Natural occurring flavonoid; antioxidant

Isoquercetin is widely distributed throughout plants. At 10 μM, seven isoquercitrin compounds considerably reduced the negative effects of H2O2 on RGC-5 cells. Cell viability increased from 63% to 83% at 10 and 50 μM, respectively, in the presence of H2O2. At the same concentration, isoquercetin (50 μM) outperforms EGCG as a neuroprotective drug [1].

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The shrub Thuja orientalis is extensively used as a herbal medicine in Korea and China. In the present study extracts of the plant were subjected to fractionation and purification, with seven compounds (myricitrin, isoquercitrin, hypoletin-7-O-β-d-xylopyranoside, quercitrin, kaempferin, kaempferol, and amentoflavone) being isolated. Of these seven compounds, isoquercitrin was found to be the most effective at attenuating the death of RGC-5 cells in culture caused by exposure to hydrogen peroxide (H2O2). It was found that an insult of H2O2 to RGC-5 cells caused them to die by apoptosis, demonstrated not only by staining dead cells for phosphatidylserine but also by the up-regulation (cleaved PARP, AIF, p53) and down-regulation (Bcl-2) of proteins associated with apoptosis and survival. Subsequent studies showed that isoquercitrin acts as a powerful antioxidant. It scavenges ROS generally as demonstrated by staining of cultures as well as the generation of individual radical species (H2O2, OHradical dot and O2radical dot−). Moreover, isoquercitrin reduced the depletion of glutathione (GSH) caused by elevation of specific radical species (H2O2, OHradical dot and O2radical dot−) in RGC-5 cells in culture and blunted the decrease in catalase and glutathione peroxidase 1 (Gpx-1) caused by exposure of RGC-5 cells to H2O2. Furthermore, isoquercitrin potently attenuated the lipid peroxidation of rat brain homogenates initiated by nitric oxide, with an IC50 value of 1.04 μM. Since isoquercitrin can be tolerated when taken orally it is suggested that this substance might reach the retina and therefore be potentially useful for treating glaucoma, in which oxidative stress is thought to play a major role in the demise of retinal ganglion cells.[1]

When animals received isoquercetin, the number of eosinophils in their blood, lung parenchyma, and BALF decreased. Only the animals treated with isoquercetin showed decreased blood neutrophil counts and lung homogenates' levels of IL-5. The quantity of monocytes did not alter [2].

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Objective: Eosinophils and cytokines are implicated in the pathogenesis of allergic diseases. In the present study, we investigate the anti-inflammatory effect of quercetin and isoquercitrin in a murine model of asthma.
Methods: BALB/c mice were immunized (ovalbumin/aluminum hydroxide, s. c.), followed by two intranasal ovalbumin challenges. From day 18 to day 22 after the first immunization, the mice received daily gavages of isoquercitrin (15 mg/kg) or quercetin (10 mg/kg). Dexamethasone (1 mg/kg, s. c.) was administered as a positive control. Leucocytes were analyzed in bronchoalveolar lavage fluid (BALF), blood and pulmonary parenchyma at 24 h after the last ovalbumin challenge. Interleukin-5 (IL-5) was analyzed in BALF and lung homogenates.
Results: In animals receiving isoquercitrin or quercetin, eosinophil counts were lower in the BALF, blood and lung parenchyma. Neutrophil counts in blood and IL-5 levels in lung homogenate were lower only in isoquercitrin-treated mice. No alterations in mononuclear cell numbers were observed.
Conclusion: Quercetin and isoquercitrin are effective eosinophilic inflammation suppressors, suggesting a potential for treating allergies [2].

Cell viability [2]
Cell viability was assessed using the MTT reduction assay modified from that of Mosmann (1983). Briefly, medium was removed from cells (in 96-well plates) and 100 μl fresh culture medium containing MTT were added at a final concentration of 0.5 mg/ml of MTT and left to incubate for 1 h at 37 °C. The medium was then removed and reduced MTT (blue formazan product) was solubilized by adding 100 μl of DMSO to each well. After agitation of the plates for 15 min, the optical density of the solubilized formazan product in each well was measured using a spectrophotometer with a 570 nm test wavelength and a 690 nm reference wavelength.

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Analysis for cell viability by propidium iodide (PI) and Hoechst 33342 [2]
Cell death was characterized by incubating cells with 8 μM Hoechst 33342 and 1.5 μM PI for 30 min at 37 °C (Hong et al., 2009). Cells were then washed twice with serum-free media and examined under fluorescence microscopy.

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Assessment of reactive oxygen species (ROS) [2]
Cells were assessed for the production of ROS using the dye DHE (Carter et al., 1994). After different treatments, cells on coverslips were stained with DHE (10 μg/ml) by incubating for 30 min at 37 °C in culture medium in a humidified chamber, and then fixed with 4% paraformaldehyde for 20 min. After washing in Tris-phosphate buffer (PBS), coverslips were mounted in PBS containing 1% glycerol and ROS were detected by their red fluorescence using a Zeiss epifluorescence microscope.
To quantify intracellular ROS, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) was used as a radical probe (Shimazawa et al., 2009). In this procedure radical species (H2O2, OHradical dot and O2radical dot−) oxidize nonfluorescent dichlorofluorescein (DCFH) to fluorescent dichlorofluorescein (DCF). Cells were pretreated with isoquercitrin or epigallocatechin 3-gallate (EGCG) for 1 h, and then DCFH-DA (10 μM) was added and the incubation continued for 20 min at 37 °C. Thereafter, the cell culture medium was replaced with fresh medium to which was added 1 mM H2O2 (H2O2 radical), 1 mM H2O2 plus 100 μM iron(II) perchlorate hexahydrate (hydroxyl radical) or KO2 at 1 mM (O2radical dot−). After an incubation period of 30 min at 37 °C, fluorescence was measured using excitation/emission wavelengths of 485/535 nm.

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Lipid peroxidation assay [2]
The procedure used was as described in previous studies (Zhang and Osborne, 2006). Following decapitation of a Wistar rat, the cerebral cortex was immediately homogenized in 10 volumes of ice-cold 0.9% saline (pH 7.0), centrifuged at 1000 × g for 10 min at 4 °C and the supernatant used for the lipid peroxidation assay by determining the amount of thiobarbituric acid reactive species (TBARS) formed. Briefly, aliquots of supernatant (0.5 ml) were preincubated at 37 °C for 5 min with 0.3 ml of 0.9% saline (pH 7.0) containing various concentrations of isoquercitrin or vehicle. Lipid peroxidation was initiated by the addition of 0.2 ml of 20 μM SNP or buffer. After 30 min of incubation at 37 °C, the test tubes were placed on ice to stop the lipid peroxidation reaction. TBARS were then determined as described by Ohkawa et al. (1979), by measuring coloured products resulting from the reaction of thiobarbituric acid, thought to be mainly malondialdehyde (a compound formed during lipid peroxidation) (Ohkawa et al., 1979). Briefly, the colour reaction was developed by the sequential addition of 0.2 ml 8.1% w/v sodium dodecyl sulphate, 1.5 ml 20% v/v acetic acid (pH 3.5) and 1.5 ml 0.8% w/v thiobarbituric acid. This mixture was incubated for 30 min in a boiling water bath. After allowing to cool, 2 ml of n-butanol:pyridine (15:1 v/v) was added and the reaction mixture was then centrifuged at 4000 × g for 10 min. Absorbance of the organic layer (at the top of the tube) was measured at 532 nm wavelength and the amount of TBARS determined using a standard curve of the malondialdehyde derivative 1,1,3,3-tetraethoxypropane (1–30 nmol). The protein concentration in whole-brain homogenate supernatant was determined using a bicinchoninic acid protein assay kit with bovine serum albumin as the standard.

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Western blot analysis [2]
The RGC-5 cells (2.0 × 104) were seeded in 60 mm2 Petri-dishes. After incubation for 24 h, the dishes were rinsed with serum-free medium, and exposed to 300 μM H2O2 in the presence or absence of 10 μM isoquercitrin for 24 h. Cells were then scraped off, suspended in lysis buffer (1 M Tris pH 7.4, 2 M NaCl, 1 M EDTA, 10% NP40, and protease inhibitor cocktail), briefly sonicated and centrifuged at 12,000 × g for 30 min at 4 °C. The protein content was then determined using a Bio-Rad Protein Assay kit.

Treatment with quercetin and isoquercitrin [2]
In order to determine the therapeutic effects of quercetin and isoquercitrin in a murine model of asthma, the following protocol was adopted. Mice immunized (on days 0 and 7) and challenged (on day 14 and 21) with ovalbumin as described above were randomly divided into 5 groups, designated Ova-only (untreated mice), Ova+vehicle (mice receiving oral propylene glycol:water, 1:1), Ova+dexamethasone (mice receiving subcutaneous injections of dexamethasone), Ova+isoquercitrin (mice receiving oral isoquercitrin) and Ova+quercetin (mice receiving oral quercetin). Equimolar amounts of quercetin (10 mg/kg) or isoquercitrin (15 mg/kg), or vehicle (propylene glycol:water, 1:1) were administered daily by gavage (0.3 mL) to each group of mice (n = 6) from days 18 to 22 after the fi rst immunization. One group treated with subcutaneous injections of dexamethasone (1 mg/kg, 0.25 mL) was used as a positive control of anti-infl ammatory activity.

[1]. Isoquercitrin is the most effective antioxidant in the plant Thuja orientalis and able to counteract oxidative-induced damage to a transformed cell line (RGC-5 cells). Neurochem Int. 2010 Dec;57(7):713-21.

[2]. Anti-inflammatory activity of quercetin and isoquercitrin in experimental murine allergic asthma. Inflamm Res. 2007 Oct;56(10):402-8.

Quercetin 3-O-beta-D-glucofuranoside is a quercetin O-glucoside in which a glucofuranosyl residue is attached at position 3 of quercetin via a beta-glycosidic linkage. It has a role as a metabolite. It is a beta-D-glucoside, a quercetin O-glucoside, a monosaccharide derivative and a tetrahydroxyflavone.
Isoquercitrin is under investigation in clinical trial NCT04622865 (Masitinib Combined With Isoquercetin and Best Supportive Care in Hospitalized Patients With Moderate and Severe COVID-19).
3-(((2S,3R,4R,5R)-5-((R)-1,2-Dihydroxyethyl)-3,4-dihydroxytetrahydrofuran-2-yl)oxy)-2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-chromen-4-one has been reported in Camellia sinensis, Caragana spinosa, and other organisms with data available.
Quercetin 3-O-glucoside is a metabolite found in or produced by Saccharomyces cerevisiae.

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In conclusion, it was demonstrated that the major compound associated with T. orientalis that blunts the negative effect of H2O2 on RGC-5 cells in culture is isoquercitrin. The neuroprotective effect of isoquercitrin seems to be related primarily to the antioxidant characteristics of the substance. Many other flavonoids (e.g. EGCG, baicalin) also have powerful antioxidant properties and attempts have been made to establish whether a relationship exists between the structure and antioxidative activity of different flavonoids (Dugas et al., 2000, Lien et al., 1999, Rice-Evans et al., 1996). One possible reason why isoquercitrin is such a powerful antioxidant might be due to it having a catechol group in its B ring, which would allow for better electron donating and thus efficient metal chelation and electron delocalization (Yang et al., 2001). However, it should be noted that flavonoids have a variety of functions, with some having no apparent antioxidant activity (e.g. genistein). The unique herbal properties of T. orientalis that allow the successful treatment of conditions such as gout, rheumatism, diarrhoea and chronic tracheitis (Zhu et al., 2004) are undoubtedly partly related to the constituent isoquercitrin. Since isoquercitrin can be taken orally and possibly crosses the blood–brain barrier to reach the retina (Paulke et al., 2008, Paulke et al., 2006), its use for treating glaucoma to attenuate ganglion cell death is certainly worthy of consideration.[1]

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A variety of pharmacological agents, such as PAF receptor antagonists and 5-lipoxygenase inhibitors, have been shown to have inhibitory effects on in vivo eosinophil accumulation. In addition, it has been demonstrated that quercetin and isoquercitrin, respectively, reduce PAF-induced and leukotriene-induced bronchoconstriction in a murine model of asthma. Therefore, it is possible that quercetin and isoquercitrin inhibit PAF and leukotriene production in cells. The results of the present study show that quercetin and isoquercitrin both attenuate infl ammation in a murine model of asthma. The effects described herein, as well as those observed by others investigators, together with the broad spectrum of the biological effects of these substances, strongly suggest that quercetin and isoquercitrin have therapeutic potential for the treatment of asthma and other allergic diseases. Therefore, the study of these two substances could lead to the development of an effective anti-asthma therapy or of a means of identifying novel anti-asthma targets.[2]

C21H20O12

464.3763

464.095

C, 54.32; H, 4.34; O, 41.34

21637-25-2

482-35-9 (EMIQ); 482-35-9 (Isoquercetin); 17-39-5 (quercetin); 21637-25-2 (Isoquercitrin)

5484006

Off-white to yellow solid powder

1.9±0.1 g/cm3

872.6±65.0 °C at 760 mmHg

225-227°

307.5±27.8 °C

0.0±0.3 mmHg at 25°C

1.803

1.75

8

12

5

33

758

5

C1=CC(=C(C=C1C2=C(C(=O)C3=C(C=C(C=C3O2)O)O)O[C@H]4[C@@H]([C@H]([C@H](O4)[C@@H](CO)O)O)O)O)O

OPJZLUXFQFQYAI-GNPVFZCLSA-N

InChI=1S/C21H20O12/c22-6-12(27)19-16(29)17(30)21(32-19)33-20-15(28)14-11(26)4-8(23)5-13(14)31-18(20)7-1-2-9(24)10(25)3-7/h1-5,12,16-17,19,21-27,29-30H,6H2/t12-,16-,17-,19-,21+/m1/s1

3-[(2S,3R,4R,5R)-5-[(1R)-1,2-dihydroxyethyl]-3,4-dihydroxyoxolan-2-yl]oxy-2-(3,4-dihydroxyphenyl)-5,7-dihydroxychromen-4-one

21637-25-2; Isotrifolin; Isoquercitroside; 0YX10VRV6J; CCRIS 7093; 3,3',4',5,7-Pentahydroxyflavone 3-beta-D-glucofuranoside; EINECS 244-488-5; quercetin 3-O-beta-D-glucofuranoside;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~125 mg/mL (~269.18 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.48 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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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.48 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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 (Please use freshly prepared in vivo formulations for optimal results.)