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An introduction is presented in which the editor discusses various reports within the issue on topics including Chinese medicine, Huperzine A, and mental depression.

Abstract: A Bernese mountain dog was diagnosed with complex partial seizures that were supported by electroencephalographic findings. Clinical signs of the problem included “star gazing,” fly snapping, licking, vacuous chewing, and ongoing anxiety. Treatment with Huperzine A, a compound isolated from Chinese club moss with NMDA receptor blocking activity, anticholinesterase activity, and anticonvulsant properties, produced useful suppression of the abnormal behavior for more than months. A relapse occurred when the dog was treated with tramadol for joint pain and the improvement that had been made was not recaptured with Huperzine A. At this stage, phenobarbital therapy was instituted and the dog improved greatly. The role of Huperzine A in controlling seizures is discussed. [Copyright &y& Elsevier]

The green tea polyphenol (–)-epigallocatechin-3-gallate (EGCG) was investigated for its enhancement effect of huperzine A on inhibiting acetylcholinesterase (AChE). The inhibitory effect of huperzine A on acetylcholinesterase is quite weak in the whole phase. EGCG hardly inhibits the AChE activity within the range 10–300 mg/kg. However, upon addition of EGCG to the huperzine A groups, a remarkably enhanced inhibitory effect was observed. The EGCG also can largely prolong the inhibitory time. These results indicate that addition of EGCG to huperzine A can reduce the dose of huperzine A required compared with huperzine A alone. The enhancement and complementary effect of EGCG on huperzine A activity may partly be due to the antioxidant property of EGCG. One of the beneficial effects of green tea is to induce a feeling of relief. It is conceivable that this function may be regulated by EGCG in the central nervous system since EGCG is distributed in the brain after oral administration. EGCG can be used as an enhanced supplement for huperzine A to treat Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Abstract: Our previous work in cells and animals showed that mitochondria are involved in the neuroprotective effect of huperzine A (HupA). In this study, the effects of HupA on isolated rat brain mitochondria were investigated. In addition to inhibiting the Aβ25-35 (40 μM)-induced decrease in mitochondrial respiration, adenosine 5′-triphosphate (ATP) synthesis, enzyme activity, and transmembrane potential, HupA (0.01 or 0.1 μM) effectively prevented Aβ-induced mitochondrial swelling, reactive oxygen species increase, and cytochrome c release. More interestingly, administration of HupA to isolated mitochondria promoted the rate of ATP production and blocked mitochondrial swelling caused by normal osmosis. These results indicate that HupA protects mitochondria against Aβ at least in part by preserving membrane integrity and improving energy metabolism. These direct effects on mitochondria further extend the noncholinergic functions of HupA. [Copyright &y& Elsevier]

Huperzine A (Hup A) is a reversible AChE inhibitor that crosses the blood-brain barrier. It is presently approved for, or is in a course of clinical trials for, the treatment of Alzheimer's disease. This compound has also been successfully tested for the pretreatment of organophosphate poisoning. Organophosphate nerve agents are potent irreversible inhibitors of acetylcholinesterase in the central and also in the peripheral compartment. In this study Hup A in a higher dose (500 μg/kg) was tested as a prophylaxis against a high, mainly centrally acting, nerve agent (soman). According to the results obtained, Hup A in this dosage was not able to protect AChE against soman in both the peripheral and central compartments. The effect of Hup A and soman was found to be additive and all animal subjects died. [ABSTRACT FROM AUTHOR]

The objective of this study was to provide an updated meta-analysis of the efficacy and safety of huperzine A (HupA) in Alzheimer’s disease (AD). We searched for randomized trials comparing HupA with placebo in the treatment of AD. The primary outcome measures were mini-mental state examination (MMSE) and activities of daily living scale (ADL). Data were extracted from four randomized clinical trials and analyzed using standard meta-analysis and meta-regression methods. Oral administration of HupA for 8–24 weeks (300–500 μg daily) led to significant improvements in MMSE and ADL. The results of meta-regression showed that the estimated effect size of MMSE and ADL was increased over the treatment time. Most adverse events were cholinergic in nature and no serious adverse events occurred. Huperzine A is a well-tolerated drug that could significantly improve cognitive performance and ADL in patients with AD. [ABSTRACT FROM AUTHOR]

Summary: Botanicals and herbs have a centuries-old tradition of use by persons with epilepsy, in many cultures around the world. At present, herbal therapies are tried by patients in developing as well as developed countries for control of seizures or adverse effects from antiepileptic drugs (AEDs), or for general health maintenance, usually without the knowledge of physicians who prescribe their AEDs. Well-designed clinical trials of herbal therapies in patients with epilepsy are scarce, and methodological issues prevent any conclusions of their efficacy or safety in this population. Furthermore, some botanicals and herbs may be proconvulsant or may alter AED metabolism. In spite of these limitations, further preclinical evaluation of botanicals and herbs and their constituent compounds using validated scientific methods is warranted based on numerous anecdotal observations of clinical benefit in patients with epilepsy and published reports showing mechanisms of action relevant to epilepsy or anticonvulsant effects in animal models of epilepsy. This review highlights the use of herbal therapies for epilepsy, outlines the role of the U.S. Food and Drug Administration in regulating herbal products, and presents the author''s approach to the scientific assessment of herbal therapies as potential therapies for patients with epilepsy. [Copyright &y& Elsevier]

A sensitive and specific liquid chromatography–electrospray ionization–tandem mass spectrometry method has been developed and validated for the quantification of huperzine A in human plasma. After the addition of trimetazidine, the internal standard (IS) and sodium hydroxide, plasma samples were extracted using 5 mL ethyl acetate. The compounds were separated on an Agilent Zorbax SB C18 column (100 mm × 2.1 mm ID, dp 3.5 μm) using an elution system of 10 mM ammonium acetate solution–methanol–formic acid (18:82:0.1, v/ v) as the mobile phase. The quantification of target compounds was obtained by using multiple reaction monitoring (MRM) transitions: m/ z 243.1, 210.1 and 267.2, 166.0 were measured in positive mode for huperzine A and IS. Linearity was established for the range of concentrations 0.01–4.0 ng mL−1 with a coefficient of correlation ( r) of 0.9991. The lower limit of quantification (LLOQ) was identifiable and reproducible at 0.01 ng mL−1. The method has been successfully applied to study the pharmacokinetics of huperzine A in healthy male Chinese volunteers. [ABSTRACT FROM AUTHOR]

Abstract: The excessive release of cytokines by the immune system contributes importantly to the pathogenesis of inflammatory diseases. Recent advances in understanding the biology of cytokine toxicity led to the discovery of the “cholinergic anti-inflammatory pathway,” defined as neural signals transmitted via the vagus nerve that inhibit cytokine release through a mechanism that requires the α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR). Vagus nerve regulation of peripheral functions is controlled by brain nuclei and neural networks, but despite considerable importance, little is known about the molecular basis for central regulation of the vagus nerve-based cholinergic anti-inflammatory pathway. Here we report that brain acetylcholinesterase activity controls systemic and organ specific TNF production during endotoxemia. Peripheral administration of the acetylcholinesterase inhibitor galantamine significantly reduced serum TNF levels through vagus nerve signaling, and protected against lethality during murine endotoxemia. Administration of a centrally-acting muscarinic receptor antagonist abolished the suppression of TNF by galantamine, indicating that suppressing acetylcholinesterase activity, coupled with central muscarinic receptors, controls peripheral cytokine responses. Administration of galantamine to α7nAChR knockout mice failed to suppress TNF levels, indicating that the α7nAChR-mediated cholinergic anti-inflammatory pathway is required for the anti-inflammatory effect of galantamine. These findings show that inhibition of brain acetylcholinesterase suppresses systemic inflammation through a central muscarinic receptor-mediated and vagal- and α7nAChR-dependent mechanism. Our data also indicate that a clinically used centrally-acting acetylcholinesterase inhibitor can be utilized to suppress abnormal inflammation to therapeutic advantage. [Copyright &y& Elsevier]

The natural product Huperzine A isolated from Huperzia serrata is a targeted inhibitor of acetylcholinesterase that has been approved for clinical use in the treatment of Alzheimer's disease. Given the large demand for natural sources of Huperzine A (Hup. A), efforts have been made to explore whether it is also produced by endophytic fungi from H. serrata and, if so, identify its biosynthetic pathway. These studies have indicated that endophytic fungi from H. serrata represent a huge and largely untapped resource for natural products (including Hup. A) with chemical structures that have been optimized by evolution for biological and ecological relevance. To date, more than three hundred endophytic fungi have been isolated from H. serrata , of which 9 strains can produce Hup. A, whilst more than 20 strains produce other important metabolites, such as polyketones, xanthones, alkaloids, steroids, triterpenoids, furanone derivatives, tremulane sesquitepenes and diterpenoids. In total, 200 secondary metabolites have been characterized in endophytic fungi from H. serrata to date. Functionally, some have cholinesterase-inhibitory or antibacterial activity. This review also considers the different classes of secondary metabolites produced by endophytic fungi, along with their possible applications. We systematically describe the taxonomy, biology, and chemistry of these secondary metabolites. It also summarizes the biosynthetic synthesis of metabolites, including that of Hup. A. The review will aid researchers in obtaining a clearer understanding of this plant-endophyte relationship to better exploit the excellent resources it offers that may be utilized by pharmaceutical industries. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: Huperzine A (HupA), a novel Lycopodium alkaloid isolated from Chinese folk medicine Huperzia serrata (Qian Ceng Ta), is a potent, selective and well-tolerated inhibitor of acetylcholinesterase (AChE). It has been proven to significantly improve the learning and memory impairment in Alzheimer''s disease (AD) and vascular dementia (VaD) patients in China. Interestingly, our recent data indicate that HupA also possesses other protective functions. This paper will give an overview on the protective effects of HupA, which includes regulating β-amyloid precursor protein (APP) metabolism, protecting against Aβ-mediated oxidative stress, apoptosis and mitochondrial dysfunction, as well as anti-inflammation. The multiple neuroprotective effects of HupA might yield additional beneficial effects in AD and VaD therapy. [Copyright &y& Elsevier]

Abstract: As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400μg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48h following the final and highest (400μg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30–40% after 100μg to >50% at 400μg, and peaking 1.5h after the last dose. Gradual recovery of AChE activity then occurs, but even 48h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5h after the final 400μg dose (5.47±2.15ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10min, followed by removal of unbound huperzine and soman from the blood by passing through a small C18 reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning. [Copyright &y& Elsevier]

Huperzine A, a reversible and selective acetylcholinesterase (AChE) inhibitor, has been reported to display neuroprotective properties. The present study investigated the protective effects of huperzine A in a rat model of transient focal cerebral ischemia created by middle cerebral artery occlusion (MCAO). Huperzine A (0.1 mg/kg), administrated intraperitoneally at the onset of occlusion and 6 h later, markedly restored regional cerebral blood flow, reduced infarct size, and decreased neurological deficit score at 24 h after reperfusion. Along with inhibiting AChE activity, huperzine A inhibited nuclear translocation of transcription factor nuclear factor-kappa B, decreased overexpression of proinflammatory factors in both ipsilateral cortex and striatum, and suppressed activation of glial cells in the ischemic penumbra. Neurological deficit and glial cells activation were also reduced by daily administration of huperzine A for 14 days. Mecamylamine, a nicotinic acetylcholine receptor (nAChR) antagonist, totally abolished the inhibitory effects of huperzine A on ischemia-induced glial cells activation. Meanwhile, mecamylamine partially reversed the infarct size-reducing effects of huperzine A. In conclusion, our results demonstrate that huperzine A exhibits neuroprotective effects against transient focal cerebral ischemia-induced brain injury and suggest that the protection mechanism may involve a cholinergic anti-inflammatory pathway, in which nAChR plays an essential role. [ABSTRACT FROM AUTHOR]

Abstract: Alzheimer’s disease (AD) is growing in impact on human health. With no known cure, AD is one of the most expensive diseases in the world to treat. Huperzine A (HupA), a anti-AD drug candidate from the traditional Chinese medicine Qian Ceng Ta (Huperzia serrata), has been shown to be a powerful and selective inhibitor of acetylcholinesterase and has attracted widespread attention because of its unique pharmacological activities and low toxicity. As a result, HupA is becoming an important lead compound for drugs to treat AD. HupA is obtained naturally from very limited and slowly growing natural resources, members of the Huperziaceae. Unfortunately, the content of HupA is very low in the raw plant material. This has led to strong interest in developing sources of HupA. We have developed a method to propagate in vitro tissues of Phlegmariurus squarrosus, a member of the Huperziaceae, that produce high levels of HupA. The in vitro propagated tissues produce even higher levels of HupA than the natural plant, and may represent an excellent source for HupA. [Copyright &y& Elsevier]

Abstract: Comparison of single and multiple dose pharmacokinetics between patches and conventional tablets of Huperzine A (Hup-A) was performed in beagle dogs to evaluate the patches’ controlled drug release characteristics in vivo, a newly developed transdermal system for treatment of Alzheimer disease. Results showed that transdermal administration of Hup-A prolonged T max value (24h vs. 3h, P <0.01), lowered C max value (3.4±0.2ngmL−1 vs. 9.8±1.0ngmL−1, P <0.01), and produced a relatively constant serum concentration within 84h after a single transdermal dose of 4mg/20cm2 Hup-A patches. Following application of the patches, Hup-A serum concentrations increased for approximately 12–24h, reaching an average C max of 3.4±0.2ngmL−1. Thereafter, a serum concentration of at least 2.1ngmL−1 was maintained for up to 84h. The serum concentration was maintained within the range of 2.4–4.3ngmL−1 during 2-week wearing period after multiple dosing, and the degree of fluctuation at the steady state of td and po administration was significantly different (0.51 vs. 1.99, P <0.01). This study indicates that Hup-A patches exhibited good controlled-release properties in vivo, maintained a relatively constant serum concentration within 3.5 d after wearing, and are suitable for twice-weekly application. [Copyright &y& Elsevier]

Amyotrophic lateral sclerosis (ALS) is a neuro-degenerative disease resulting from the progressive loss of motor neurons in the spinal cord and brain. To date, clinically effective neuroprotective agents have not been available. The current study demonstrates for the first time that huperzine A, a potential neuroprotective agent, has the ability to protect a motor neuron-like cell line and motor neurons in spinal cord organotypic cultures from toxin-induced cell death. The neuroblastoma-spinal motor neuron fusion cell line, NSC34 and rat spinal cord organotypic cultures (OTC) were exposed to cell death inducers for 24 h or 14 d, respectively, with and without pre-treatment with huperzine A. The inducers used here include: staurosporine, thapsigargin, hydrogen peroxide (H2O2), carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and L-(-)-threo-3-hydroxyaspartic acid (THA). These agents were selected as they induce apoptosis/ necrosis via mechanisms implicated in patients with generalized motor neuron disease. Cell death was determined in NSC34 cells by metabolic activity, caspase activity/expression and by nuclear morphology and in the OTCs, using immunohistochemistry and Western blot analysis. Nuclear staining of NSC34 cells revealed cell death induced by staurosporine, thapsigargin, H2O2 and CCCP. This induction was significantly reduced with 2 h pre-treatment with 10 µM huperzine A (maximum, 35% rescue; p<0.05) following exposure to staurosporine, thapsigargin and H2O2 but not with CCCP. These data were supported by the metabolic assays and caspase activity. In addition, pretreatment with huperzine A dramatically improved motor neuron survival, based on choline acetyltransferase (ChAT) expression analysis in OTCs following exposure to THA, and compared to THA-treated control cultures. These studies are currently being extended to include other inducers and with additional compounds as potential drug therapies that could be used in combination for the treatment of patients with ALS. [ABSTRACT FROM AUTHOR]

Abstract: Alpha-secretase (α-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-β (Aβ) sequence, resulting in the release of a secreted fragment of APP (αAPPs) and precluding Aβ generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Aβ generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0–10 μM) increased αAPPs release. Therefore, we evaluated two α-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-α converting enzyme (TACE)/ADAM17 inhibited the Hup A–induced rise in αAPPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic α-secretase pathway. Hup A had no effect on Aβ generation in this cell line. The steady-state levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A–induced αAPPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimer’s disease (AD). [Copyright &y& Elsevier]

Abstract: Huperzine A (HupA), isolated originally from a traditional Chinese medicine Qiang Ceng Ta, whole plant of Huperzia serrata (Thunb. ex Murray) Trev., a member of the Huperziaceae family, has attracted intense attention since its marked anticholinesterase activity was discovered by Chinese scientists. Several members of the Huperziaceae (Huperzia and Phlegmariurus species) have been used as medicines in China for contusions, strains, swellings, schizophrenia, myasthenia gravis and organophosphate poisoning. HupA has been marketed in China as a new drug for Alzheimer''s disease (AD) treatment and its derivative ZT-1 is being developed as anti-AD new drug candidate both in China and in Europe. A review of the chemistry, bioactivities, toxicology, clinical trials and natural resources of HupA source plants is presented. [Copyright &y& Elsevier]

Abstract: This paper presents to investigate the levels of Huperzine A in plasma and CSF of rats after three different kinds of administrations and to find out whether intranasal administration is the best root to transfer the drug into the CNS. The drugs of two doses (167 and 500μg/kg) were administered to male Sprague–Dawley rats intravenously, intranasally and intragastricly, respectively. Series plasma and cerebrospinal fluid (CSF) samples were collected from femoral artery and cisterna magna for 6h. The drug concentrations were determined by HPLC-fluorescence method. The AUCplasma and the AUCCSF of intranasal administration were 90.3% and 127.7% in low dose group (167μg/kg) and 91.3% and 69.4% in high dose group (500μg/kg) compared with intravenous administration. The AUCplasma and the AUCCSF of intragastric administration were 98.9% and 52.1% in high dose group (500μg/kg) compared with intravenous administration. [Copyright &y& Elsevier]

Abstract: A simple reversed-phase high-performance liquid chromatography (HPLC)-fluorescence method for the determination of Huperzine A in rat plasma was developed and validated. Separation was achieved on Kromasil C(8) column (5μm, 150mm×4.6mm i.d.). The mobile phase, methanol–water–triethanol amine (45:55:0.05, v/v/v), was delivered at a flow rate of 1.0ml/min. The eluent was monitored by a fluorescence detector with excitation wavelength at 310nm and emission wavelength at 370nm. No interfering peaks were observed in rat blank plasma. The relationship between Huperzine A concentration and peak-area ratio of Huperzine A to the IS was linear over the range of 2.5–250ng/ml. The intra- and inter-day coefficients of variation were ≤10.1% and ≤14.1%, respectively. The method and extraction recovery of Huperzine A were 101.9–108.9% and 73.5–84.6%, respectively. Huperzine A was found to be stable for at least 5h at RT and 1 week at −20°C. The method was applied to a pharmacokinetic study of Huperzine A in rats following intranasal administration. [Copyright &y& Elsevier]

Abstract: Huperzine A, a reversible acetylcholinesterase inhibitor for the treatment of Alzheimer disease (HupA), was studied using an (MALDI TOF MS) instrument in MALDI mode. The formation of a HupA dimmer in a vacuum was observed and several matrices were found that were able to inhibit its formation. The structures of the neutral and protonated form of the HupA molecule were calculated and optimized using a Hyperchem program. Detection limit using MALDI TOF MS in the model sample was 5.3pg. MALDI TOF MS was also applied to the direct detection of the drug in medical preparations and in human serum. The limit of detection in plasma was 14.2pg with a signal-to-noise ratio of 3:1. However, the sensitivity was not as high as it usually is in MALDI. Therefore, a new method for the derivatization of HupA was developed using fluorescent labelling with rhodamine B isothiocyanate (RBITC). A limit of detection using capillary electrophoresis laser induced fluorescence detection (CE-LIF) equal to 4×10−9 moll−1 was reached. [Copyright &y& Elsevier]

Abstract: The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase, which makes the enzyme essentially intractable. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. This strategy was successfully tested in animal experiments, but its efficacy still awaits complete understanding. In particular, it is unclear whether survival is improved by a higher residual activity of acetylcholinesterase during the acute phase, when the reversible and irreversible inhibitors are present together. In previous experiments with carbamate pre-treatment and paraoxon challenge we noticed an increased residual activity of erythrocyte acetylcholinesterase compared to non-pre-treatment. This result was encouraging to also test for comparable effects when using soman. Immobilized human erythrocytes were continuously perfused for real-time measurement of acetylcholinesterase activity by a modified Ellman method using 0.45mM acetylthiocholine. After having established the inhibition rate constant of soman, we tested the prophylactic potential of physostigmine, pyridostigmine and huperzine A. Pre-treatment with the reversible inhibitors inhibited the enzyme by 20–95%. Additional perfusion with 10nM soman for 30min resulted in a residual activity of 1–5%, at low and high pre-inhibition, respectively. The residual activity was markedly higher than in the absence of reversibly blocking agents (0.1%). After discontinuation of soman and the reversible inhibitors, enzyme activity recovered up to 30% following pre-inhibition by 50%. The experimental data agreed with computer simulations when feeding the kinetic-based model with the established rate constants. The results with soman essentially agreed with those obtained previously with paraoxon. [Copyright &y& Elsevier]

Abstract: The protective effects of huperzine A against oxygen–glucose deprivation (OGD)-induced injury in C6 cells were investigated. OGD for 6h and reoxygenation for 6h enhanced phosphorylation and degradation of IκBα and nuclear translocation of nuclear factor-kappa B (NF-κB), triggered overexpression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and nitric oxide (NO) in C6 cells. Along with inhibiting acetylcholinesterase activity, treatment with 1μM huperzine A inhibited activation of NF-κB, attenuated iNOS, COX-2 and NO overexpression, and promoted survival in C6 cells subjected to OGD insult. The protective effects of huperzine A were partly mediated by “cholinergic anti-inflammatory pathway” through α7 nicotinic acetylcholine receptor. [Copyright &y& Elsevier]

Abstract: The objective of the present work was to further study the in vitro characteristics, in vivo pharmacokinetics and pharmacodynamics of huperzine A (HupA) loaded biodegradable microspheres designed for sustained release of HupA over several weeks. A conventional o/w emulsion-solvent evaporation method was used to incorporate HupA, which is of interest in the palliative treatment of Alzheimer''s disease (AD), into end-group uncapped poly(d,l-lactide-co-glycolide) (PLG-H). A prolonged in vitro drug release profile was observed, with a complete release of the incorporated drug within 5–6 weeks. The in vivo pharmacokinetics study of HupA loaded microspheres showed sustained plasma HupA concentration–time profile after subcutaneous injection into rats. The pharmacodynamics evaluated by determination of the activity of acetylcholinesterase in the rat cortex also showed a prolonged pharmacological response. Both the in vitro release and in vivo pharmacological responses correlated well with the in vivo pharmacokinetics profile. The results suggest the potential use of HupA-loaded biodegradable microspheres for treatment of AD over long periods. [Copyright &y& Elsevier]

Aim: To determine the uptake extent of Huperzine A (Hup A) into the brain after intranasal administration of Hup A in situ gel to rats, and to compare the pharmacokinetic parameters between intranasal administration and iv and po. Methods: Hup A was administered to male Sprague-Dawley rats via nasal, iv and oral routes at the dose of 166.7, 166.7, and 500 μg/kg, respectively. Blood and brain tissue samples including the cerebrum, hippocampus, cerebellum and olfactory bulb were collected, and the concentrations of Hup A in the samples were assayed by HPLC. The area under the concentration–time curve (AUC0→6h) and the ratio of the AUCbrain to the AUCplasma (drug targeting efficiency, DTE) were calculated to evaluate the brain targeting efficiency of the drug via 3 administration routes. Results: The AUC00←6h of the drug in the cerebrum, hippocampus, cerebellum, left olfactory bulb and right olfactory bulb after intranasal administration of the Hup A in situ gel were 1.5,1.3,1.0,1.2, and 1.0 times of those after iv administration of the injection, and 2.7, 2.2, 1.9, 3.1, and 2.6 times of those after administration of the oral formulation. The AUCbrain0←6h/AUCplasma0←6h of Hup A in the cerebrum, hippocampus and left olfactory bulb following the intranasal administration dose were significantly higher ( P < 0.05) than the iv dose. Conclusion: Intranasal delivery showed a viable, non-invasive strategy for delivering the drug into brain. [ABSTRACT FROM AUTHOR]

Abstract: The pH-zone refining centrifugal partition chromatography technique was used to separate the two acetylcholinesterase inhibitors huperzines A and B from a crude alkaloid extract of the club moss Huperzia serrata. Complete co-elution of huperzines A and B was initially observed with the well-known methyl tert-butyl ether–acetonitrile–water (4:1:5, v/v/v) solvent system with triethylamine (8mM) as the displacer and methane sulfonic acid (6mM) as the retainer. An efficient biphasic system was designed on the basis of solvent association that provided selectivity in the elution mode: n-heptane/ethyl acetate/n-propanol/water (5:15:35:45, v/v/v/v). Lowering the bridge solvent content (n-propanol) of this system increased the polarity difference between the two phases thus adapting it to the pH-zone refining mode. Thus, the purification of these compounds was achieved using the biphasic system n-heptane/ethyl acetate/n-propanol/water (10:30:15:45, v/v/v/v) with triethylamine (8mM) as the displacer and methane sulfonic acid (6mM) as the retainer. [Copyright &y& Elsevier]

Recently, there has been an escalating interest in exploring novel pharmacological approaches in Alzheimer Dementia and Schizophrenia. We reviewed the pharmacology of Huperzine A, an alkaloid isolated from Huperzia Serrata. The dual action of Huperzine in inhibiting cholinesterase activity and modulating the excitatory amino acid neurotransmitter NMDA (N-methyl-D-aspartic acid) makes the compound highly appealing as target in pharmaceutical development as a cognitive enhancer agent. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to investigate the pharmacokinetics and in vitro/in vivo correlation (IVIVC) of huperzine A loaded poly(lactic-co-glycolic acid) (PLGA) microspheres in dogs. Several huperzine A loaded PLGA microspheres were prepared by an O/W method and three of them (single dose) were injected intramuscularly (i.m.) or subcutaneously (s.c.) to five beagle dogs, respectively. With the increase of the molecular weight of PLGA and the particle size of microspheres, the in vitro and in vivo release periods of huperzine A were prolonged. After s.c. injection, the release of huperzine A from microspheres was faster than that after i.m. injection. The IVIVC models of huperzine A loaded PLGA microspheres were established successfully and after i.m. administration the linear relationship between the in vitro and the in vivo releases was better than that after s.c. administration. It was also found when the particle size of the microspheres was smaller; the values of correlation coefficient were higher. [Copyright &y& Elsevier]

Aim: To assess the effects of cholinesterase inhibitors huperzine A, donepezil and rivastigmine on cerebral neurotransmitters in the cortex and hippocampus in freely-moving rats. Methods: Double-probe cerebral microdialysis and HPLC with electrochemical detection were used to detect neurotransmitters. Results: Our results showed that huperzine A (0.25, 0.5, and 0.75 umol/kg, po) dose-dependently elevated extracellular acetylcholine (ACh) levels in the medial prefrontal cortex (mPFC) and hippocampus. Oral administration of donepezil (5.4 μmol/kg) or rivastigmine (1 μmol/kg) also elicited significant increases in ACh in the mPFC and hippocampus. The time course of cortical acetylcholinesterase (AChE) inhibition with the 3 inhibitors mirrored the increases of ACh at the same dose. The marked elevation of ACh after oral administration of huperzine A (0.5 μmol/kg) and donepezil (5.4 μmol/kg) was associated with a significantly increased release of dopamine (DA) in the mPFC or hippocampus. None of the 3 inhibitors affected norepinephrine (NE) and 5-hydroxytryptamine (5-HT) levels in the mPFC and hippocampus. The effects of huperzine A and rivastigmine did not depend on the route of administration, but donepezil was less efficacious by the oral route than by ip injection. The ability of huperzine A to increase ACh levels was unchanged when tests were performed after multiple oral administration of the drug at 0.5 μmol/kg, once per day for 30 d. Conclusion: The present findings showed that, in molar terms, huperzine A had similar potency on increasing mPFC ACh and DA levels as compared to the 11- and 2-fold dosages of donepezil and rivastigmine, respectively, and had longer lasting effects after oral dosing. [ABSTRACT FROM AUTHOR]

Abstract: In organophosphate poisoning, the underlying mechanism of the therapeutic efficacy of carbamate prophylaxis, which was successfully tested in animal experiments, still awaits complete understanding. In particular, it is unclear whether survival is improved by increased acetylcholinesterase activity during the acute phase, when both carbamate and organophosphate are present. This question should be solved experimentally by means of a dynamically working in vitro model. Immobilized human erythrocytes were continuously perfused while acetylcholinesterase activity was monitored in real-time by a modified Ellman method. The concentrations of reversible inhibitors and of paraoxon were varied to assess the influence of both components on the enzyme activity under steady-state conditions. Physostigmine, pyridostigmine and huperzine A were tested for their prophylactic potential. Upon pretreatment with these reversible inhibitors the enzyme was inhibited by 20–90%. Additional perfusion with 1μM paraoxon for 30min resulted in a residual activity of 1–4%, at low and high pre-inhibition, respectively. The residual activity was significantly higher than in the absence of reversibly blocking agents (0.3%). After discontinuing paraoxon, the activity increased even in the presence of the reversible blockers. Stopping the reversibly blocking agents resulted in 10–35% recovery of the enzyme activity, depending on the degree of pre-inhibition. The experimental results agreed with computer simulations upon feeding with the essential reaction rate constants, showing that physostigmine was somewhat superior to pyridostigmine in enhancing residual activity in the presence of 1μM paraoxon for 30min. The model predicts that inhibitors with a faster dissociation rate, e.g. huperzine A, may be superior in case of a ‘hit-and-run’ poison such as soman. [Copyright &y& Elsevier]

Abstract: This study is to investigate the effects of huperzine A on memory deficits, neuronal damage and neurotrophic factors production after transient cerebral ischemia and reperfusion in mice, as well as the potential downstream signaling pathway. Bilateral common carotid occlusion (BCCAo) combined with systemic hypotension induced severe memory deficits in a water maze task and neuronal degeneration in cerebral cortex and hippocampus in mice. Oral administration of huperzine A (0.2 mg/kg, once per day, started 2 days before surgery and lasted for 7 days after surgery) markedly attenuated the memory deficits and neuronal damage. Meanwhile, huperzine A significantly increased the mRNA and protein levels of NGF, BDNF and TGF-β1, and potentiated phosphorylation of MAPK/ERK 1/2 in both cerebral cortex and hippocampus compared with transient cerebral ischemia and reperfusion group. This study provides evidence for the protective effects of huperzine A against transient cerebral ischemia and reperfusion in mice, and suggests potentially important roles that neurotrophic factors might play in these effects. It also indicates that the MAPK/ERK pathway might be involved in the in vivo neurotrophic effects of huperzine A against transient cerebral ischemia and reperfusion. [Copyright &y& Elsevier]

Abstract: The Huperziaceae is comprised of two genera, Huperzia and Phlegmariurus. Because of the content of Lycopodium alkaloids like huperzine A, which are used to treat a number of human ailments, plants of the Huperziaceae are experiencing a rapid decline in China, mostly due to over-harvesting. Because of this trend, we engaged from 1995 to 2001 in an investigation of the natural resources of the Huperziaceae in China. The main objectives of this study were: to catalog Huperziaceae plant resources including the occurrence, general distribution, and abundance of the various Huperziaceae species in China; and to determine traditional use and pharmaceutical values of each species. Twenty-nine species, 2 varieties, and 2 forma of Huperzia and 19 species of Phlegmariurus were identified through field investigation, collection, visits with local traditional doctors, and review of specimens in herbaria and of the literature. Ethnobotanical studies of these plants revealed that 33 of these species are used by the local communities for medicinal purposes. One species, Huperzia serrata, is one of the most popular. As a result, it is observing the greatest decline, mandating a change in collection practice and general attitude towards these plants. Introduction of conservation plans and training of the local communities regarding appropriate collection practices of these plants and their marketing in China are required to reverse the trend of decline among these species. In addition, development of cultivation or other propagation practices, such as in vitro propagation, would have the added benefits of socio-economic uplift of the local communities and sustainability of this important source of huperzine A. [Copyright &y& Elsevier]

Abstract: A rapid, simple and reliable high performance liquid chromatography (HPLC) method has been established for the analysis of the major alkaloids in Huperzia serrata, a traditional Chinese medicine (TCM) herb. After chromatographic separation on a reversed-phase C18 column with methanol-ammonium acetate (pH 6.0; 80mM, 30/70, v/v) as the mobile phase, nine alkaloid compounds in the alkaloid extracts of H. serrata were identified by online diode array detection–MS and by comparing with data from literature and standard samples. One compound in the extract, huperzine A, is a drug for treating Alzheimer''s disease. Its content was quantified by HPLC coupled with UV-vis. The method was the validated. The recovery rates were 96.8–97.7% with R.S.D <2.44%. The intra- and inter-day precisions, expressed as R.S.D., ranged from 0.53% to 1.51%. Good linear regression was observed in the concentration range of 5–100μg/ml (r =0.9997). The results demonstrate that this method is simple, selective, and suitable for the quality control of this TCM herb. [Copyright &y& Elsevier]

A rapid, facile, and environmentally friendly process was developed for purification of huperzine A (HupA) and huperzine B (HupB) from the Chinese traditional herb Huperzia serrata . The process consisted of two steps of preparative low-pressure liquid chromatography (LPLC) on two polystyrene based resins, successively. The first step removed a large amount of impurities and captured HupA and HupB using Amberlite XAD-4 from the herbal extraction prepared by 1% aqueous H 2 SO 4 . This step was more efficient than multi-cycle liquid-liquid extraction as an initial separation step. The second step was able to separate HupA and HupB, employing a novel uniform polystyrene based porous microsphere named PST as the packing material. The PST column demonstrated a better separation and shorter run time compared with a commercial C 18 column. The mobile phases used in both LPLC's simply consisted of water and ethanol that are considered to be of low toxicity. Combination of the XAD-4 and PST chromatography and one step of crystallization enabled purification of HupA and HupB from 0.18% and 0.08% to 98.2% and 98.8%, respectively, with total recoveries of 82.8% and 84.3%. [ABSTRACT FROM AUTHOR]

Abstract: The synthesis of the core structure of huperzine A by cyclisation of 2-pyridylmethyl radicals is described. (2-Methylpyridin-3-yl)cyclohexenols are directly selenated at the benzylic position by deprotonation/selenation and the products undergo either 5-exo-trig or 6-exo-trig radical cyclisations giving access to hexahydroindenopyridines and the bicyclo[3.3.1]nonane core of huperzine A, respectively. [Copyright &y& Elsevier]

Abstract: ZT-1 is a novel acetylcholinesterase (AChE) inhibitor. It is rapidly transformed to Huperzine A (Hup A) in vitro. A simple and rapid HPLC-UV method for the simultaneous determination of ZT-1 and its metabolite Hup A in plasma is described. The chromatographic separations were achieved on a C18 ODS column (250mm×4.6mm ID) using methanol-1mmol/L ammonium acetate (70:30,v/v) as mobile phase. The flow rate was 0.7mL/min, the detection wavelength was 313nm and the column temperature was kept at 35°C. Plasma samples were prepared as rapidly as possible and extracted immediately with 5mL of chloroform:iso-propyl alcohol mixture (v/v, 9:1).The retention times of ZT-1 and Huperzine A (Hup A) were 18.7 and 14.4min, respectively. The mean absolute recoveries of two analytes were >90%. Quantification limits were all 0.02nmol/mL for ZT-1 and Hup A. This analytical method was reliable and convenient procedure that meets the criteria for the pharmacokinetic evaluation of ZT-1 on experimental animals. [Copyright &y& Elsevier]

Huperzine A (HupA), a novel alkaloid isolated from the Chinese herb Huperzia serrata, is a potent, highly specific and reversible inhibitor of acetylcholinesterase (AChE). Compared with tacrine, donepezil, and rivastigmine, HupA has better penetration through the blood-brain barrier, higher oral bioavailability, and longer duration of AChE inhibitory action. HupA has been found to improve cognitive deficits in a broad range of animal models. HupA possesses the ability to protect cells against hydrogen peroxide, β-amyloid protein (or peptide), glutamate, ischemia and staurosporine-induced cytotoxicity and apoptosis. These protective effects are related to its ability to attenuate oxidative stress, regulate the expression of apoptotic proteins Bcl-2, Bax, P53, and caspase-3, protect mitochondria, upregulate nerve growth factor and its receptors, and interfere with amyloid precursor protein metabolism. Antagonizing effects of HupA on N-me-thyl-D-aspartate receptors and potassium currents may also contribute to its neuroprotection as well. Pharmacokinetic studies in rodents, canines, and healthy human volunteers indicated that HupA was absorbed rapidly, distributed widely in the body, and eliminated at a moderate rate with the property of slow and prolonged release after oral administration. Animal and clinical safety tests showed that HupA had no unexpected toxicity, particularly the dose-limiting hepatotoxicity induced by tacrine. The phase IV clinical trials in China have demonstrated that HupA significantly improved memory deficits in elderly people with benign senescent forgetfulness, and patients with Alzheimer disease and vascular dementia, with minimal peripheral cholinergic side effects and no unexpected toxicity. HupA can also be used as a protective agent against organophosphate intoxication. [ABSTRACT FROM AUTHOR]

Abstract: Cholinesterases (ChEs) are classified as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selected inhibitors. The activities of AChE in red blood cells (RBC-AChE) and BChE in serum can be used as potential biomarkers of suppressed and/or heightened activity in the central and peripheral nervous systems. Exposure to organophosphate (OP) chemical warfare agents (CWAs), pesticides, anesthetics, and a variety of drugs such as cocaine, as well as some neurodegenerative and liver disease states, selectively reduces AChE or BChE activity. In humans, the toxicity of pesticides is well documented. Therefore, blood cholinesterase activity can be exploited as a tool for confirming exposure to these agents and possible treatments. Current assays for measurement of RBC-AChE and serum BChE require several labor-intensive processing steps, suffer from wide statistical variation, and there is no inter-laboratory conversion between methods. These methods, which determine only the serum BChE or RBC-AChE but not both, include the Ellman, radiometric, and ΔpH (modified Michel) methods. In contrast, the Walter Reed Army Institute of Research Whole Blood (WRAIR WB, US Patent #6,746,850) cholinesterase assay rapidly determines the activity of both AChE and BChE in unprocessed (uncentrifuged) whole blood, uses a minimally invasive blood sampling technique (e.g., blood from a finger prick), and is semi-automated for high-throughput using the Biomek 2000 robotic system. To date, the WRAIR whole blood assay was used to measure AChE and BChE activities in human blood from volunteers in FDA clinical trials. In the first FDA study, 24 human subjects were given either 30mg PB orally (n =19) or placebo (n =5). Blood samples were obtained pre-dosing and 2.5, 5, 8, and 24h post-dosing. The samples were analyzed for AChE and BChE activity using the WRAIR WB robotic system, and for PB concentration by HPLC. We found that maximal inhibition of AChE (26.2%) and concentration of PB (17.1ng/mL) occurred at 2.5h post-PB dosing. AChE activity returned to almost 100% of pre-dose values by 6h. A dose-dependent linear correlation was found between the amount of PB measured in the blood and the inhibition of AChE. Following soman (GD) exposure, recovered AChE activity was similar to levels that were reversibly protected by the PB administration. Therefore, the WRAIR ChE WB data clearly supports the conclusion that PB is an effective pre-treatment drug for nerve agent exposure (GD). In the second FDA human study for the treatment of Alzheimer''s disease, the WRAIR ChE WB assay was used to determine the RBC-AChE and serum BChE profile of healthy elderly volunteers receiving Huperzine A. Huperzine A is a plant-derived reversible and selective AChE inhibitor compared to BChE, and is a more potent inhibitor of AChE than PB. Huperzine A is available as a nutraceutical, a natural supplement reported to improve memory, and has a variety of neuroprotective effects. Individuals received an increasing dose regimen of huperzine A (final dose 200μg after 4 weeks), which produced more than 50% inhibition of RBC-AChE. Huperzine A was well tolerated by these patients at doses that sequestered more RBC-AChE than PB, and thus warrants further study as a prophylaxis for OP poisoning in addition to Alzheimer''s therapy. Due to the documented use of OPs by terrorists and in warfare around the globe, Federal, State, and local authorities need a reliable, fast, inexpensive, and standard method for confirming such an assault in order to initiate appropriate containment, decontamination, and treatment measures. This assay is ideal for prescreening military personnel for atypical ChE activities that would preclude their deployment to areas of potential CWA exposure. The WRAIR WB ChE assay will fulfill the requirement for rapid and reliable monitoring of such exposure in military and civilian populations. [Copyright &y& Elsevier]

To develop a long-acting injectable huperzine A-PLGA microsphere for the chronic therapy of Alzheimer's disease, the microsphere was prepared by using an o/w emulsion solvent extraction evaporation method based on a series of formulation design of the emulsion. The dialysis method was used for release analysis. The encapsulation efficiency and release amount of the microspheres were determined by a UV/VIS spectrophotometer. The morphology of the microspheres was observed by scanning electron microscopy. The distribution of the drug within microspheres was observed by a confocal laser scanning microscope. The results indicated that the PLGA 15?000 microspheres possessed a smooth and round appearance with average particle size of 50?µm or so. The encapsulation percentages of microspheres prepared from PLGA 15?000, 20?000 and 30?000 were 62.75%, 27.52% and 16.63%, respectively. The drug release percentage during the first day decreased from 22.52% of PLGA 30?000 microspheres to 3.97% of PLGA 15?000 microspheres, the complete release could be prolonged to 3 weeks. The initial burst release of microspheres with higher molecular weight PLGA could be explained by the inhomogeneous distribution of drug within microspheres. The encapsulation efficiency of the microspheres improved as the polymer concentration increased in the oil phase and PVA concentration decreased in the aqueous phase. The burst release could be controlled by reducing the polymer concentration. Evaporation temperature had a large effect on the drug release profiles. It had better be controlled under 30°C. Within a certain range of particle size, encapsulation efficiency decreased and drug release rate increased with the reducing of the particle size. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this study was to prepare microspheres containing Huperzine A, which is used for patients suffering from Alzheimer''s disease because of its potent anticholineestase activity, and to clarify in vitro and in vivo release characteristics of them. The preparation and in vitro and in vivo release studies of Huperzine A loaded microspheres were described. By spray drying method, Huperzine A was encapsulated successfully in the microspheres which were spherical with a non-porous and smooth surface. In vitro studies showed that the release of Huperzine A from microspheres was depended on the properties of polymers and the release medium. Counter-ionic interaction between the primary amine group of Huperzine A and the carboxylic terminal group of PLG polymers improves the encapsulation of Huperzine A, reducing the initial burst and extending the sustained release. High molecular weight of PLG polymer leads to a negative influence on sustained release of Huperzine A due to less carboxylic terminal groups. Acidic medium also reduces the initial burst and sustained the release due to decreased swelling of the polymeric matrix. In vivo experiment showed, after intramuscular injection, that the plasma concentration of Huperzine A reached the max. at 2 h, then fell rapidly to a stable and near constant level of 0.5 to 2.5 ng/ml within 2 weeks, until the drug was exhausted from the microspheres. It indicates the potential of a 2-week sustained release system of Huperzine A. [Copyright &y& Elsevier]

Abstract: Our previous study demonstrated that huperzine A, a selective acetylcholinesterase inhibitor, stimulates the synthesis of nerve growth factor (NGF) in cultured rat cortical astrocytes. The present studies are designed to examine if huperzine A exerts its neuroprotective activity against oxidative stress damage through increasing the synthesis of NGF in SHSY5Y neuroblastoma cells. Transient exposure of the cells to 200 μM H2O2 triggered a significant reduction of cell viability and decreased the mRNA and protein levels of NGF, neurotrophin receptor P75 (P75NTR) receptor and tyrosine kinase A (TrkA) receptor. Incubation of cells with 10 μM huperzine A prior to H2O2 exposure significantly elevated their survival and restored the mRNA and protein levels of NGF, P75NTR receptor and TrkA receptor. These neuroprotective effects of huperzine A on H2O2-induced cytotoxicity were blocked by the TrkA receptor phosphorylation inhibitor K252α, and were antagonized by the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) inhibitor, PD98059. The present results indicate that the cytoprotective effect of huperzine A is mediated at least partly by up-regulated NGF and NGF receptors. The results also show that the MAP/ERK kinase signal pathway is crucial for huperzine A to protect against H2O2-induced damage in SHSY5Y cells. [Copyright &y& Elsevier]

Huperzine A-loaded microspheres composed of poly(D,L-lactide-co-glycolide) were prepared by an O/W emulsion solvent evaporation method. The characterization of the microspheres such as drug loading, size, shape and release profile was described. The in vitro release in the initial 7 days was nearly linear with 10% released per day. Thereafter drug release rate became slow gradually and about 90% drug released at day 21. The in vitro release rate determined by dialysis bag method had a good correlation with the in vivo release rate. Huperzine A aqueous solution was intramuscularly injected (i.m.) at 0.4 mg/kg and microspheres were intramuscularly injected at 8.4 mg eq huperzine A/kg in rats. The maxium plasma concentration (Cmax) after i.m. microspheres was only 32% of that after i.m. solution. Drug in plasma could be detectd until day 14 and about 5% of administered dose was residued at the injection site at day 14. The relative bioavailability of huperzine A microspheres over a period of 14 days was 94.7%. Inhibition of acyecholinesterase activity (AchE) in rat's cortex, hippocampus and striatum could sustain for about 14 days. In conclusion, huperzine A-loaded microspheres possessed a prolonged and complete drug release with significant inhibition of AchE for 2 weeks in rats. [ABSTRACT FROM AUTHOR]

Abstract: Huperzine A, natural product from Huperzia serrata, is quite an important compound used to treat the Alzheimer''s disease as a food supplement and also proposed as a prospective and prophylactic antidote against organophosphate poisoning. In this work, simple and fast capillary electrophoresis (CE) procedure with UV detection (at 230nm) for determination of Huperzine A was developed and optimized. Capillary electrophoresis determination of Huperzine A was optimized using a combination of the experimental design (ED) and the artificial neural networks (ANN). In the first stage of optimization, the experiments were done according to the appropriate ED. Data evaluated by ANN allowed finding the optimal values of several analytical parameters (peak area, peak height, and analysis time). Optimal conditions found were 50mM acetate buffer, pH 4.6, separation voltage 10kV, hydrodynamic injection time 10s and temperature 25°C. The developed method shows good repeatability as relative standard division (R.S.D.=0.9%) and it has been applied for determination of Huperzine A in various pharmaceutical products and in biological liquids. The limit of detection (LOD) in aqueous media was 0.226ng/ml and 0.233ng/ml for determination in the serum. [Copyright &y& Elsevier]