Madecassic acid targets the NF-κB pathway. It inhibits LPS-induced activation of nuclear factor-κB (NF-κB) by abrogating inhibitory kappa B-α (IκB-α) degradation and phosphorylation, and subsequently blocking p65 protein translocation to the nucleus. [1]

LPS-induced expression of COX-2 and iNOS is inhibited by madecassic acid (150 μM) for a 24-hour period [1].

---

Madecassic acid (50, 100, or 150 μM) concentration-dependently inhibited LPS (1 μg/mL)-induced NO production in RAW 264.7 cells with an IC50 of 78.56 μM. [1]

Madecassic acid inhibited LPS-induced PGE2 production with an IC50 of 102.12 μM. [1]

Madecassic acid reduced LPS-induced TNF-α, IL-1β, and IL-6 production in a concentration-dependent manner, with IC50 values of 124.35 μM, 58.50 μM, and 90.76 μM, respectively. [1]

Madecassic acid (50–150 μM) significantly decreased LPS-induced iNOS and COX-2 protein levels, as determined by Western blotting. [1]

Madecassic acid concentration-dependently reduced LPS-induced iNOS, COX-2, TNF-α, IL-1β, and IL-6 mRNA expression, as shown by RT-PCR. [1]

Madecassic acid (50–150 μM) suppressed LPS-induced NF-κB-DNA binding activity in a dose-dependent manner, as measured by EMSA. [1]

Madecassic acid significantly reduced LPS-induced NF-κB-dependent luciferase reporter gene expression. [1]

Madecassic acid blocked LPS-induced IκB-α degradation and IκB-α phosphorylation in a concentration-dependent manner, as determined by Western blotting. [1]

Madecassic acid attenuated the LPS-induced translocation of p65 (NF-κB active subunit) from the cytosol to the nucleus. [1]

Madecassic acid at concentrations up to 150 μM showed no cytotoxic effects on RAW 264.7 cells in the presence or absence of LPS, as assessed by MTT assay. [1]

Reduce the amount of γδT17 cells and IL-17 levels in respiratory inflammatory tissues using madecassic acid (facial, 25 mg/kg, 10 days) to alleviate respiratory inflammation [2].

---

Electrophoretic mobility shift assay (EMSA) was performed to assess NF-κB-DNA binding. RAW 264.7 macrophages (1×10^6 cells/mL) were preincubated with various concentrations of Madecassic acid (50, 100, or 150 μM) for 1 h and then stimulated with LPS (1 μg/mL) for 1 h. Nuclear extracts were prepared and mixed with double-stranded NF-κB oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') end-labeled with [γ-32P]dATP. Binding reactions were carried out at 37°C for 30 min in reaction buffer containing 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 4% glycerol, 1 μg poly(dI-dC), and 1 mM DTT. DNA-protein complexes were separated from free probe on native 5% polyacrylamide gels at 100 V in 0.5× TBE buffer. Gels were dried and exposed to X-ray film. [1]

Luciferase reporter gene assay was performed to measure NF-κB activity. RAW 264.7 cells were transiently transfected with pNF-κB-Luc reporter plasmid using a transfection reagent. At 24 h post-transfection, cells were pretreated with Madecassic acid for 1 h and then stimulated with LPS (1 μg/mL) for 4 h. Cells were lysed and luciferase activities were determined using a luciferase assay system and a luminometer, normalized to protein concentration. [1]

Western Blot Analysis[1]
Cell Types: RAW 264.7 mouse macrophages
Tested Concentrations: 50, 100, 150 μM
Incubation Duration: 24 h followed by treatment with LPS (1 µg/mL) for 4 hrs (hours)
Experimental Results: Inhibition of LPS in a dose-dependent manner Induced iNOS and COX-2 protein expression.

---

RAW 264.7 murine macrophage cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 units/mL), and streptomycin sulfate (100 μg/mL) at 37°C in a humidified 5% CO2 atmosphere. Cells were preincubated with Madecassic acid (50, 100, or 150 μM) for 1 h and then stimulated with LPS (1 μg/mL) for indicated times. [1]

Cell viability was measured by MTT assay. After 24 h treatment with Madecassic acid (50, 100, or 150 μM) in the presence or absence of LPS, MTT solution was added to cells and incubated. The formazan crystals were dissolved, and absorbance was measured. [1]

Nitrite accumulation (indicator of NO synthesis) was measured in culture media using the Griess reaction. After 24 h treatment, culture supernatants were mixed with sulfanilamide and N-(1-naphthyl)ethylenediamine, and absorbance was read at 540 nm. [1]

PGE2, TNF-α, IL-1β, and IL-6 levels in macrophage culture media were quantified by enzyme immunoassay (EIA) kits following the manufacturer's instructions. [1]

Western blot analysis: Cellular proteins were extracted from control and Madecassic acid-treated RAW 264.7 cells using lysis buffer containing HEPES, NaCl, EDTA, Nonidet P-40, phenylmethylsulfonyl fluoride, dithiothreitol, Na fluoride, Na orthovanadate, leupeptin, and aprotinin. Forty micrograms of protein were separated by 10% SDS-PAGE and electroblotted onto nitrocellulose membranes. Membranes were blocked with 5% skim milk, incubated with primary antibodies (iNOS, COX-2, IκB-α, p-IκB-α, p65, β-actin, PARP) overnight at 4°C, then with horseradish peroxidase-conjugated secondary antibody for 1 h, and signals were visualized using an enhanced chemiluminescence system. [1]

RT-PCR: Total RNA was isolated using an RNA extraction kit. One microgram of RNA was reverse-transcribed using MuLV reverse transcriptase, dNTP, and oligo(dT) primers. PCR was performed for iNOS, COX-2, TNF-α, IL-1β, IL-6, and β-actin using specific primers. PCR products were electrophoresed on 2% agarose gel and visualized by ethidium bromide staining and UV irradiation. [1]

Animal/Disease Models: 2.5% DSS-induced colitis in mice [2]
Doses: 25 mg/kg/day
Route of Administration: po (oral gavage), 10 days
Experimental Results: The number of γδT17 cells in the colon diminished. Reduce IL-17 expression in colon tissue.

---

Madecassic acid at concentrations of 50, 100, or 150 μM did not affect the viability of RAW 264.7 cells after 24 h treatment (with or without LPS), as determined by MTT assay, indicating no cytotoxic effects in vitro. [1]

[1]. Anti-inflammatory effects of madecassic acid via the suppression of NF-kappaBpathway in LPS-induced RAW 264.7 macrophage cells. Planta Med. 2010 Feb;76(3):251-7.

[2]. Madecassic acid alleviates colitis-associated colorectal cancer by blocking the recruitment of myeloid-derived suppressor cells via the inhibition of IL-17 expression in γδT17 cells. Biochem Pharmacol. 2022.

It has been reported that plants of the genus Centella, Centella erecta, and Centella asiatica contain 2,3,6,23-tetrahydroxyarbutin-12-ene-28-ic acid, and relevant data are available. See also: Madecaic acid (note moved to).

---

Madecassic acid is the aglycone of madecassoside, a triterpenoid saponin from Centella asiatica. In this study, Madecassic acid exhibited stronger anti-inflammatory effects than madecassoside in LPS-induced RAW 264.7 macrophages, suggesting that the in vivo anti-inflammatory activity of madecassoside may be due to its metabolic conversion to Madecassic acid. The anti-inflammatory mechanism involves downregulation of iNOS, COX-2, TNF-α, IL-1β, and IL-6 via suppression of NF-κB activation through inhibition of IκB-α phosphorylation and degradation, and blockade of p65 nuclear translocation. [1]

C30H48O6

504.6985

504.345

C, 71.39; H, 9.59; O, 19.02

18449-41-7

258809

White to off-white solid powder

1.2±0.1 g/cm3

641.7±55.0 °C at 760 mmHg

270ºC (dec.)

355.9±28.0 °C

0.0±4.3 mmHg at 25°C

1.591

4.99

5

6

2

36

963

0

O([H])C1([H])C([H])([H])[C@@]2(C([H])([H])[H])[C@]3(C([H])([H])[H])C([H])([H])C([H])([H])[C@@]4(C(=O)O[H])C([H])([H])C([H])([H])C([H])(C([H])([H])[H])C([H])(C([H])([H])[H])[C@@]4([H])C3=C([H])C([H])([H])[C@]2([H])[C@@]2(C([H])([H])[H])C([H])([H])C([H])(C([H])([C@@](C([H])([H])[H])(C([H])([H])O[H])[C@@]21[H])O[H])O[H]

PRAUVHZJPXOEIF-AOLYGAPISA-N

InChI=1S/C30H48O6/c1-16-9-10-30(25(35)36)12-11-28(5)18(22(30)17(16)2)7-8-21-26(3)13-20(33)24(34)27(4,15-31)23(26)19(32)14-29(21,28)6/h7,16-17,19-24,31-34H,8-15H2,1-6H3,(H,35,36)/t16-,17+,19-,20-,21-,22+,23-,24+,26-,27+,28-,29-,30+/m1/s1

(1S,2R,4aS,6aR,6aS,6bR,8R,8aR,9R,10R,11R,12aR,14bS)-8,10,11-trihydroxy-9-(hydroxymethyl)-1,2,6a,6b,9,12a-hexamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid

Madecassic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~62.5 mg/mL (~123.84 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.12 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.

---

Solubility in Formulation 2: ≥ 2.08 mg/mL (4.12 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (4.12 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.

---

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