Plasmodium; anti-malarial
The study demonstrates that artemisinin exerts neuroprotective effects by activating the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway.

Artemisinin (3.125-100 μM) concentration-dependently suppresses
Aβ25-35 induced cytotoxicity in PC12 cells. Artemisinin (25 μM)
suppresses Aβ25-35-induced LDH release, apoptosis and ROS production,
attenuates Aβ-induced mitochondrial membrane potential loss and caspase
3/7 activity increase, and stimulates the phosphorylation of ERK1/2 in a
time- and concentration-dependent manner in PC12 cell. ERK 1/2 pathway
mediates the protect effects of artemisinin in PC12 cells[1].
Artemisinin shows cytotoxic activity in MCF-7/Dox cell line with IC50 of
3.7±0.4 μg/mL after 24 h treatment. Besides, Artemisinin treatment of
MCF-7 cells, sensitive and resistant to Dox and DDP, causes a decrease
in expression of proteins such as LF, FTH1, and HEP. Artemisinin
activates p38 MAPK-kinase cascade regardless of the oxidative stress due
to inhibition of VEGF expression and cell migration[2]. Artemisinin
(50, 100 or 200 mg) significantly inhibits isoflurane-induced
hippocampal neuronal loss, decreases caspase-3-positive cell counts and
also cleaves caspase-3 expression, and modulates the expression of
apoptosis pathway proteins. Artemisinin modulates JNK/ERK 1/2 signalling
and histone acetylation[3]. Artemisinin inhibits HCV replication in a
dose-dependent manner with EC50 value of 167±38 µM. Artemisinin and its
most potent analogues partially inhibit the in vitro replication of HCV
by induction of reactive oxygen species (ROS)[4]. Artemisinin
significantly inhibits VSMC proliferation in a dose-dependent manner.
Artemisinin (1 mM) for 72 h significantly reduces the expression of
proliferating cell nuclear antigen messenger RNA[5].

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Artemisinin (at concentrations of 12.5 µM and 25 µM) significantly protected rat pheochromocytoma (PC12) cells from β-amyloid peptide fragment Aβ25-35-induced cytotoxicity, as measured by the MTT cell viability assay. Pre-treatment with 25 µM artemisinin for 1 hour attenuated Aβ25-35 (0.3 µM, 24 h)-induced cell death, increasing viability compared to Aβ-treated controls.
Post-treatment with artemisinin (25 µM or 50 µM for 24 h) after a 30-minute exposure to Aβ25-35 also rescued PC12 cells from toxicity, indicating both protective and rescue effects.
Pre-treatment with 25 µM artemisinin significantly reduced Aβ25-35-induced lactate dehydrogenase (LDH) release from PC12 cells.
Hoechst 33342 nuclear staining showed that 25 µM artemisinin pre-treatment decreased the percentage of apoptotic nuclei with condensed chromatin induced by Aβ25-35.
Artemisinin (25 µM) pre-treatment significantly attenuated the intracellular reactive oxygen species (ROS) production induced by Aβ25-35 in PC12 cells.
Pre-treatment with 25 µM artemisinin prevented the loss of mitochondrial membrane potential (ΔΨm) induced by Aβ25-35, as assessed by the JC-1 fluorescence ratio (red/green).
Artemisinin (25 µM) pre-treatment significantly reduced the activation of executioner caspases-3/7 induced by Aβ25-35 in PC12 cells.
Western blot analysis showed that artemisinin treatment stimulated the phosphorylation of ERK1/2 in PC12 cells in a time-dependent (5-80 min) and concentration-dependent (3.15-50 µM) manner, but did not affect the phosphorylation of Akt.
The neuroprotective effects of artemisinin against Aβ25-35 were blocked by pre-incubation with the specific MEK/ERK pathway inhibitor PD98059 (5-20 µM), but not by the PI3K/Akt pathway inhibitor LY294002, confirming the central role of ERK1/2 activation.
Artemisinin (25 µM) pre-treatment also protected PC12 cells from cytotoxicity induced by the longer, more physiologically relevant amyloid peptide Aβ1-42 (0.3 µM).

Artemisinin (50, 100 or 200 mg/kg b.wt/day, p.o.) prevents
isoflurane-induced working memory impairments as observed in T-maze
test. Artemisinin enhances spatial navigation and memory of rats exposed
to isoflurane. Artemisinin-treated rats exhibit markedly better
performance in comparison with isoflurane-alone-exposed rats[3].

Reactive Oxygen Species Measurements[2]
Intracellular and mitochondrial ROS production were assessed by a fluorometric assay using 2′,7′-dichlorofluorescein diacetate (H2DCFDA) and MitoTracker Red CMXRos, respectively. After 12 h treatment of artemisinin and followed by 12 h treatment of glutamate, cells were incubated in 10 μmol/L H2DCFDA or 0.25 μmol/L MitoTracker Red CMXRos for 30 min at 37°C. The fluorescence was then observed via a fluorescence microscope. The fluorescence was detected with 530/485-nm and 579/599-nm excitation/emission wave lengths.[2]

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Mitochondrial Membrane Potential (ΔΨm) Measurement[2]
The mitochondrial membrane potential was detected using Tetramethylrhodamine, Ethyl Ester (TMRE) mitochondrial membrane potential assay kit (Abcam, USA). Cells were loaded with 20 nM of TMRE working solution for 20 min at 37°C. The fluorescent images were observed and obtained on a Zeiss fluorescence microscope. Fluorescence intensity was measured using a Tecan Infinite F200 plate reader with 594/575-nm excitation/emission.

For this purpose, cells are cultivated in 96-well plates in DMEM,
supplemented with insulin. The artemisinin, Dox, and DDP are added to
media at different concentrations and the cells are cultivated for
either 24 or 48 h. For this purpose artemisinin is diluted in 0.01% DMSO
in media. After this time, 10 µL of the MTT dye solution (5 mg/mL in
phosphate buffer saline) is added to the cells; the cells are incubated
at the same conditions for 3 h. After centrifugation (1500 rpm, 5 min)
the supernatant is removed. 100 μL of dimethyl sulfoxide is added to
each well, to dissolve formazan. The absorption is measured, using a
multi-well spectrophotometer at a wavelength of 540 nm.

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Cell viability assay (MTT): PC12 cells were seeded in 96-well plates coated with poly-L-lysine and serum-starved. After pre-treatment with artemisinin or vehicle for 1 hour, cells were incubated with or without Aβ peptides (Aβ25-35 or Aβ1-42) for 24 hours. MTT reagent was then added and incubated for 3 hours. The formed formazan crystals were dissolved in DMSO, and the absorbance was measured at 570 nm to assess cell viability.
Lactate dehydrogenase (LDH) release assay: PC12 cells were seeded in 96-well plates. After treatment, the activity of LDH released into the culture medium was measured using a fluorescent assay kit according to the manufacturer's protocol. Fluorescence intensity (excitation 560 nm/emission 590 nm) was measured and normalized to controls.
Apoptosis detection by Hoechst 33342 staining: After treatment, PC12 cells were fixed and stained with the DNA-binding dye Hoechst 33342. Cells were visualized under a fluorescence microscope. Nuclei exhibiting condensed or fragmented chromatin were counted as apoptotic. The percentage of apoptotic cells relative to total cells was calculated.
Intracellular reactive oxygen species (ROS) measurement: After treatment, PC12 cells were incubated with a cell-permeable ROS-sensitive fluorescent probe (CellROX Deep Red Reagent) for 1 hour in the dark. Cells were washed, and fluorescence (excitation 640 nm/emission 665 nm) was observed and quantified using image analysis software.
Mitochondrial membrane potential (ΔΨm) assay: PC12 cells seeded in black 96-well plates were treated and then incubated with the cationic dye JC-1. The fluorescence intensity ratio of red aggregates (excitation 560 nm/emission 595 nm) to green monomers (excitation 485 nm/emission 535 nm) was measured using a microplate reader. The ratio reflects the mitochondrial membrane potential.
Caspase-3/7 activity assay: After treatment, PC12 cells were lysed. Caspase-3/7 activity in the lysates was measured using a commercial luminescent assay kit. The lysate was incubated with a proluminescent caspase-3/7 substrate, and the generated luminescence was measured.
Western blot analysis: Treated PC12 cells were lysed. Equal amounts of protein were separated by SDS-PAGE, transferred to a membrane, and probed with specific primary antibodies against phospho-ERK1/2, total ERK1/2, phospho-Akt, total Akt, and loading controls (GAPDH or β-tubulin). Protein bands were visualized using chemiluminescence and quantified.

Separate group of rat pups (total rat pups 80; n = 16 per group)
is administered artemisinin (50, 100 or 200 mg/kg body weight) via oral
gavage, every day from P2 to P21. On P7, the pups are exposed to
isoflurane (0.75% in 30% oxygen or air) for 6 h in a
temperature-controlled chamber. Animals that are not exposed to
anaesthesia nor given artemisinin served as control group, while rats
that receive isoflurane alone are grouped as anaesthetic-controls.

In cell-based assays, artemisinin at concentrations up to 50 µM did not show intrinsic cytotoxicity to PC12 cells without Aβ damage, as the survival rate of the cell group treated with artemisinin alone was comparable to that of the solvent control group.

[1]. Artemisinin protects PC12 cells against β-amyloid-induced apoptosis through activation of the ERK1/2 signaling pathway. Redox Biol. 2017 Apr 4;12:625-633.

[2]. Artemisinin Prevents Glutamate-Induced Neuronal Cell Death Via Akt Pathway Activation. Front Cell Neurosci. 2018 Apr 20;12:108.

(+)-Artemisinin is a sesquiterpene lactone extracted from Artemisia annua and used to treat multidrug-resistant Plasmodium falciparum malaria. It is both an antimalarial drug and a plant metabolite. It is a sesquiterpene lactone and an organic peroxide. Artemisinin has been used in clinical trials investigating schizophrenia, malaria, Plasmodium falciparum, and Plasmodium falciparum infection. Artemisinin has been reported in Artemisia lancea, Artemisia annua, and several other organisms with relevant data. Increasing evidence suggests that abnormal deposition of β-amyloid (Aβ) is a major cause of Alzheimer's disease (AD). Therefore, clearing Aβ is considered an important strategy for treating AD. Utilizing cultured neuronal cells to counteract Aβ peptide toxicity to discover candidate drugs is considered an effective method for developing drugs to treat Alzheimer's disease (AD). We have previously demonstrated that the FDA-approved antimalarial drug artemisinin has recently shown neuroprotective effects. In this study, we aimed to investigate the protective effect of artemisinin against β-amyloid toxicity in PC12 neurons and its potential mechanisms. Our research showed that clinically relevant concentrations of artemisinin can protect PC12 cells from Aβ25-35-induced cell death. Further investigation revealed that artemisinin significantly alleviated Aβ25-35-induced cell death by restoring abnormal changes in nuclear morphology, lactate dehydrogenase, intracellular reactive oxygen species (ROS), mitochondrial membrane potential, and apoptosis caspase activity. Western blotting analysis showed that artemisinin activated extracellular signal-regulated kinases ERK1/2 but not the Akt survival signaling pathway. Consistent with the effects of ERK1/2, pre-incubation of cells with the ERK1/2 pathway inhibitor PD98059 blocked the effects of artemisinin, while the PI3K inhibitor LY294002 had no such effect. Furthermore, Aβ1-42 can cause PC12 cell death, while artemisinin can inhibit the cytotoxicity of Aβ1-42 on PC12 cells. In summary, these results are the first to suggest that artemisinin may exert a potential protective effect against β-amyloid damage by activating the ERK1/2 pathway. Our findings offer potential applications of artemisinin in the prevention and treatment of Alzheimer's disease (AD). [1]

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Artemisinin is an antimalarial drug that has been used for nearly half a century. Recently, there have been reports of novel biological effects of artemisinin on cancer, inflammation-related diseases, and cardiovascular diseases. However, the neuroprotective effect of artemisinin against glutamate-induced oxidative stress has not been studied. This study investigated the protective effect of artemisinin against oxidative damage in the HT-22 mouse hippocampal cell line. We found that artemisinin pretreatment reduced the production of reactive oxygen species (ROS), alleviated glutamate-induced mitochondrial membrane potential collapse, and rescued HT-22 cells from glutamate-induced cell death. In addition, our study showed that artemisinin can activate the Akt/Bcl-2 signaling pathway, and that the Akt-specific inhibitor MK2206 can block the neuroprotective effect of artemisinin. In summary, our study shows that artemisinin prevents glutamate-induced oxidative damage to HT-22 neurons by activating the Akt signaling pathway. [2] Artemisinin is a sesquiterpene lactone, originally isolated from Artemisia annua L., and is a first-line antimalarial drug. This study suggests that artemisinin has potential applications in the prevention and treatment of Alzheimer's disease due to its neuroprotective effect against β-amyloid toxicity (a key pathological feature of Alzheimer's disease). Its mechanism of action involves inhibiting Aβ-induced oxidative stress, mitochondrial dysfunction, and caspase-dependent apoptosis, mainly mediated by activating the ERK1/2 cell survival signaling pathway rather than the PI3K/Akt pathway. This study points out that artemisinin can easily cross the blood-brain barrier, which is a... This property makes it an ideal ingredient for potential central nervous system therapeutics.

C15H22O5

282.3322

282.146

C, 63.81; H, 7.85; O, 28.33

63968-64-9

Artemisinin-d3;176652-07-6

White to off-white solid powder

1.2±0.1 g/cm3

389.9±42.0 °C at 760 mmHg

156-157ºC

172.0±27.9 °C

0.0±0.9 mmHg at 25°C

1.533

2.27

O1[C@@]23[C@]4([H])OC([C@]([H])(C([H])([H])[H])[C@]2([H])C([H])([H])C([H])([H])[C@@]([H])(C([H])([H])[H])[C@]3([H])C([H])([H])C([H])([H])C(C([H])([H])[H])(O1)O4)=O

BLUAFEHZUWYNDE-NNWCWBAJSA-N

InChI=1S/C15H22O5/c1-8-4-5-11-9(2)12(16)17-13-15(11)10(8)6-7-14(3,18-13)19-20-15/h8-11,13H,4-7H2,1-3H3/t8-,9-,10+,11+,13-,14-,15-/m1/s1

(3R,5aS,6R,8aS,9R,12S,12aR)-3,6,9-trimethyloctahydro-12H-3,12-epoxy[1,2]dioxepino[4,3-i]isochromen-10(3H)-one

qinghaosu; NSC-369397; NSC369397; Arteannuin; Huanghuahaosu; Artemisinine; Artemisine; (+)-Artemisinin; NSC 369397

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)

DMSO : 50~56 mg/mL (177.10~198.34 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.37 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 20.8 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: ≥ 2.08 mg/mL (7.37 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 20.8 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: ≥ 2.08 mg/mL (7.37 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 3% DMSO+ 97% Corn oil: 6mg/ml (21.25mM)

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