Natural flavonoid

Quercetin (5-50 μM; 24-72 h) gradually raises the toxicity of colorectal cancer cells by inhibiting their growth [1]. In DLD-1 cells, quercetin (5-50 μM; 24-72 h) and quercetin (50 μM; 48-72 h) cause cellular inflammation and disruption of the mitochondrial membrane potential. The outer leaflet receives the translocation of phospholipid serine (PS) from the inner leaflet [1].

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Quercetin, which is the most abundant bioflavonoid compound, is mainly present in the glycoside form of quercitrin. Although different studies indicated that quercitrin is a potent antioxidant, the action of this compound is not well understood. In this study, we investigated whether quercitrin has apoptotic and antiproliferative effects in DLD-1 colon cancer cell lines. Time and dose dependent antiproliferative and apoptotic effects of quercitrin were subsequently determined by WST-1 cell proliferation assay, lactate dehydrogenase (LDH) cytotoxicity assay, detection of nucleosome enrichment factor, changes in caspase-3 activity, loss of mitochondrial membrane potential (MMP) and also the localization of phosphatidylserine (PS) in the plasma membrane. There were significant increases in caspase-3 activity, loss of MMP, and increases in the apoptotic cell population in response to quercitrin in DLD-1 colon cancer cells in a time- and dose-dependent manner. These results revealed that quercitrin has antiproliferative and apoptotic effects on colon cancer cells. Quercitrin activity supported with in vivo analyses could be a biomarker candicate for early colorectal carcinoma[1].

Quercetin (50 and 100 mg/kg; gavage, once) shows effective protective effects against brain injury in mice by inhibiting carbon tetrachloride-induced oxidative intermediates and activation [2].

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Quercitrin is one of the primary flavonoid compounds present in vegetables and fruits. The aim of the present study was to evaluate the effects of quercitrin against carbon tetrachloride (CCl4) induced brain injury and further to elucidate its probable mechanisms. ICR mice received CCl4 intraperitoneally with or without quercitrin co-administration for 4 weeks. Our data showed that quercitrin significantly suppressed the elevation of reactive oxygen species (ROS) production and malondialdehyde (MDA) content, reduced tissue plasminogen activator (t-PA) activity, enhanced the antioxidant enzyme activities and abrogated cytochrome P450 2E1 (CYP2E1) induction in mouse brains. Quercitrin also prevented CCl4 induced cerebral function disorders associated with its ability to inhibit the activities of monoamine oxidase (MAO), acetylcholine esterase (AChE) and the N-methyl-d-aspartate receptor 2B subunit (NR2B). In addition, western blot analysis showed that quercitrin suppressed the release of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). Taken together, our findings suggested that quercitrin may be a potential candidate to be developed as a neuroprotective agent [2].

Biochemical analysis [2]
ROS was measured as described previously, based on the oxidation of 2′7′-dichlorodihydrofluorescein diacetate to 2′7′-dichloro-fluorescein. ROS formation was quantified from a DCF-standard curve and data are expressed as pmol DCF formed per min per mg protein. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), monoamine oxidase (MAO) and acetylcholine esterase (AChE) in the hippocampus homogenates were measured using the commercial kits. The malondialdehyde (MDA) concentration in the hippocampus homogenates was measured by the thiobarbituric acid reactive substances (TBARS) assay in accordance with the manufacturer's instructions and the absorbance was measured at a wavelength of 532 nm.

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Functionally active t-PA activity assay [2]
Functionally active t-PA was determined using the active t-PA ELISA kit according to the manufacturer's instructions. The amount of color development is directly proportional to the concentration of active t-PA in the sample.

Cell proliferation assay [1]
Cell Types: DLD-1 colon cancer cell line
Tested Concentrations: 5, 10, 25 and 50 μM
Incubation Duration: 24, 48 and 72 hrs (hours)
Experimental Results: Colorectal cancer cell proliferation diminished in a time- and dose-dependent manner.

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Apoptosis analysis [1]
Cell Types: DLD-1 colon cancer cell line
Tested Concentrations: 50 μM
Incubation Duration: 48 and 72 hrs (hours)
Experimental Results: Apoptosis was induced, and caspase-3 enzyme activity increased in a time- and dose-dependent manner.

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Measurement of Cell Growth and Cytotoxicity [1]
To detect the effect of quercitrin on cell viability after treatment, a WST-1 cell proliferation assay was performed. The WST-1 conversion assay is based on the mitochondrial function of intact cells that enables them to metabolise the stable tetrazolium salt WST-1 (4-[3- (4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3- benzene disulfonate) to a soluble violet formazan product. Quanities of 1x104 cells/well were seeded into 96-well plates containing 100 μl of the growth medium in the absence or presence of increasing concentrations of quercitrin and then incubated at 37 °C in 5 % CO2 for 24, 48 and 72 h. After the incubation period, cells were treated with 10 μl of WST-1 for 4 h. Dye accumulation was measured at 450 nm using a Multiscan ELISA reader. Viability was calculated by subtraction of the mean values without WST-1 from those with WST-1 substrate and was expressed as a percentage of control. Data was confirmed by three other independent experiments. Cytotoxic effects of quercitrin in the dose and time dependent manner were colorimetrically determined with a “CytoTox 96R Non-Radioactive Cytotoxicity Assay” kit. Cell treatment to prepare for cytotoxicity test was done as described for the WST-1 assay. Culture medium (10 μl) was then transferred to a 96- well microtiter plate. The levels of lactate dehydrogenase (LDH) were determined by adding 50 μl fresh substrate mix, incubating in a dark at room temperature for 30 min, then adding 50 μl stop solution, and measuring optical density (OD) at 490 nm with a microplate reader. The natural color of chemicals at 490 nm was corrected by subtracting the OD values of the corresponding chemical×concentration medium that were treated and measured in triplicates in the same manner as with the cells. Data was confirmed by three other independent experiments.

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Detection of Apoptotic Nucleosomes and Necrotic DNA Release [1]
The Cell Death Detection Elisa plus (CDDE) kit is a photometric enzyme-immunoassay to determine the quality and quantity of DNA in terms of necrosis and cytoplasmic histone-associated DNA fragments (mono- and oligonucleosomes) related to apoptosis in the supernatant of treated cells. Application according to manufacturer’s instruction provides concurrent detection of apoptosis and necrosis in the same well. Colour development of samples was determined by enrichment factor of the amount of DNA fragments in cytoplasm or cell supernatant indicating apoptosis or necrosis, respectively, and expressed relative to untreated cells. In order to investigate possible interference with the assay, particles were added to the cells as two fold higher than the final concentration used in above experiment (160 μg/cm2 ). Subsequently, the samples were centrifuged (10 min, 200 g) conformance with the CDDE kit protocol. The supernatant was then mixed 1:1 with lysate of 50 μm quercitrin treated cells for 48 and 72 h using the lysis buffer included in the kit, and analysed according to manufacturer’s instructions.

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Determination of Changes in Mitochondrial Membrane Potential [1]
We also examined the loss of MMP, another important sign of apoptosis, in response to quercitrin treatment for 48 h in DLD1 and MRC5 cells using the JC-1 Mitochondrial Membrane Potential Detection Kit. This kit uses JC-1, a unique cationic dye, to signal the loss of the MMP. JC-1 accumulates in the mitochondria which stain red in nonapoptotic cells, while in apoptotic cells the MMP collapses, and thus the JC-1 remains in the cytoplasm as a monomer that stains green under fluorescent light. The cells (5×105 cells/2 mL) were induced to undergo apoptosis and collected by centrifugation at 1,000 rpm for 10 min. Supernatants were removed, pellets were homogenized by 200 μl of medium, and 20 μl of JC-1 dye was added onto the cells; then, the cells were incubated at 37 °C in 5 % CO2 for 30 min. Then, they were centrifuged at 400 g for 5 min, supernatants were removed, and 200 μl of assay buffer was added onto the pellets and vortexed. Then, this step was repeated once more. Afterwards, all pellets were resuspended with 320 μl assay buffer and 100 μl from each of them was added into the 96-well plate as triplicates. In healthy cells, the aggregate in red form has absorption/emission maxima of 560/595 nm, whereas in apoptotic cells the monomeric green form has absorption/emission maxima of 485/535 nm. The plate was read at these wavelengths using a fluorescence Elisa reader. The green/red (485/560) values were calculated to determine the changes in MMP.

Animal/Disease Models: Male ICR mice carbon tetrachloride (CCl4)-induced brain injury [2]
Doses: 50 and 100 mg/kg
Route of Administration: po (oral gavage); 50 and 100 mg/kg, once
Experimental Results: Dose-dependent Dramatically diminished the levels of ROS and malondialdehyde (MDA) concentration in hippocampal homogenates, and also dose-dependently diminished the levels of CYP2E1 in the brain. Increase the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). Inhibits N-methyl-D-aspartate receptor 2B subunit (NR2B) levels and the activities of monoamine oxidase (MAO) and acetylcholinesterase (AChE) in mouse brain.

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Male ICR mice (20–25 g) were maintained in an animal facility at 20–22 °C under 40–60% relative humidity and a 12/12 h (light/dark) cycle. After acclimation for 1 week, forty mice were randomly divided into four groups. Brain injury was induced by an intraperitoneal (i.p.) injection of 2 ml of CCl4 in olive oil (1 : 1, v/v) per kg body weight twice weekly for up to 4 weeks.13 Mice in Group 1 were given two weekly injections of peanut oil (vehicle control) and received water containing 0.5% carboxymethylcellulose sodium by oral gavage; mice in Group 2 were injected with CCl4 and received water containing 0.5% carboxymethylcellulose sodium by oral gavage; mice in Group 3 and Group 4 were injected with CCl4, as in group 2, and administered with quercitrin (50 and 100 mg per kg body-weight, suspended in 0.5% carboxymethylcellulose sodium daily) by oral gavage, respectively. The choice of quercitrin dose is based on previous findings.[2]

Metabolism / Metabolites
YIELDS QUERCETIN IN ASPERGILLUS; WESTLAKE DWS, CAN J MICROBIOL 9: 211 (1963); YIELDS QUERCITRIN QUINONE PROBABLY IN POLYPORUS; PICKARD MA & WESTLAKE DWS, CAN J BIOCHEM 48: 1351 (1970). /FROM TABLE/
Quercitrin has known human metabolites that include L-Rhamnose, Quercetin, and alpha-L-Rhamnose.

5280459 mouse LD50 intraperitoneal 200 mg/kg National Technical Information Service., AD277-689
5280459 rabbit LD intravenous >150 mg/kg Journal of the American Pharmaceutical Association, Scientific Edition., 41(119), 1952

[1]. Apoptotic Effects of Quercitrin on DLD-1 Colon Cancer Cell Line. Pathol Oncol Res. 2015 Apr;21(2):333-8.

[2]. Quercitrin offers protection against brain injury in mice by inhibiting oxidative stress and inflammation. Food Funct. 2016 Jan;7(1):549-56.

Quercitrin is a quercetin O-glycoside that is quercetin substituted by a alpha-L-rhamnosyl moiety at position 3 via a glycosidic linkage. It has a role as an antioxidant, an antileishmanial agent, an EC 1.1.1.184 [carbonyl reductase (NADPH)] inhibitor, an EC 1.1.1.21 (aldehyde reductase) inhibitor, an EC 1.14.18.1 (tyrosinase) inhibitor and a plant metabolite. It is a monosaccharide derivative, a tetrahydroxyflavone, an alpha-L-rhamnoside and a quercetin O-glycoside. It is a conjugate acid of a quercitrin-7-olate.
Quercitrin has been reported in Persicaria muricata, Camellia sinensis, and other organisms with data available.

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In conclusion, taken together, all these data showed the antiproliferative and apoptotic effects of quercitrin on colon cancer cells. Based on the above results, quercitrin appears to activate specific intracellular death-related pathways in DLD1 cells, leading to a loss of mitochondrial cell potential and inducing apoptosis. Our findings suggest the possible use of quercitrin against human colon cancer[1]

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In conclusion, this is the first report on the fact that quercitrin has protective effects on brain injury due to exposure to CCl4. The primary mechanisms of this effect could be due to attenuation of brain oxidative stress, suppression of inflammation, and amelioration of neurotransmitter dysfunction (Fig. 8). Quercitrin decreased the activities of ROS, CYP2E1, MDA, t-PA, NR2B, AChE and MAO, and increased the activities of antioxidant enzymes in mouse brains exposed to CCl4. Quercitrin also suppressed the levels of key pro-inflammatory cytokines triggered by an inflammatory stimulus. Overall, despite the positive effects of quercitrin on CCl4-induced brain injury, more in-depth mechanistic studies are needed to support these beneficial effects.[2]

C21H20O11

448.38

448.1

C, 56.25; H, 4.50; O, 39.25

522-12-3

5280459

Light yellow to yellow solid powder

1.8±0.1 g/cm3

814.0±65.0 °C at 760 mmHg

174-183ºC

288.3±27.8 °C

0.0±3.1 mmHg at 25°C

1.776

2.17

7

11

3

32

741

5

C[C@H]1[C@@H]([C@H]([C@H]([C@@H](O1)OC2=C(OC3=CC(=CC(=C3C2=O)O)O)C4=CC(=C(C=C4)O)O)O)O)O

OXGUCUVFOIWWQJ-HQBVPOQASA-N

InChI=1S/C21H20O11/c1-7-15(26)17(28)18(29)21(30-7)32-20-16(27)14-12(25)5-9(22)6-13(14)31-19(20)8-2-3-10(23)11(24)4-8/h2-7,15,17-18,21-26,28-29H,1H3/t7-,15-,17+,18+,21-/m0/s1

2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxychromen-4-one

Thujin; 522-12-3; Quercetin 3-rhamnoside; Quercitroside; Quercetrin; Quercimelin; Thujin; Quercetin 3-L-rhamnoside; Quercitrin

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 : ~125 mg/mL (~278.78 mM)

Solubility in Formulation 1: ≥ 2.58 mg/mL (5.75 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 25.8 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 (4.64 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.)