5-LOX; COX-2
Cyclooxygenase-2 (COX-2): direct inhibition of COX-2 enzymatic activity (44.8% inhibition at 50 μM in COX-2-preinduced RAW 264.7 cells). [1]
5-Lipoxygenase (5-LOX): inhibition of 5-LOX-mediated leukotriene production (80.0% inhibition at 10 μM, 97.0% inhibition at 50 μM in A23187-treated RBL-1 cells). [1]
Tyrosinase: inhibition of intracellular tyrosinase activity (46.9±4.5% of control at 30 μM in melan-a cells). [2]
Microphthalmia-associated transcription factor (MITF): down-regulation of MITF protein expression (51.5±8.5% inhibition) and mRNA expression (32.3±1.0% reduction) in melan-a cells. [2]

Pectolinarigenin (1-50 μM) significantly inhibited COX-2-mediated PGE2 production in LPS-treated RAW 264.7 cells: at 10 μM, PGE2 concentration was 1.8±0.1 mM (90.1% inhibition); at 25 μM, 1.0±0.0 mM (95.8% inhibition); at 50 μM, 0.6±0.0 mM (99.0% inhibition). It also inhibited 5-LOX-mediated LT (LTC4/D4/E4) production in A23187-treated RBL-1 cells: at 10 μM, LT concentration was 536.4±142.1 pg/ml (80.0% inhibition); at 50 μM, 113.3±13.1 pg/ml (97.0% inhibition). No significant cytotoxicity was observed up to 50 μM as measured by MTT assay. [1]
Pectolinarigenin (50 μM) inhibited PGE2 production from COX-2-preinduced RAW 264.7 cells by 44.8% when arachidonic acid (100 μM) was added as substrate, indicating direct inhibition of COX-2 enzymatic activity. [1]
Western blot analysis showed that Pectolinarigenin (10, 25, 50 μM) did not reduce COX-2 expression in LPS-treated RAW 264.7 cells. EMSA analysis demonstrated that Pectolinarigenin did not affect NF-κB activation. [1]
In melan-a cells, Pectolinarigenin (30 μM) reduced melanin content to 31.5±2.2% of control (17.4±6.2% reduction? Actually the paper says "reduced the melanin content to 31.5±2.2%" but careful: "to 31.5±2.2%" means remaining percentage? Actually Fig1B: pectolinarigenin reduced melanin content to 31.5±2.2% compared with controls - need to interpret. The text says "reduced the melanin content to 17.4±6.2% and 31.5±2.2% respectively" – likely a mistake. Based on context, pectolinarin reduced to 17.4%, pectolinarigenin to 31.5%. So inhibition by pectolinarigenin is about 68.5%? But we'll state exactly as per text: reduced to 31.5±2.2% of control. [2]
Intracellular tyrosinase activity in melan-a cells was suppressed by Pectolinarigenin (30 μM) to 46.9±4.5% of the control group. [2]
Western blotting in melan-a cells showed that Pectolinarigenin (30 μM) reduced protein expression of tyrosinase by 37.9±3.2%, TRP-1 by 42.0±2.6%, TRP-2 by 38.3±3.8%, and MITF by 51.5±8.5% compared to control. [2]
Q-PCR analysis in melan-a cells demonstrated that Pectolinarigenin (30 μM) reduced mRNA expression of tyrosinase by 55.0±6.1%, TRP-1 by 10.7±3.3%, and MITF by 32.3±1.0% relative to control. [2]
In a reconstructed human skin model (Neoderm®-ME), Pectolinarigenin (30 μM for 2 days) significantly reduced melanin content to 20.8% of the control group and decreased L-DOPA content as visualized by Fontana-Masson and L-DOPA staining. [2]

Oral administration of Pectolinarigenin (20-100 mg/kg) inhibited arachidonic acid-induced mouse ear edema: at 20 mg/kg, ear thickness increase was 0.058±0.017 mm (18.7% inhibition); at 100 mg/kg, 0.048±0.016 mm (34.7% inhibition). Reference drug indomethacin at 20 mg/kg gave 0.015±0.021 mm (88.0% inhibition). [1]
Pectolinarigenin (20-100 mg/kg, oral) inhibited carrageenan-induced mouse paw edema: at 20 mg/kg, paw volume increase was 0.135±0.023 ml (13.2% inhibition); at 100 mg/kg, 0.120±0.051 ml (21.1% inhibition). Prednisolone at 20 mg/kg gave 0.078±0.021 ml (48.7% inhibition). [1]
Pectolinarigenin (20 mg/kg, oral twice, 1 h prior to each IgE and antigen injection) inhibited passive cutaneous anaphylaxis in rats: absorbance was 0.648±0.228 (30.8% inhibition) compared to allergen-treated control (0.807±0.139). Prednisolone (20 mg/kg×2) gave 0.439±0.256 (71.3% inhibition). [1]

For direct COX-2 inhibition measurement, RAW 264.7 cells were incubated with LPS (1 μg/ml) for 24 h to induce COX-2 expression. After complete washing with serum-free DMEM, test compounds (including Pectolinarigenin) and arachidonic acid (100 μM) were added to the COX-2-preinduced cells. Following 15 minutes of incubation, PGE2 concentration in the media was measured using an ELISA kit. Pectolinarigenin at 50 μM showed 44.8% inhibition of PGE2 production. [1]
For 5-LOX activity assay, rat basophilic leukemia (RBL-1) cells were plated in 96-well plates. Test compounds (Pectolinarigenin) were added and preincubated for 10 minutes. To activate 5-LOX, A23187 (3 μM) was added and cells were incubated for 15 minutes. The concentration of 5-LOX products (cysteinyl leukotrienes LTC4/D4/E4) in the media was measured using an ELISA kit. Pectolinarigenin inhibited LT production with 80.0% inhibition at 10 μM and 97.0% at 50 μM. [1]
For intracellular tyrosinase activity measurement, melan-a cells were treated with or without Pectolinarigenin (30 μM) for 72 h. Cells were washed, lysed in 1% Triton X-100, and chilled on ice for 10 min. After centrifugation, the supernatant was collected as enzyme source. The reaction mixture contained 100 μl of 0.1 M phosphate buffer (pH 6.5), 100 μl of 20 mM L-DOPA, and 40 μg of cell lysate in a 96-well plate. Initial absorbance was measured at 490 nm, and the mixture was incubated at room temperature for 1 h before final absorbance measurement. Intracellular tyrosinase activity was estimated as the ratio to control. Pectolinarigenin reduced tyrosinase activity to 46.9±4.5% of control. [2]

RAW 264.7 cells (mouse macrophage-like cell line) were cultured in DMEM with 10% FBS and 1% antibiotics under 5% CO2 at 37°C. Cells were plated in 96-well plates (2×10^5 cells/well). After pre-incubation for 2 h, test compounds (Pectolinarigenin) and LPS (1 μg/ml) were added and incubated for 24 h. Cell viability was assessed by MTT assay. PGE2 concentration in the media was measured using an ELISA kit. Pectolinarigenin inhibited PGE2 production in a concentration-dependent manner (1-50 μM). [1]
For COX-2 preinduction experiment, RAW 264.7 cells were incubated with LPS (1 μg/ml) for 24 h, then washed with serum-free DMEM. Test compounds (Pectolinarigenin) and arachidonic acid (100 μM) were added, and after 15 min PGE2 was measured. [1]
RBL-1 cells (rat basophilic leukemia) were cultured in RPMI 1640 with 10% FBS, 2 mM glutamine and 1% antibiotics under 5% CO2 at 37°C. Cells were plated in 96-well plates, test compounds (Pectolinarigenin) added and preincubated for 10 min, then A23187 (3 μM) added for 15 min. LT concentration in media was measured by ELISA. [1]
Melan-a cells were grown in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 200 nM TPA at 37°C in 5% CO2. For melanin content measurement, cells were seeded in 24-well plates (1×10^5 cells/well), incubated for 24 h, then treated with or without Pectolinarigenin (30 μM) for 72 h. Cells were washed, lysed with 2 N NaOH, and absorbance measured at 475 nm. [2]
For Western blotting, melan-a cells were harvested and homogenized at 4°C in lysis buffer. Protein concentrations were determined by BCA assay. 20 μg of protein was separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% skim milk and incubated with primary antibodies against tyrosinase, TRP-1, TRP-2, MITF, and GAPDH, followed by HRP-conjugated secondary antibodies. Bands were visualized using enhanced chemiluminescence. Pectolinarigenin (30 μM) reduced protein expression of these melanogenesis-related proteins. [2]
For Q-PCR analysis, total RNA was extracted using an RNeasy mini kit. cDNA was amplified using reverse transcription. SYBR green-based quantitative PCR was performed with specific primers for MITF, tyrosinase, TRP-1, TRP-2, and β-actin. Data were analyzed using the 2^-ΔΔCT method. Pectolinarigenin (30 μM) reduced mRNA levels of tyrosinase, TRP-1, and MITF. [2]
Reconstructed human skin model (Neoderm®-ME) consisting of human epidermal melanocytes and keratinocytes was incubated in serum-free maintenance medium with Pectolinarigenin (30 μM) for 2 days. Skin tissues were fixed in 10% formalin, embedded in paraffin, and stained for melanin using Fontana-Masson and L-DOPA staining methods. Melanin and L-DOPA contents were quantified using ImageJ software. [2]

Male ICR mice (4 weeks old, specific pathogen-free) were used for arachidonic acid-induced ear edema. Pectolinarigenin dissolved in DMSO (0.05 ml/mouse) was administered orally 1 h prior to arachidonic acid treatment. Arachidonic acid (2% in acetone, 20 μl/ear) was topically applied to mouse ears. One hour later, ear thickness was measured using a dial thickness gauge. [1]
Male ICR mice were used for carrageenan-induced paw edema. Pectolinarigenin dissolved in DMSO was administered orally. One hour later, 1% λ-carrageenan (w/v) dissolved in pyrogen-free sterile saline (0.05 ml per paw) was injected into the right hind paw. After 5 h, paw volume was measured using a plethysmometer. The increase in paw volume from the initial non-treated paw volume was regarded as edema. [1]
Male Sprague-Dawley rats were used for passive cutaneous anaphylaxis (PCA). The backs of the rats were shaved and injected intradermally with monoclonal anti-dinitrophenyl (DNP) mouse IgE (100 μl/site, 1:1000 dilution). After 48 h, PCA was induced by intravenous injection of the antigen (1 mg of DNP-bovine serum albumin) in PBS containing 1% Evans blue. Pectolinarigenin was orally administered twice, 1 h prior to each IgE and antigen injection. Thirty minutes after antigen challenge, the skin was removed, and dye that leaked into the skin was extracted and quantified. [1]

No significant cytotoxicity was observed for Pectolinarigenin in RAW 264.7 cells at concentrations up to 50 μM as measured by MTT assay. [1]
Pectolinarigenin (30 μM) showed no cytotoxicity in melan-a cells as determined by MTT assay. [2]

[1]. Anti-inflammatory activity of pectolinarigenin and pectolinarin isolated from Cirsium chanroenicum. Biol Pharm Bull. 2008 Nov;31(11):2063-7.

[2]. Pectolinarigenin, an aglycone of pectolinarin, has more potent inhibitory activities on melanogenesis than pectolinarin.Biochem Biophys Res Commun. 2017 Nov 4;493(1):765-772.

Pectolinarigenin is a dimethoxyflavonoid, a 6,4'-dimethyl ether derivative of baicalin. It is a plant metabolite. It is a dimethoxyflavonoid and a dihydroxyflavonoid. Its function is related to that of baicalin. Pectolinarigenin has been reported to be found in sage, bees, and other organisms with relevant data.

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Pectolinarigenin is a flavonoid with 5,7-dihydroxy-4',6-dimethoxyflavone structure. It is the aglycone of pectolinarin (pectolinarigenin 7-rhamnosyl-(1→6)-glucoside). The compound possesses anti-inflammatory activity by inhibiting eicosanoid formation (PGE2 and leukotrienes) through direct inhibition of COX-2 and 5-LOX enzymes, without affecting COX-2 expression or NF-κB activation. It also exhibits anti-melanogenic activity by down-regulating MITF and tyrosinase-related gene expression. [1][2]
The C-6 methoxyl group in Pectolinarigenin is suggested to be important for 5-LOX inhibitory activity, as flavonoids with 5,7-dihydroxyl substitution and C-6 hydroxyl/methoxyl group possess 5-LOX inhibitory activity. [1]
Orally administered Pectolinarigenin and its glycoside pectolinarin show similar in vivo anti-inflammatory potencies, possibly because pectolinarin is hydrolyzed to the aglycone form (Pectolinarigenin) in the intestine before entering the bloodstream. [1]
Pectolinarigenin has potential as a new anti-inflammatory/anti-allergic agent because COX inhibitors are used as anti-inflammatory agents and 5-LOX inhibitors show anti-allergic activity. [1]
Microwave irradiation under 1% acetic acid conditions can efficiently convert pectolinarin to Pectolinarigenin (increased pectolinarigenin content by 66.3% compared to control, while pectolinarin content decreased to 14.5%). [2]

C17H14O6

314.2895

314.079

520-12-7

5320438

Light yellow to yellow solid

1.4±0.1 g/cm3

565.5±50.0 °C at 760 mmHg

220-223°

212.3±23.6 °C

0.0±1.6 mmHg at 25°C

1.646

2.64

2

6

3

23

468

0

O1C(=C([H])C(C2C(=C(C(=C([H])C1=2)O[H])OC([H])([H])[H])O[H])=O)C1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])[H]

GPQLHGCIAUEJQK-UHFFFAOYSA-N

InChI=1S/C17H14O6/c1-21-10-5-3-9(4-6-10)13-7-11(18)15-14(23-13)8-12(19)17(22-2)16(15)20/h3-8,19-20H,1-2H3

5,7-dihydroxy-6-methoxy-2-(4-methoxyphenyl)chromen-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 : ~33.33 mg/mL (~106.05 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (3.18 mM) (saturation unknown) in 2% DMSO + 98% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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: 1 mg/mL (3.18 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication (<60°C).
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.)