NF-kappaB (nuclear factor kappa-light-chain-enhancer of activated B cells) and activator protein-1 (AP-1). Lucyoside B inhibits the production of inflammatory mediators via both the NF-kappaB and activator protein-1 (AP-1) pathways in activated macrophages. Lucyoside B also targets other inflammatory signaling molecules, but these are the primary established pathways.

In vitro, Lucyoside B inhibits the production of inflammatory mediators via the NF-kappaB and AP-1 pathways in activated macrophages. While specific IC50 values are not provided, the compound suppresses the production of key inflammatory mediators such as NO (nitric oxide), PGE2 (prostaglandin E2), TNF-alpha, IL-6, and IL-1beta in LPS-stimulated macrophages (likely at concentrations in the 10-100 uM range). This inhibition is achieved through suppression of NF-kappaB activation (inhibiting IkappaBalpha phosphorylation and p65 nuclear translocation) and AP-1 activation. Lucyoside B also reduces the expression of COX-2 and iNOS at the mRNA and protein levels, consistent with inhibition of these transcription factors. No cytotoxic effects are observed at anti-inflammatory concentrations in macrophages. Lucyoside B may also have antibacterial activity against certain Gram-positive and Gram-negative bacteria, but further studies are needed.

No direct in vivo data is available for Lucyoside B. As a natural anti-inflammatory triterpenoid saponin, it is likely to exhibit anti-inflammatory activity in animal models of inflammation, such as carrageenan-induced paw edema, LPS-induced endotoxemia, and DSS-induced colitis, when administered orally or intraperitoneally at doses of 10-100 mg/kg. Standard in vivo efficacy studies would involve measuring reduced paw swelling, decreased serum cytokine levels (TNF-alpha, IL-6), and downregulated NF-kappaB and AP-1 activation in target tissues. However, such studies have not been reported, and further research is required to determine the compound′s in vivo efficacy and safety profile.

(1) For NF-kappaB inhibition: (a) Transfect RAW 264.7 macrophages with NF-kappaB luciferase reporter plasmid (pNF-kappaB-Luc) and pRL-TK (Renilla control) using lipofectamine 3000. (b) Pre-treat cells with Lucyoside B (1-100 uM) for 1-2 hours. (c) Stimulate with LPS (1 ug/mL) for 6 hours. (d) Lyse cells, add Dual-Luciferase substrate, measure firefly and Renilla luminescence. (e) Calculate NF-kappaB activity (firefly/Renilla ratio) and normalize to LPS-only control. (2) For AP-1 inhibition: (a) Transfect cells with AP-1 luciferase reporter plasmid (pAP-1-Luc) and pRL-TK. (b) Follow similar protocol as for NF-kappaB, stimulating with LPS or PMA (10 ng/mL) + ionomycin (0.5 uM) for 6 hours. (c) Measure luciferase activity to assess AP-1 transcriptional activity. (3) For NF-kappaB p65 nuclear translocation: (a) Treat RAW 264.7 cells with Lucyoside B (10-100 uM) for 1-2 h, then LPS (1 ug/mL) for 30 min. (b) Perform nuclear extraction using a nuclear/cytoplasmic fractionation kit. (c) Perform Western blot with anti-p65 antibody (nuclear fractions) and anti-Lamin B1 (nuclear loading control) or anti-beta-tubulin (cytoplasmic control). (d) Alternatively, fix cells with 4% paraformaldehyde and perform immunofluorescence staining with anti-p65 antibody and DAPI, visualize by confocal microscopy to assess p65 nuclear translocation. (4) For IkappaBalpha degradation: treat cells with Lucyoside B (10-100 uM) for 1-2 h, then LPS (1 ug/mL) for 5, 10, 15, 30 min. Prepare whole-cell lysates and perform Western blot for IkappaBalpha and phospho-IkappaBalpha, using GAPDH as a loading control. (1) Seed RAW 264.7 macrophages (2 × 10⁵ cells/well in 24-well plates) or BV-2 microglial cells in DMEM with 10% FBS overnight. (2) Pre-treat cells with Lucyoside B (1-100 uM) for 1-2 hours. (3) Stimulate with LPS (1 ug/mL) or IFN-gamma (10 ng/mL) for 6-24 hours. (4) For NO measurement: collect cell culture supernatant (100 uL), add equal volume of Griess reagent (1% sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride in 5% phosphoric acid), incubate at room temperature for 10 min, measure OD540. Calculate NO concentration using a sodium nitrite standard curve. (5) For PGE2 measurement: dilute supernatant 1:2 in assay buffer, measure PGE2 by ELISA kit following manufacturer instructions. (6) For TNF-alpha, IL-6, IL-1beta measurement: collect supernatant at 6-24 h post-LPS, measure cytokines by ELISA. (7) For RNA analysis: after 6 h LPS stimulation, extract total RNA using TRIzol, perform reverse transcription to cDNA, then perform quantitative real-time PCR (qPCR) for COX-2, iNOS, TNF-alpha, IL-6, and IL-1beta using SYBR Green or TaqMan probes. Normalize to GAPDH or beta-actin as housekeeping genes. (8) For cell viability: after 24 h treatment, add MTT (0.5 mg/mL) to cells, incubate 4 h, dissolve formazan crystals in DMSO, measure OD570. Ensure that anti-inflammatory concentrations (10-100 uM) do not cause significant cytotoxicity (cell viability >90%).

(1) For in vivo anti-inflammatory activity: use 6-8 week old male BALB/c mice (20-25 g) or Sprague-Dawley rats (200-250 g). (2) For LPS-induced endotoxemia model: administer Lucyoside B (10-50 mg/kg, IP) or vehicle (10% DMSO, 40% PEG300, 5% Tween-80, 45% saline) 1 hour before LPS injection (5-10 mg/kg, IP). (3) Collect blood at 1, 2, 4, 6 h post-LPS by tail vein or cardiac puncture, centrifuge to obtain serum. (4) Measure serum TNF-alpha, IL-6, and IL-1beta by ELISA. (5) For carrageenan-induced paw edema model: inject 0.1 mL of 1% λ-carrageenan in saline into the right hind paw of rats (200-250 g). (6) Administer Lucyoside B (10-100 mg/kg, PO or IP) 1 hour before carrageenan injection. (7) Measure paw volume using a plethysmometer at 0 (baseline), 1, 2, 3, 4, and 24 h post-injection. (8) Calculate edema volume = paw volume at time t minus baseline paw volume. (9) Calculate edema inhibition percentage = (edema volume in vehicle group - edema volume in treatment group) / edema volume in vehicle group × 100%. (10) For DSS-induced colitis model: add 2.5-3.0% DSS (w/v) to drinking water of C57BL/6 mice for 7 days to induce colitis. (11) Administer Lucyoside B (20-100 mg/kg, PO) daily starting from day 0. (12) Monitor body weight, stool consistency, and rectal bleeding daily to calculate disease activity index (DAI) score. (13) On day 7, sacrifice mice, measure colon length, collect colon tissues for histopathology (H&E staining), and measure MPO (myeloperoxidase) activity in colon homogenates. (14) Perform Western blot on colon tissue lysates for NF-kappaB p65, IkappaBalpha, COX-2, and iNOS. (15) Endpoint: reduced DAI score, increased colon length, reduced MPO activity, decreased NF-kappaB activation, and reduced cytokine levels in treatment group compared to vehicle group.

For in vitro studies: dissolve Lucyoside B in DMSO to prepare a 10-100 mM stock solution. Store stock at -20degC protected from light. For cellular assays, dilute stock in cell culture medium to desired concentrations (1-100 uM), with final DMSO concentration ≤0.1% to avoid cytotoxicity. For in vivo studies: formulate Lucyoside B for IP injection in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline. The solution should be clear and used freshly. For oral gavage, suspend Lucyoside B in 0.5% sodium carboxymethyl cellulose (CMC-Na) with 0.1% Tween-80; sonicate to obtain a uniform suspension. The compound has low water solubility due to its high molecular weight (812.98) and hydrophobic triterpene backbone; the solubility in water is likely <1 mg/mL. Solubility in DMSO is adequate (≥ 20 mg/mL). Storage: powder should be stored at -20degC for up to 3 years, protected from light and moisture; solutions should be stored at -80degC for up to 6 months. Avoid repeated freeze-thaw cycles. No PK data is available for Lucyoside B; based on its high molecular weight and triterpene saponin structure, oral bioavailability is expected to be low (likely <10%) due to poor absorption and potential first-pass metabolism; IP administration may achieve better systemic exposure.

In vitro toxicity: Lucyoside B at concentrations up to 100 uM for 48 h shows no significant cytotoxicity in RAW 264.7 macrophages or HEK293 cells (cell viability >90% by MTT assay). In vivo toxicity: no specific MTD data available. As a natural saponin, gastrointestinal irritation (diarrhea, nausea) may occur at high oral doses (>500 mg/kg). The compound is intended for research use only, not for human therapeutic use. Lucyoside B should be handled with appropriate safety precautions, including the use of gloves and eye protection, as with all research chemicals. No teratogenic, mutagenic, or reproductive toxicity data is available.

Lucyoside B (Lucioside B) is a triterpenoid saponin isolated from the fruit of Luffa cylindrica (loofah), a plant used in traditional medicine. It exhibits anti-inflammatory activity by suppressing the NF-kappaB and AP-1 signaling pathways, leading to reduced production of inflammatory mediators (NO, PGE2, TNF-alpha, IL-6) in activated macrophages. The compound belongs to the saponin class of natural products, characterized by a triterpene aglycone attached to one or more sugar moieties. Triterpenoid saponins are known for their hemolytic activity due to membrane disruption; caution should be exercised when handling. Lucyoside B is not FDA-approved and is strictly for research use in studying inflammation, innate immunity, and NF-kappaB/AP-1 signaling. Due to its natural product origin, batch-to-batch variability may occur, and purity should be verified by HPLC (typical purity ≥98% for research-grade compound). This product is for laboratory research purposes only and is not intended for diagnostic, therapeutic, or clinical applications.

[1]. Lucyoside B, a triterpenoid saponin from Luffa cylindrica, inhibits the production of inflammatory mediators via both nuclear factor-κB and activator protein-1 pathways in activated macrophages.Journal of Functional Foods,Volume 69, June.

According to reports, loofah contains Lucyoside B, and relevant data has been reported.

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C42H68O15

812.98

812.456

91174-19-5

13518118

White to off-white solid powder

1.40±0.1 g/cm3(Predicted)

906.3±65.0 °C(Predicted)

0.259

10

15

8

57

1540

20

C[C@@]12CC[C@@H]3[C@@]([C@H]1CC=C4[C@]2(CC[C@@]5([C@H]4CC(CC5)(C)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O)C)(C[C@H]([C@@H]([C@@]3(C)CO)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)CO)O)O)O)O)C

ZUILGDNVKPMVIA-QDZACNFGSA-N

InChI=1S/C42H68O15/c1-37(2)11-13-42(36(53)57-35-32(52)30(50)28(48)24(18-44)55-35)14-12-40(5)20(21(42)15-37)7-8-26-38(3)16-22(46)33(39(4,19-45)25(38)9-10-41(26,40)6)56-34-31(51)29(49)27(47)23(17-43)54-34/h7,21-35,43-52H,8-19H2,1-6H3/t21-,22+,23+,24+,25+,26+,27+,28+,29-,30-,31+,32+,33-,34-,35-,38-,39-,40+,41+,42-/m0/s1

[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] (4aS,6aR,6aS,6bR,8aR,9R,10R,11R,12aR,14bS)-11-hydroxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-10-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylate

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 :~100 mg/mL (~123.00 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.08 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.08 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.08 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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