- `4-Hydroxylonchocarpin` targets tyrosinase, tyrosinase-related proteins (TRP-1, TRP-2), and MAPK phosphatase (MKP-1). It inhibits tyrosinase activity with an IC₅₀ of 25.3 μM, and downregulates the expression of TRP-1, TRP-2 [1]

- Antimelanogenic effect in B16F10 melanoma cells: When B16F10 cells were treated with `4-Hydroxylonchocarpin` (10, 50, 100 μM) for 48 hours (with α-MSH 100 nM to stimulate melanin production), melanin content was reduced in a concentration-dependent manner. At 50 μM, melanin content decreased by 42 ± 3% compared to the α-MSH-stimulated control. Western blot analysis showed that 50 μM `4-Hydroxylonchocarpin` downregulated the protein expression of TRP-1 (to 0.4-fold of control), TRP-2 (to 0.3-fold), and microphthalmia-associated transcription factor (MITF, to 0.2-fold) [1]
- Regulation of MAPK signaling pathway: `4-Hydroxylonchocarpin` (50 μM) treatment for 48 hours in B16F10 cells increased the phosphorylation levels of ERK1/2 (to 2.1-fold of control) and JNK (to 1.8-fold), while decreasing MKP-1 protein expression (to 0.3-fold). RT-PCR further confirmed that 50 μM `4-Hydroxylonchocarpin` reduced MKP-1 mRNA expression to 0.4-fold of the control, indicating modulation of the MAPK pathway via MKP-1 inhibition [1]
- Tyrosinase activity inhibition (cell-free assay): In a cell-free system using mushroom tyrosinase, `4-Hydroxylonchocarpin` (0-100 μM) inhibited tyrosinase activity with an IC₅₀ of 25.3 μM. At the maximum tested concentration (100 μM), the inhibition rate of tyrosinase activity reached 68 ± 4% [1]

- The assay was conducted in 50 mM phosphate buffer (pH 6.8) with a total volume of 200 μL. Each reaction mixture contained 10 units of mushroom tyrosinase, 0.5 mM L-tyrosine (substrate), and `4-Hydroxylonchocarpin` at concentrations of 0, 10, 25, 50, 75, or 100 μM. The mixture was incubated at 37℃ for 30 minutes to allow the oxidation of L-tyrosine to dopachrome. After incubation, the absorbance at 475 nm (characteristic absorbance of dopachrome) was measured using a microplate reader. The inhibition rate of tyrosinase was calculated using the formula: [(Absorbance of control - Absorbance of treatment) / Absorbance of control] × 100%. The IC₅₀ value was determined by plotting the inhibition rate against the logarithm of `4-Hydroxylonchocarpin` concentration and fitting a concentration-response curve [1]

- B16F10 Cell Melanin Content Assay: B16F10 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin, maintained at 37℃ in a 5% CO₂ incubator. Cells were seeded into 6-well plates at a density of 2×10⁵ cells per well and allowed to adhere overnight. The next day, 100 nM α-MSH was added to each well to stimulate melanin synthesis, followed by the addition of `4-Hydroxylonchocarpin` (10, 50, 100 μM). After 48 hours of treatment, cells were washed twice with PBS, harvested by trypsinization, and centrifuged at 1,000 × g for 5 minutes. The cell pellet was resuspended in 100 μL of 1 N NaOH containing 10% DMSO, then heated at 80℃ for 1 hour to fully lyse cells and solubilize melanin. The absorbance of the lysate at 405 nm was measured, and melanin content was quantified using a standard curve generated with synthetic melanin [1]
- B16F10 Cell Western Blot Assay: B16F10 cells were treated with 50 μM `4-Hydroxylonchocarpin` for 48 hours, then washed with cold PBS and lysed in RIPA buffer (containing protease and phosphatase inhibitors) on ice for 30 minutes. The cell lysate was centrifuged at 12,000 × g for 15 minutes at 4℃, and the supernatant was collected to measure protein concentration using a protein assay kit. Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated overnight at 4℃ with primary antibodies against TRP-1, TRP-2, MITF, MKP-1, phosphorylated ERK1/2 (p-ERK1/2), total ERK1/2, phosphorylated JNK (p-JNK), total JNK, and β-actin (internal control). After washing with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an ECL chemiluminescence reagent, and band intensity was quantified using densitometry software [1]
- B16F10 Cell RT-PCR Assay: B16F10 cells were treated with 50 μM `4-Hydroxylonchocarpin` for 24 hours, then total RNA was extracted using an RNA isolation reagent. The extracted RNA was reverse-transcribed into cDNA using a reverse transcription kit with oligo(dT) primers. PCR amplification was performed using specific primers for MKP-1 and GAPDH (internal reference gene). The PCR reaction conditions were: initial denaturation at 94℃ for 5 minutes, followed by 30 cycles of denaturation at 94℃ for 30 seconds, annealing at 58℃ for 30 seconds, and extension at 72℃ for 45 seconds, with a final extension at 72℃ for 10 minutes. The PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide for visualization under UV light [1]

In vitro cytotoxicity of B16F10 cells: B16F10 cells were seeded at a density of 5 × 10³ cells per well in 96-well plates and cultured overnight. 4-hydroxygentianine at concentrations of 10, 25, 50, 75, or 100 μM was added, and the cells were cultured for another 48 hours. After treatment, 20 μL of MTT solution (5 mg/mL) was added to each well, and the cells were cultured at 37°C for another 4 hours. The supernatant was carefully removed, and 150 μL of DMSO was added to each well to dissolve formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and cell viability was calculated relative to the untreated control group. At all tested concentrations (up to 100 μM), cell viability remained above 85 ± 2%, indicating no significant cytotoxicity [1].

[1]. Antimelanogenic effect of 4-hydroxylonchocarpin through the inhibition of tyrosinase-related proteins and MAPK phosphatase. Exp Dermatol. 2016 Jul;25(7):574-6.

Isobavachromene has been reported to exist in Mundulea sericea, Dorstenia dinklagei, and other organisms with relevant data. 4-Hydroxyloncaprine is a natural isoflavone compound isolated from legumes. Its anti-melanogenesis mechanism involves two complementary pathways: 1) direct inhibition of tyrosinase activity (IC₅₀ = 25.3 μM), tyrosinase is a key enzyme in melanin synthesis; 2) indirect inhibition of melanin-related protein (TRP-1, TRP-2, MITF) expression by inhibiting MKP-1, thereby activating the ERK1/2 and JNK MAPK signaling pathways [1] - Due to its strong anti-melanogenesis activity and low cytotoxicity to B16F10 cells, 4-hydroxygentianine has potential application value in the development of cosmetics for skin whitening and therapeutic drugs for treating hyperpigmentation disorders such as melasma and freckles [1]

C20H18O4

322.3545

322.12

56083-03-5

5889042

Yellow to orange solid powder

1.3±0.1 g/cm3

535.1±50.0 °C at 760 mmHg

203-204℃

193.7±23.6 °C

0.0±1.5 mmHg at 25°C

1.651

5.99

2

4

3

24

514

0

CC1(C=CC2=C(O1)C=CC(=C2O)C(=O)/C=C/C3=CC=C(C=C3)O)C

IQHPDUUSMBMDGN-WEVVVXLNSA-N

InChI=1S/C20H18O4/c1-20(2)12-11-16-18(24-20)10-8-15(19(16)23)17(22)9-5-13-3-6-14(21)7-4-13/h3-12,21,23H,1-2H3/b9-5+

(E)-1-(5-hydroxy-2,2-dimethylchromen-6-yl)-3-(4-hydroxyphenyl)prop-2-en-1-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 : ~50 mg/mL (~155.11 mM)

Solubility in Formulation 1: 2.5 mg/mL (7.76 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.
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 (7.76 mM) 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; with ultrasonication.
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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 (Please use freshly prepared in vivo formulations for optimal results.)