Macrophage activation markers (CD80, CD86); pro-inflammatory cytokines (TNF-α, IL-1β); migration-related proteins (COX-2, MCP-1); osteoclastogenesis-related transcription factors (NFATc1, DC-STAMP). [1]

LA (0-80 μM) showed no cytotoxicity in RAW 264.7 cells and bone marrow-derived macrophages (BMDMs) after 24 h treatment with 1 μg/mL LPS, as measured by MTT assay. [1]
- LA suppressed LPS-induced activation of RAW 264.7 cells in a dose-dependent manner. The percentages of CD80+ and CD86+ cells induced by LPS (25.8%) were reduced to 10.8% by 80 μM LA, as assayed by flow cytometry. [1]
- LA reduced LPS-induced secretion of TNF-α and IL-1β in both RAW 264.7 cells and BMDMs in a dose-dependent manner, measured by ELISA. [1]
- LA inhibited LPS-induced migration of RAW 264.7 cells in a dose-dependent manner using a transwell assay. The number of migrated cells decreased with increasing LA concentration. Curcumin (3 μM) was used as a positive control. [1]
- LA downregulated the expression of COX-2 and MCP-1 proteins in LPS-stimulated RAW 264.7 cells in a dose-dependent manner, as determined by Western blotting. Relative to the LPS group, COX-2 expression decreased from 1.00 to 0.13, and MCP-1 from 3.23 to 1.15 after 80 μM LA treatment. [1]
- LA inhibited RANKL-mediated osteoclastogenesis in RAW 264.7 cells. TRAP staining showed that the number of TRAP-positive multinucleated cells (≥3 nuclei) was decreased by LA in a dose-dependent manner. [1]
- LA decreased the mRNA levels of NFATc1 and DC-STAMP in RANKL-stimulated RAW 264.7 cells in a dose-dependent manner, as shown by RT-PCR. [1]

In collagen-induced arthritis (CIA) DBA/1J mice, daily intraperitoneal injection of 50 mg/kg LA for 21 days significantly reduced arthritis incidence (20% in LA group vs. 100% in RA group on day 30), clinical scores, and paw thickness compared to the RA group. [1]
- LA treatment significantly lowered serum levels of TNF-α and IL-1β in CIA mice on days 20, 32, 39, and 43 after the first immunization, as measured by ELISA. [1]
- LA suppressed the uptake of 18F-FDG in the hind lower limbs of CIA mice on day 43, as assessed by microPET/CT imaging. The relative 18F-FDG uptake ratio was 0.64 for LA-treated mice vs. 2.27 for RA mice. [1]
- LA ameliorated bone erosion and reduced synovial macrophage infiltration in CIA mice. H&E staining showed less cartilage damage and synovial hyperplasia; CD68 immunohistochemistry revealed fewer CD68+ infiltrating macrophages in the LA group compared to the RA group. [1]
- Ex vivo Western blotting of hind paw proteins from CIA mice showed that LA suppressed the elevated expressions of VEGF, COX-2, MCP-1, granzyme B, and IL-10, while restoring the decreased TGF-β level compared to the RA group. [1]

MTT cytotoxicity assay: RAW 264.7 cells or BMDMs (5×10⁴/well) were cultured in 96-well plates for 24 h, then treated with 0-80 μM LA for 24 h. MTT reagent was added, and absorbance was measured by an ELISA reader. No cytotoxicity was observed. [1]
- Flow cytometry for CD80/CD86: RAW 264.7 cells or BMDMs (1×10⁶/well in 6-well plates) were pretreated with 0-80 μM LA for 1 h, then stimulated with 1 μg/mL LPS for 24 h. Cells were stained with CD80-APC and CD86-FITC antibodies and analyzed by flow cytometry. The percentage of positive cells was determined. [1]
- ELISA for TNF-α and IL-1β: Culture supernatants from LPS-stimulated RAW 264.7 cells or BMDMs treated with LA were collected. TNF-α and IL-1β levels were measured by ELISA following the kit instructions. [1]
- Transwell migration assay: RAW 264.7 cells (in upper inserts, 5 μm pore size) were pretreated with 0-80 μM LA for 1 h, then 1 μg/mL LPS was added to the lower chamber. After 4 h incubation, membranes were fixed with methanol:acetic acid (3:1), stained with hematoxylin, and migrated cells were counted under a microscope. [1]
- Western blotting for protein expression: RAW 264.7 cells (1×10⁶/well in 6-well plates) were pretreated with 0-80 μM LA for 1 h, then stimulated with 1 μg/mL LPS for 24 h. Cells were lysed, proteins separated by SDS-PAGE, transferred to PVDF membranes, blocked, and incubated with primary antibodies against MCP-1, COX-2, etc., followed by HRP-conjugated secondary antibodies. Signals were detected by chemiluminescence and quantified by ImageJ. [1]
- Osteoclast differentiation and TRAP staining: RAW 264.7 cells (1×10⁴/well in 96-well plates) were cultured in α-MEM with 100 ng/mL RANKL for 72 h to induce differentiation, then treated with 0-80 μM LA for another 48 h. Cells were fixed with 3.7% paraformaldehyde and stained for TRAP. TRAP-positive multinucleated cells (≥3 nuclei) were counted as osteoclasts. [1]
- RT-PCR for NFATc1 and DC-STAMP: RAW 264.7 cells (1×10⁶/well in 6-well plates) were cultured with 100 ng/mL RANKL and 0-80 μM LA for three days. Total RNA was extracted, reverse transcribed to cDNA, and amplified with specific primers for NFATc1, DC-STAMP, c-FOS, and β-actin. PCR products were run on 1.2% agarose gel. [1]

Collagen-induced arthritis (CIA) model: 8-week-old DBA/1J mice were injected intradermally at the base of the tail with 100 μL of an emulsion containing equal volumes of complete Freund’s adjuvant (with 5 mg/mL heat-killed M. tuberculosis) and bovine type II collagen. A booster injection was given on day 21. [1]
- Grouping and treatment: Mice were randomly divided into four groups (n=7/group): normal (no CIA induction), RA (CIA induction without treatment), LA-treated (50 mg/kg lupeol acetate), and curcumin-treated (100 mg/kg, positive control). LA was dissolved in 0.1% DMSO and administered intraperitoneally daily for 21 days (from day 21 to day 43 post first immunization). [1]
- Clinical assessment: Paw thickness was measured three times per week with a digital caliper. Clinical score (0-4 per paw, max 16 per mouse) was determined based on redness and swelling. [1]
- 18F-FDG microPET/CT imaging: On days 20, 25, 32, 39, and 43, mice were anesthetized with 1-3% isoflurane and injected intravenously with 18.5 MBq/100 μL of 18F-FDG. 40 min post-injection, images were acquired on a SPECT/PET/CT scanner. Relative 18F-FDG uptake ratio was calculated as (mean of two hind paws - background)/background, with muscle as background. [1]
- Histopathology and immunohistochemistry: On day 43, mice were sacrificed, and ankles were removed, fixed, paraffin-embedded, sectioned (5 μm), and stained with H&E for histopathological examination. Arthritis score (0-3 per limb, max 12 per mouse) was assessed. For IHC, sections were stained with anti-CD68 antibody to evaluate macrophage infiltration. [1]
- Ex vivo Western blotting: Hind paws of sacrificed mice were collected, and total proteins were extracted. Western blotting was performed to detect VEGF, COX-2, MCP-1, granzyme B, IL-10, and TGF-β, with β-actin as internal control. [1]

No general toxicity was observed in any group (n=7 per group) based on body weight changes throughout the experiment. [1]
- LA (0-80 μM) showed no cytotoxicity in RAW 264.7 cells and BMDMs after 24 h treatment with 1 μg/mL LPS, as determined by MTT assay. [1]

[1]. Lupeol acetate ameliorates collagen-induced arthritis and osteoclastogenesis of mice through improvement of microenvironment. Biomed Pharmacother. 2016;79:231-240.

Lupeol acetate is an organic molecular entity and a metabolite. It has been reported to be present in hybrid amaranth, creeping amaranth, and other organisms with relevant data.

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Lupeol acetate is a derivative of lupeol, a triterpene found in fruits and vegetables (mango, green pepper, strawberries). It exhibits better bioavailability than lupeol and has anti-inflammatory and anti-arthritic properties. [1]
- The study demonstrates that LA suppresses the progression of rheumatoid arthritis (RA) by inhibiting macrophage activation and osteoclastogenesis through downregulation of TNF-α, IL-1β, MCP-1, COX-2, VEGF, and granzyme B, while upregulating TGF-β. [1]
- LA (50 mg/kg, i.p.) showed comparable or better efficacy than curcumin (100 mg/kg, i.p.) in the CIA mouse model, suggesting LA may have potential as an alternative medicine for anti-rheumatoid arthritis. [1]
- The study also used 18F-FDG PET/CT imaging as a non-invasive method to assess inflammatory activity in arthritis. [1]

C32H52O2

468.7541

468.397

1617-68-1

92157

White to off-white solid powder

1.01g/cm3

502.7ºC at 760mmHg

218ºC

254.7ºC

3.1E-10mmHg at 25°C

1.524

8.595

0

2

3

34

872

10

CC(=C)[C@@H]1CC[C@]2([C@H]1[C@H]3CC[C@@H]4[C@]5(CC[C@@H](C([C@@H]5CC[C@]4([C@@]3(CC2)C)C)(C)C)OC(=O)C)C)C

ODSSDTBFHAYYMD-YOJQYFTNSA-N

InChI=1S/C32H52O2/c1-20(2)22-12-15-29(6)18-19-31(8)23(27(22)29)10-11-25-30(7)16-14-26(34-21(3)33)28(4,5)24(30)13-17-32(25,31)9/h22-27H,1,10-19H2,2-9H3/t22-,23+,24-,25+,26-,27+,29+,30-,31+,32+/m0/s1

[(1R,3aR,5aR,5bR,7aR,9S,11aR,11bR,13aR,13bR)-3a,5a,5b,8,8,11a-hexamethyl-1-prop-1-en-2-yl-1,2,3,4,5,6,7,7a,9,10,11,11b,12,13,13a,13b-hexadecahydrocyclopenta[a]chrysen-9-yl] acetate

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)

Ethanol :≥ 2 mg/mL (~4.27 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.)