Effects of tenuifolin extracted from radix polygalae on learning and memory: a behavioral and biochemical study on aged and amnesic mice.
Although normal cognitive changes take place when a person becomes older, aging in humans is generally associated with deterioration of cognitive performance and, in particular, of learning and memory. These cognitive deficits can cause debilitating consequences due to aging. There are a number of herbal medicines which are reported to improve brain function including intelligence.
In the present study, improving effects of tenuifolin, extracted from Radix Polygalae (RP), on learning and memory in aged and dysmnesia mice were determined using step-down type passive avoidance test or Y type maze trial. Oral administration of tenuifolin (0.02, 0.04, 0.08g/kg [d.sup.-1], for 15d) evidently improved the latency and number of errors in aged and dysmnesia mice. The levels of cortical acetylcholine esterase (AChE) activity and hippocampal neurotransmitters in aged mice given tenuifolin (0.02, 0.04, 0.08g/kg [d.sup.-1], for 15d) were also investigated, and increased levels of norepinephrine (NE), dopamine (DA), decreased activity of AChE were found. However, serotonin (5-HT) had no significant difference from that of aged mice given distilled water. The evident improvement of learning and memory of aged mice is carried out by the effects of tenuifolin on the three stages of memory process, that is, acquisition, consolidation and retrieval. This may do so by relatively increasing the levels of NE, DA in the hippocampus and by decreasing the activity of AChE in the cortex.
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Keywords: Tenuifolin; Learning and memory; Ethology; Neurotransmitter; Acetylcholine esterase; Mouse
There are normal cognitive changes that take place when a person becomes older. Aging in humans is associated with deterioration of cognitive performance and, in particular, of learning and memory, and cognitive deficits are the debilitating consequences of aging. For more than a millennium, herbal remedies have been used apparently safely and effectively in Asian countries, especially in China, Japan and Korea, as a treatment for alleviating various symptoms of cognitive deficits and facilitating learning and memory. Radix Polygalae (RP), the root of Polygala tenuifolia Willd., has been used as a treatment for illnesses of the brain and for its actions of tranquilization and promoting intelligence. In combination with other herbal drugs, RP is one of the most prescribed herbal remedies for treating various kinds of cognitive disorders such as cerebrovascular diseases, aging, senile dementia including AD (Chen et al., 2002, 2004; Tian et al., 2004; Yan and Li, 2006). Anecdotal clinical experiences support the notion that RP is safe and effective in treating and/or alleviating symptoms of these diseases. Among 75 of the most famous Chinese complex prescriptions characterized by promoting intelligence in past dynasties in China, more than half of these prescriptions contain RP (Liu and Liu, 2005).
Tenuifolin is mostly the mixture of saponins (Jia et al., 2004) extracted from RP. Most saponins in RP are the derivates from presenegenin (structure, see Fig. 1). Tenuifolin is shown to improve learning and memory ability of dementia-like rats induced by combined injection of [alpha]-amyloid peptide and ibotenic acid into the right nucleus basalis magnocellularis. The muscarinic receptor density and the choline acetytransferase activity in their brain are markedly enhanced, whereas acetylcholinesterase activity is significantly inhibited (Chen et al., 2002). Tenuifolin can also increase total protein and total antioxidant capacity in the brain tissues of senescence-accelerated mice, decrease acetylcholinesterase activity in the brain and interleukin-2 in their blood serum (Tian et al., 2004). As far as we are aware, no reports have been issued on the effects of tenuifolin from RP on learning and memory in naturally aged mice, amnesiac mice by using step-down and Y-maze tasks and, on the levels of hippocampal 5-HT, NE, DA, cortical AChE activity of naturally aged mice.
[FIGURE 1 OMITTED]
In the current experiments, we investigated the effects of MST from RP on cognitive functions of young, aged animals and chemical-induced dysmnesia subjects. Learning and memory parameters in these subjects were evaluated using step-down type passive avoidance task and Y type maze trial. In addition, this study also examined the effects of MST on the levels of monoaminergic neurotransmitters (NE, DA and 5-HT), together with AChE activity in the brain tissues of aged mice, all of which are known to be important to mediation of learning and memory processes.
Materials and methods
Drug and administration
RP was purchased from Chengdu Tong-ren-tang Pharmaceutical Group, and identified as the root of Polygala tenuifolia Willd. by Professor D-G Wan, a pharmacognosist, from The Pharmacy Faculty of Chengdu University of Traditional Chinese Medicine (Chengdu, China). A voucher specimen was deposited. One kilogram of P. tenuifolia was extracted with 1000 ml of ethanol (95%, v/v) in a 50[degrees]C water bath three times for 2 h each. The extract was incubated at 4[degrees]C for 24 h and then centrifuged. The sediment was resuspended in 100 ml methanol, filtered and cleared with activated carbon. The filtered phase was concentrated to 1/2 of the original volume under reduced pressure, then the same volume of acetone was added, and the sample was filtered again. The sediment was washed with acetone, freeze-dried, and appeared as yellow brown powder (Jia et al., 2004). The content of tenuifolin in the above powder was determined by thin-layer chromatography scanning (TLCS) method with the dehydroxypresenegenin as reference (National Committee of Pharmacopoeia. Pharmacopoeia of PR China, 2005) and the average content was 72.52% (RSD = 1.22%, n = 5).
Tenuifolin was dissolved in distilled water prior to administration. Three groups of animals (n = 10) were orally administered 0.02, 0.04, 0.08 g/kg [d.sup.-1] of tenuifolin by intubation, respectively, for 15 days. Two more groups of animals (normal and control groups) were orally administered distilled water, and they were run concurrently with tenuifolin-treated groups, all of which were given in a volume of 10 ml/kg body weight irrespective of dose.
Animals and grouping
Animals, obtained from Experimental Animal Center of Chengdu University of Traditional Chinese Medicine (Chengdu, China), were housed in a regulated environment (20 [+ or -]1[degrees]C), with a 12-h light and 12-h dark cycle (08:00-20:00, light) and were grouped as follows: young and aged male Kunming mice, (30 days of age, 18-22g; 22-24 months of age, 40-50 g, respectively), Grade II, Certificate No. 2000-7. Food and water were given adlibitum, except for the duration of the experimental session. On the day of the experiment, animals were brought to the experimental room and allowed to habituate to the environmental conditions for a period of approximately 60 min before the beginning of the experiment. All animal treatments were strictly in accordance with international ethical guidelines concerning the Care and Use of Laboratory Animals, and the experiments were carried out under the approval of the Committee of Experimental Animal Administration of the University.
In the experiments on the effects of MST on learning performances in aged mice by step-down test and by Y-maze trial, 40 aged mice were divided into four groups (n = 10) ad libitum, namely, three MST-treated groups (0.02, 0.04, 0.08 g/kg[d.sup.-1]) and one control group. Another ten young mice served as normal group. In the experiments on the effects of MST on dysmnesia mice which were induced by scopolamine, sodium nitrite or ethanol, 40 young subjects were divided into four groups (n = 10) ad libitum, viz. three MST-treated groups (0.02, 0.04, 0.08g/kg[d.sup.-1]) and one control group. Another ten young subjects served as normal group. In the experiments on the effects of MST on the levels of 5-HT, NE, and DA in the hippocampus of aged mice, and on the levels of cortical AChE activity in aged mice, 40 aged mice were divided into four groups (n = 10) ad libitum, viz. three tenuifolin-treated groups (0.02, 0.04, 0.08g/kg[d.sup.-1]) and one control group. Another ten young mice served as normal group.
Chemicals and modeling
Scopolamine (Mingxing pharmaceutical factory, Guangzhou, China) and sodium nitrite (Chengdu chemical reagent factory, China) were dissolved in sterile 0.9% saline, respectively. Ethanol was diluted to a concentration of 30% (v/v) with distilled water. All chemicals were administered intraperitoneally in a volume of 5 ml/kg body weight irrespective of dose. Control and normal animals received respective solvent injections, and they were run concurrently with drugtreated groups. In experiments on the effects of tenuifolin on dysmnesia animal models, scopolamine (lmg/kg, i.p) was administered 30 min before the training trial and induced memory acquisition impairment of mouse; sodium nitrite (120 mg/kg, i.p.) was injected immediately after the training trial and induced memory consolidation impairment of mouse; and finally 30% alcohol (1.5g/kg, i.p.) was injected 30 min before testing trial and induced memory retrieval impairment of mice.
The method described by Xu et al. (2002) and Zhang et al. (2007) served as the reference. The step-down apparatus consisted of an acrylic box (20 X 20 X 20 cm high) with a stainless-steel grid floor and a wooden platform (4 X 4 X 4 cm) was fixed at the center of the box. Electric shocks (36V) were delivered to the grid floor for 6 s with an isolated pulse stimulator. At the beginning of the training trial, mice were placed in the box to adapt for 3 min. After 3 min, electric shocks were delivered and the mice jumped on the platform to avoid the noxious stimulation, and the shocks were maintained for 5 min. After a 24h interval, the mice were again placed on the platform, and the latency to step down on the grid with all four paws for the first time and the number of errors subjected to shocks within 5 min were measured as learning performances.
The method described by Xu et al. (2002) and Zhang et al. (2007) served as the reference. The Y-maze apparatus with a conductive grid floor consisted of three identical arms (40l X 10w X 20h cm) made of dark opaque Plexiglas and these three arms were symmetrically disposed at 120[degrees] to each other. Arms 1 and 3 were in the non-safety zone (shocks were administered via these) and arm 2 was a safety zone (on the top of which there was an insulated grid floor of 10 X 15 cm). Rats were placed on the top of arm 1 and a fixed resistance shock source was connected to an automatically operated switch and electric shocks (36 V) were applied. After shocking, the mice escaped from foot shocks by accidentally entering the top of arm 2 and this was counted as one practice and the mice were repeatedly trained for this procedure a further 10 times. After a 24h interval the mice were successively tested for 10 times and their latency to enter safety zone (i.e., insulated grid floor) from non-safety zone for the first time and the number of errors displayed by entering the non-safety zone within 10 times were recorded as learning performances.
Assays of NE, DA, 5-HT levels and AChE activity
For determination of the levels of 5-HT, NE, and DA in the hippocampus and cortical AChE activity, three groups of aged mice (n = 10) received 0.02, 0.04 or 0.08 g/kg[d.sup.-1] po of tenuifolin for 15 d before decapitation. Two more groups of animals (n = 10), serving as normal group (young mice) and control group (aged mice), were given orally distilled water by intubation, and run concurrently with tenuifolin-treated groups. Animals were decapitated and skulls were split on ice and salt mixture and the hippocampi and cortices were isolated respectively. The hippocampi were weighed, and homogenized in ice-cold n-butanol solution (5ml/g tissue) according to Miller et al. (1970) and Biochemistry Group of Acupuncture and Meridian Research Institute of TCM academy (1975). Homogenization was performed using an ice-cold homogenizer for 1 min and a 20% homogenate was made which was then centrifuged at 3000 rpm for 5 min. Supernatant (2.5 ml) was then transferred to a tube containing 1.6ml of 0.2N acetic acid and 5 ml n-heptane. After mixing on a vortex mixer for 30s, the tubes were centrifuged at 3000 rpm for 5min. The aqueous phases were used for the estimation of 5-HT, NE, and DA levels employing the fluorospectrophotometry (850 type, HIITACHI Corp. Japan) reported by Ciarlone (1978). The cortices were isolated, weighed, and homogenized in phosphate buffer, pH 8.0 (1 ml/40 mg tissue), for 1 min and a 4% homogenate was made. Estimation of the cortical AChE activity was performed with some modifications according to the assay described by Ellman et al. (1961) and Zhengxin Ni (1990).
The data were analyzed using a statistical package (SPSS10.0). The data for multiple comparisons were performed by one-way ANOVA followed by Dunnett's t-test. p < 0.05 was considered statistically significant and all results are presented as the mean[+ or -] s.e.m.
Results and discussion
Ethologic investigation of learning and memory is at present one of the most reliable targets reflecting levels of animal intelligence. Many nootropic studies investigate the animal's behavior changes using step-down, step-through, maze test, etc., which are often applied to the determination of capabilities of passive avoidance and spatial memory in animals. The results in Table 1 and 2 demonstrated improvements in learning performances in aged mice receiving tenuifolin by an increased latency and a decreased number of errors in the stepdown test, and by a shortened latency and a decreased number of errors in the Y-maze test. However, tenuifolin at a dosage of 0.02 g/kg did not improve learning performances in aged mice in the step-down and Y-maze test, which had no statistical significance compared with the control group.
Table 1. Effects of tenuifolin on memory performances in aged mice by step-down test Group N Dose (g/kg Latency (second) Number of errors [d.sup.-1] (time/5 min) Normal 10 Distilled 150.50[+ or -] 1.90[+ or -] water 23.30 0.38 Control 10 Distilled 70.90[+ or -] 4.50[+ or -] water 12.05 **** 0.60 **** Tenuifolin 10 0.08 139.20[+ or -] 2.10[+ or -] 18.06 * 0.53 ** Tenuifolin 10 0.04 115.60[+ or -] 2.20[+ or -] 17.51 0.36 ** Tenuifolin 10 0.02 98.70[+ or -] 3.70[+ or -] 13.11 0.58 In the control group containing aged mice, the latencies significantly shortened and the number of errors markedly increased compared with the normal group. In contrast, in aged mice treated by MST (0.04, 0.08 g/kg[d.sup.-1]) for 15 days, learning performances were manifestly improved, except at the lower dose group of MST (0.02 g/kg[d.sup.-1]), which was similar to that observed in the control group.**** P < 0.01 compared with normal mice; * p < 0.05, ** p < 0.0l compared with control aged mice. Data are expressed as the mean[+ or -]s.e.m. Table 2. Effects of tenuifolin on memory performances in aged mice by Y-maze task Group N Dose (g/kg Latency (second) Number of errors [d.sup-1]) (time/10 times) Normal 10 Distilled 11.40[+ or -] 1.50[+ or -] water 2.08 0.22 Control 10 Distilled 31.1[+ or -] 4.90[+ or -] water 4.51 **** 0.69 **** Tenuifolin 10 0.08 16.10[+ or -] 1.90[+ or -] 3.11 ** 0.35 ** Tenuifolin 10 0.04 16.00[+ or -] 2.10[+ or -] 2.30 ** 0.46 ** Tenuifolin 10 0.02 20.60[+ or -] 3.60[+ or -] 3.92 0.76 In aged mice of control group, the latencies were significantly prolonged and the number of errors markedly increased in contrast to young mice. However, in tenuifolin-treated groups (0.04, 0.08 g/kg [d.sup.-1] still for 15 days), learning performances were manifestly improved, except at the lower dose group of tenuifolin-treated groups (0.02 g/kg [d.sup.-1]), which had no statistical significance compared with control group.**** p < 0.001 compared with normal mice; ** p < 0.01, *** p < 0.001 compared with control aged mice. Data are expressed as the mean [+ or -] s.e.m.
Generally, memory as measured by changes in an animal's behavior some time after learning is considered to be a process that has several stages, including acquisition, consolidation and retrieval (Abel and Lattal, 2001). The use of pharmacological, genetic and lesion approaches has helped to define the brain systems and molecular processes important for these different stages of memory. Some chemical agents, such as scopolamine, sodium nitrite and ethanol, impair memory in animals trained on a step-down type passive avoidance and a radial maze type task, which are used to measure the three stages of memory process depending on drug-treated period. In the present study, nice given scopolamine or 30% ethanol displayed poor performances, whose latency shortened and the number of errors were increased as determined by the step-down test; administration of sodium nitrite in mice evidently increased the latency and the number of errors. As can be seen in Tables 3-5, the administration of tenuifolin improved cognitive behavior in dysmnesia animals to a great degree. Tenuifolin (0.02, 0.04, and 0.08 g/kg) showed a bell-shaped effect on acquired learning of mice with scopolamine-induced dysmnesia. The observed results of tenuifolin at the same doses on consolidation memory stages also revealed a bell-shaped effect. However, tenuifolin only at a dosage of 0.08 g/kg improved the latencies and number of errors of mice with ethanol-induced dysmnesia.
Table 3. Effects of tenuifolin on dysmnesia in mice induced by scopolamine Group N Dose (g/kg Latency (second) Number of errors [d.sup-1]) (time/10 times) Normal 10 Distilled 158.50[+ or -] 1.80[+ or -] water 30.08 0.61 Control 10 Distilled 41.80[+ or -] 3.80[+ or -] water 8.03 **** 0.61 **** Tenuifolin 10 0.08 115.40[+ or -] 2.10[+ or -] 32.24 0.46 * Tenuifolin 10 0.04 144.00[+ or -] 1.70[+ or -] 32.18 * 0.42 * Tenuifolin 10 0.02 84.10[+ or -] 3.50[+ or -] 21.86 0.62 A 1 mg/kg dose of scopolamine was injected ip 30 min before the training trial in the induced memory acquisition impairment model, and this impaired the step-down type passive avoidance test performance of mice. Mice of the control group displayed poor performances, whose latencies shortened and the number of errors increased as determined by the step-down test. In contrast, tenuifolin dosages of 0.04, 0.08 g/kg, significantly decreased the number of errors, and a dosage of 0.04 g/kg increased the latencies, which had statistical significance compared with control group.**** p < 0.05 compared with normal mice; * p < 0.05 compared with control mice. Data are expressed as the mean [+ or -]s.e.m. Table 4. Effects of tenuifolin on dysmnesia in mice induced by sodium nitrite Group N Dose (g/kg Latency (second) Number of errors [d.sup-1]) (time/10 times) Normal 10 Distilled 12.80[+ or -] 1.80[+ or -] water 1.91 0.33 Control 10 Distilled 38.40[+ or -] 5.50[+ or -] water 7.31 **** 0.76 **** Tenuifolin 10 0.08 20.60[+ or -] 2.80[+ or -] 4.20 * 0.70 ** Tenuifolin 10 0.04 18.40[+ or -] 2.30[+ or -] 2.94 ** 0.45 ** Tenuifolin 10 0.02 22.90[+ or -] 3.00[+ or -] 3.27 * 0.54 * Sodium nitrite impaired the Y-maze type test performances of mice. Tenuifolin produced an overall statistically significant improvement in performances of mice at doses of 0.02, 0.04, 0.08 g/kg, that is to say, the latencies evidently shortened and the number of errors markedly decreased compared with the control group **** p < 0.001 compared with normal mice; * p < 0.05, ** p < 0.01 compared with control mice. Data are expressed as the mean [+ or -]s.e.m. Table 5. Effects of tenuifolin on dysmnesia in mice induced by ethanol Group N Dose (g/kg Latency (second) Number of errors [d.sup-1]) (time/10 times) Normal 10 Distilled 169.40[+ or -] 1.50[+ or -] water 26.29 0.37 Control 10 Distilled 68.90[+ or -] 3.10[+ or -] water 14.18 **** 0.53 **** Tenuifolin 10 0.08 157.20[+ or -] 1.40[+ or -] 28.60 * 0.34 * Tenuifolin 10 0.04 141.50[+ or -] 1.90[+ or -] 25.25 0.46 ** Tenuifolin 10 0.02 103.80[+ or -] 2.80[+ or -] 20.95 0.49 30 min before testing trial, mice were intraperitoneally injected with 30% alcohol, which evidently impaired the step-down type passive avoidance test performances. Tenuifolin (0.02, 0.04, 0.08 g/kg) increased step-down latencies and decreased the number of errors in dose-dependent manner, but only memory performances of the tenuifolin-treated group at a dosage of 0.08 g/kg were differed significantly from that of the control group. **** p < 0.05 compared with normal mice; * p < 0.05 compared with control mice. Data are expressed as the mean [+ or -]s.e.m.
Learning and memory is one of the most important functions of the brain, which is associated with complex neurophysiologic and neurochemical changes. Many neurotransmitters, including acetylcholine (ACh), dopamine (DA), norepinephrine (NE), and, serotonin (5-HT) play an important role in the learning and memory processes (Blokland, 1996; Trond, 2003). ACh has been related to attentional processes (Himmelheber et al., 2000) and plays an important role in cognitive processing. DA has been associated with motivational processes (Wilson et al., 1995) and has a special role in appetitively motivated tasks. Central serotonin (5-HT) has been linked to emotional processes (Hashimoto et al., 1999) and plays a particular role in emotionally related tasks, and despite the lack of functional specialization, the serotonergic system plays a significant role in learning and memory (Buhot et al., 2000). Whilst NE has been relevant to learning and memory consolidation, possibly by acting as a regulation of signals (Crow, 1968; Kety, 1970). Cognitive deficits induced by various lesions to the locus ceruleus are reversible by the administration of drugs that enhance noradrenergic neurotransmission.
Aging is often accompanied by some alterations in the neurotransmitter systems of humans and other mammals (Arranz et al., 1996; Magnone et al., 2000; Monica et al., 2003). Most of the studies on brain physiology in aging have been performed in rodents and the results do not always show consistent changes in the neurochemical parameters. Some of the discrepancies observed may be due to species or strain differences. Nevertheless, the published work appears to agree with respect to several aspects such as the reductions of the levels of neurotransmitters, including DA, NE, 5-HT, which have been demonstrated in the aging brain (Habib and Ewan, 2001; Monica et al., 2003). Our findings (Table 6) demonstrate that reductions in the levels of DA, NE, 5-HT significantly decreased in aging brain, which are consistent with earlier reports (Habib and Ewan, 2001; Monica et al., 2003). However, the most significant result of our study is that the administration of tenuifolin caused significant increases in the levels of DA, NE in the hippocampus of aged mice. On the other hand, the decreased levels of 5-HT in hippocampus of aged mice were not reversed by the administration of tenuifolin, which had no statistical differences between control group and tenuifolin-treated groups (0.02, 0.04, 0.08 g/kg). It seems to suggest that the improvement of tenuifolin on cognitive function is not directly influenced by the levels of 5-HT in hippocampus of aged mice.
Table 6. Effects of tenuifolin on the hippocampal 5-HT, NE, and DA levels in aged mice Group N Dose (g/kg [d.sup.-1] 5-HT (ng/g) Normal 10 Distilled water 560.70 [+ or -] 46.69 Control 10 Distilled water 358.50 [+ or -] 37.43 **** Tenuifolin 10 0.08 399.80 [+ or -] 61.68 Tenuifolin 10 0.04 453.20 [+ or -] 54.89 Tenuifolin 10 0.02 397.10 [+ or -] 50.13 Group NE (ng/g) DA (ng/g) Normal 1030.20 [+ or -] 27.71 786.70 [+ or -] 58.40 Control 667.30 [+ or -] 53.62 **** 509.50 [+ or -] 54.64 **** Tenuifolin 950.60 [+ or -] 77.29 * 809.90 [+ or -] 71.80 ** Tenuifolin 936.90 [+ or -] 89.98 * 756.00 [+ or -] 63.83 * Tenuifolin 852.80 [+ or -] 93.53 683.70 [+ or -] 51.13 In the control group consisting of aged mice, the levels of hippocampal NE, DA, and 5-HT were significantly decreased compared to those observed in the mice in the normal group. When the aged mice were given tenuifolin at dosages of 0.04, 0.08 g/kg the levels of NE and DA were similar to those observed in the normal group, this difference being statistically significant. At a tenuifolin dosage of 0.02 g/kg only DA levels were significantly increased when compared to the values observed in the control group. However, at tenuifolin dosages of 0.02, 0.04, 0.08 g/kg although there was some improvement in the levels of 5-HT, this had no statistical significance when compared to the values observed in the control group. **** p < 0.05, **** p < 0.01 compared with normal mice; * p < 0.05, ** p < 0.01 compared with control aged mice. Data are expressed as the mean [+ or -] s.e.m.
Although age-related reductions of AChE activity are found in previous studies (Sirvio et al., 1988, 1989), numerous experiments show no substantial changes in the activity of these enzymes in the brain of aged rats (Aubert et al., 1995; Birthelmer et al., 2003; Colombo and Gallagher, 1998). Our present data (Fig. 2) are in line with the latter findings. However, the markedly decreased activity of AChE in the cortex of aged mice was found after the administration of tenuifolin at dosages of 0.02, 0.04, 0.08 g/kg. The previous investigation also demonstrates that tenuifolin evidently inhibits AChE activity (Park et al., 2002).
[FIGURE 2 OMITTED]
In summary, the administration of tenuifolin significantly enhances learning performances in aged mice by the step-down and Y-maze tasks. Tenuifolin also ameliorates memory deficits in amnesic mice induced by chemical agents. The improvement of learning and memory of aged mice is mediated by the effects of tenuifolin on the three stages of memory process, that is, acquisition, consolidation and retrieval. This may do so by relatively increasing the levels of NE, DA in the hippocampus and by decreasing the activity of AChE in the cortex. Since a desirable cognitive effect has been reported for glutamate (Ohno and Watanabe, 1996; Trond, 2003), further studies should be directed towards investigating the effect of tenuifolin on glutamatergic neurotransmission in brain areas implicated in the control of learning and memory processes. In addition, the investigation of tenuifolin promoting intelligence should be made at the molecular level.
The authors are grateful to Dr. Baokang Huang, Dr. Qiaoyang Zhang and Professor Hanchen Zheng (Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China) for technical assistance.
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Hong Zhang (a), Ting Han (a), Lei Zhang (a), Cheng-Hao Yu (b), De-Guang Wan (b), Khalid Rahman (c), Lu-Ping Qin (a), *, Cheng Peng (b), *
(a) Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China
(b) School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 610075, PR China
(c) School of Biomolecular Sciences, Faculty of Science, Liverpool John Moores University, Liverpool L3 3AF, England, UK
* Corresponding authors. Tel./fax: +862125070394 (Lu-Ping Qin), +862887769954 (Cheng Peng).
E-mail addresses: Ipqin@smmu.edu.cn (L.-P. Qin), firstname.lastname@example.org (C. Peng).