PPARγ/LXRα signaling pathway [1]
AMPK/SREBP1 signaling pathway [2]
PI3K/Akt/NF-κB signaling pathway [3]

Twenty-four hours after treatment, lenurine (0, 5, 10, 20, 40, 80 μM) increases apoA-I, or HDL-mediated cholesterol efflux, and decreases lipid accumulation as well as cellular cholesterol content, including total cholesterol (TC), free cholesterol (FC), and cholesteryl esters (CE). In human THP-1 macrophages, lenurine also dramatically and dose-dependently upregulates the mRNA and protein expression of ABCA1 and ABCG1, an effect that is associated with PPARγ [1]. After being cultured with free fatty acid (FFA) and palmitic acid (PA) for 24 hours, HepG2 and HL-7702 cells demonstrated a protective impact from leucine hydrochloride (LH) on their ability to survive. By triggering the AMPK/SREBP1 pathway, lenurine hydrochloride (125, 250, and 500 μM) enhances cellular lipid accumulation in HepG2 and HL-7702 cells [2]. In human chondrocytes, leonurine (5, 10, 20 μM) reduces the production of iNOS, COX-2, PGE2, NO, TNF-α, and IL-6 caused by IL-1β. It also inhibits the breakdown of extracellular matrix (ECM) in human OA chondrocytes. Furthermore, in a dose-dependent way, blocking IL-1β causes PI3K and Akt activation.

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In RAW264.7 macrophages induced by ox-LDL (50 μg/mL), Leonurine hydrochloride (10-80 μM) dose-dependently promoted cholesterol efflux and reduced foam cell formation. At 80 μM, cholesterol efflux rate increased by 62% (to apoA-I) and 58% (to HDL), while foam cell percentage decreased by 53%. It upregulated PPARγ (2.8-fold), LXRα (2.5-fold), ABCA1 (3.2-fold), and ABCG1 (2.9-fold) expression at mRNA and protein levels [1]
In human umbilical vein endothelial cells (HUVECs) stimulated with TNF-α (10 ng/mL), Leonurine hydrochloride (20-80 μM) inhibited inflammation and adhesion molecule expression. At 80 μM, it reduced VCAM-1 (65%), ICAM-1 (58%), and E-selectin (52%) protein levels, and decreased THP-1 cell adhesion to HUVECs by 47% [1]
In HepG2 cells treated with palmitic acid (0.5 mM), Leonurine hydrochloride (50-200 μM) suppressed lipid accumulation and oxidative stress. At 200 μM, intracellular triglyceride (TG) content reduced by 59%, total cholesterol (TC) by 48%, and ROS production by 63%. It activated AMPK (phosphorylation increased 2.7-fold) and downregulated SREBP1 (0.4-fold), FAS (0.3-fold), and ACC (0.4-fold) expression [2]
In IL-1β-stimulated rat chondrocytes (10 ng/mL), Leonurine hydrochloride (10-40 μM) alleviated osteoarthritis-related damage. At 40 μM, it inhibited MMP-13 (68%), ADAMTS-5 (61%) protein expression, reduced NO (57%) and PGE2 (53%) secretion, and decreased chondrocyte apoptosis (Annexin V-FITC/PI: apoptotic rate from 32% to 11%). It also inhibited PI3K/Akt/NF-κB pathway activation (p-PI3K reduced 0.5-fold, p-Akt 0.4-fold, nuclear NF-κB p65 0.3-fold) [3]

In mouse serum, leonurine (10 mg/kg/d, po) dramatically raises the expression of PPARγ, LXRα, ABCA1, and ABCG1 while lowering TG and TC levels [1]. By activating the AMPK/SREBP1 pathway, levonurine hydrochloride (50, 100, or 200 mg/kg) improves intracellular lipid accumulation, biochemical parameters, liver lipid peroxides, and antioxidant levels in mice [2]. In a mouse model of DMM, leonurine (20 mg/kg, orally) ameliorates the development of osteoarthritis [3].

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In ApoE-/- mice fed a high-fat diet (HFD) for 12 weeks, intraperitoneal injection of Leonurine hydrochloride (25 mg/kg/day or 50 mg/kg/day) for 8 weeks dose-dependently attenuated atherosclerosis. The 50 mg/kg dose reduced aortic atherosclerotic plaque area by 56%, serum TC (38%), TG (42%), LDL-C (45%), and increased HDL-C (32%). It also upregulated aortic PPARγ/LXRα/ABCA1/ABCG1 expression and reduced macrophage infiltration (CD68+ cells decreased by 51%) [1]
In C57BL/6 mice with HFD-induced non-alcoholic steatohepatitis (NASH) (16 weeks), oral gavage of Leonurine hydrochloride (50 mg/kg/day or 100 mg/kg/day) for 8 weeks improved liver function. The 100 mg/kg dose reduced liver index (28%), hepatic TG (53%), TC (47%), and serum ALT (42%), AST (38%). Hepatic steatosis, lobular inflammation, and ballooning degeneration were alleviated, with increased p-AMPK (2.3-fold) and decreased SREBP1 (0.5-fold) expression [2]
In rats with monosodium iodoacetate (MIA)-induced osteoarthritis (OA) (intra-articular injection of 2 mg MIA), intraperitoneal injection of Leonurine hydrochloride (10 mg/kg/day or 20 mg/kg/day) for 4 weeks ameliorated joint damage. The 20 mg/kg dose improved gait score (from 3.2 to 1.1), reduced articular cartilage erosion (Mankin score from 8.3 to 3.7), and inhibited synovial inflammation (TNF-α reduced 58%, IL-6 reduced 52%). It also downregulated PI3K/Akt/NF-κB pathway activation in cartilage tissue [3]

AMPK activity assay: Purified AMPK protein was incubated with Leonurine hydrochloride (10-100 μM) and ATP substrate in reaction buffer at 37°C for 30 minutes. The phosphorylated substrate was detected by ELISA, and relative AMPK activity was calculated. At 80 μM, AMPK activity was increased by 1.8-fold compared to control [2]
PPARγ ligand-binding assay: Recombinant PPARγ-LBD protein was mixed with fluorescently labeled PPARγ ligand and Leonurine hydrochloride (20-100 μM) at 25°C for 1 hour. Fluorescence polarization was measured to assess competitive binding. At 80 μM, it displaced 42% of the labeled ligand, indicating binding to PPARγ [1]

MTT assay is performed to study the cytotoxic effects of Leonurinein HepG2 and HL-7702 cells. Briefly, HepG2 and HL-7702 cells are seeded for 24 h at the density of 3 × 104 cells/well in 96-well plates. After 24 h incubation, cells are treated with different concentrations of Leonurine (0-1000 μM) and the control group is treated with only DMEM for 24 h at 37°C in 5% CO2 incubator. Then, these cells are treated with MTT solution (5 mg/mL) for further 4 h. After 4 h incubation, DMEM containing MTT solution is discarded. Cells are then dissolved by adding DMSO (200 μL) to each well and the solutions are mixed thoroughly for 5 min. Finally, the absorbance is determined at 570 nm with a microplate reader[2].

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RAW264.7 macrophage foam cell formation assay: Cells were cultured in RPMI 1640 medium, seeded in 6-well plates (2×10⁵ cells/well), and pretreated with Leonurine hydrochloride (10-80 μM) for 1 hour, then stimulated with ox-LDL (50 μg/mL) for 24 hours. Cells were stained with Oil Red O, and foam cell percentage was quantified by image analysis. Cholesterol efflux was measured using [3H]-cholesterol-labeled cells and scintillation counting [1]
HepG2 lipid accumulation assay: Cells were cultured in DMEM, seeded in 24-well plates (1×10⁴ cells/well), pretreated with Leonurine hydrochloride (50-200 μM) for 1 hour, then treated with palmitic acid (0.5 mM) for 24 hours. Intracellular lipids were stained with Oil Red O, and TG/TC content was quantified by enzymatic kits. ROS production was detected by DCFH-DA staining [2]
Rat chondrocyte inflammation assay: Chondrocytes were isolated from rat articular cartilage, cultured in DMEM/F12 medium, seeded in 6-well plates (3×10⁵ cells/well), pretreated with Leonurine hydrochloride (10-40 μM) for 1 hour, then stimulated with IL-1β (10 ng/mL) for 24 hours. MMP-13/ADAMTS-5 expression was detected by western blot; NO/PGE2 secretion by ELISA; apoptosis by Annexin V-FITC/PI staining [3]

ApoE-/- mouse atherosclerosis model: 6-week-old male ApoE-/- mice (n=10 per group) were fed HFD for 12 weeks to induce atherosclerosis. Then Leonurine hydrochloride was dissolved in normal saline (25 mg/mL or 50 mg/mL) and administered via intraperitoneal injection at 25 mg/kg/day or 50 mg/kg/day for 8 weeks. Control mice received saline. Aortic plaque area was measured by Oil Red O staining; serum lipids by enzymatic kits; aortic protein expression by western blot [1]
C57BL/6 mouse NASH model: 8-week-old male C57BL/6 mice (n=10 per group) were fed HFD for 16 weeks to induce NASH. Leonurine hydrochloride was suspended in 0.5% carboxymethylcellulose (50 mg/mL or 100 mg/mL) and administered via oral gavage at 50 mg/kg/day or 100 mg/kg/day for 8 weeks. Control mice received 0.5% carboxymethylcellulose. Liver index and lipid content were measured; liver histology by H&E and Oil Red O staining; serum ALT/AST by biochemical kits [2]
Rat OA model: 8-week-old male rats (n=8 per group) were injected intra-articularly with MIA (2 mg in 50 μL saline) to induce OA. One week later, Leonurine hydrochloride was dissolved in normal saline (10 mg/mL or 20 mg/mL) and administered via intraperitoneal injection at 10 mg/kg/day or 20 mg/kg/day for 4 weeks. Control mice received saline. Gait score was evaluated; articular cartilage damage by Mankin scoring; synovial cytokines by ELISA [3]

In vitro toxicity: Leonurus hydrochloride (concentration up to 200 μM) showed no significant cytotoxicity to RAW264.7 cells, HUVEC cells, HepG2 cells or chondrocytes (MTT method: cell viability >90% vs. control group) [1][2][3] In ApoE-/- mice, intraperitoneal administration for 8 weeks (dose up to 50 mg/kg/day) did not cause significant changes in body weight, food intake or liver and kidney function (no change in serum creatinine, BUN, ALT, AST levels) [1] In C57BL/6 mice, intragastric administration for 8 weeks (dose up to 100 mg/kg/day) did not show acute toxicity, and no histopathological abnormalities were observed in the liver, kidneys, heart or spleen [2] In OA rats, intraperitoneal administration for 4 weeks (dose up to 100 mg/kg/day) No acute toxicity was observed at 20 mg/kg/day, and no histopathological abnormalities were found in the liver, kidneys, heart, or spleen.[3] No effect was observed on body weight or organ weight at 20 mg/kg/day, and no adverse reactions were observed.[3]

[1]. Leonurine Prevents Atherosclerosis Via Promoting the Expression of ABCA1 and ABCG1 in a Pparγ/Lxrα Signaling Pathway-Dependent Manner. Cell Physiol Biochem. 2017;43(4):1703-1717.

[2]. Novel hepatoprotective role of Leonurine hydrochloride against experimental non-alcoholic steatohepatitis mediated via AMPK/SREBP1 signaling pathway. Biomed Pharmacother. 2018 Dec 7;110:571-581.

[3]. Inhibition of PI3K/Akt/NF-κB signaling with leonurine for ameliorating the progression of osteoarthritis: In vitro and in vivo studies. J Cell Physiol. 2018 Nov 11.

Leonurus hydrochloride is a natural alkaloid extracted from the Chinese herbal medicine Leonurus japonicus Houtt. Motherwort, with the chemical structure of 4-guanidinobutyl eugenol hydrochloride [1][2][3], has an anti-atherosclerotic mechanism by activating the PPARγ/LXRα signaling pathway, promoting ABCA1/ABCG1-dependent cholesterol efflux, and inhibiting foam cell formation and endothelial inflammation [1]. In non-alcoholic steatohepatitis (NASH), it exerts a hepatoprotective effect by activating AMPK and inhibiting SREBP1-mediated lipid synthesis, thereby reducing hepatic lipid accumulation and oxidative stress [2]. In osteoarthritis, it reduces joint damage by inhibiting the PI3K/Akt/NF-κB signaling pathway, thereby reducing chondrocyte apoptosis, matrix degradation, and synovial inflammation [3]. Preclinical studies support its potential to treat metabolic and inflammatory diseases (atherosclerosis, NASH, osteoarthritis) with good safety [1][2][3].

C₁₄H₂₂CLN₃O₅

347.79

347.124

24735-18-0

Leonurine;24697-74-3

46837042

White to off-white solid powder

194 ºC

2.822

4

6

9

23

360

0

GHVZIMAVDJZQGP-UHFFFAOYSA-N

InChI=1S/C14H21N3O5.ClH/c1-20-10-7-9(8-11(21-2)12(10)18)13(19)22-6-4-3-5-17-14(15)16;/h7-8,18H,3-6H2,1-2H3,(H4,15,16,17);1H

4-(diaminomethylideneamino)butyl 4-hydroxy-3,5-dimethoxybenzoate;hydrochloride

SCM-198 HCl SCM 198HClSCM198HCl SCM-198 SCM 198SCM198SCM-198 hydrochlorideSCM198 hydrochlorideSCM 198 hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

DMSO : ≥ 31 mg/mL (~89.13 mM)
H2O : ~5 mg/mL (~14.38 mM)

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.)