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Anti-amnesic effect of alkaloid fraction from Lycopodiella cernua (L.) Pic. Serm. on scopolamine-induced memory impairment in mice
Neuroscience Letters 575 (2014) 42–46
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Anti-amnesic effect of alkaloid fraction from Lycopodiella cernua (L.) Pic. Serm. on scopolamine-induced memory impairment in mice Nguyen Ngoc Chuong a , Bui Huu Trung b , Tran Cong Luan a , Tran Manh Hung b,c,∗ , Nguyen Hai Dang d , Nguyen Tien Dat d a
Research Center of Ginseng and Medicinal Materials, 41-Dinh Tien Hoang, District 1, Ho Chi Minh City, Viet Nam Faculty of Chemistry, University of Science, Vietnam National University-Ho Chi Minh City, 227 Nguyen Van Cu, District 5, Ho Chi Minh City, Viet Nam c College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, South Korea d Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay District, Hanoi, Viet Nam b
h i g h l i g h t s • • • •
The Lycopodiella cernua alkaloid fraction (VLC) inhibited AChE activity in vitro. VLC significantly reversed cognitive impairments in mice by passive avoidance test. VLC reduced escape latency and prolonged swimming times in mice by water maze task. Vietnamese L. cernua inhibited AChE activity and might be useful for AD.
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Article history: Received 11 December 2013 Received in revised form 13 April 2014 Accepted 14 May 2014 Available online 23 May 2014 Keywords: Lycopodiella cernua Licopodiaceae Acetylcholinesterase Scopolamine Amnesic
a b s t r a c t Lycopodiella cernua (L.) Pic. Serm. (Licopodiaceae) has been used in Vietnamese folk medicine for treating central nervous system conditions. In this study, the alkaloid fraction from the methanol extract of this plant (VLC) was evaluated for in vitro acetylcholinesterase (AChE) inhibitory activity in cognition-relevant brain areas of mice. In in vivo study, the cognitive-enhancing effect of VLC on amnesic mice induced by scopolamine was investigated by assessing a passive avoidance and a Morris water maze test. VLC inhibited AChE activity in mouse frontal cortex, hippocampus and striatum with IC50 values of 26.7, 32.2 and 25.7 g/mL, respectively. Administration of VLC (10, 20, 50 and 100 mg/kg, p.o.) significantly reversed cognitive impairments in mice by passive avoidance test. Treating with VLC (50 mg/kg) reduced escape latencies in training trials and prolonged swimming times in the target quadrant during the probe trial in the water maze task (P < 0.05). These results indicated that L. cernua originated from Vietnam has anti-cholinesterase activity and might be useful for the treatment of cognitive impairment. © 2014 Elsevier Ireland Ltd. All rights reserved.
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease with many cognitive and neuropsychiatric manifestations which result in progressive disability and eventual incapacitation. A decrease in acetylcholine in the brain of patients with AD appears to be a critical element in producing dementia [1]. Loss of cholinergic cells, particularly in the basal forebrain, is accompanied by loss of the neurotransmitter acetylcholine. One approach is to inactivate acetylcholinesterase, the enzyme that cleaves synaptic acetylcholine and terminates neuronal signaling.
∗ Corresponding author at: Faculty of Chemistry, University of Science, Vietnam National University-Ho Chi Minh City, 227 Nguyen Van Cu, District 5, Ho Chi Minh City, Viet Nam. Tel.: +84 83 829 2646; fax: +84 83 829 2646. E-mail address: [email protected] (T.M. Hung). http://dx.doi.org/10.1016/j.neulet.2014.05.031 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Acetylcholinesterase (AChE) inhibitors increase the availability of acetylcholine in central cholinergic synapses and are the most promising currently available drugs for the treatment of AD [2]. AChE inhibitors from general chemical classes such as physostigmine, tacrine, donepezil, galanthamine, huperzine A and heptylphysostigmine have been tested for the symptomatic treatment of AD. Although there have been a number of reports on the designing and development of synthetic AChE inhibitors, few have been reported on searching for AChE inhibitors derived from plants [3,4]. In our screening program to search for AChE inhibitors from plants, a methanol extract of Lycopodiella cernua (Licopodiaceae) exhibited significant AChE inhibitory activity (85.5% at 100 g/mL). As reported in literature, Huperzia serrata (Huperziaceae), a famous traditional Chinese medicine, has been used for over 1000 years in China for treating contusions, strains, swellings, schizophrenia,
N.N. Chuong et al. / Neuroscience Letters 575 (2014) 42–46
myasthenia gravis, and organophosphate poisoning [5]. Since the anticholinesterase activity of huperzine A was reported [6], many attempts have been made to look for its derivatives that would possess higher activity, longer duration of action but lower toxicity. In China, a chemotaxonomic analysis of Lycopodium alkaloids in Huperzia and related genera was performed, those species are from the Huperziaceae, Lycopodiaceae, and Sellaginellaceae [7]. Until now, many Lycopodium alkaloids such as huperzines A and B, serratine, serratinine, lycodoline, lucidioline, lycopodine, lyconnotine, annotinine and cernuine were isolated [5]. Among the isolates, huperzines A and B have been proven to be potent, reversible and selective AChE inhibitors. Since huperzine A displayed a remarkable activity on memory and learning [8], Lycopodium alkaloids have become a new potential source of pharmacologically interesting natural products all over the world [7,9–15]. In Vietnam, some plants belonging to Lycopodiaceae species such as H. serrata (Thunb.) Trevis (Thach Tung Rang Cua), H. squarrosa (Forst.) Trevis (Rau Rong), Lycopodium complanatum L. (Thach Tung) and L. cernua (L.) Franco & Vasc (Thach Tung Det) are mainly distributed in the mountainous areas of Da Lat, Lam Dong province. These species have been used in the treatment of contusions, strains, swellings, schizophrenia, myasthenia and memory dysfunction in folk medicine [16]. Until now, there has been no report on the antiAD of these plants. The present study evaluates the AChE inhibitory effects of the extract of L. cernua in the cognition-relevant brain areas of mice and its effects on the scopolamine induced memory impairment in mice. The plant L. cernua was collected in the Da Lat, Lam Dong province, Vietnam in April 2012, and was identified by Prof. Tran Cong Luan, Department of Oriental Medicine, Ho Chi Minh city University of Medicine and Pharmacy. Voucher specimens (TCL-00112) was deposited at the Herbarium of the Research Center of Ginseng and Medicinal Materials, Ho Chi Minh city, Vietnam. The dried and ground aerial parts of L. cernua (4.0 kg) were separately extracted with n-hexane using a Soxhlet extractor. Afterwards, the defatted plant materials were exhaustively macerated with methanol and the resulting crude extracts were dried under reduced pressure. The extract was then suspended in 5% HCl and partitioned with CH2 Cl2 . The aqueous layers were alkalinized until pH 11–12 with 0.1 N NaOH and were extracted again with CH2 Cl2 . The organic layers were combined, dried over Na2 SO4 and were evaporated to give the crude alkaloid fraction (VLC, 1.95 g). Adult Sprague–Dawley male rats, weighing 200–250 g, and male ICR mice, weighing 20–25 g, were purchased from Daehan Biolink, Korea. The animals were housed 5 or 6 per cage, allowed access to water and food ad libitum, and maintained in a constant temperature (23 ± 1 ◦ C) and humidity (60 ± 10%) environment under a 12-h light/dark cycle (light on 07.30–19.30 h). Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Catholic University of Daegu, Korea. Rats were decapitated, the brain was rapidly dissected on ice into cortex, hippocampus and striatum, and then the dissected parts were weighed and homogenized in five volumes of cold 75 mM sodium phosphate buffer, pH 7.4. The homogenates were centrifuged at 13,000 × g for 30 min at 4 ◦ C; supernatants used as acetylcholinesterase sources were divided into aliquots and stored at −20 ◦ C. The AChE inhibitory activity was measured by the principle of the Ellman method with some modifications [17]. Enzyme samples in 10 mM phosphate buffer, pH 7.5, were incubated for 150 s at 37 ◦ C with 0.3 mM acetylthiocholine iodide in the presence of 50 M tetraisopropyl pyrophosphoramide, a selective inhibitor of butyrylcholinesterase, and 5,5 -dithiobis-(2-nitrobenzoic acid) for color development (all chemicals from Sigma). Protein concentration was measured with the Coomassie brilliant blue protein-binding method [18] using bovine serum albumin as standard. AChE activity was expressed in
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international units (1 IU = 1 M acetylthiocholine hydrolyzed per second) per mg protein. The extract was initially dissolved in 0.02% dimethyl sulfoxide (DMSO) and diluted to various concentrations in sodium phosphate buffer (100 mM, pH 8.0) immediately before use. An aliquot of diluted VLC (5–200 g/mL) solution was then mixed with sodium phosphate buffer (100 mM, pH 8.0), acetylthiocholine iodide solution (75 mM) and Ellman’s reagent (10 mM 5,5 -dithio-bis [2-nitrobenzoic acid] and 15 mM sodium bicarbonate) and was reacted at room temperature for 30 min. Absorbance was measured at 410 nm immediately after adding the enzyme source to the reaction mixtures by using a spectrophotometer (Shimadzu UV-1240, Tokyo, Japan). Readings were taken at 30 s intervals for 5 min. The concentration of compound required to inhibit acetylcholinesterase activity by 50% (IC50 ) was calculated using an enzyme inhibition dose response curve. Beberine [19] and tacrine were used as positive controls. Passive avoidance task was carried out in identical illuminated and nonilluminated boxes. The illuminated compartment (20 cm × 20 cm × 20 cm) contained an 100 W bulb. The floor of nonilluminated compartment (20 cm × 20 cm × 20 cm) was composed of 2 mm stainless steel rods spaced 1 cm apart. These compartments were separated by a guillotine door (5 cm × 5 cm). For the acquisition trial, mice were initially placed in the illuminated compartment and the door between the two compartments was opened 10 s later. When the mice entered the dark compartment, the door automatically closed and an electrical foot shock (0.5 mA) of 3 s durations was delivered through the stainless steel rods. One hour before the acquisition trial, the mice were administered with VLC (10, 20, 50, and 100 mg/kg, p.o.) or tacrine (10.0 mg/kg, p.o). After a delay of 60 min, the amnesia was induced by subcutaneous administration of scopolamine (1.0 mg/kg, i.p.). The control animals were administered with 10% Tween 80 solution only. Twenty-four hours after the acquisition trial, the mice were again placed in the illuminated compartment for the retention trials. The time taken for a mouse to enter the dark compartment after door opening was measured as latency times in both acquisition and retention trials. If the mice stayed in the light compartment for more than 180 s, it was concluded that the mice had memorized the passive avoidance training after the training trial [19]. A spatial test was performed by the method of Morris with a minor modification. The water maze is a circular pool (90 cm in diameter and 45 cm in height) with a featureless inner surface. The pool was filled to a depth of 30 cm with water containing 500 mL of milk (20 ± 1 ◦ C). The pool was divided into four quadrants of equal area. A white platform (6 cm in diameter and 29 cm high) was then placed in one of the pool quadrants. The first experimental day was dedicated to swimming training for 60 s without the submerged platform. During the five subsequent days, the mice were given two daily trials with an inter-trial interval of 30 min in the presence of the platform in place. When a mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 120 s, it was placed on the platform for 10 s. The animal was taken to its home cage and was allowed to dry up under an infrared lamp after each trial [19]. During each trial session, the time taken to find the hidden platform (latency) was recorded. One day after the last training trial sessions, the mice were subjected to a probe trial session in which the platform was removed from the pool, allowing the mice to swim for 120 s to search for it. A record was kept of the swimming time in the pool quadrant where the platform had previously been placed. VLC (50 mg/kg, p.o.) or tacrine (10 mg/kg, p.o.) was given 1 h before the first trial session at every consecutive day. Memory impairment was induced in mice with scopolamine (1 mg/kg, i.p.) at 60 min after treatment of test samples. The control group received 10% Tween 80 solution only. The results are expressed as mean values ± S.D. Statistical analysis was performed using one-way ANOVA. A P < 0.05 was considered statistically significant.
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Table 1 Inhibition of VLC on brain enzyme sources. Test samples
VLC Berberineb Tacrineb a b
IC50 (g/mL)a Brain cortex
Hippocampus
Striatum
26.7 ± 5.5 14.1 ± 1.2 0.09 ± 0.02
32.2 ± 4.1 14.2 ± 0.8 0.15 ± 0.04
25.7 ± 5.1 15.8 ± 2.8 0.09 ± 0.01
Results are the mean of three replications. Reference controls.
For investigating anticholinesterase activity of VLC, mouse brain cortex, hippocampus and striatum were selected for the in vitro assay [20]. The results (IC50 ) were summarized in Table 1. VLC showed a strong inhibitory activity against AChE in mouse frontal cortex, hippocampus and striatum with IC50 values of 26.7 ± 5.5, 32.2 ± 4.1 and 25.7 ± 5.1 g/mL, respectively. Berberine, a natural active alkaloid from Corydalis turtschaninovii [19], showed a potent inhibitory activity with IC50 values ranging from 14.1 to 15.8 g/mL. Tacrine, which was effectively used as a positive control, exhibited the inhibitory activity with IC50 values ranging from 0.09 to 0.15 g/mL (Table 1). Since AChE inhibitors have been used to reduce the cholinergic deficit in AD and based on the remarkable effect of VLC in in vitro assay, we assessed whether VLC (10, 20, 50 and 100 g/mL) improves memory function by passive avoidance learning, which is largely dependent on long-term memory. When placed into the bright side of a step-through box, mice quickly entered the dark compartment. Mice were conditioned using a mild foot shock after entering the dark compartment and hesitated to re-enter the dark compartment when tested 24 h later. By using this shuttle box system, the Tween-treated control mice stayed in the light compartment for 220 ± 10 s after the passive avoidance training. Meanwhile, the mice given scopolamine (1.0 mg/kg body weight) stayed in the light compartment for only 20 ± 3 s before moving into the dark compartment. Thus, the step-through latency of the scopolamine-treated mice was significantly shorter than that of normal control group (Fig. 1, P < 0.05). In the tacrine plus scopolamine-treated mice, the step-through latency was significantly greater than that of the scopolamine-treated mice (160 ± 12 s vs. 22 ± 3 s). Moreover, the reduction in the stepthrough latency induced by scopolamine was significantly reversed by VLC at the dose of 20 and 50 mg/kg, p.o (P < 0.05) at 148 ± 15 and 167 ± 21 s, respectively. At the dose of 10 mg/kg, VLC did not exhibit
a significant effect compared to that of the vehicle group (28 ± 5 s vs. 22 ± 3 s). However, at the highest dose of 100 mg/kg, the latency time decreased significantly compare to that of 50 mg/kg treated group (115 ± 14 s vs. 22 ± 3 s, P < 0.001). Latency time during the acquisition trial was not affected by any drug treatment (Fig. 1). The effect of VLC (50 mg/kg, p.o.) on spatial learning (long-term or short-term memory) was evaluated using the Morris water maze test. The Tween-treated control group rapidly learned the location of the platform (Fig. 2A). The scopolamine-treated group exhibited longer escape latencies (the time taken to find the platform) throughout training days than did the control group (P < 0.001). VLC (50 mg/kg, p.o.) significantly attenuated the effect of scopolamine on escape latency (P < 0.05), as did tacrine (P < 0.05). On the day of probe testing, a significant group effect was observed on swimming times in the probe test (Fig. 2B). Swimming times within the platform quadrant for the scopolamine-treated group were significantly lower than those of normal control group (Fig. 2B, P < 0.05). Moreover, the shorter swimming times within the platform quadrant induced by scopolamine were significantly reversed by VLC and/or tacrine (P < 0.05). In the past decade, treatment for AD has largely involved replacement of neurotransmitters that are known to be lacking, mostly based on the inhibition of AChE, an important approach that was being developed on the cholinergic hypothesis of the disease. One of the most remarkable biochemical changes in AD patients is a reduction of acetylcholine levels in the hippocampus and cortex of the brain. The use of AChE inhibitors is the most commonly treatment for the symptoms of mild AD, which helps improving the cholinergic deficit [21,22]. AChE inhibitors including tacrine, donepezil and rivastigmine were approved by the U.S. Food and Drug Administration for the symptomatic treatment of patients with mild to moderate AD. Some potent inhibitors of AChE are derived from natural sources, most of them belong to the chemical class of alkaloids, including galanthamine and huperzine A. Huperzine A, a novel Lycopodium alkaloid discovered from H. serrata, has been found to inhibit AChE selectively and is well tolerate properties may be especially suitable for AD treatment. Several studies reported on the in vitro and in vivo anticholinesterasic activities of the alkaloids Lycopodium genus collected in Asia, Euro, Australia and South America [10–15]. The well known alkaloid contents containing in decoctions of the entire plants have been used in folk medicine for treating CNS-related conditions such as nerve tonics and motor disorders [23]. In the present study, the effect of improving memory deficit of VLC was evaluated using
Fig. 1. Effect of a single administration of VLC on scopolamine-induced memory deficits as determined by the passive avoidance task. At 60 min before acquisition trials, VLC (10, 20, 50 and 100 mg/kg, p.o.), tacrine (10 mg/kg, p.o.) or vehicle (same volume of 10% Tween 80) solution was administered to mice. Memory impairment was induced by administering scopolamine (1 mg/kg, i.p.). Five different animals were used per treatment group. Acquisition trials were carried out 30 min after a single scopolamine treatment. 24 h after the acquisition trials, retention trials were carried out for 3 min. Data represent means ± S.E.M (*P < 0.001 vs. vehicle control group, **P < 0.05 vs. scopolamine-treated group).
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Fig. 2. Effect of VLC on latency time during the training trial sessions (A), and on swimming time (B) during the probe trial session of the Morris water maze task in scopolamine-induced memory deficits mice. At 60 min before training trial sessions, VLC (50 mg/kg, p.o.), tacrine (10 mg/kg, p.o.) or vehicle (same volume of 10% Tween 80) solution was administered to mice. Memory impairment was induced by scopolamine treatment (1 mg/kg, i.p.). Five different animals were used per treatment group. The training trial and the probe trial sessions were performed as described in materials and methods. Data represent means ± S.E.M (*P < 0.001 vs. vehicle control group, **P < 0.05 vs. scopolamine-treated group).
the amnesia mouse model induced by scopolamine, which is a muscarinic receptor antagonist and causes amnesia in animals by blocking cholinergic neurotransmission. Cholinergic interneurons in the striatum contain even richer source of acetylcholinesterase and would also be affected strongly by enzyme inhibitors. Acute and systemic administration of scopolamine in young animals provides the appropriate memory deficits related to the cholinergic deficit in AD or senile CNS dysfunction. The scopolamine induced amnesic model has been widely used to provide a pharmacological model of memory dysfunction for screening potential cognition enhancing agents [24]. In our in vitro study, the alkaloid fraction from Vietnamese L. cernue exhibited the AChE inhibitory activity. In in vivo experiments, the cognitive-enhancing activity of VLC on the scopolamine-induced memory impairments in mice was investigated by using passive avoidance test and the Morris water maze test. The passive avoidance test is generally used to evaluate the treatments on the three stages of memory, such as learning acquisition, memory retention, and the retrieval process [22]. Administration of scopolamine significantly shortened the step-through latency in the retention trial, demonstrating that the central cholinergic neuronal system plays an important role in learning acquisition (Fig. 1). Treatment of VLC (10–100 mg/kg) prolonged dose-dependently the step-through latency shortened
by scopolamine. These results suggested that the anti-amnesic effect of VLC on scopolamine-induced memory impairment may be related to the mediation of the cholinergic nervous system. To confirm the effects of VLC on other stages of memory, the mice treated with scopolamine showed a more prolonged escape latency than the mice of the control group in the Morris water maze test. VLC treated groups (50 mg/kg) significantly reduced the escape latency during the trial sessions. In the probe test, VLC treatment decreased the escape latency and significantly increased the number of times of visiting the platform site in comparison to the control group. The Morris water maze test investigating spatial learning and memory has been used in detecting changes of the central cholinergic system. If the animals spent more time and swam a longer distance in the pool quadrant where the platform had previously been placed during the training session, this would indicate that the animals learned from prior experience with the Morris water maze test, showing the spatial memory improvement. Therefore, these results suggest that VLC can repair the long-term memory in scopolamine induced memory impairments. To elucidate the underlying mechanisms of memory enhancing effects of VLC, the AChE inhibitory activities were assessed by using the AChE in mouse cognition brain areas (frontal cortex, hippocampus and striatum). VLC inhibited AChE activity in a dose dependent manner. These results suggest
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that the ability to alleviate scopolamine-induce memory impairment or the anti-amnesic activity of the alkaloid fraction is, in part, mediated by the AChE inhibition. It would be interesting to indicate that although some alkaloid extracts of Lycopodiaceae species have been already tested for in vitro and in vivo AChE inhibitory activity, no study has been carried out on the anti-AChE effect of the Vietnamese species. Therefore, in order to identify the AChE inhibitors, the active compounds in the extracts are currently being investigated. As expected, both of huperzines A and B were not detected in this plant (see supporting information), pointing out that the biological activities found above may be due to other Lycopodium alkaloids. This is in accordance with previous results that the chemical study on the extract of South America L. cernua revealing a number of Lycopodium alkaloids belonging to the lycopodane, flabellidane and cernuane groups. The presence of huperzines A and B were not detected in the extracts. In conclusion, the cognitive enhacing activity of Vietnamese L. cernua might result from the inhibition of AChE. This result suggests that VLC may be used as a medication for neurodegenerative diseases. However, more studies should be preserved. Acknowledgment This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.99-2011.19. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2014.05.031. References [1] R. Becker, E. Giacobini, R. Elble, M. McIlhany, K. Sherman, Potential pharmacotherapy of Alzheimer disease. A comparison of various forms of physostigmine administration, Acta Neurol. Scand. Suppl. 116 (1988) 19–32. [2] E. Giacobini, in: E. Giacobini (Ed.), Cholinesterase Inhibitors: From the Calabar Bean to Alzheimer Therapy, M. Dunitz, Thonex, Geneva, 2000, pp. 181–226. [3] M.H. Oh, P.J. Houghton, W.K. Whang, J.H. Cho, Screening of Korean herbal medicines used to improve cognitive function for anti-cholinesterase activity, Phytomedicine 11 (2004) 544–548.
[4] P.J. Houghton, Y. Ren, M.J. Howes, Acetylcholinesterase inhibitors from plants and fungi, Nat. Prod. Rep. 23 (2006) 181–199. [5] X. Ma, C. Tan, D. Zhu, D.R. Gang, P. Xiao, Huperzine A from Huperzia species—an ethnopharmacolgical review, J. Ethnopharmacol. 113 (2007) 15–34. [6] J.S. Liu, Y.L. Zhu, C.M. Yu, Y.Z. Zhou, Y.Y. Han, F.W. Wu, B.F. Qi, The structures of huperzine A and B, two new alkaloids exhibiting marked anticholinesterase activity, Can. J. Chem. 64 (1986) 837–839. [7] X. Ma, D.R. Gang, The lycopodium alkaloids, Nat. Prod. Rep. 21 (2004) 752–772. [8] R.W. Zhang, X.C. Tang, Y.Y. Han, G.W. Sang, Y.D. Zhang, Y.X. Ma, C.L. Zhang, R.M. Yang, Drug evaluation of huperzine A in the treatment of senile memory disorders, Zhongguo Yao Li Xue Bao 12 (1991) 250–252. [9] M.G. Ortega, A.M. Agnese, J.L. Cabrera, Anticholinesterase activity in an alkaloid extract of Huperzia saururus, Phytomedicine 11 (2004) 539–543. [10] M.G. Vallejo, M.G. Ortega, J.L. Cabrera, V.P. Carlini, S. Rubiales de Barioglio, A.M. Agnese, Huperzia saururus increases memory retention in rats, J. Ethnopharmacol. 111 (2007) 685–687. [11] E.L. Konrath, B.M. Neves, C.S. Passos, P.S. Lunardi, M.G. Ortega, J.L. Cabrera, C.A. Gonc¸alves, A.T. Henriques, Huperzia quadrifariata and Huperzia reflexa alkaloids inhibit acetylcholinesterase activity in vivo in mice brain, Phytomedicine 19 (2012) 1321–1324. [12] A.I. Calderón, J. Simithy-Williams, R. Sanchez, A. Espinosa, I. Valdespino, M.P. Gupta, Lycopodiaceae from Panama: a new source of acetylcholinesterase inhibitors, Nat. Prod. Res. 27 (2013) 500–505. [13] W.H. Lim, J.Q. Goodger, A.R. Field, J.A. Holtum, I.E. Woodrow, Huperzine alkaloids from Australasian and southeast Asian Huperzia, Pharm. Biol. 48 (2010) 1073–1078. [14] R. Ho, N. Marsousi, P. Eugster, J.P. Bianchini, P. Raharivelomanana, Detection by UPLC/ESI-TOF-MS of alkaloids in three Lycopodiaceae species from French Polynesia and their anticholinesterase activity, Nat. Prod. Commun. 4 (2009) 1349–1352. [15] E.S. Halldorsdottir, J.W. Jaroszewski, E.S. Olafsdottir, Acetylcholinesterase inhibitory activity of lycopodane-type alkaloids from the Icelandic Lycopodium annotinum ssp. Alpestre, Phytochemistry 71 (2010) 149–157. [16] P.H. Ho, Cay Co Viet Nam: An Illustrated Flora of Viet Nam, 2, Nha Xuat Ban Tre, Hanoi, 1999, pp. 24. [17] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–90. [18] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [19] T.M. Hung, M. Na, N.T. Dat, T.M. Ngoc, U. Youn, H.J. Kim, B.S. Min, J. Lee, K. Bae, Cholinesterase inhibitory and anti-amnesic activity of alkaloids from Corydalis turtschaninovii, J. Ethnopharmacol. 119 (2008) 74–80. [20] Q. Zhao, X.C. Tang, Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine, donepezil, rivastigmine and physostigmine, Eur. J. Pharmacol. 455 (2002) 101–107. [21] E. Giacobini, The cholinergic system in Alzheimer disease, Prog. Brain Res. 84 (1990) 321–332. [22] J.E. LeDoux, Emotional memory systems in the brain, Behav. Brain Res. 58 (1993) 69–79. [23] A.A. Izzo, F. Capasso, Herbal medicines to treat Alzheimer’s disease, Trends Pharmacol. Sci. 28 (2007) 47–48. [24] U. Ebert, W. Kirch, Scopolamine model of dementia: electroencephalogram findings and cognitive performance, Eur. J. Clin. Invest. 28 (1998) 944–949.
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Anti-amnesic effect of alkaloid fraction from Lycopodiella cernua (L.) Pic. Serm. on scopolamine-induced memory impairment in mice Nguyen Ngoc Chuong a , Bui Huu Trung b , Tran Cong Luan a , Tran Manh Hung b,c,∗ , Nguyen Hai Dang d , Nguyen Tien Dat d a
Research Center of Ginseng and Medicinal Materials, 41-Dinh Tien Hoang, District 1, Ho Chi Minh City, Viet Nam Faculty of Chemistry, University of Science, Vietnam National University-Ho Chi Minh City, 227 Nguyen Van Cu, District 5, Ho Chi Minh City, Viet Nam c College of Pharmacy, Catholic University of Daegu, Gyeongsan 712-702, South Korea d Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Cau Giay District, Hanoi, Viet Nam b
h i g h l i g h t s • • • •
The Lycopodiella cernua alkaloid fraction (VLC) inhibited AChE activity in vitro. VLC significantly reversed cognitive impairments in mice by passive avoidance test. VLC reduced escape latency and prolonged swimming times in mice by water maze task. Vietnamese L. cernua inhibited AChE activity and might be useful for AD.
a r t i c l e
i n f o
Article history: Received 11 December 2013 Received in revised form 13 April 2014 Accepted 14 May 2014 Available online 23 May 2014 Keywords: Lycopodiella cernua Licopodiaceae Acetylcholinesterase Scopolamine Amnesic
a b s t r a c t Lycopodiella cernua (L.) Pic. Serm. (Licopodiaceae) has been used in Vietnamese folk medicine for treating central nervous system conditions. In this study, the alkaloid fraction from the methanol extract of this plant (VLC) was evaluated for in vitro acetylcholinesterase (AChE) inhibitory activity in cognition-relevant brain areas of mice. In in vivo study, the cognitive-enhancing effect of VLC on amnesic mice induced by scopolamine was investigated by assessing a passive avoidance and a Morris water maze test. VLC inhibited AChE activity in mouse frontal cortex, hippocampus and striatum with IC50 values of 26.7, 32.2 and 25.7 g/mL, respectively. Administration of VLC (10, 20, 50 and 100 mg/kg, p.o.) significantly reversed cognitive impairments in mice by passive avoidance test. Treating with VLC (50 mg/kg) reduced escape latencies in training trials and prolonged swimming times in the target quadrant during the probe trial in the water maze task (P < 0.05). These results indicated that L. cernua originated from Vietnam has anti-cholinesterase activity and might be useful for the treatment of cognitive impairment. © 2014 Elsevier Ireland Ltd. All rights reserved.
Alzheimer’s disease (AD) is the most common age-related neurodegenerative disease with many cognitive and neuropsychiatric manifestations which result in progressive disability and eventual incapacitation. A decrease in acetylcholine in the brain of patients with AD appears to be a critical element in producing dementia [1]. Loss of cholinergic cells, particularly in the basal forebrain, is accompanied by loss of the neurotransmitter acetylcholine. One approach is to inactivate acetylcholinesterase, the enzyme that cleaves synaptic acetylcholine and terminates neuronal signaling.
∗ Corresponding author at: Faculty of Chemistry, University of Science, Vietnam National University-Ho Chi Minh City, 227 Nguyen Van Cu, District 5, Ho Chi Minh City, Viet Nam. Tel.: +84 83 829 2646; fax: +84 83 829 2646. E-mail address: [email protected] (T.M. Hung). http://dx.doi.org/10.1016/j.neulet.2014.05.031 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Acetylcholinesterase (AChE) inhibitors increase the availability of acetylcholine in central cholinergic synapses and are the most promising currently available drugs for the treatment of AD [2]. AChE inhibitors from general chemical classes such as physostigmine, tacrine, donepezil, galanthamine, huperzine A and heptylphysostigmine have been tested for the symptomatic treatment of AD. Although there have been a number of reports on the designing and development of synthetic AChE inhibitors, few have been reported on searching for AChE inhibitors derived from plants [3,4]. In our screening program to search for AChE inhibitors from plants, a methanol extract of Lycopodiella cernua (Licopodiaceae) exhibited significant AChE inhibitory activity (85.5% at 100 g/mL). As reported in literature, Huperzia serrata (Huperziaceae), a famous traditional Chinese medicine, has been used for over 1000 years in China for treating contusions, strains, swellings, schizophrenia,
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myasthenia gravis, and organophosphate poisoning [5]. Since the anticholinesterase activity of huperzine A was reported [6], many attempts have been made to look for its derivatives that would possess higher activity, longer duration of action but lower toxicity. In China, a chemotaxonomic analysis of Lycopodium alkaloids in Huperzia and related genera was performed, those species are from the Huperziaceae, Lycopodiaceae, and Sellaginellaceae [7]. Until now, many Lycopodium alkaloids such as huperzines A and B, serratine, serratinine, lycodoline, lucidioline, lycopodine, lyconnotine, annotinine and cernuine were isolated [5]. Among the isolates, huperzines A and B have been proven to be potent, reversible and selective AChE inhibitors. Since huperzine A displayed a remarkable activity on memory and learning [8], Lycopodium alkaloids have become a new potential source of pharmacologically interesting natural products all over the world [7,9–15]. In Vietnam, some plants belonging to Lycopodiaceae species such as H. serrata (Thunb.) Trevis (Thach Tung Rang Cua), H. squarrosa (Forst.) Trevis (Rau Rong), Lycopodium complanatum L. (Thach Tung) and L. cernua (L.) Franco & Vasc (Thach Tung Det) are mainly distributed in the mountainous areas of Da Lat, Lam Dong province. These species have been used in the treatment of contusions, strains, swellings, schizophrenia, myasthenia and memory dysfunction in folk medicine [16]. Until now, there has been no report on the antiAD of these plants. The present study evaluates the AChE inhibitory effects of the extract of L. cernua in the cognition-relevant brain areas of mice and its effects on the scopolamine induced memory impairment in mice. The plant L. cernua was collected in the Da Lat, Lam Dong province, Vietnam in April 2012, and was identified by Prof. Tran Cong Luan, Department of Oriental Medicine, Ho Chi Minh city University of Medicine and Pharmacy. Voucher specimens (TCL-00112) was deposited at the Herbarium of the Research Center of Ginseng and Medicinal Materials, Ho Chi Minh city, Vietnam. The dried and ground aerial parts of L. cernua (4.0 kg) were separately extracted with n-hexane using a Soxhlet extractor. Afterwards, the defatted plant materials were exhaustively macerated with methanol and the resulting crude extracts were dried under reduced pressure. The extract was then suspended in 5% HCl and partitioned with CH2 Cl2 . The aqueous layers were alkalinized until pH 11–12 with 0.1 N NaOH and were extracted again with CH2 Cl2 . The organic layers were combined, dried over Na2 SO4 and were evaporated to give the crude alkaloid fraction (VLC, 1.95 g). Adult Sprague–Dawley male rats, weighing 200–250 g, and male ICR mice, weighing 20–25 g, were purchased from Daehan Biolink, Korea. The animals were housed 5 or 6 per cage, allowed access to water and food ad libitum, and maintained in a constant temperature (23 ± 1 ◦ C) and humidity (60 ± 10%) environment under a 12-h light/dark cycle (light on 07.30–19.30 h). Animal treatment and maintenance were carried out in accordance with the Principle of Laboratory Animal Care (NIH publication No. 85-23, revised 1985) and the Animal Care and Use Guidelines of Catholic University of Daegu, Korea. Rats were decapitated, the brain was rapidly dissected on ice into cortex, hippocampus and striatum, and then the dissected parts were weighed and homogenized in five volumes of cold 75 mM sodium phosphate buffer, pH 7.4. The homogenates were centrifuged at 13,000 × g for 30 min at 4 ◦ C; supernatants used as acetylcholinesterase sources were divided into aliquots and stored at −20 ◦ C. The AChE inhibitory activity was measured by the principle of the Ellman method with some modifications [17]. Enzyme samples in 10 mM phosphate buffer, pH 7.5, were incubated for 150 s at 37 ◦ C with 0.3 mM acetylthiocholine iodide in the presence of 50 M tetraisopropyl pyrophosphoramide, a selective inhibitor of butyrylcholinesterase, and 5,5 -dithiobis-(2-nitrobenzoic acid) for color development (all chemicals from Sigma). Protein concentration was measured with the Coomassie brilliant blue protein-binding method [18] using bovine serum albumin as standard. AChE activity was expressed in
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international units (1 IU = 1 M acetylthiocholine hydrolyzed per second) per mg protein. The extract was initially dissolved in 0.02% dimethyl sulfoxide (DMSO) and diluted to various concentrations in sodium phosphate buffer (100 mM, pH 8.0) immediately before use. An aliquot of diluted VLC (5–200 g/mL) solution was then mixed with sodium phosphate buffer (100 mM, pH 8.0), acetylthiocholine iodide solution (75 mM) and Ellman’s reagent (10 mM 5,5 -dithio-bis [2-nitrobenzoic acid] and 15 mM sodium bicarbonate) and was reacted at room temperature for 30 min. Absorbance was measured at 410 nm immediately after adding the enzyme source to the reaction mixtures by using a spectrophotometer (Shimadzu UV-1240, Tokyo, Japan). Readings were taken at 30 s intervals for 5 min. The concentration of compound required to inhibit acetylcholinesterase activity by 50% (IC50 ) was calculated using an enzyme inhibition dose response curve. Beberine [19] and tacrine were used as positive controls. Passive avoidance task was carried out in identical illuminated and nonilluminated boxes. The illuminated compartment (20 cm × 20 cm × 20 cm) contained an 100 W bulb. The floor of nonilluminated compartment (20 cm × 20 cm × 20 cm) was composed of 2 mm stainless steel rods spaced 1 cm apart. These compartments were separated by a guillotine door (5 cm × 5 cm). For the acquisition trial, mice were initially placed in the illuminated compartment and the door between the two compartments was opened 10 s later. When the mice entered the dark compartment, the door automatically closed and an electrical foot shock (0.5 mA) of 3 s durations was delivered through the stainless steel rods. One hour before the acquisition trial, the mice were administered with VLC (10, 20, 50, and 100 mg/kg, p.o.) or tacrine (10.0 mg/kg, p.o). After a delay of 60 min, the amnesia was induced by subcutaneous administration of scopolamine (1.0 mg/kg, i.p.). The control animals were administered with 10% Tween 80 solution only. Twenty-four hours after the acquisition trial, the mice were again placed in the illuminated compartment for the retention trials. The time taken for a mouse to enter the dark compartment after door opening was measured as latency times in both acquisition and retention trials. If the mice stayed in the light compartment for more than 180 s, it was concluded that the mice had memorized the passive avoidance training after the training trial [19]. A spatial test was performed by the method of Morris with a minor modification. The water maze is a circular pool (90 cm in diameter and 45 cm in height) with a featureless inner surface. The pool was filled to a depth of 30 cm with water containing 500 mL of milk (20 ± 1 ◦ C). The pool was divided into four quadrants of equal area. A white platform (6 cm in diameter and 29 cm high) was then placed in one of the pool quadrants. The first experimental day was dedicated to swimming training for 60 s without the submerged platform. During the five subsequent days, the mice were given two daily trials with an inter-trial interval of 30 min in the presence of the platform in place. When a mouse located the platform, it was permitted to remain on it for 10 s. If the mouse did not locate the platform within 120 s, it was placed on the platform for 10 s. The animal was taken to its home cage and was allowed to dry up under an infrared lamp after each trial [19]. During each trial session, the time taken to find the hidden platform (latency) was recorded. One day after the last training trial sessions, the mice were subjected to a probe trial session in which the platform was removed from the pool, allowing the mice to swim for 120 s to search for it. A record was kept of the swimming time in the pool quadrant where the platform had previously been placed. VLC (50 mg/kg, p.o.) or tacrine (10 mg/kg, p.o.) was given 1 h before the first trial session at every consecutive day. Memory impairment was induced in mice with scopolamine (1 mg/kg, i.p.) at 60 min after treatment of test samples. The control group received 10% Tween 80 solution only. The results are expressed as mean values ± S.D. Statistical analysis was performed using one-way ANOVA. A P < 0.05 was considered statistically significant.
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Table 1 Inhibition of VLC on brain enzyme sources. Test samples
VLC Berberineb Tacrineb a b
IC50 (g/mL)a Brain cortex
Hippocampus
Striatum
26.7 ± 5.5 14.1 ± 1.2 0.09 ± 0.02
32.2 ± 4.1 14.2 ± 0.8 0.15 ± 0.04
25.7 ± 5.1 15.8 ± 2.8 0.09 ± 0.01
Results are the mean of three replications. Reference controls.
For investigating anticholinesterase activity of VLC, mouse brain cortex, hippocampus and striatum were selected for the in vitro assay [20]. The results (IC50 ) were summarized in Table 1. VLC showed a strong inhibitory activity against AChE in mouse frontal cortex, hippocampus and striatum with IC50 values of 26.7 ± 5.5, 32.2 ± 4.1 and 25.7 ± 5.1 g/mL, respectively. Berberine, a natural active alkaloid from Corydalis turtschaninovii [19], showed a potent inhibitory activity with IC50 values ranging from 14.1 to 15.8 g/mL. Tacrine, which was effectively used as a positive control, exhibited the inhibitory activity with IC50 values ranging from 0.09 to 0.15 g/mL (Table 1). Since AChE inhibitors have been used to reduce the cholinergic deficit in AD and based on the remarkable effect of VLC in in vitro assay, we assessed whether VLC (10, 20, 50 and 100 g/mL) improves memory function by passive avoidance learning, which is largely dependent on long-term memory. When placed into the bright side of a step-through box, mice quickly entered the dark compartment. Mice were conditioned using a mild foot shock after entering the dark compartment and hesitated to re-enter the dark compartment when tested 24 h later. By using this shuttle box system, the Tween-treated control mice stayed in the light compartment for 220 ± 10 s after the passive avoidance training. Meanwhile, the mice given scopolamine (1.0 mg/kg body weight) stayed in the light compartment for only 20 ± 3 s before moving into the dark compartment. Thus, the step-through latency of the scopolamine-treated mice was significantly shorter than that of normal control group (Fig. 1, P < 0.05). In the tacrine plus scopolamine-treated mice, the step-through latency was significantly greater than that of the scopolamine-treated mice (160 ± 12 s vs. 22 ± 3 s). Moreover, the reduction in the stepthrough latency induced by scopolamine was significantly reversed by VLC at the dose of 20 and 50 mg/kg, p.o (P < 0.05) at 148 ± 15 and 167 ± 21 s, respectively. At the dose of 10 mg/kg, VLC did not exhibit
a significant effect compared to that of the vehicle group (28 ± 5 s vs. 22 ± 3 s). However, at the highest dose of 100 mg/kg, the latency time decreased significantly compare to that of 50 mg/kg treated group (115 ± 14 s vs. 22 ± 3 s, P < 0.001). Latency time during the acquisition trial was not affected by any drug treatment (Fig. 1). The effect of VLC (50 mg/kg, p.o.) on spatial learning (long-term or short-term memory) was evaluated using the Morris water maze test. The Tween-treated control group rapidly learned the location of the platform (Fig. 2A). The scopolamine-treated group exhibited longer escape latencies (the time taken to find the platform) throughout training days than did the control group (P < 0.001). VLC (50 mg/kg, p.o.) significantly attenuated the effect of scopolamine on escape latency (P < 0.05), as did tacrine (P < 0.05). On the day of probe testing, a significant group effect was observed on swimming times in the probe test (Fig. 2B). Swimming times within the platform quadrant for the scopolamine-treated group were significantly lower than those of normal control group (Fig. 2B, P < 0.05). Moreover, the shorter swimming times within the platform quadrant induced by scopolamine were significantly reversed by VLC and/or tacrine (P < 0.05). In the past decade, treatment for AD has largely involved replacement of neurotransmitters that are known to be lacking, mostly based on the inhibition of AChE, an important approach that was being developed on the cholinergic hypothesis of the disease. One of the most remarkable biochemical changes in AD patients is a reduction of acetylcholine levels in the hippocampus and cortex of the brain. The use of AChE inhibitors is the most commonly treatment for the symptoms of mild AD, which helps improving the cholinergic deficit [21,22]. AChE inhibitors including tacrine, donepezil and rivastigmine were approved by the U.S. Food and Drug Administration for the symptomatic treatment of patients with mild to moderate AD. Some potent inhibitors of AChE are derived from natural sources, most of them belong to the chemical class of alkaloids, including galanthamine and huperzine A. Huperzine A, a novel Lycopodium alkaloid discovered from H. serrata, has been found to inhibit AChE selectively and is well tolerate properties may be especially suitable for AD treatment. Several studies reported on the in vitro and in vivo anticholinesterasic activities of the alkaloids Lycopodium genus collected in Asia, Euro, Australia and South America [10–15]. The well known alkaloid contents containing in decoctions of the entire plants have been used in folk medicine for treating CNS-related conditions such as nerve tonics and motor disorders [23]. In the present study, the effect of improving memory deficit of VLC was evaluated using
Fig. 1. Effect of a single administration of VLC on scopolamine-induced memory deficits as determined by the passive avoidance task. At 60 min before acquisition trials, VLC (10, 20, 50 and 100 mg/kg, p.o.), tacrine (10 mg/kg, p.o.) or vehicle (same volume of 10% Tween 80) solution was administered to mice. Memory impairment was induced by administering scopolamine (1 mg/kg, i.p.). Five different animals were used per treatment group. Acquisition trials were carried out 30 min after a single scopolamine treatment. 24 h after the acquisition trials, retention trials were carried out for 3 min. Data represent means ± S.E.M (*P < 0.001 vs. vehicle control group, **P < 0.05 vs. scopolamine-treated group).
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Fig. 2. Effect of VLC on latency time during the training trial sessions (A), and on swimming time (B) during the probe trial session of the Morris water maze task in scopolamine-induced memory deficits mice. At 60 min before training trial sessions, VLC (50 mg/kg, p.o.), tacrine (10 mg/kg, p.o.) or vehicle (same volume of 10% Tween 80) solution was administered to mice. Memory impairment was induced by scopolamine treatment (1 mg/kg, i.p.). Five different animals were used per treatment group. The training trial and the probe trial sessions were performed as described in materials and methods. Data represent means ± S.E.M (*P < 0.001 vs. vehicle control group, **P < 0.05 vs. scopolamine-treated group).
the amnesia mouse model induced by scopolamine, which is a muscarinic receptor antagonist and causes amnesia in animals by blocking cholinergic neurotransmission. Cholinergic interneurons in the striatum contain even richer source of acetylcholinesterase and would also be affected strongly by enzyme inhibitors. Acute and systemic administration of scopolamine in young animals provides the appropriate memory deficits related to the cholinergic deficit in AD or senile CNS dysfunction. The scopolamine induced amnesic model has been widely used to provide a pharmacological model of memory dysfunction for screening potential cognition enhancing agents [24]. In our in vitro study, the alkaloid fraction from Vietnamese L. cernue exhibited the AChE inhibitory activity. In in vivo experiments, the cognitive-enhancing activity of VLC on the scopolamine-induced memory impairments in mice was investigated by using passive avoidance test and the Morris water maze test. The passive avoidance test is generally used to evaluate the treatments on the three stages of memory, such as learning acquisition, memory retention, and the retrieval process [22]. Administration of scopolamine significantly shortened the step-through latency in the retention trial, demonstrating that the central cholinergic neuronal system plays an important role in learning acquisition (Fig. 1). Treatment of VLC (10–100 mg/kg) prolonged dose-dependently the step-through latency shortened
by scopolamine. These results suggested that the anti-amnesic effect of VLC on scopolamine-induced memory impairment may be related to the mediation of the cholinergic nervous system. To confirm the effects of VLC on other stages of memory, the mice treated with scopolamine showed a more prolonged escape latency than the mice of the control group in the Morris water maze test. VLC treated groups (50 mg/kg) significantly reduced the escape latency during the trial sessions. In the probe test, VLC treatment decreased the escape latency and significantly increased the number of times of visiting the platform site in comparison to the control group. The Morris water maze test investigating spatial learning and memory has been used in detecting changes of the central cholinergic system. If the animals spent more time and swam a longer distance in the pool quadrant where the platform had previously been placed during the training session, this would indicate that the animals learned from prior experience with the Morris water maze test, showing the spatial memory improvement. Therefore, these results suggest that VLC can repair the long-term memory in scopolamine induced memory impairments. To elucidate the underlying mechanisms of memory enhancing effects of VLC, the AChE inhibitory activities were assessed by using the AChE in mouse cognition brain areas (frontal cortex, hippocampus and striatum). VLC inhibited AChE activity in a dose dependent manner. These results suggest
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that the ability to alleviate scopolamine-induce memory impairment or the anti-amnesic activity of the alkaloid fraction is, in part, mediated by the AChE inhibition. It would be interesting to indicate that although some alkaloid extracts of Lycopodiaceae species have been already tested for in vitro and in vivo AChE inhibitory activity, no study has been carried out on the anti-AChE effect of the Vietnamese species. Therefore, in order to identify the AChE inhibitors, the active compounds in the extracts are currently being investigated. As expected, both of huperzines A and B were not detected in this plant (see supporting information), pointing out that the biological activities found above may be due to other Lycopodium alkaloids. This is in accordance with previous results that the chemical study on the extract of South America L. cernua revealing a number of Lycopodium alkaloids belonging to the lycopodane, flabellidane and cernuane groups. The presence of huperzines A and B were not detected in the extracts. In conclusion, the cognitive enhacing activity of Vietnamese L. cernua might result from the inhibition of AChE. This result suggests that VLC may be used as a medication for neurodegenerative diseases. However, more studies should be preserved. Acknowledgment This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 104.99-2011.19. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.neulet. 2014.05.031. References [1] R. Becker, E. Giacobini, R. Elble, M. McIlhany, K. Sherman, Potential pharmacotherapy of Alzheimer disease. A comparison of various forms of physostigmine administration, Acta Neurol. Scand. Suppl. 116 (1988) 19–32. [2] E. Giacobini, in: E. Giacobini (Ed.), Cholinesterase Inhibitors: From the Calabar Bean to Alzheimer Therapy, M. Dunitz, Thonex, Geneva, 2000, pp. 181–226. [3] M.H. Oh, P.J. Houghton, W.K. Whang, J.H. Cho, Screening of Korean herbal medicines used to improve cognitive function for anti-cholinesterase activity, Phytomedicine 11 (2004) 544–548.
[4] P.J. Houghton, Y. Ren, M.J. Howes, Acetylcholinesterase inhibitors from plants and fungi, Nat. Prod. Rep. 23 (2006) 181–199. [5] X. Ma, C. Tan, D. Zhu, D.R. Gang, P. Xiao, Huperzine A from Huperzia species—an ethnopharmacolgical review, J. Ethnopharmacol. 113 (2007) 15–34. [6] J.S. Liu, Y.L. Zhu, C.M. Yu, Y.Z. Zhou, Y.Y. Han, F.W. Wu, B.F. Qi, The structures of huperzine A and B, two new alkaloids exhibiting marked anticholinesterase activity, Can. J. Chem. 64 (1986) 837–839. [7] X. Ma, D.R. Gang, The lycopodium alkaloids, Nat. Prod. Rep. 21 (2004) 752–772. [8] R.W. Zhang, X.C. Tang, Y.Y. Han, G.W. Sang, Y.D. Zhang, Y.X. Ma, C.L. Zhang, R.M. Yang, Drug evaluation of huperzine A in the treatment of senile memory disorders, Zhongguo Yao Li Xue Bao 12 (1991) 250–252. [9] M.G. Ortega, A.M. Agnese, J.L. Cabrera, Anticholinesterase activity in an alkaloid extract of Huperzia saururus, Phytomedicine 11 (2004) 539–543. [10] M.G. Vallejo, M.G. Ortega, J.L. Cabrera, V.P. Carlini, S. Rubiales de Barioglio, A.M. Agnese, Huperzia saururus increases memory retention in rats, J. Ethnopharmacol. 111 (2007) 685–687. [11] E.L. Konrath, B.M. Neves, C.S. Passos, P.S. Lunardi, M.G. Ortega, J.L. Cabrera, C.A. Gonc¸alves, A.T. Henriques, Huperzia quadrifariata and Huperzia reflexa alkaloids inhibit acetylcholinesterase activity in vivo in mice brain, Phytomedicine 19 (2012) 1321–1324. [12] A.I. Calderón, J. Simithy-Williams, R. Sanchez, A. Espinosa, I. Valdespino, M.P. Gupta, Lycopodiaceae from Panama: a new source of acetylcholinesterase inhibitors, Nat. Prod. Res. 27 (2013) 500–505. [13] W.H. Lim, J.Q. Goodger, A.R. Field, J.A. Holtum, I.E. Woodrow, Huperzine alkaloids from Australasian and southeast Asian Huperzia, Pharm. Biol. 48 (2010) 1073–1078. [14] R. Ho, N. Marsousi, P. Eugster, J.P. Bianchini, P. Raharivelomanana, Detection by UPLC/ESI-TOF-MS of alkaloids in three Lycopodiaceae species from French Polynesia and their anticholinesterase activity, Nat. Prod. Commun. 4 (2009) 1349–1352. [15] E.S. Halldorsdottir, J.W. Jaroszewski, E.S. Olafsdottir, Acetylcholinesterase inhibitory activity of lycopodane-type alkaloids from the Icelandic Lycopodium annotinum ssp. Alpestre, Phytochemistry 71 (2010) 149–157. [16] P.H. Ho, Cay Co Viet Nam: An Illustrated Flora of Viet Nam, 2, Nha Xuat Ban Tre, Hanoi, 1999, pp. 24. [17] G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–90. [18] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254. [19] T.M. Hung, M. Na, N.T. Dat, T.M. Ngoc, U. Youn, H.J. Kim, B.S. Min, J. Lee, K. Bae, Cholinesterase inhibitory and anti-amnesic activity of alkaloids from Corydalis turtschaninovii, J. Ethnopharmacol. 119 (2008) 74–80. [20] Q. Zhao, X.C. Tang, Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine, donepezil, rivastigmine and physostigmine, Eur. J. Pharmacol. 455 (2002) 101–107. [21] E. Giacobini, The cholinergic system in Alzheimer disease, Prog. Brain Res. 84 (1990) 321–332. [22] J.E. LeDoux, Emotional memory systems in the brain, Behav. Brain Res. 58 (1993) 69–79. [23] A.A. Izzo, F. Capasso, Herbal medicines to treat Alzheimer’s disease, Trends Pharmacol. Sci. 28 (2007) 47–48. [24] U. Ebert, W. Kirch, Scopolamine model of dementia: electroencephalogram findings and cognitive performance, Eur. J. Clin. Invest. 28 (1998) 944–949.