IL-1β IL-10

Effect of Kaempferol-3-O-β-d-glucuronide (K3G) on LPS-induced production of pro-inflammatory cytokines and cell viability[1]
The concentrations of IL-1β released by the LPS stimulated RAW264.7 macrophages and Balb/c mice decreases with increase in the concentration of K3G. Fig. 3(A–D) indicates that K3G significantly decreased the concentration of IL-1β in RAW 264.7 cells being 205.7 pg/mL for 10 μM and 217.4 pg/mL at 5 μM respectively. K3G didn,t affect the viability of RAW 264.7 cells (Fig. 4). In Balb/c mice, the value of IL-1β is 173.8 pg/mL for 2 mg/kg and 73.6 pg/mL for 5 mg/kg. The value of IL-10 indicating anti-inflammatory activity increased with the concentration of K3G in both LPS treated RAW 264.7 cells and mice model and the results are quite comparable to that of the standard drug dexamethasone. The results indicate that K3G treatment in LPS-stimulated cells significantly inhibited the levels of IL-1β. Maximum inhibition was observed at 10 μM. K3G inhibited gene expression of TNF-α and IL-6 in RAW 264.7 cells with reference to housekeeping gene, GAPDH when studied through RT-PCR. Nevertheless, the three concentrations (1.0, 5.0 and 10.0 μM) were selected for subsequent assays. K3G significantly inhibited pro-inflammatory cytokines.

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Effects of K3G on LPS-induced NO, PGE2 and LTB4 production[1]
The inhibitory effect of K3G on the production of NO, PGE2 and LTB4 was investigated in LPS-activated RAW 264.7 cells. The results showed that they were also inhibited in a dose-dependent manner with maximum inhibition at a concentration of 10 μM and is 47.2%, 460.2 pg/mL and 678.6 pg/mL for NO, PGE2 and LTB4 respectively. The inhibition of K3G for all these inflammatory mediators was quite comparable to that of positive control drug dexamethasone 51.9%, 460.2 pg/mL and 541.9 pg/mL for NO, PGE2 and LTB4 respectively (Fig. 6A–C). K3G significantly inhibited gene expression of COX-2 and iNOS in RAW 264.7 cells with reference to housekeeping gene, GAPDH when studied through RT-PCR (Fig. 5B).

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Effects of K3G on LPS-induced NF-κB p65 expression[1]
RAW 264.7 cells were pretreated with K3G (5 and 10 μM) for 1 h and stimulated with LPS (1 µg/mL) for 24 h. Effect on phosphorylation of NF-kB p65 was studied in comparison to dexamethasone (0.5 μM). It is clear from Fig. 9 that treatment with LPS increased the phosphorylation of NF-κB p65 in the RAW 264.7 cells. However, pretreatment with K3G considerably inhibited this phosphorylation.

Effect of K3G on body weight and lymphoid organ weight[1]
K3G doses didn't show any undesirable changes in treated animals. Table 2 shows that the changes in weight of spleen and kidney were insignificant when compared to control (normal saline treated) (Group I). At doses of 1 and 2 mg/kg, there was slight increase in relative organ weight of thymus but no effect at 5 mg/kg. In case of relative organ weight of liver, a significant increase was observed at doses of 2 and 5 mg/kg while slight at 1 mg/kg. To check whether the increase in liver weight is not due to any abnormality, liver function tests (LFT) were performed and the results show no negative signs (Table 3).

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Histopathology[1]
Microscopic examination of liver of vehicle treated animals showed central vein and normal hepatocytic architecture. Liver of LPS toxicated mice (Fig. 10) showed central vein surrounded by chronic inflammation with focal hepatocytic necrosis. The inflammatory infiltrate is rich in lymphocyte and few neutrophils in the perivascular region. K3G at a concentration of 2 mg/kg (Fig. 10C) showed normalization of necrosis, vascular conjection and declined neutrophil infiltration. At 5 mg/kg (Fig. 10C), the morphology is nearly normal.

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Carrageenan-induced paw oedema and phagocytic index[1]
The effect of K3G on carrageenan induced paw oedema was measured over a period of 10 h at doses 2 and 5 mg/kg (Fig. 8). The maximum inhibition of 48% was observed at 5 mg/kg. The results were comparable to the standard drug dexamethasone. The phagocytic index (Table 4) also show that phagocytosis increased with increase in the concentration of K3G and is significant when compared with the standard drug dexamethasone.

Determination of PGE2 & LTB4 production[1]
RAW 264.7 cells (2 × 105 cells/well) were treated with K3G for 1 h and induced with LPS (1 μg/mL) for 24 h. The inhibitory effect of each concentration of K3G on the production of PGE2 & LTB4 from the LPS treated RAW 264.7 cells was determined. Supernatants were then harvested and assayed for both the mediators by ELISA. PGE2 and LTB4 concentration was quantified using a competitive enzyme immunoassay kits according to the manufacturer's instructions. The inhibition of PGE2 and LTB4 was measured relative to that of the control treatment. All experiments were performed in triplicate.

Effect of K3G on cytokine production in RAW264.7 cells[1]
The inhibitory effect of K3G on the production of IL-1β and IL-10 were determined by mouse enzyme-linked immunosorbent assay kit (ELISA). RAW 264.7 cells were seeded at a density of 2 × 105 cells/well in 96-well plate and incubated overnight. The cells were treated with K3G for 1 h followed with addition of 1 μg/mL LPS for 24 h. The supernatants were assayed according to the manufacturer protocol (Invitrogen). The experiment was carried out in triplicate.

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MTT assay for measurement of cell viability[1]
MTT staining method was used to assess cell viability. RAW 264.7 cells (16,000 cells/well) were treated with different concentrations of K3G for 48 h in flat bottomed 96-well microtitre plates. 20 μL MTT solution (2.5 mg/mL) prepared in PBS, pH 7.4 was added 4 h before culture termination. The culture medium was eliminated by adding 100 μL of 100% DMSO to each well to solubilize the formazan. At 570 nm absorbance was measured using Synergy Mx plate reader. LPS induced group is used as control in calculating percentage cell viability.

Effect of K3G on cytokine production in Balb/c mice[1]
Mice were fasted overnight and drugged with K3G at different concentrations (1 mg/kg, 2 mg/kg and 5 mg/kg) for 1 h and LPS (0.5 mg/kg) was administered via i.p. Blood was obtained after 3 h from the retro-orbital plexus. The collected serum was stored at -80 degree until use for ELISA to study the inhibition of IL-1β and IL-10 cytokines using Invitrogen (Mouse) kits.

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Efficacy of K3G against carrageenan induced paw oedema[1]
30 Balb/c mice were divided in 6 groups comprising 5 animals each. Mice were fasted overnight and treated with different doses of K3G (2 mg/kg and 5 mg/kg). After 1 h 1% Carrageenan solution (100 μL) in PBS was injected at subplantar region of left hind paw of all animal groups. Volume of the injected paw was measured using plethysmometer at 1 h, 3 h, 6 h, 8 h and 10 h. The percentage inhibition of paw oedema was calculated by using the following formula: Vc — volume of paw oedema in control groupVt — volume of paw oedema in treated group.

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Macrophage phagocytosis by carbon clearance method[1]
Balb/c mice were divided into five groups of five mice each. Drugs were administered in various groups as shown in Table 4 for five days. On day six, all the groups were given 0.1 mL of carbon ink suspension through the tail vein. Blood was collected from the retro-orbital plexus at 0 and 30 min immediately after the injection of carbon suspension. Blood was lysed with 2 mL of 0.1% sodium carbonate and the absorbance was measured at 675 nm. The rate of carbon clearance, termed as phagocytic index, was calculated by using equation

[1]. Kaempferol-3-o-β-d-glucuronate exhibit potential anti-inflammatory effect in LPS stimulated RAW 264.7 cells and mice model. Int Immunopharmacol. 2018;57:62-71.

[2]. Absorption of kaempferol from endive, a source of kaempferol-3-glucuronide, in humans. Eur J Clin Nutr. 2004;58(6):947-954.

Kaempferol 3-O-glucuronide is a kaempferol O-glucuronide that is kaempferol with a beta-D-glucosiduronic acid residue attached at the 3-position. It has a role as a metabolite. It is a kaempferol O-glucuronide and a trihydroxyflavone.
Kaempferol 3-glucuronide has been reported in Myrothamnus flabellifolia, Diospyros cathayensis, and other organisms with data available.

C21H18O12

462.36

462.079

C, 54.55; H, 3.92; O, 41.52

22688-78-4

5318759

White to yellow solid powder

1.9±0.1 g/cm3

876.8±65.0 °C at 760 mmHg

189-191 °C

309.8±27.8 °C

0.0±0.3 mmHg at 25°C

1.789

2.3

7

12

4

33

790

5

FNTJVYCFNVUBOL-UHFFFAOYSA-N

InChI=1S/C21H18O12/c22-8-3-1-7(2-4-8)17-18(13(25)12-10(24)5-9(23)6-11(12)31-17)32-21-16(28)14(26)15(27)19(33-21)20(29)30/h1-6,14-16,19,21-24,26-28H,(H,29,30)

6-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid

22688-78-4; Kaempferol 3-glucuronide; Kaempferol-O-glucuronide; Kaempferol 3-O-; A-D-glucuronide; Kaempferol 3-O-beta-D-glucuronide; Kaempferol-3-beta-O-glucuronide; Kaempferol 3-O-glucuronide;

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 (216.28 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.41 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 (5.41 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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 (Please use freshly prepared in vivo formulations for optimal results.)