Acetylcholinesterase (AChE) (Ki = 0.025 μM; IC50 = 0.086 μM) [2]
Butyrylcholinesterase (BuChE) (weaker affinity compared to AChE) [2]
N-methyl-D-aspartate (NMDA) receptor [3]

(-)-Huperzine A (1 μM; 2 hours) attenuates neuronal damage caused by Aβ23-35 (20 μM) [2]. (-)-Huperzine A (100 μM) reversibly suppresses NMDA-induced currents (IC50=126 μM) in whole-cell voltage-clamp recordings in CA1 pyramidal neurons that had been acutely separated from the rat hippocampal [3].

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In rat dissociated hippocampal neurons, (−)-Huperzine A (HupA) inhibited NMDA-induced current in a concentration-dependent manner. The inhibition was reversible, and the effect was more pronounced at higher NMDA concentrations [3]
(−)-Huperzine A (HupA) showed potent and selective inhibition of AChE, with a much lower inhibitory effect on BuChE. It bound to the active site of AChE, preventing the hydrolysis of acetylcholine [2]
In vitro models relevant to neuronal degeneration, (−)-Huperzine A (HupA) exerted neuroprotective effects by reducing oxidative stress and inhibiting neuronal apoptosis [4]

In rats with degeneration brought on by icv infusion of beta-amyloid-(1-40), (-)-Huperzine A (0.1-0.2 mg/kg; intraperitoneal injection; daily; for 12 days) lessens neuronal damage and cognitive dysfunction [5].

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In rats injected with β-amyloid protein-(1-40) (Aβ1-40), intraperitoneal administration of (−)-Huperzine A (HupA) (0.15 and 0.3 mg/kg) attenuated cognitive dysfunction as evaluated by the Morris water maze test. It also reduced neuronal degeneration in the hippocampus and cerebral cortex, along with decreased oxidative damage markers [5]
Acute administration of (−)-Huperzine A (HupA) (5 and 10 mg/kg, ip) to rats did not cause significant changes in liver histology, but slightly increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels at the higher dose; these changes were transient [1]
In various animal models of memory impairment (e.g., scopolamine-induced), (−)-Huperzine A (HupA) improved learning and memory functions by increasing acetylcholine levels in the brain [2]

Prepare AChE-containing homogenates from appropriate tissues. Dilute the homogenate to a working concentration and add to reaction tubes with different concentrations of (−)-Huperzine A (HupA), followed by incubation at 37°C for 15 minutes. Add acetylthiocholine iodide as the substrate and continue incubation for 30 minutes. Stop the reaction with a chromogenic reagent, measure the absorbance at a specific wavelength, and calculate the enzyme inhibition rate to determine IC50 or Ki values [2]
For BuChE inhibition assay, use butyrylthiocholine iodide as the substrate and follow a similar procedure as the AChE assay. Compare the inhibitory potency of (−)-Huperzine A (HupA) on BuChE with that on AChE [2]

Isolate hippocampi from neonatal rats, dissociate into single neurons by enzymatic digestion and mechanical trituration, and seed them in culture dishes coated with appropriate substrates. After culturing for 7-14 days to allow neuron maturation, treat the cells with different concentrations of (−)-Huperzine A (HupA) for a specified time. Use patch-clamp technique to record NMDA-induced current changes before and after drug treatment [3]
Cultivate neuronal cells relevant to neurodegeneration models, expose them to neurotoxic stimuli (e.g., Aβ1-40), and co-treat with (−)-Huperzine A (HupA) at various concentrations. Detect cell viability using a colorimetric assay, measure apoptotic rates by flow cytometry, and assess oxidative stress markers (e.g., reactive oxygen species, malondialdehyde) through specific detection kits [4]

Animal/Disease Models: Male SD (Sprague-Dawley) rats (220-280 g)[5]
Doses: 0.1 mg/kg, 0.2 mg/kg
Route of Administration: intraperitoneal (ip)injection, daily, for 12 days
Experimental Results: Partly reversed the down-regulation of anti-apoptotic Bcl-2 and the up-regulation of pro-apoptotic Bax and P53 proteins and decreased the apoptosis that normally followed b-amyloid injection; alleviated the cognitive dysfunction induced by b-amyloid protein-(1-40).

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Acute liver effect study: Adult rats are randomly divided into control and treatment groups. (−)-Huperzine A (HupA) is dissolved in normal saline and administered intraperitoneally at doses of 5 and 10 mg/kg. The control group receives an equal volume of normal saline. Rats are sacrificed 24 hours after administration, and serum is collected to measure ALT and AST levels; liver tissues are harvested for histopathological examination [1]
Aβ1-40-induced cognitive impairment model: Adult rats are anesthetized and stereotaxically injected with Aβ1-40 into the hippocampus to establish the model. One week after model establishment, (−)-Huperzine A (HupA) is administered intraperitoneally at doses of 0.15 and 0.3 mg/kg once daily for 14 consecutive days. The control group receives the same volume of vehicle. Cognitive function is evaluated using the Morris water maze test before sacrifice; brain tissues are collected for histological and biochemical analyses [5]
Scopolamine-induced memory impairment model: Rats are given (−)-Huperzine A (HupA) via oral gavage at doses of 0.1-0.5 mg/kg 30 minutes before scopolamine injection. Scopolamine is administered intraperitoneally to induce memory impairment. Learning and memory functions are assessed using the passive avoidance test or Y-maze test [2]

Oral absorption: (−)-Huperzine A (HupA) is well absorbed after oral administration in animals and humans, with an oral bioavailability of approximately 36-40% [2]. Distribution: It is widely distributed in tissues, with higher concentrations in the brain, liver, and kidneys. The brain/plasma concentration ratio is approximately 0.6-0.8 [4]. Metabolism: It is mainly metabolized in the liver by cytochrome P450 enzymes, producing a variety of metabolites with reduced activity [2]. Excretion: The elimination half-life (t1/2) in humans is approximately 4-6 hours, and in rats it is approximately 2-3 hours. It is mainly excreted by the kidneys, with approximately 20-30% of the dose excreted unchanged in the urine [4].

The acute oral LD50 of (−)-huperzine A (HupA) is approximately 13.6 mg/kg in mice and approximately 8.5 mg/kg in rats [2]. Acute high-dose administration (10 mg/kg, intraperitoneal injection) in rats caused mild and transient increases in liver enzymes, but no significant hepatocellular necrosis or fibrosis was observed [1]. Chronic toxicity studies in animals showed no significant adverse effects on major organs (liver, kidneys, heart) at therapeutic doses. Common mild side effects included nausea, diarrhea, and dizziness, which were dose-related [4]. The plasma protein binding rate of (−)-huperzine A (HupA) is approximately 55–60% [2].

[1]. Acute effects of huperzine A and tacrine on rat liver. Acta Pharmacol ogica Sinica, 2003, 24(3):247-250.

[2]. Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine. Acta Pharmacol Sin. 2006 Jan;27(1):1-26.

[3]. Huperzine A, a nootropic alkaloid, inhibits N-methyl-D-aspartate-induced current in rat dissociated hippocampal neurons.Neuroscience. 2001;105(3):663-9.

[4]. The pharmacology and therapeutic potential of (−)-huperzine A. J Exp Pharmacol. 2012; 4: 113–123.

[5]. Huperzine A attenuates cognitive dysfunction and neuronal degeneration caused by beta-amyloid protein-(1-40) in rat. Eur J Pharmacol. 2001 Jun 15;421(3):149-56.

Huperzine A is a sesquiterpene alkaloid isolated from the genus Huperzia serrata and has been shown to possess neuroprotective activity. It is also a potent acetylcholinesterase inhibitor and has attracted considerable attention as a potential treatment for Alzheimer's disease. It possesses multiple functions, including as an EC 3.1.1.7 (acetylcholinesterase) inhibitor, neuroprotective agent, plant metabolite, and nootropic agent. It is a sesquiterpene alkaloid, pyridone, primary amino compound, and organic heterocyclic tricyclic compound. It is the conjugate base of huperzine A(1+). Huperzine A is a naturally occurring sesquiterpene alkaloid found in extracts of the genus Huperzia serrata. This plant has been used in China for centuries to treat swelling, fever, and blood disorders. Recent clinical trials in China have demonstrated its neuroprotective effects. Currently, huperzine A is being investigated as a potential treatment for neurodegenerative diseases, particularly Alzheimer's disease.
Huperzine A has been reported to exist in Aspergillus versicolor, Aspergillus niger, and other organisms with relevant data.

Drug Indications
Its application/treatment in Alzheimer's disease is under investigation.

Mechanism of Action
Huperzine A has been identified as an acetylcholinesterase inhibitor. This is the same mechanism of action as drugs used to treat Alzheimer's disease, such as galantamine and donepezil.

Pharmacodynamics
Huperzine A is an alkaloid extracted from Huperzia serrulata (sold as a herbal product in the United States). Its potential as an acetylcholinesterase inhibitor is currently being investigated. Clinical trials in China have shown that Huperzine A is comparable in efficacy to currently marketed drugs and may even be safer in terms of side effects.

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(−)-Huperzine A (HupA) is a natural alkaloid isolated from Chinese medicinal herbs of the genus Huperzia (e.g., Huperzia serrulata)[2]
Its main mechanism of action is selective and reversible inhibition of acetylcholinesterase (AChE), thereby increasing the level of acetylcholine in the central nervous system and improving cholinergic cognitive impairment[4]
It also exerts neuroprotective effects by regulating NMDA receptors, reducing oxidative stress, inhibiting neuronal apoptosis, and preventing Aβ aggregation[5]
Clinically, it is used to treat mild to moderate Alzheimer's disease and other cognitive impairments[2]
It has less hepatotoxicity than synthetic AChE inhibitors (e.g., tacrine)[1]

C15H18N2O

242.32

242.141

102518-79-6

854026

White to off-white solid powder

1.6±0.1 g/cm3

479.5±25.0 °C at 760 mmHg

211-216oC

243.8±23.2 °C

0.0±1.2 mmHg at 25°C

1.741

-0.22

2

2

0

18

551

2

C/C=C/1\[C@@H]2CC3=C([C@]1(CC(=C2)C)N)C=CC(=O)N3

ZRJBHWIHUMBLCN-YQEJDHNASA-N

InChI=1S/C15H18N2O/c1-3-11-10-6-9(2)8-15(11,16)12-4-5-14(18)17-13(12)7-10/h3-6,10H,7-8,16H2,1-2H3,(H,17,18)/b11-3+/t10-,15+/m0/s1

(1R,9R,13E)-1-amino-13-ethylidene-11-methyl-6-azatricyclo[7.3.1.02,7]trideca-2(7),3,10-trien-5-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% propylene glycol, 5% Tween 80, 65% D5W:30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)