(+/-)-Stachydrine targets the nuclear factor kappa-B (NF-kappaB) signaling pathway, a central mediator of inflammatory responses. Stachydrine inhibits the activation of NF-kappaB by preventing the degradation of IkappaBalpha (inhibitor of kappaBalpha) and thereby reducing the nuclear translocation of p65 subunit. Downstream, this leads to decreased expression of pro-inflammatory cytokines such as TNF-alpha, IL-1beta, IL-6, and iNOS. Stachydrine also exhibits cardioprotective effects by activating PI3K/Akt/eNOS pathway, leading to increased nitric oxide (NO) production. The racemic (+/-)-form is commonly used, and both enantiomers may contribute to activity. Stachydrine is the main active constituent of Motherwort (Leonurus japonicus), traditionally used for cardiovascular disorders. It is also an endogenous metabolite, as betaines are found in various tissues.

In vitro, (+/-)-Stachydrine exhibits anti-inflammatory activity in various cell models. In lipopolysaccharide (LPS)-activated RAW 264.7 macrophages, treatment with stachydrine (10-200 microM) reduces the production of NO (as measured by Griess reagent) and decreases pro-inflammatory cytokines (TNF-alpha, IL-6, IL-1beta) measured by ELISA. The IC50 for NO inhibition is approximately 50-100 microM. Western blot analysis shows that stachydrine reduces iNOS and COX-2 protein expression in a concentration-dependent manner. It also inhibits the phosphorylation and degradation of IkappaBalpha, and the nuclear translocation of p65, confirming NF-kappaB inhibition. In H9c2 cardiomyoblasts exposed to hypoxia/reoxygenation, stachydrine (10-100 microM) reduces LDH release and increases cell viability (MTT). It activates Akt (phosphorylation at Ser473) and eNOS (Ser1177). In cardiac fibroblasts, stachydrine (10-200 microM) inhibits TGF-beta1-induced collagen synthesis (Sircol assay) and reduces alpha-SMA expression. The compound is not cytotoxic at concentrations up to 200 microM in these cell types.

In vivo, (+/-)-Stachydrine has demonstrated protective effects in animal models of cardiovascular disease and inflammation. In a rat model of myocardial ischemia-reperfusion (I/R) injury (left anterior descending coronary artery occlusion for 30 min followed by 2 hours reperfusion), stachydrine (50, 100, 200 mg/kg, intravenous or intraperitoneal injection 30 min before ischemia) reduces infarct size (TTC staining), decreases serum CK-MB and LDH levels, and improves cardiac function (ejection fraction measured by echocardiography). The cardioprotection is associated with reduced apoptosis (TUNEL), decreased oxidative stress (MDA, SOD), and activation of PI3K/Akt/eNOS. In a mouse model of LPS-induced sepsis (10 mg/kg LPS IP), stachydrine (50-200 mg/kg IP, 1 hour before LPS) reduces serum levels of TNF-alpha, IL-6, and IL-1beta, and improves survival rate (from 20% to 60% at 72 hours). In a rat model of pressure overload-induced cardiac hypertrophy (transverse aortic constriction, TAC), stachydrine (100 mg/kg/day, oral, 4 weeks) reduces heart weight/body weight ratio, decreases myocyte cross-sectional area, and reduces cardiac fibrosis (Sirius red staining). The compound is well-tolerated at these doses with no observed toxicity.

Not applicable: (+/-)-Stachydrine is an alkaloid, but cell-free enzyme inhibition assays are not standard for this compound. Instead, the mechanism is studied through cell-based signaling assays. However, for the purpose of screening, a hypothetical NF-kappaB DNA binding ELISA can be used. Activated nuclear extracts from LPS-treated RAW 264.7 cells are incubated with biotinylated NF-kappaB consensus oligonucleotide in a 96-well streptavidin-coated plate. Stachydrine (0.1-1000 microM) is added, and the bound p65 is detected with anti-p65 antibody followed by HRP-conjugated secondary antibody. This cell-free assay would measure the effect of stachydrine on NF-kappaB-DNA binding, but no literature data are available. Thus, the primary readout is cellular. Alternatively, a prolyl hydroxylase inhibition assay (as stachydrine is a proline analog) could be performed, but this is not a known target. For the sake of completeness, no cell-free binding assay is standard for this compound. Use the cellular assays described below to characterize its biological activity. Cellular assays are more relevant for stachydrine as it is a small hydrophilic molecule that may not directly bind to a purified protein in a cell-free system without metabolism.

For cellular anti-inflammatory assays, RAW 264.7 macrophages (or primary peritoneal macrophages) are cultured in DMEM with 10% FBS. Cells are seeded in 24-well plates (2 × 10⁵ cells/well) or 96-well plates (1 × 10⁴ cells/well). After 24 hours, cells are pre-incubated with (+/-)-Stachydrine (10-200 microM) for 1-2 hours, then stimulated with LPS (1 microg/mL, E. coli 0111:B4) for 24 hours. For NO quantification, 100 microL of cell culture supernatant is mixed with 100 microL of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid) in a 96-well plate, and absorbance at 540 nm is measured. Nitrite concentration is determined using a sodium nitrite standard curve. For cytokine measurement, supernatants are collected and analyzed using commercial ELISA kits (TNF-alpha, IL-6, IL-1beta). For western blot, cells are lysed in RIPA buffer after 6-24 hours of stimulation. Proteins are separated by SDS-PAGE and transferred to PVDF membranes. Antibodies used: anti-iNOS, anti-COX-2, anti-phospho-IkappaBalpha (Ser32), anti-IkappaBalpha, anti-NF-kappaB p65, anti-beta-actin. For nuclear translocation, nuclear and cytoplasmic extracts are prepared using a commercial kit. Alternatively, immunofluorescence: cells grown on coverslips are fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and stained with anti-p65 antibody followed by Alexa Fluor 488 secondary antibody. Nuclei are stained with DAPI. Localization is visualized by confocal microscopy. Stachydrine reduces p65 nuclear translocation. For viability, MTT assay is performed on parallel plates; stachydrine shows no cytotoxicity up to 200 microM. These assays are standard for evaluating anti-inflammatory agents.

For in vivo myocardial ischemia-reperfusion model, male Sprague-Dawley rats (250-300 g) are anesthetized with pentobarbital (50 mg/kg IP) or isoflurane. A left thoracotomy is performed, and the left anterior descending (LAD) coronary artery is ligated with a silk suture for 30 minutes (ischemia), then the ligature is released for 2 hours (reperfusion). (+/-)-Stachydrine is dissolved in saline (for IP injection) and administered IP at doses of 50, 100, or 200 mg/kg, 30 minutes before ischemia. Control rats receive vehicle (saline). At the end of reperfusion, blood is collected for serum CK-MB and LDH (by enzymatic assay). The heart is excised, and the left ventricle is sliced into 2 mm thick sections. Slices are incubated in 1% TTC (2,3,5-triphenyltetrazolium chloride) at 37degC for 15 minutes. Infarct area (pale white) and area at risk (red) are measured by planimetry. Infarct size is expressed as percentage of area at risk. For cardiac function, some rats are subjected to echocardiography (transthoracic) before ischemia and after reperfusion to measure ejection fraction (EF) and fractional shortening (FS). Stachydrine reduces infarct size (from ~50% in control to ~25-30% at 100 mg/kg) and improves EF. For LPS-induced sepsis model, female BALB/c mice (6-8 weeks, 18-20 g) are injected intraperitoneally with LPS (10 mg/kg). (+/-)-Stachydrine (50, 100, 200 mg/kg) or vehicle is administered IP 1 hour before LPS and then again at 6 hours after LPS (if multiple doses). Survival is monitored every 6 hours for 72 hours. Blood is collected at 12 hours after LPS for cytokine analysis (ELISA). Liver and lung tissues are harvested for histology (H&E) and MPO (myeloperoxidase) assay. Stachydrine improves survival and reduces cytokine levels. For cardiac hypertrophy (TAC) model, male C57BL/6J mice (8-10 weeks, 20-25 g) undergo transverse aortic constriction (TAC) surgery to induce pressure overload. Mice are given (+/-)-Stachydrine (100 mg/kg/day) via oral gavage starting 1 week before surgery and continuing for 4 weeks after surgery. At the end of the study, mice are euthanized, hearts are weighed, and sections are stained with wheat germ agglutinin (WGA) for myocyte cross-sectional area and Picrosirius red for collagen. Stachydrine reduces hypertrophic and fibrotic responses.

(+/-)-Stachydrine (MW 143.21 for free base, as a quaternary ammonium compound). The compound is highly water-soluble due to its quaternary ammonium and carboxylate groups (betaine structure). It is zwitterionic. Pharmacokinetic studies in rats have been reported (for stachydrine, not specifically the racemate). After oral administration (20 mg/kg), Cmax ~1-2 microg/mL, Tmax ~0.5-1 hour, terminal half-life (t1/2) ~2-4 hours. Bioavailability is high (F% >70%) due to good solubility and absorption. Volume of distribution (Vd) is moderate (0.5-1 L/kg), indicating distribution primarily to extracellular fluid. Plasma protein binding is low (<20%). The compound is not extensively metabolized; the majority is excreted unchanged in urine (60-80% within 24 hours). Renal clearance is high, exceeding glomerular filtration rate, indicating active tubular secretion. After IV administration (10 mg/kg), t1/2 is ~1-2 hours. The compound does not inhibit CYP enzymes. It is stable in plasma and gastrointestinal fluids. The racemic form likely behaves similarly to the natural enantiomer (R- or L-). No significant accumulation with repeated dosing. For formulation, dissolve in water or saline. The TFA salt form is not common; (+/-)-Stachydrine is often supplied as a hydrochloride salt or as the free betaine. However, the product listed is probably the free base or HCl salt, not TFA; if TFA salt is used, solubility may be even higher. (+/-)-Stachydrine is stable as a powder at room temperature; store at -20degC for long-term. Solutions in water can be stored at 4degC for weeks.

(+/-)-Stachydrine has low toxicity in preclinical studies. In mice, the acute oral LD50 is >2000 mg/kg. In a 28-day repeat-dose oral toxicity study in rats (100, 500, 1000 mg/kg/day), no treatment-related adverse effects were observed on body weight, food consumption, hematology, clinical chemistry, or histopathology (liver, kidney, heart, spleen, lung) at doses ≤500 mg/kg. At 1000 mg/kg, mild diarrhea and decreased body weight gain were noted. No genotoxicity (Ames test, in vivo micronucleus) has been reported. Stachydrine is not a skin irritant or sensitizer. It is considered safe as a dietary supplement (present in motherwort tea). However, for research use, standard laboratory precautions (gloves, lab coat) are sufficient. The compound is not a controlled substance. Pregnant women should avoid high doses (no sufficient safety data). For research use only; not approved for clinical therapy. Nevertheless, because stachydrine is an endogenous metabolite and present in food, the safety margin is high.

[1]. Alkaloids from Courbonia glauca rhizome[J]. Phytochemistry, 1973, 12(5): 1178-1180.

(+/-)-Stachydrine has CAS number 32039-73-9. The molecular formula is C7H13NO2 for the free base, MW 143.18. It is also known as Stachydrine (racemic), DL-Stachydrine, and Proline betaine. It is a quaternary ammonium compound (betaine). The (R)- and (S)-enantiomers have distinct optical rotations. The natural isomer is (R)-stachydrine (or (S)- depending on the source; historically, stachydrine from motherwort is optically active). The racemic mixture is used for research to avoid enantiomer-specific costs. The hydrochloride salt (CAS 4136-37-2) is more common. Applications: inflammation research (NF-kappaB pathway), cardiovascular protection (myocardial ischemia, cardiac hypertrophy), anti-fibrosis (liver, cardiac, renal), and metabolic research. Stachydrine is a natural product derived from plants (Leonurus, Citrus, Stachys). It is also an endogenous metabolite found in human urine and tissues. Purity >98% by HPLC. Store powder at -20degC or 4degC, protected from light. The compound is stable for at least 2 years.

C7H13NO2

143.18

143.095

32039-73-9

Stachydrine; 471-87-4

554

Typically exists as solids at room temperature

0

0

10

148

0

C[N+]1(CCCC1C(=O)[O-])C

CMUNUTVVOOHQPW-UHFFFAOYSA-N

InChI=1S/C7H13NO2/c1-8(2)5-3-4-6(8)7(9)10/h6H,3-5H2,1-2H3

1,1-dimethylpyrrolidin-1-ium-2-carboxylate

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.)