioral tests (open-field test and Morris water maze) were carried out.

Open-field test

The open-field test was carried out after subcutaneous injection of D-gal for 8 weeks. The test was performed as described previously [17], with minor modifications. The open-field apparatus consisted of a square wooden arena (30  × 30  × 15 cm), with black inner walls. The floor of the wooden arena was divided equally into 25 squares marked by black lines. The test was performed in the breeding room between 9:00 a. m. and 4:00 p. m. Each mouse was placed individually in the center of the arena and allowed to explore freely. The number of both line crossings (squares crossed with all paws) and rearings (rising of the front paws) was recorded during a test period of 3 min. The apparatus was cleaned with a detergent and dried after occupancy by each mouse.

Morris water maze test

Twenty-four hours after the open-field test, the Morris water maze test was conducted as described previously [16], with minor modifications. The experimental apparatus (Water Mazes; TSE Systems) consisted of a blank circular water tank (100 cm in diameter, 35 cm in height), containing water (23 ± 1 °C) to a depth of 15.5 cm, which was rendered opaque by adding ink. Four poles along the perimeter of the pool conceptually divided the maze into four equal quadrants. A platform (4.5 cm in diameter, 14.5 cm in height) was submerged 1 cm below the water surface and placed at the midpoint of one quadrant. The pool was located in a test room, which contained various prominent visual cues (e.g., pictures, lamps, etc.). Twenty-four hours before the spatial training, each mouse received four pretraining sessions: the mouse was put on the platform for 20 sec, given a 30-sec free swim, and then assisted to the platform where it was allowed to rest for another 20 sec. Spatial training to find the hidden platform in the water maze was conducted for four consecutive days. Each day, the mouse was placed in the water facing the pool wall at one of three randomized starting positions (in three different quadrants that did not contain the platform). The mice were given 60 sec to find the platform and allowed to stay on it for 20 sec. Animals that failed to find the platform within 60 sec were gently guided to the platform and allowed to stay on it for 20 sec, and the escape latency (finding the submerged platform) was recorded as 60 sec. Each mouse performed four trials per day, and the intertrial interval was 60 sec. The escape latency to the platform of each trial was recorded, and the average value of the four trials was calculated. Twenty-four hours after the last day of spatial training, the probe test was carried out by removing the platform and allowing each mouse to swim freely for 60 sec. The time that each mouse spent swimming in the target quadrant (where the platform was once hidden) and the number of times (frequency) that it crossed over the platform site were recorded.

Preparation of the brain tissue samples

Twenty-four hours after the completion of the behavioral tests, the mice were sacrificed, and their brains were rapidly harvested. Each brain was washed with cold sterile physiological saline and stored at −80 °C until use. For biochemical analysis, 10 % (w/v) brain homogenate was prepared in sodium phosphate buffer (0.1 M PBS, pH 7.4) containing a protease inhibitor cocktail (Sigma-Aldrich), using a potter homogenizer at a speed of 1200 rpm. The homogenate was centrifuged at 9000 × g for 15 min at 4 °C. The supernatant was collected and stored at −80 °C until use.

Assay of the MDA level

The MDA level in the brain was measured according to the method described in a previous study [21]. In brief, an aliquot (100 µL) of brain homogenate was mixed with 1.5 mL of 20 % (v/v) acetic acid, 1.5 mL of 0.8 % (w/v) thiobarbituric acid, and 200 µL of 8 % (w/v) sodium dodecyl sulfate. Each reaction mixture was heated for 60 min at 95 °C and cooled to room temperature. Then the mixture was extracted with 5 mL of n-butanol. The mixture was centrifuged at 3000 × g for 10 min, the n-butanol layer was collected, and its absorbance was measured at 532 nm using a spectrophotometer.

Assay of the GSH level

The GSH level in the brain was measured following a method previously described [21]. Briefly, an aliquot (100 µL) of brain homogenate was mixed with 200 µL of trichloroacetic acid (25 %, v/v) and 200 µL of saline. The mixture was centrifuged at 3000 × g for 10 min at 4 °C. An aliquot (200 µL) of supernatant was mixed with 1 mL of phosphate buffer (100 mM, pH 8.0) and 50 µL of 5,5′-dithiobis-2-nitrobenzoic acid (3 mM). The solution was left to stand at room temperature for 5 min, and absorbance was measured at 412 nm using a spectrophotometer.

Assay of the Ach level

The Ach level in the brain was measured according to a method previously described [18]. Briefly, 0.8 mL of brain homogenate was mixed with 1.4 mL of distilled water, to which 0.2 mL of calabarine sulfate (1.54 mmol) was then added. Next, 0.8 mL of trichloracetic acid (1.84 M) was added and mixed in thoroughly. The mixture was centrifuged at 3000 × g for 15 min. An aliquot (1.0 mL) of the supernatant was mixed with 1.0 mL of alkaline hydroxylamine solution (prepared by mixing equal volumes of 3.5 M sodium hydroxide and 2 M hydroxylamine hydrochloride solutions). Each reaction solution was incubated at room temperature for 15 min. Subsequently, 0.5 mL of hydrochloride acid (4 M) and 0.5 mL of ferric chloride (0.37 M) were added, and the solution was shaken violently. Absorbance was measured at 540 nm using a spectrophotometer.

Measurement of the AChE activity

The activity of AChE was determined according to a method described in previous studies [22], with minor modifications. Briefly, the reaction mixture consisted of 50 µL of brain homogenate, 2.85 mL of phosphate buffer (0.1 M, pH 8.0), and 50 µL of dithiobisnitrobenzoic acid (DTNB, 10 mM). After adding 20 µL of the substrate acetylthiocholine iodide (75 mM), the change in absorbance was monitored at 412 nm for 10 min using a spectrophotometer. Enzyme activity is expressed as nmol of substrate hydrolyzed per minute per mg of protein.

Protein assay

Protein concentration was determined by using bovine serum albumin as the standard.

Statistical analysis

Data are expressed as the mean plus or minus the standard error of the mean (mean ± SEM). Group differences in the escape latency in the Morris water maze training task were analyzed using two-way analysis of variance (ANOVA) with repeated measures, the factors being treatment and training day. The other data were analyzed using one-way ANOVA followed by Dunnett's test to detect intergroup differences. GraphPad Prism software was used to perform the statistical analysis (Version 4.0; GraphPad Software, Inc.). A difference was considered statistically significant if the p value was less than 0.05.

Results

Based on the spontaneous exploration of a novel environment, the open-field test is one of the most widely used behavioral tests [17]. As shown in [Fig. 2], among mice given subcutaneous injections of D-gal for 8 weeks, the number of crossings [F (5, 54) = 8.044, p < 0.01] ([Fig. 2 A]) and rearings [F (5, 54) = 3.411, p < 0.01] ([Fig. 2 B]) significantly decreased, compared to controls (i.e., non-D-gal-treated mice). This result suggests the impairment of exploration activity in mice treated with D-gal. Among D-gal-treated mice given EUR (200 or 400 mg/kg) for 8 weeks, the number of crossings (p < 0.05 and p < 0.01, respectively) and rearings (p < 0.01 and p < 0.01, respectively) significantly increased, compared to D-gal-treated controls. This result suggests that EUR could reverse the reduction in the locomotor activity induced by D-gal. Vitamin E treatment (100 mg/kg) also significantly increased the number of crossings (p < 0.01) and rearings (p < 0.01) of D-gal-treated mice.

The Morris water maze is sensitive in revealing impairments in spatial learning and memory. During the training period, the performance of all groups of mice improved, as indicated by the shortened escape latency across successive days ([Fig. 3 A]). A significant difference was found in mean latency between training days [F (3, 216) = 326.9, p < 0.001] and between treatments [F (5, 216) = 18.95, p < 0.001], but no interaction was observed between training day and treatment [F (15, 216) = 326.9, p > 0.05]. However, the D-gal-treated mice took longer to find the platform throughout the training period compared to the control mice [day 1, F (5, 54) = 0.5437, p > 0.05; day 2, F (5, 54) = 4.855, p < 0.01; day 3, F (5, 54) = 11.76, p < 0.01; day 4, F (5, 54) = 16.46, p < 0.01]. This result reveals that the D-gal-treated mice had significant cognitive impairment. EUR (200 mg/kg) treatment significantly reduced the mean latency to find the platform from the third day onwards (p < 0.05) among the D-gal-treated mice, compared to the D-gal-treated controls. EUR (400 mg/kg) and vitamin E (100 mg/kg) treatment also significantly shortened the mean latency to find the platform from the third day onwards (p < 0.01 and p < 0.01, respectively) in the D-gal-treated mice, compared to the D-gal-treated controls, but EUR (100 mg/kg) had no effect in the training trials among the D-gal-treated mice.

In the probe test, the number of target crossings ([Fig. 3 B]) and time spent in the target quadrant ([Fig. 3 C]) were significantly reduced among the D-gal-treated mice [F (5, 54) = 3.727, p < 0.01 and F (5, 54) = 4.874, p < 0.01, respectively], compared to the controls. EUR (200 or 400 mg/kg) treatment led to a significant increase in the number of target crossings (p < 0.05 and p < 0.01, respectively) in the D-gal-treated mice, compared to the D-gal-treated controls. In addition, D-gal-treated mice in the EUR (200 or 400 mg/kg) treatment groups spent a longer time in the target quadrant (p < 0.05 and p < 0.01, respectively), compared to the D-gal-treated controls. Vitamin E (100 mg/kg) treatment also significantly increased the number of target crossings and time spent in the target quadrant (p < 0.01 and p < 0.01, respectively) among D-gal-treated mice. Taken together, these results indicate that EUR and vitamin E can improve the spatial learning and memory abilities of D-gal-treated mice.

The antioxidant status of brain tissue was assessed by measuring the levels of GSH ([Fig. 4 A]) and MDA ([Fig. 4 B]). The level of GSH in the brain of D-gal-treated mice significantly decreased [F (5, 54) = 7.431, p < 0.01], compared to controls. Among the D-gal-treated mice treated with EUR (200 and 400 mg/kg) for 8 weeks, GSH levels in the brain tissue significantly increased (p < 0.05 and p < 0.01, respectively), compared to the D-gal-treated controls. In addition, the level of MDA in the brain of D-gal-treated mice significantly increased [F (5, 54) = 13.33, p < 0.01], compared to controls. This increase was attenuated among the D-gal-treated mice treated with EUR at dosages of 200 and 400 mg/kg (p < 0.01 and p < 0.01, respectively), compared to the D-gal-treated controls. Vitamin E treatment (100 mg/kg) also significantly decreased GSH depletion (p < 0.01) and MDA production (p < 0.01) in the brain of D-gal-treated mice. All of these results demonstrate that EUR enhanced the ability of the mouse brain to protect itself against oxidative stress induced by D-gal, as expected for an antioxidant drug.

The effect of EUR treatment on the cholinergic system in postmortem brain tissue was evaluated by determining the level of Ach ([Fig. 5 A]) and the activity of AChE ([Fig. 5 B]). D-gal was found to significantly decrease [F (5, 54) = 7.542, p < 0.01] the level of Ach in the brain of treated mice, compared to controls. Treatment with EUR at daily doses of 200 or 400 mg/kg for 8 weeks significantly increased the level of Ach in the brain of D-gal-treated mice (p < 0.05 and p < 0.01, respectively), compared to D-gal-treated controls. D-gal significantly increased the AChE activity [F (5, 54) = 14.95, p < 0.01] in the brain of treated mice, compared to controls. Treatment with EUR (200 or 400 mg/kg) for 8 weeks significantly decreased the AChE activity in the brain of D-gal-treated mice (p < 0.05 and p < 0.01, respectively), compared to D-gal-treated controls. Vitamin E (100 mg/kg) also showed similar effects to those of EUR on the Ach level and AChE activity in the brain of D-gal-treated mice. Taken together, these results suggest that the effect of EUR on the dysfunction of the cholinergic system induced by long-term exposure to D-gal is similar to that of vitamin E, the reference compound.

Discussion

Animal models play a pivotal role in understanding the pathology and therapeutics of AD [23]. Although no animal model can fully mimic the human pathologic spectrum of the disease, the long-term subcutaneous injection of D-gal in mice has been shown to induce several characteristics of AD, including the overproduction of free radicals and impairment of the basal forebrain cholinergic system, leading to the decreased expression of memory-related protein and deterioration of learning and memory function [15], [16], [17], [18], [19], [24], [25]. In addition, long-term injection of D-gal can impair neurogenesis in the dentate gyrus, a process similar to natural aging, in mice [15]. Thus, the D-gal-induced senescent model can be used for evaluating the efficacy of anti-dementia treatment through behavioral tests such as the open-field test and Morris water maze task [16], [25].

In the open-field test, normal animals usually show increased locomotor activity in a novel environment, driven by the instinct for exploration [26]. However, after long-term injection of D-gal, animals display decreased locomotor activity and impairment of novelty-induced exploratory behavior [16], [25]. In this study, subcutaneous injection of D-gal into mice for 8 weeks caused a significant decrease in the number of crossings and rearings in the open-field test. Long-term treatment with EUR was shown to reverse the D-gal-induced abnormal locomotor activity. However, it is possible that the beneficial effect of EUR was due to its improvement on motivational impairments induced by D-gal. Therefore, the Morris water maze test, especially for the measurement on “time spent in the target quadrant” ([Fig. 3 C]), is important to demonstrate the protecting effect of EUR against cognitive deficits induced by D-gal. The Morris water maze test, which is sensitive in revealing impairments of spatial learning and memory, revealed significant levels of such impairments in the D-gal treated mice. This observation is consistent with previous reports [18], [25]. Long-term treatment with EUR was found to have a positive effect on the D-gal-induced learning and memory impairment of treated mice; namely, EUR was able to reverse learning and memory deficits induced by D-gal.

The formation of reactive oxygen species (ROS) has been proposed to be an important step leading to neuronal death in a variety of age-related neurodegenerative disorders such as AD and Parkinson's disease [27], [28]. Oxidative damage resulting from ROS is thought to play a key role in the aging process [16]. Excessive ROS production can cause oxidative damage to cellular macromolecules and induce aging and the development of degenerative diseases [29]. In this study, D-gal was found to impair the antioxidant status of brain tissue, presumably through the overproduction of ROS. This observation is consistent with that of previous studies [18], [25]. The ability of EUR to inhibit D-gal-induced GSH depletion and MDA production (i.e., lipid peroxidation) is attributed, at least in part, to its antioxidant activity [6]. In other words, the protection offered by EUR against oxidative stress to the brain may be involved in the action mechanism of EUR in ameliorating the impairments of learning and memory.

Accumulating evidence shows that learning and memory deficits in a number of neurodegenerative disorders are correlated with the degeneration of cholinergic neurons [20], [30]. The cholinergic system plays an essential role in the cognitive functions of attention, learning, and memory. Ach, a neurotransmitter in the central nervous system, plays a major role in modulating these functions [31]. AChE is one of the key cholinergic enzymes in the nervous system, terminating nerve impulses by catalyzing the hydrolysis of the neurotransmitter Ach. It is known that Ach and AChE regulate neuritic outgrowth and the survival of cultured neurons [32], [33]. Therefore, preventing the decrease of the level of Ach, and the activation of AChE, may be an effective treatment strategy for age-related neurodegenerative diseases. The results of this study revealed that subcutaneous injection of D-gal in mice for 8 weeks significantly decreased the level of Ach and increased the AChE activity in the brain of the treated mice. Long-term treatment with EUR inhibited Ach depletion and AChE activation. Thus, the beneficial effect of EUR in improving cognitive deficits may be mediated by the inhibition of AChE activity.

In conclusion, the results of this study showed that long-term treatment with EUR significantly improved cognitive deficits induced by D-gal in mice and suggest that this action may be mediated, at least in part, by the inhibition of AChE activity and the enhancement of antioxidant activity in brain tissue. However, further investigation is needed to reveal the detailed mechanism of EUR in the treatment of AD.

Acknowledgements

This study was supported by a Direct Grant for Research from the Chinese University of Hong Kong.

Conflict of Interest

The authors declare that there are no conflicts of interest.

References

• 1 Ivins K J, Ivins J K, Sharp J P, Cotman C W. Multiple pathways of apoptosis in PC12 cells. CrmA inhibits apoptosis induced by beta-amyloid.  J Biol Chem. 1999;  274 2107-2112
  CrossrefPubMedSearch in Google Scholar
• 2 Wimo A, Winblad B, Aguero Torres H, von Strauss E. The magnitude of dementia occurrence in the world.  Alzheimer Dis Assoc Disord. 2003;  17 63-67
  CrossrefPubMedSearch in Google Scholar
• 3 Inglis F. The tolerability and safety of cholinesterase inhibitors in the treatment of dementia.  Int J Clin Pract Suppl. 2002;  127 45-63
  PubMedSearch in Google Scholar
• 4 Oken B S, Storzbach D M, Kaye J A. The efficacy of Ginkgo biloba on cognitive function in Alzheimer disease.  Arch Neurol. 1998;  55 1409-1415
  CrossrefPubMedSearch in Google Scholar
• 5 Kuramochi T, Chu J, Suga T. Gou-teng (from Uncaria rhynchophylla Miquel)-induced endothelium-dependent and -independent relaxations in the isolated rat aorta.  Life Sci. 1994;  54 2061-2065
  CrossrefPubMedSearch in Google Scholar
• 6 Hsieh C L, Tang N Y, Chiang S Y, Hsieh C T, Lin J G. Anticonvulsive and free radical scavenging actions of two herbs, Uncaria rhynchophylla (MIQ) Jack and Gastrodia elata BI., in kainic acid-treated rats.  Life Sci. 1999;  65 2071-2082
  CrossrefPubMedSearch in Google Scholar
• 7 Watanabe H, Zhao Q, Matsumoto K, Tohda M, Murakami Y, Zhang S H, Kang T H, Mahakunakorn P, Maruyama Y, Sakakibara I, Aimi N, Takayama H. Pharmacological evidence for antidementia effect of Choto-san (Gouteng-san), a traditional Kampo medicine.  Pharmacol Biochem Behav. 2003;  75 635-643
  CrossrefPubMedSearch in Google Scholar
• 8 Tabuchi M, Yamaguchi T, Lizuka S, Imamura S, Ikarashi Y, Kase Y. Ameliorative effects of yokukansan, a traditional Japanese medicine, on learning and non-cognitive disturbances in the Tg2576 mouse model of Alzheimer's disease.  J Ethnopharmacol. 2009;  122 157-162
  CrossrefPubMedSearch in Google Scholar
• 9 Kim D Y, Jung J A, Kim T H, Seo S W, Jung S K, Park C S. Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-γ down-regulation in NC/Nga mice.  J Ethnopharmacol. 2009;  122 567-572
  CrossrefPubMedSearch in Google Scholar
• 10 Jung J W, Ahn N Y, Oh H R, Lee B K, Lee K J, Kim S Y, Cheong J H, Ryu J H. Anxiolytic effects of the aqueous extract of Uncaria rhynchophylla.  J Ethnopharmacol. 2006;  108 193-197
  CrossrefPubMedSearch in Google Scholar
• 11 Shim J S, Kim H G, Ju M S, Choi J G, Jeong S Y, Oh M S. Effects of the hook of Uncaria rhynchophylla on neurotoxicity in the 6-hydroxydopamine model of Parkinson's disease.  J Ethnopharmacol. 2009;  126 361-365
  CrossrefPubMedSearch in Google Scholar
• 12 Fujiwara H, Iwasaki K, Furukawa K, Seki S, He M, Maruyama M, Tomita N, Kudo Y, Higuchi M, Saido T C, Maeda S, Takashima A, Hara M, Ohizumi Y, Arai H. Uncaria rhynchophylla, a Chinese medicinal herb, has potent antiaggregation effects on Alzheimer's beta-amyloid proteins.  J Neurosci Res. 2006;  84 427-433
  CrossrefPubMedSearch in Google Scholar
• 13 Hellström-Lindahl E, Viitanen M, Marutle A. Comparison of Abeta levels in the brain of familial and sporadic Alzheimer's disease.  Neurochem Int. 2009;  55 243-252
  CrossrefPubMedSearch in Google Scholar
• 14 Zhang C, Wang S Z, Zuo P P, Cui X. Protective effect of tetramethylpyrazine on learning and memory function in D-galactose lesioned mice.  Chin Med Sci J. 2004;  19 180-184
  PubMedSearch in Google Scholar
• 15 Cui X, Zuo P, Zhang Q, Li X, Hu Y, Long J, Packer L, Liu J. Chronic systemic D-galactose exposure induces memory loss, neurodegeneration and oxidative damage in mice: protective effects of R-alpha-lipoic acid.  J Neurosci Res. 2006;  83 1584-1590
  CrossrefPubMedSearch in Google Scholar
• 16 Lu J, Zheng Y L, Luo L, Wu D M, Sun D X, Feng Y J. Quercetin reverses D-galactose induced neurotoxicity in mouse brain.  Behav Brain Res. 2006;  171 251-260
  CrossrefPubMedSearch in Google Scholar
• 17 Lu J, Zheng Y L, Wu D M, Luo L, Sun D X, Shan Q. Ursolic acid ameliorates cognition deficits and attenuates oxidative damage in the brain of senescent mice induced by D-galactose.  Biochem Pharmacol. 2007;  74 1078-1090
  CrossrefPubMedSearch in Google Scholar
• 18 Zhong S Z, Ge Q H, Qu R, Li Q, Ma S P. Paeonol attenuates neurotoxicity and ameliorates cognitive impairment induced by D-galactose in ICR mice.  J Neurol Sci. 2009;  277 58-64
  CrossrefPubMedSearch in Google Scholar
• 19 Zhang Q, Li X K, Cui X, Zuo P P. D-galactose injured neurogenesis in the hippocampus of adult mice.  Neurol Res. 2005;  27 552-556
  CrossrefPubMedSearch in Google Scholar
• 20 Lu J, Wu D M, Hu B, Cheng W, Zheng Y L, Zhang Z F, Ye Q, Fan S H, Shan Q, Wang Y J. Chronic administration of troxerutin protects mouse brain against D-galactose-induced impairment of cholinergic system.  Neurobiol Learn Mem. 2010;  93 157-164
  CrossrefPubMedSearch in Google Scholar
• 21 Mao Q Q, Ip S P, Ko K M, Tsai S H, Xian Y F, Che C T. Effects of peony glycosides on mice exposed to chronic unpredictable stress: further evidence for antidepressant-like activity.  J Ethnopharmacol. 2009;  124 316-320
  CrossrefPubMedSearch in Google Scholar
• 22 Ellman G L, Courtney K O, Andres V J, Feather-Stone R M. A new and rapid colorimetric determination of acetylcholinesterase activity.  Biochem Pharmacol. 1961;  7 88-95
  CrossrefPubMedSearch in Google Scholar
• 23 Barry D G, Mary J S, Davis S H, Shujath M A, Sandi L S, George P, Robert S, Richard W S. APP transgenesis: approaches toward the development of animal models for Alzheimer disease neuropathology.  Neurobiol Aging. 1996;  17 153-171
  CrossrefPubMedSearch in Google Scholar
• 24 Lei M, Su Y, Hua X, Ding J, Han Q, Hu G, Xiao M. Chronic systemic injection of D-galactose impairs the septohippocampal cholinergic system in rats.  Neuroreport. 2008;  19 1611-1615
  CrossrefPubMedSearch in Google Scholar
• 25 Long J, Wang X, Gao H, Liu Z, Liu C, Miao M, Cui X, Packer L, Liu J. D-galactose toxicity in mice is associated with mitochondrial dysfunction: protecting effects of mitochondrial nutrient R-alpha-lipoic acid.  Biogerontology. 2007;  8 373-381
  CrossrefPubMedSearch in Google Scholar
• 26 Luo D D, An S C, Zhang X. Involvement of hippocampal serotonin and neuropeptide Y in depression induced by chronic unpredicted mild stress.  Brain Res Bull. 2008;  72 8-12
  PubMedSearch in Google Scholar
• 27 Castegna A, Aksenov M, Aksenova M, Thongboonkerd V, Klein J B, Pierce W M, Booze R, Markesbery W R, Butterfield D A. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1.  Free Radic Biol Med. 2002;  33 562-571
  CrossrefPubMedSearch in Google Scholar
• 28 De Iuliis A, Grigoletto J, Recchia A, Giusti P, Arslan P. A proteomic approach in the study of an animal model of Parkinson's disease.  Clin Chim Acta. 2005;  357 202-209
  CrossrefPubMedSearch in Google Scholar
• 29 Johnson F B, Sinclair D A, Guarente L. Molecular biology of aging.  Cell. 1999;  96 291-302
  CrossrefPubMedSearch in Google Scholar
• 30 Muthuraju S, Maiti P, Solanki P, Sharma A K, Amitabh, Singh S B, Prasad D, Ilavazhagan G. Acetylcholinesterase inhibitors enhance cognitive functions in rats following hypobaric hypoxia.  Behav Brain Res. 2009;  203 1-14
  CrossrefPubMedSearch in Google Scholar
• 31 Bartus R T, Dean 3rd R L, Beer B, Lippa A S. Cholinergic hypothesis of geriatric memory dysfunction.  Science. 1982;  217 408-414
  CrossrefPubMedSearch in Google Scholar
• 32 Lipton S A, Kater S B. Neurotransmitter regulation of neuronal outgrowth, plasticity and survival.  Trends Neurosci. 1989;  12 265-270
  CrossrefPubMedSearch in Google Scholar
• 33 Small D H. A non-classical action of acetylcholinesterase and acetylcholine in the regulation of neurite outgrowth.  J Neurochem. 1995;  65 (Suppl.) S126C
  PubMedSearch in Google Scholar

Dr. Siu-Po Ip

School of Chinese Medicine
The Chinese University of Hong Kong

Shatin, Hong Kong

China

Phone: +852 31 63 44 57

Fax: +852 31 63 44 59

Email: paulip@cuhk.edu.hk

References

• 1 Ivins K J, Ivins J K, Sharp J P, Cotman C W. Multiple pathways of apoptosis in PC12 cells. CrmA inhibits apoptosis induced by beta-amyloid.  J Biol Chem. 1999;  274 2107-2112
  CrossrefPubMedSearch in Google Scholar
• 2 Wimo A, Winblad B, Aguero Torres H, von Strauss E. The magnitude of dementia occurrence in the world.  Alzheimer Dis Assoc Disord. 2003;  17 63-67
  CrossrefPubMedSearch in Google Scholar
• 3 Inglis F. The tolerability and safety of cholinesterase inhibitors in the treatment of dementia.  Int J Clin Pract Suppl. 2002;  127 45-63
  PubMedSearch in Google Scholar
• 4 Oken B S, Storzbach D M, Kaye J A. The efficacy of Ginkgo biloba on cognitive function in Alzheimer disease.  Arch Neurol. 1998;  55 1409-1415
  CrossrefPubMedSearch in Google Scholar
• 5 Kuramochi T, Chu J, Suga T. Gou-teng (from Uncaria rhynchophylla Miquel)-induced endothelium-dependent and -independent relaxations in the isolated rat aorta.  Life Sci. 1994;  54 2061-2065
  CrossrefPubMedSearch in Google Scholar
• 6 Hsieh C L, Tang N Y, Chiang S Y, Hsieh C T, Lin J G. Anticonvulsive and free radical scavenging actions of two herbs, Uncaria rhynchophylla (MIQ) Jack and Gastrodia elata BI., in kainic acid-treated rats.  Life Sci. 1999;  65 2071-2082
  CrossrefPubMedSearch in Google Scholar
• 7 Watanabe H, Zhao Q, Matsumoto K, Tohda M, Murakami Y, Zhang S H, Kang T H, Mahakunakorn P, Maruyama Y, Sakakibara I, Aimi N, Takayama H. Pharmacological evidence for antidementia effect of Choto-san (Gouteng-san), a traditional Kampo medicine.  Pharmacol Biochem Behav. 2003;  75 635-643
  CrossrefPubMedSearch in Google Scholar
• 8 Tabuchi M, Yamaguchi T, Lizuka S, Imamura S, Ikarashi Y, Kase Y. Ameliorative effects of yokukansan, a traditional Japanese medicine, on learning and non-cognitive disturbances in the Tg2576 mouse model of Alzheimer's disease.  J Ethnopharmacol. 2009;  122 157-162
  CrossrefPubMedSearch in Google Scholar
• 9 Kim D Y, Jung J A, Kim T H, Seo S W, Jung S K, Park C S. Oral administration of Uncariae rhynchophylla inhibits the development of DNFB-induced atopic dermatitis-like skin lesions via IFN-γ down-regulation in NC/Nga mice.  J Ethnopharmacol. 2009;  122 567-572
  CrossrefPubMedSearch in Google Scholar
• 10 Jung J W, Ahn N Y, Oh H R, Lee B K, Lee K J, Kim S Y, Cheong J H, Ryu J H. Anxiolytic effects of the aqueous extract of Uncaria rhynchophylla.  J Ethnopharmacol. 2006;  108 193-197
  CrossrefPubMedSearch in Google Scholar
• 11 Shim J S, Kim H G, Ju M S, Choi J G, Jeong S Y, Oh M S. Effects of the hook of Uncaria rhynchophylla on neurotoxicity in the 6-hydroxydopamine model of Parkinson's disease.  J Ethnopharmacol. 2009;  126 361-365
  CrossrefPubMedSearch in Google Scholar
• 12 Fujiwara H, Iwasaki K, Furukawa K, Seki S, He M, Maruyama M, Tomita N, Kudo Y, Higuchi M, Saido T C, Maeda S, Takashima A, Hara M, Ohizumi Y, Arai H. Uncaria rhynchophylla, a Chinese medicinal herb, has potent antiaggregation effects on Alzheimer's beta-amyloid proteins.  J Neurosci Res. 2006;  84 427-433
  CrossrefPubMedSearch in Google Scholar
• 13 Hellström-Lindahl E, Viitanen M, Marutle A. Comparison of Abeta levels in the brain of familial and sporadic Alzheimer's disease.  Neurochem Int. 2009;  55 243-252
  CrossrefPubMedSearch in Google Scholar
• 14 Zhang C, Wang S Z, Zuo P P, Cui X. Protective effect of tetramethylpyrazine on learning and memory function in D-galactose lesioned mice.  Chin Med Sci J. 2004;  19 180-184
  PubMedSearch in Google Scholar
• 15 Cui X, Zuo P, Zhang Q, Li X, Hu Y, Long J, Packer L, Liu J. Chronic systemic D-galactose exposure induces memory loss, neurodegeneration and oxidative damage in mice: protective effects of R-alpha-lipoic acid.  J Neurosci Res. 2006;  83 1584-1590
  CrossrefPubMedSearch in Google Scholar
• 16 Lu J, Zheng Y L, Luo L, Wu D M, Sun D X, Feng Y J. Quercetin reverses D-galactose induced neurotoxicity in mouse brain.  Behav Brain Res. 2006;  171 251-260
  CrossrefPubMedSearch in Google Scholar
• 17 Lu J, Zheng Y L, Wu D M, Luo L, Sun D X, Shan Q. Ursolic acid ameliorates cognition deficits and attenuates oxidative damage in the brain of senescent mice induced by D-galactose.  Biochem Pharmacol. 2007;  74 1078-1090
  CrossrefPubMedSearch in Google Scholar
• 18 Zhong S Z, Ge Q H, Qu R, Li Q, Ma S P. Paeonol attenuates neurotoxicity and ameliorates cognitive impairment induced by D-galactose in ICR mice.  J Neurol Sci. 2009;  277 58-64
  CrossrefPubMedSearch in Google Scholar
• 19 Zhang Q, Li X K, Cui X, Zuo P P. D-galactose injured neurogenesis in the hippocampus of adult mice.  Neurol Res. 2005;  27 552-556
  CrossrefPubMedSearch in Google Scholar
• 20 Lu J, Wu D M, Hu B, Cheng W, Zheng Y L, Zhang Z F, Ye Q, Fan S H, Shan Q, Wang Y J. Chronic administration of troxerutin protects mouse brain against D-galactose-induced impairment of cholinergic system.  Neurobiol Learn Mem. 2010;  93 157-164
  CrossrefPubMedSearch in Google Scholar
• 21 Mao Q Q, Ip S P, Ko K M, Tsai S H, Xian Y F, Che C T. Effects of peony glycosides on mice exposed to chronic unpredictable stress: further evidence for antidepressant-like activity.  J Ethnopharmacol. 2009;  124 316-320
  CrossrefPubMedSearch in Google Scholar
• 22 Ellman G L, Courtney K O, Andres V J, Feather-Stone R M. A new and rapid colorimetric determination of acetylcholinesterase activity.  Biochem Pharmacol. 1961;  7 88-95
  CrossrefPubMedSearch in Google Scholar
• 23 Barry D G, Mary J S, Davis S H, Shujath M A, Sandi L S, George P, Robert S, Richard W S. APP transgenesis: approaches toward the development of animal models for Alzheimer disease neuropathology.  Neurobiol Aging. 1996;  17 153-171
  CrossrefPubMedSearch in Google Scholar
• 24 Lei M, Su Y, Hua X, Ding J, Han Q, Hu G, Xiao M. Chronic systemic injection of D-galactose impairs the septohippocampal cholinergic system in rats.  Neuroreport. 2008;  19 1611-1615
  CrossrefPubMedSearch in Google Scholar
• 25 Long J, Wang X, Gao H, Liu Z, Liu C, Miao M, Cui X, Packer L, Liu J. D-galactose toxicity in mice is associated with mitochondrial dysfunction: protecting effects of mitochondrial nutrient R-alpha-lipoic acid.  Biogerontology. 2007;  8 373-381
  CrossrefPubMedSearch in Google Scholar
• 26 Luo D D, An S C, Zhang X. Involvement of hippocampal serotonin and neuropeptide Y in depression induced by chronic unpredicted mild stress.  Brain Res Bull. 2008;  72 8-12
  PubMedSearch in Google Scholar
• 27 Castegna A, Aksenov M, Aksenova M, Thongboonkerd V, Klein J B, Pierce W M, Booze R, Markesbery W R, Butterfield D A. Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1.  Free Radic Biol Med. 2002;  33 562-571
  CrossrefPubMedSearch in Google Scholar
• 28 De Iuliis A, Grigoletto J, Recchia A, Giusti P, Arslan P. A proteomic approach in the study of an animal model of Parkinson's disease.  Clin Chim Acta. 2005;  357 202-209
  CrossrefPubMedSearch in Google Scholar
• 29 Johnson F B, Sinclair D A, Guarente L. Molecular biology of aging.  Cell. 1999;  96 291-302
  CrossrefPubMedSearch in Google Scholar
• 30 Muthuraju S, Maiti P, Solanki P, Sharma A K, Amitabh, Singh S B, Prasad D, Ilavazhagan G. Acetylcholinesterase inhibitors enhance cognitive functions in rats following hypobaric hypoxia.  Behav Brain Res. 2009;  203 1-14
  CrossrefPubMedSearch in Google Scholar
• 31 Bartus R T, Dean 3rd R L, Beer B, Lippa A S. Cholinergic hypothesis of geriatric memory dysfunction.  Science. 1982;  217 408-414
  CrossrefPubMedSearch in Google Scholar
• 32 Lipton S A, Kater S B. Neurotransmitter regulation of neuronal outgrowth, plasticity and survival.  Trends Neurosci. 1989;  12 265-270
  CrossrefPubMedSearch in Google Scholar
• 33 Small D H. A non-classical action of acetylcholinesterase and acetylcholine in the regulation of neurite outgrowth.  J Neurochem. 1995;  65 (Suppl.) S126C
  PubMedSearch in Google Scholar

Dr. Siu-Po Ip

School of Chinese Medicine
The Chinese University of Hong Kong

Shatin, Hong Kong

China

Phone: +852 31 63 44 57

Fax: +852 31 63 44 59

Email: paulip@cuhk.edu.hk