- Ikarisoside A targets inducible nitric oxide synthase (iNOS) in LPS-stimulated RAW 264.7 cells, with an IC₅₀ value of approximately 12.5 μM for inhibiting NO production (a downstream product of iNOS)[1]
- Ikarisoside A targets nicotinic acetylcholine receptors (nAChRs) in cultured bovine adrenal medullary cells, with an IC₅₀ value of ~8.3 μM for inhibiting acetylcholine-induced catecholamine secretion[3]

Ikarisoside A has a concentration-dependent effect on the expression of nitric oxide (NO) and inducible nitric oxide synthase (iNOS) in LPS-stimulated RAW 264.7 cells and mouse bone marrow-derived macrophages (BMM). generate. Furthermore, proinflammatory cytokines like interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) are less released when ikariseside A is present. Furthermore, Ikarisoside A has the ability to suppress nuclear factor kappaB (NF-kappaB) and p38 kinase [1]. In both bone marrow-derived macrophages and RANKL-stimulated RAW 264.7 cells, isokarisoside A is a strong inhibitor of osteoclastogenesis. Osteoclast-specific genes, including matrix metalloproteinase 9 (MMP9), tartrate-resistant acid phosphatase (TRAP), receptor activator of NF-κB (RANK), and cathepsin K, were downregulated in response to Ikarisoside A inhibition. Furthermore, on calcium phosphate-coated plates, Ikarisoside A inhibits RAW 264.7 cells' ability to reabsorb substances. Moreover, RANKL-mediated activation of NF-kappaB, JNK, and Akt is inhibited by isokarisoside A [2].

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1. In LPS-stimulated RAW 264.7 cells: Treatment with Ikarisoside A (5–25 μM) for 24 hours reduced nitric oxide (NO) production in a dose-dependent manner. At 25 μM, NO production was inhibited by 78% compared to the LPS-only control. Western blot analysis showed that Ikarisoside A (10–25 μM) decreased iNOS protein expression by 42–68% and suppressed phosphorylation of p38 kinase (by 35–59%) and nuclear translocation of NF-κB p65 subunit (by 45–72%)[1]
2. In RANKL-induced RAW 264.7 cells (osteoclastogenic differentiation model): Ikarisoside A (1–10 μM) inhibited osteoclast formation in a dose-dependent way. At 10 μM, the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was reduced by 65%. It also decreased mRNA expression of osteoclast markers (TRAP, cathepsin K, MMP-9) by 40–62% and suppressed phosphorylation of JNK (by 38–55%) and NF-κB p65 nuclear translocation (by 41–63%)[2]
3. In cultured bovine adrenal medullary cells: Ikarisoside A (3–30 μM) inhibited acetylcholine (100 μM)-induced catecholamine (norepinephrine + epinephrine) secretion in a dose-dependent manner. At 30 μM, secretion was reduced by 71%. It also suppressed acetylcholine-induced inward currents (mediated by nAChRs) by 58% at 10 μM, without affecting K⁺ or Ca²⁺ channel activities[3]

1. iNOS activity-related NO detection assay: RAW 264.7 cells were seeded in 24-well plates and stimulated with LPS (1 μg/mL) for 1 hour, then treated with Ikarisoside A (5–25 μM) for 24 hours. Culture supernatants (100 μL) were mixed with an equal volume of Griess reagent, incubated at room temperature for 15 minutes, and absorbance was measured at 540 nm. NO concentration was calculated using a sodium nitrite standard curve[1]
2. nAChR-mediated current recording assay: Bovine adrenal medullary cells were enzymatically dissociated and plated on glass coverslips. Whole-cell patch-clamp recordings were performed at room temperature. Acetylcholine (100 μM) was applied to induce inward currents, and Ikarisoside A (3–30 μM) was pre-incubated for 5 minutes before acetylcholine application. Current amplitudes were recorded and normalized to the control (acetylcholine alone)[3]

1. RAW 264.7 cell culture and iNOS-related assay: Cells were cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin, maintained at 37°C with 5% CO₂. For Western blot: Cells were lysed after treatment, proteins separated by SDS-PAGE, transferred to membranes, and probed with antibodies against iNOS, phospho-p38, total p38, or NF-κB p65. Band intensity was quantified by densitometry. For NF-κB nuclear translocation: Cells were fixed, permeabilized, stained with NF-κB p65 antibody and DAPI, then observed under a fluorescence microscope to count nuclear p65-positive cells[1]
2. RAW 264.7 osteoclast differentiation assay: Cells were seeded in 96-well plates and treated with RANKL (50 ng/mL) plus Ikarisoside A (1–10 μM) for 5 days. On day 5, cells were fixed, stained with TRAP reagent, and TRAP-positive multinucleated cells (≥3 nuclei) were counted under a microscope. For qPCR: Total RNA was extracted, reverse-transcribed to cDNA, and amplified with primers for TRAP, cathepsin K, and MMP-9; GAPDH was used as an internal control[2]
3. Bovine adrenal medullary cell culture and catecholamine assay: Adrenal medullary cells were isolated from bovine adrenal glands, cultured in DMEM/F12 medium with 10% FBS for 24 hours. Cells were pre-treated with Ikarisoside A (3–30 μM) for 10 minutes, then stimulated with acetylcholine (100 μM) for 15 minutes. Culture supernatants were collected, and catecholamine levels were measured by high-performance liquid chromatography (HPLC) with electrochemical detection[3]

1. In RAW 264.7 cells: treatment with up to 25 μM icaroside A for 24 hours showed no significant cytotoxicity, and MTT assay showed cell viability >90% (compared to the untreated control group) [1]
2. In RAW 264.7 cells (osteoclast differentiation model): treatment with up to 10 μM icaroside A for 5 days did not affect cell viability, and MTT assay showed cell viability >92% (compared to the control group) [2]
3. In cultured bovine adrenal medullary cells: treatment with up to 30 μM icaroside A for 1 hour showed no cytotoxicity, and trypan blue exclusion assay showed cell survival >93% [3]

[1]. Ikarisoside A inhibits inducible nitric oxide synthase in lipopolysaccharide-stimulated RAW 264.7 cells via p38 kinase and nuclear factor-kappaB signaling pathways. Eur J Pharmacol. 2008 Dec 28;601(1-3):171-8.

[2]. Choi HJ, eta l. Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-kappaB signaling pathways. Eur J Pharmacol. 2010 Jun 25;636(1-3):28-35.

[3]. Ikarisoside A inhibits acetylcholine-induced catecholamine secretion and synthesis by suppressing nicotinic acetylcholine receptor-ion channels in cultured bovine adrenal medullary cells. Naunyn Schmiedebergs Arch Pharmacol. 2015;388(12):1259-1269.

It has been reported that Epimedium sagittatum, Epimedium sempervirens, and other organisms with relevant data contain β-caryoside II.

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1. Icaryoside A is a natural compound isolated from Epimedium species (e.g., Epimedium koreanum), which have traditionally been used in herbal medicine[1][2][3].
2. Its anti-inflammatory activity (cited from [1]) is mediated by inhibition of the p38/NF-κB signaling pathway, thereby reducing iNOS expression and NO production—key mediators of the inflammatory response[1].
3. Its anti-osteoclastogenic activity (cited from [2]) involves inhibition of the JNK/NF-κB pathway, which is essential for RANKL-induced osteoclast differentiation, suggesting its potential use in the treatment of bone resorption-related diseases (e.g., osteoporosis)[2]. 4. Its effect on catecholamine secretion (cited from [3]) is through direct inhibition of nAChR without interfering with other ion channels, indicating that it has specificity for nAChR-mediated signal transduction in adrenal medullary cells [3].

C26H28O10

500.49500

500.168

55395-07-8

5481982

Light yellow to yellow solid powder

1.5±0.1 g/cm3

782.1±60.0 °C at 760 mmHg

264.7±26.4 °C

0.0±2.8 mmHg at 25°C

1.697

4.47

6

10

5

36

859

5

C[C@H]1[C@@H]([C@H]([C@H]([C@@H](O1)OC2=C(OC3=C(C(=CC(=C3C2=O)O)O)CC=C(C)C)C4=CC=C(C=C4)O)O)O)O

RPLMLWBOUPDPQF-GULSFEPBSA-N

InChI=1S/C26H28O10/c1-11(2)4-9-15-16(28)10-17(29)18-20(31)25(36-26-22(33)21(32)19(30)12(3)34-26)23(35-24(15)18)13-5-7-14(27)8-6-13/h4-8,10,12,19,21-22,26-30,32-33H,9H2,1-3H3/t12-,19-,21+,22+,26-/m0/s1

5,7-dihydroxy-2-(4-hydroxyphenyl)-8-(3-methylbut-2-enyl)-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-one

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 (~199.80 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.00 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 2: ≥ 2.08 mg/mL (4.16 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.

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