Inducible nitric oxide synthase (iNOS); Protein kinase C-theta (PKC-θ) [1][2]

Anemonin (4) concentration-dependently inhibited NO production in LPS-activated RAW 264.7 macrophages with an IC50 value of 5.37 ± 0.39 μM. At 100 μM, it completely reversed NO production induced by LPS. [2]
Anemonin (2.5-30 μM) significantly and concentration-dependently suppressed LPS-induced iNOS protein expression in RAW 264.7 cells as measured by Western blotting and densitometry. [2]
Anemonin (2.5-30 μM) gradually decreased LPS-induced iNOS mRNA expression in RAW 264.7 cells at 6 hours of incubation, as measured by semi-quantitative RT-PCR. [2]
Anemonin (30 μM, up to 150 min) did not exhibit NO-scavenging activity in a sodium nitroprusside solution, as it did not affect nitrite accumulation. [2]
In HT-29 cells, anemonin (2.5, 5, and 10 μM) had no significant effect on cell viability or apoptosis (CCK-8 and flow cytometry), indicating no cytotoxicity. [1]
Anemonin (2.5, 5, and 10 μM) dose-dependently downregulated LPS-induced mRNA and protein levels of IL-1β, TNF-α, and IL-6 in HT-29 cells. [1]
Anemonin did not affect PRKCQ gene transcription but significantly inhibited PKC-θ protein translation or protein stability. [1]
In HT-29 cells transfected with PRKCQ overexpression plasmid (pcPRKCQ), anemonin's inhibitory effects on cytokine production (IL-1β, TNF-α, IL-6) were significantly reversed. [1]
Molecular docking predicted that anemonin (Molecule ID: MOL001883) docks with PKC-θ (protein ID: 4FKD) with a binding free energy of -5.9 kcal/mol. [1]

In a DSS-induced acute UC mouse model, anemonin (2, 5, and 10 mg/kg, intraperitoneal injection) dose-dependently prevented body weight loss and colon length shortening, and significantly inhibited the disease activity index (DAI) score. [1]
HE staining showed that anemonin dose-dependently attenuated DSS-induced histopathological changes in colon tissues. [1]
Anemonin dose-dependently inhibited DSS-induced mRNA, protein, and release of IL-1β, TNF-α, and IL-6 in mouse colon tissues (RT-qPCR, ELISA, western blotting). [1]
Anemonin significantly inhibited PKC-θ protein expression in colon tissues of DSS-induced mice in a dose-dependent manner. [1]
In rats, anemonin (5-30 μM) significantly reversed LPS-induced vascular hyperactivity to phenylephrine in endothelium-denuded aortic rings, restoring maximal contraction from 1.60 ± 0.23 g to 3.09 ± 0.14 g. [2]
Anemonin (10, 100 μM) did not affect acetylcholine-induced endothelial NO-dependent vasorelaxation in endothelium-intact rat aortic rings, indicating no effect on eNOS activity. [2]

NO measurement: RAW 264.7 cells (5 × 10⁵ cells/mL) were grown to confluence on 24-well plates. After 24 h, the medium was changed to serum-free media for 4 h. Anemonin (2.5-30 μM), vehicle, or positive controls (L-NNA or aminoguanidine at 100 μM) were added in the presence of LPS (200 ng/mL) for 24 h. Nitrite concentration in the culture medium was determined spectrophotometrically using Griess reagent as an index of NO production. [2]
Western blot assay for iNOS protein: RAW 264.7 cells were challenged by LPS (200 ng/mL) for 24 h in the presence of anemonin (2.5-30 μM) or vehicle. Cell lysates were sonicated and centrifuged at 3000 × g for 20 min at 4°C. Cytoplasmic protein concentration was determined by Bradford method. Proteins were separated by SDS-PAGE, transferred to membranes, incubated with mouse monoclonal anti-iNOS antibody (1:1000) at 4°C overnight, then with HRP-conjugated secondary antibody for 1.5 h, and detected by chemiluminescence. [2]
RT-PCR assay for iNOS mRNA: RAW 264.7 cells (5 × 10⁶ cells per 6-cm dish) were exposed for 6 h to anemonin (2.5-30 μM) or vehicle in the presence of LPS (200 ng/mL). Total RNA was isolated by TRIzol. RT-PCR was performed using primers for iNOS (384-bp product) and GAPDH (452-bp product, housekeeping control). PCR products were analyzed on 2% agarose gel and visualized by UV transillumination. [2]
NO-scavenging activity assay: Sodium nitroprusside solution (5 mM) was incubated with anemonin (2.5-30 μM) or vehicle in microcentrifuge tubes under light at room temperature. Nitrite levels were determined at 0, 30, 60, 90, 120, and 150 min using Griess reagent. [2]
Vascular tension experiment: Endothelium-denuded rat aortic rings were incubated in MEM containing LPS (300 ng/mL) plus anemonin (5-30 μM) or vehicle for 6 h. Rings were then fixed in organ chambers containing oxygenated Krebs' solution under passive tension of 1.8 g. Concentration-response curves for phenylephrine (1 nM to 100 μM) were constructed. For eNOS function assessment, endothelium-intact aortic rings pre-contracted with phenylephrine (0.3 μM) were treated with cumulative concentrations of acetylcholine (10 nM to 10 μM) in the presence of anemonin (10, 100 μM), L-NNA (100 μM), aminoguanidine (100 μM), or vehicle for 20 min. [2]
Molecular docking: AutoDock Vina software was used to dock anemonin (Molecule ID: MOL001883) with PKC-θ (protein ID: 4FKD). Discovery Studio 4.5 Client software was used to further analyze interaction results. Binding free energy was calculated as -5.9 kcal/mol. [1]

Cell viability assay (alamarBlue): After NO measurement, 10% alamarBlue in DMEM was added to each well containing RAW 264.7 cells. Plates were incubated at 37°C in 5% CO₂ for 3 h. Absorbance was read spectrophotometrically at 570 and 600 nm. [2]
HT-29 cell culture and treatment: HT-29 cells were cultured in DMEM with 10% FBS and 1% streptomycin/penicillin at 37°C in 5% CO₂. Cells (5 × 10⁴ cells/mL) were seeded in 96-well plates (100 μL per well). After 24 h, cells were treated with LPS (1 μg/mL) for 24 h, then with anemonin (2.5, 5, and 10 μM) for 48 h. [1]
Cell viability (CCK-8) and apoptosis (flow cytometry): HT-29 cell viability was detected using CCK-8 assay. Apoptosis was detected using an annexin V-FITC apoptosis detection kit and flow cytometry. [1]
RT-qPCR for inflammatory factors: Total RNA was extracted from colon tissues and HT-29 cells using TRIzol reagent, reverse-transcribed using an RT-PCR kit, and real-time PCR performed with SYBR Green PCR kit using GAPDH as internal control. Primers used included those for IL-1β, TNF-α, IL-6, PRKCQ, PTPN2, and GAPDH. The 2-ΔΔCt method was used to analyze data. [1]
Western blotting for inflammatory factors and PKC-θ: Proteins were extracted using RIPA total protein lysate, separated on 10% SDS-PAGE gels, transferred to PVDF membranes, incubated with primary antibodies (1:1000; anti-IL-1β, anti-TNF-α, anti-IL-6, anti-PKC-θ) at 4°C overnight, then with HRP-conjugated secondary antibody (1:2000) for 1.5 h, and detected using ECL western reagent. [1]
Cell transfection: HT-29 cells were seeded at 1.5 × 10⁵ cells/cm². Lipofectamine 2000 reagent was used for transfection with PRKCQ overexpression plasmid (pcPRKCQ) or empty pcDNA3.1 plasmid. The Lipo2000/DNA complex was formed for 20 min at room temperature, then 100 μL was added to cells for 4-6 h, followed by culture in medium with 1% streptomycin/penicillin and 10% FBS for 24 h. [1]

DSS-induced acute UC mouse model: C57BL/6 mice were administered 3% (w/v) DSS in drinking water for 7 days. Anemonin (2, 5, and 10 mg/kg) was intraperitoneally injected daily during DSS administration. Sterile water was used for the control group. On day 9, mice were sacrificed for experiments. Body weight and disease activity index (DAI) were recorded. DAI = (weight loss [%] + characterization of feces + hematochezia)/3. [1]
AAV vector infection: One week before DSS administration, mice were anesthetized with 3% pentobarbital sodium. A soft catheter was inserted into the mouse anus at a depth of 4 cm. Sterile saline containing 0.2 mL of AAV-PRKCQ or AAV-null (4 × 10¹⁰ viral genome) was instilled into the mouse colon via catheter. Mice were placed upside down for 1 min to allow distribution throughout the colon. [1]
HE staining: Colon tissue slices were dewaxed, incubated with hematoxylin for 2 min, stained with eosin, dehydrated, and embedded for microscopic analysis. [1]
ELISA for inflammatory factors: IL-1β, IL-6, and TNF-α levels in colon tissues were measured using ELISA kits according to manufacturer's instructions. Optical density was measured at 450 nm using a microplate reader. [1]
Rat thoracic aortic ring experiment: Male Sprague-Dawley rats (280-350 g) were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and killed by exsanguination. Thoracic aorta was isolated, cleaned, and divided into 3-mm-long rings. Rings were placed in 24-well plates with 1 mL MEM containing LPS (300 ng/mL) plus anemonin (5-30 μM) or vehicle, and incubated at 37°C for 6 h. Endothelium-denuded rings were then fixed isometrically in organ chambers containing oxygenated Krebs' solution under passive tension of 1.8 g. Vascular tension was recorded via a force displacement transducer. Concentration-response curves for phenylephrine (1 nM to 100 μM) were constructed. For eNOS studies, endothelium-intact rings were pre-contracted with phenylephrine (0.3 μM), then cumulative concentrations of acetylcholine (10 nM to 10 μM) were applied in the presence of anemonin (10, 100 μM), L-NNA (100 μM), aminoguanidine (100 μM), or vehicle for 20 min. [2]

In RAW 264.7 macrophages, anemonin at 100 μM did not interfere with cell viability in the presence of LPS, as measured by alamarBlue assay. [2]
In HT-29 cells, anemonin (2.5, 5, and 10 μM) had no significant effect on cell viability (CCK-8) or apoptosis (flow cytometry), indicating no cytotoxicity. [1]
In rats, anemonin (10, 100 μM) did not affect baseline tension of aortic rings, indicating no direct vasoactive effects. [2]

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- Source Plants: Found in Pulsatilla (Anemone pulsatilla and related species) and Clematis flammula.
- Chemical Constituents: Contains ranunculin, protoanemonin, anemonin, triterpene saponins, and flavonoids.
- Toxicity/Irritation: The fresh plant is extremely irritating to the skin, gastrointestinal tract, and mucous membranes.
- Allergic Potential: Allergic reactions to pulsatilla have been reported.
- Traditional Use (Lactation): Homeopathic preparations are reportedly used for sore nipples and mastitis, either to reduce an overabundant milk supply or to increase milk supply.
- Lack of Clinical Evidence: No scientifically valid clinical trials support these uses for lactation. Other agents may be preferred in nursing mothers due to a lack of information.
- Traditional Use (Wound Healing): In Tunisian traditional medicine, Clematis flammula has been used to treat skin diseases, including mycotic infections.
- Wound Healing Study: Anemonin was isolated from Clematis flammula and incorporated into a cream.
- Major Compound: GC-MS analysis identified protoanemonin (86.74%) as the major compound, which partially dimerizes to form anemonin.
- Wound Healing Efficacy: The study results provide strong support for the effective wound healing activity of anemonin cream, suggesting it is a promising candidate as a therapeutic agent in tissue repair.

[1]. Anti-inflammatory effects of anemonin on acute ulcerative colitis via targeted regulation of protein kinase C-θ. Chin Med. 2022 Mar 28;17(1):39.

[2]. Anemonin, from Clematis crassifolia, potent and selective inducible nitric oxide synthase inhibitor. J Ethnopharmacol. 2008 Mar 28;116(3):518-27.

Anemonin is a butenolactone. It has been reported that camphor from Anemonin is found in Andean Anemonin, and relevant data is available for reference.

C10H8O4

192.17

192.042

C, 62.50; H, 4.20; O, 33.30

508-44-1

46173847

Typically exists as solid at room temperature

1.45

535.7ºC at 760 mmHg

157-158ºC

300.7ºC

1.61

0.483

0

4

0

14

357

2

O1C(C([H])=C([H])[C@@]21C([H])([H])C([H])([H])[C@@]12C([H])=C([H])C(=O)O1)=O

JLUQTCXCAFSSLD-NXEZZACHSA-N

InChI=1S/C10H8O4/c11-7-1-3-9(13-7)5-6-10(9)4-2-8(12)14-10/h1-4H,5-6H2/t9-,10-/m1/s1

(5S,6S)-4,7-dioxadispiro[4.0.46.25]dodeca-1,9-diene-3,8-dione

508-44-1; Pulsatilla camphor; Anemonine; Pulsatilla camphor; trans-Anemonin; DTXSID70903913; G99XG5B674; (5S,6S)-4,7-dioxadispiro(4.0.46.25)dodeca-1,9-diene-3,8-dione;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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