IL-8; EP; IL-6

Pectin causes rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) to express higher amounts of Bax and lower levels of Bcl-2. PI3K/Akt in RA-FLSs is inactivated by pectin [2].

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- Anti-inflammatory activity (COX-2/5-LOX inhibition): Unlike its aglycone, Pectolinarin exhibited no significant inhibitory activity on COX-2-mediated PGE2 production in LPS-treated RAW 264.7 cells or on 5-LOX-mediated leukotriene (LT) production in A23187-treated RBL-1 cells at concentrations up to 50 μM. For example, at 50 μM, PGE2 concentration was 18.0±0.7 mM (0% inhibition) and LT concentration was 2550.4±879.1 pg/ml (no significant inhibition reported). [1]
- Anti-melanogenesis activity: In melan-a cells, treatment with Pectolinarin at 30 μM for 72 hours reduced melanin content to 17.4±6.2% of the control group, showing an inhibitory effect on melanin synthesis without cytotoxicity. However, it did not inhibit intracellular tyrosinase activity; instead, it increased it relative to control. Pectolinarin did not suppress the protein expression of melanogenesis-related proteins (MITF, Tyrosinase, TRP-1, TRP-2) in western blotting, nor did it suppress the mRNA expression of Tyrosinase, TRP-1, or MITF as measured by Q-PCR. [2]

- Anti-inflammatory activity (mouse ear edema): Oral administration of Pectolinarin at 100 mg/kg significantly inhibited arachidonic acid (AA)-induced mouse ear edema, with an ear thickness increase of 0.048±0.013 mm (34.7% inhibition). Lower doses (4 and 20 mg/kg) showed less or non-significant inhibition (0.1% and 16.0% inhibition, respectively). [1]
- Anti-inflammatory activity (mouse paw edema): Oral administration of Pectolinarin at 4, 20, and 100 mg/kg showed some inhibition of carrageenan (CGN)-induced mouse paw edema, with paw volume increases of 0.160±0.022 ml (no % inhibition calculated), 0.142±0.026 ml (6.6% inhibition), and 0.128±0.040 ml (15.8% inhibition) respectively, though these effects were not statistically significant at the doses tested. [1]
- Anti-allergic activity (PCA in rats): Oral administration of Pectolinarin at 20 mg/kg twice significantly inhibited passive cutaneous anaphylaxis (PCA) in rats, with an absorbance of 0.662±0.085 (28.2% inhibition) compared to the allergen-treated control group (0.807±0.139). [1]

- 5-LOX mediated LT production assay: RBL-1 cells were cultured and plated in 96-well plates. Test compounds, including Pectolinarin, were added and pre-incubated for 10 minutes. To activate 5-LOX, the calcium ionophore A23187 (3 μM) was added and the cells were incubated for 15 minutes. The culture media was then collected, and the concentration of the 5-LOX products (cysteinyl leukotrienes LTC4/D4/E4) was measured using an ELISA kit, following the manufacturer's instructions. [1]
- COX-2 mediated PGE2 production assay (cell-based): RAW 264.7 cells were plated in 96-well plates. After pre-incubation for 2 hours, test compounds (Pectolinarin) and LPS (1 μg/ml) were added and incubated for 24 hours. From the media, the PGE2 concentration was measured using an ELISA kit according to the manufacturer's instructions. [1]

- COX-2 mediated PGE2 production in RAW 264.7 cells: RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics. Cells were plated in 96-well plates (2x10^5 cells/well). After pre-incubation for 2 hours, test compounds (including Pectolinarin) and LPS (1 μg/ml) were added and incubated for 24 hours. Cell viability was assessed by MTT assay. PGE2 concentration in the media was measured using an ELISA kit. [1]
- 5-LOX mediated LT production in RBL-1 cells: Rat basophilic leukemia (RBL-1) cells were cultured in RPMI 1640 with 10% FBS, 2 mM glutamine, and 1% antibiotics. Cells were plated in 96-well plates for 2 hours. Test compounds (Pectolinarin) were added and pre-incubated for 10 minutes. A23187 (3 μM) was added to activate 5-LOX, and cells were incubated for 15 minutes. The concentration of cysteinyl leukotrienes (LTC4/D4/E4) in the media was measured using an ELISA kit. [1]
- Melanin content measurement in melan-a cells: Melan-a cells were seeded in 24-well plates (1x10^5 cells/well) and incubated for 24 hours. Cells were then treated with or without Pectolinarin (30 μM). After 72 hours, cells were washed with PBS and lysed with 2N NaOH. The lysed cells were transferred to 96-well plates, and absorbance was measured at 475 nm to determine melanin content. [2]
- Intracellular tyrosinase activity assay in melan-a cells: Melan-a cells were seeded in 60-mm dishes (4x10^5 cells/dish) for 24 hours, then treated with or without Pectolinarin (30 μM). After 72 hours, cells were washed, lysed in 1% Triton X-100, and centrifuged. The supernatant was collected as the enzyme source. The reaction mixture, containing 100 μL of 0.1 M phosphate buffer (pH 6.5), 100 μL of 20 mM L-DOPA, and 40 μg of cell lysate, was incubated for 1 hour at room temperature. Absorbance was measured at 490 nm initially and after 1 hour to estimate tyrosinase activity. [2]
- Western blotting in melan-a cells: Melan-a cells were harvested and homogenized in lysis buffer at 4°C. After centrifugation, 20 μg of protein was separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% skim milk and then incubated with primary antibodies against Tyrosinase, TRP-1, TRP-2, and MITF, followed by HRP-conjugated secondary antibodies. Immuno-reactive bands were visualized using a chemiluminescence substrate and an imaging system. GAPDH was used as a loading control. [2]
- Quantitative real-time PCR (Q-PCR) in melan-a cells: Total RNA was extracted using an RNeasy mini kit. cDNA was amplified using a reverse transcription system. SYBR green-based quantitative PCR was performed using a real-time PCR system with specific primers for MITF, Tyrosinase, TRP1, TRP2, and β-actin (control). The data were analyzed using the 2-ΔΔCT method. [2]

- Arachidonic acid-induced ear edema in mice: Test compounds, including Pectolinarin (dissolved in DMSO), were administered orally (0.05 ml/mouse) to mice 1 hour prior to arachidonic acid treatment. Arachidonic acid (2% in acetone, 20 μl/ear) was topically applied to the ears. One hour later, ear thickness was measured using a dial thickness gauge to assess edema. [1]
- Carrageenan-induced paw edema in mice: Test compounds (Pectolinarin, dissolved in DMSO) were administered orally to mice. One hour later, 1% λ-carrageenan (w/v) dissolved in sterile saline (0.05 ml/paw) was injected into the right hind paw. After 5 hours, paw volume was measured using a plethysmometer. The increase in paw volume from the initial untreated paw volume was regarded as edema. [1]
- Passive cutaneous anaphylaxis (PCA) in rats: The backs of rats were shaved and injected intradermally with monoclonal anti-DNP mouse IgE (100 μl/site, 1:1000 dilution). After 48 hours, PCA was induced by intravenous injection of the antigen (1 mg DNP-bovine serum albumin) in PBS containing 1% Evans blue. Test compounds, including Pectolinarin (dissolved in DMSO), were administered orally twice, 1 hour prior to each IgE and antigen injection. Thirty minutes after antigen challenge, the skin was removed, and the leaked dye was extracted and quantified. [1]

- The provided texts do not contain quantitative ADME/pharmacokinetic parameters (e.g., half-life, bioavailability, volume of distribution) for Pectolinarin. However, it is discussed that orally administered flavonoid glycosides like Pectolinarin may enter the circulation through the intestinal wall, probably after hydrolysis of their glycosidic portion. The corresponding flavonoid aglycone (pectolinarigenin) and its methylated derivatives are found in the bloodstream, exerting in vivo pharmacological activity. This is suggested as the reason why Pectolinarin shows in vivo anti-inflammatory activity despite lacking activity in cell culture experiments. [1]

- Cytotoxicity: In anti-inflammatory assays using RAW 264.7 and RBL-1 cells, Pectolinarin showed no significant cytotoxicity at concentrations up to 50 μM (and up to 25 μg/ml for extracts/fractions containing it) as measured by MTT assay. [1]
- Cytotoxicity in melan-a cells: Pectolinarin at 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]. Pectolinarin inhibits proliferation, induces apoptosis, and suppresses inflammation in rheumatoid arthritis fibroblast-like synoviocytes by inactivating the phosphatidylinositol 3 kinase/protein kinase B pathway.J Cell Biochem. 2019 Sep;120(9):15202-15210.

Pectolinarin is a disaccharide derivative formed by replacing the Pectolinarin aglycone at position 7 with a 6-O-(6-deoxy-α-L-mannopyranosyl)-β-D-glucopyranosyl group via a glycosidic bond. It possesses various functions, including inducing apoptosis, anti-inflammation, as a plant metabolite, antitumor activity, inhibition of EC 3.4.22.69 (the main protease of SARS coronavirus), and as an antioxidant. It is a dimethoxyflavonoid, rutin, glycosyloxyflavonoid, disaccharide derivative, and monohydroxyflavanone. Its functions are related to the Pectolinarin aglycone. Pectolinarin has been reported in Cirsium subcoriaceum, Viburnum cotinifolium, and other organisms with relevant data. See also: Red clover (Trifolium pratense) flower (partial).

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- Mechanism of in vivo activity: While Pectolinarin did not inhibit COX-2/5-LOX in cell culture experiments, it showed in vivo anti-inflammatory activity. This phenomenon may be caused by the inaccessibility of flavonoid glycosides to the inside of the cell due to their hydrophilicity. Orally administered Pectolinarin is thought to be hydrolyzed to its aglycone, pectolinarigenin, which is the active form found in the bloodstream. [1]
- Role in plant extract: Pectolinarin is a major compound in Cirsium setidens water extract, but its aglycone form, pectolinarigenin, is present at a lower amount. Methods such as microwave irradiation under 1% acetic acid were identified as efficient for converting Pectolinarin from C. setidens hot-water extract into pectolinarigenin, which has more potent anti-melanogenesis activity. [2]
- Comparison with aglycone: There are significant differences in the anti-melanogenesis effects of Pectolinarin and its aglycone, pectolinarigenin. Pectolinarigenin showed more potent inhibitory activity on melanin synthesis and suppressed tyrosinase activity and melanogenesis-related protein/gene expression, whereas Pectolinarin did not. [2]

C29H34O15

622.5713

622.189

28978-02-1

168849

Off-white to light yellow solid

1.6±0.1 g/cm3

896.4±65.0 °C at 760 mmHg

292.4±27.8 °C

0.0±0.3 mmHg at 25°C

1.683

1.71

7

15

8

44

1000

10

O1[C@@]([H])([C@]([H])([C@@]([H])([C@]([H])([C@]1([H])C([H])([H])O[C@]1([H])[C@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])[H])O1)O[H])O[H])O[H])O[H])O[H])O[H])OC1C([H])=C2C(C(C([H])=C(C3C([H])=C([H])C(=C([H])C=3[H])OC([H])([H])[H])O2)=O)=C(C=1OC([H])([H])[H])O[H]

DUXQKCCELUKXOE-CBBZIXHGSA-N

InChI=1S/C29H34O15/c1-11-20(31)23(34)25(36)28(41-11)40-10-18-21(32)24(35)26(37)29(44-18)43-17-9-16-19(22(33)27(17)39-3)14(30)8-15(42-16)12-4-6-13(38-2)7-5-12/h4-9,11,18,20-21,23-26,28-29,31-37H,10H2,1-3H3/t11-,18+,20-,21+,23+,24-,25+,26+,28+,29+/m0/s1

5-hydroxy-6-methoxy-2-(4-methoxyphenyl)-7-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxymethyl]oxan-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 : ~31.25 mg/mL (~50.20 mM)

Solubility in Formulation 1: 2.08 mg/mL (3.34 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.34 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 20.8 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: ≥ 2.08 mg/mL (3.34 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 20.8 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.)