Myricetin-3-o-galactoside exhibited cytotoxic effects on human chronic myelogenous leukemia (CML) K562 cell line with an IC50 (median growth inhibitory concentration) value of 220 µg/ml. Cytotoxicity index percentages at various concentrations were: 21% at 50 µg/ml, 41% at 100 µg/ml, 48% at 200 µg/ml, 57% at 400 µg/ml, and 62% at 800 µg/ml [1].
The compound demonstrated anti-lipid peroxidation activity in K562 cells induced by H₂O₂. Inhibition of malondialdehyde (MDA) formation increased with increasing concentrations (110-440 µg/ml), with inhibition rates ranging from 30-92%. The IC50 value for lipid peroxidation inhibition was 160 µg/ml. At 440 µg/ml, the compound displayed 92% MDA inhibition. Cell viability was evaluated to confirm that MDA reduction was due to antioxidant effect rather than cell death [1].
Myricetin-3-o-galactoside showed DPPH radical scavenging activity with percentage decreases in absorbance of 88%, 76%, 80%, 76%, and 18% at concentrations of 100, 30, 10, 3, and 1 µg/ml respectively, with an IC50 value of 2.3 µg/ml [1].
The compound inhibited xanthine oxidase activity in a concentration-dependent manner. At 100 µg/ml, it showed 57% inhibition of uric acid production. An increase in xanthine oxidase activity was observed at 200 and 300 µg/ml, indicating a pro-oxidant effect at higher doses [1].
In the SOS chromotest using E. coli PQ37, Myricetin-3-o-galactoside showed antimutagenic activity against aflatoxin B1 (AFB1, 5 µg/assay with microsomal activation) and nifuroxazide (10 µg/assay without microsomal activation). At concentrations of 2, 10, and 50 µg/assay, it decreased the induction factor (IF) of AFB1 by 96%, 90%, and 80% respectively, and decreased the IF of nifuroxazide by 25%, 61%, and 78% respectively [1].
In the Comet assay using K562 cells, Myricetin-3-o-galactoside (220 µg/ml for 24 h) was not genotoxic, with a tail extent moment (TEM) of 3.79 compared to negative control TEM of 2.64 (no significant difference). The compound reduced H₂O₂-induced (30 µM for 2 h) genotoxicity by 38% at 220 µg/ml [1].
Using cDNA microarray analysis of K562 cells stressed with H₂O₂ (50 µM) and treated with Myricetin-3-o-galactoside (220 µg/ml for 24 h before H₂O₂ exposure), the expression of 39 transcripts was modulated. Thioredoxin peroxidase (AOE372) and thioredoxin (TXN) were more expressed in cells treated with both H₂O₂ and the compound than in cells treated with H₂O₂ only. Glutathione peroxidase 1 (GPX1), selenoprotein W (SEPW1), DNA-3-methyladenine glycosidase (MPG), poly ADP-ribose polymerase (PARP), and Src homology 2 domain containing (SHC1) were less expressed. DNA repair-related transcripts including xeroderma pigmentosum complementation group C (XPC), ligase 4 (LIG4), replication protein 14 kD (RPA3), proliferative cell nuclear antigen (PCNA), and DNA damage inducible transcript 3 (DDIT3) were induced. X-ray repair cross-complementing defective repair in Chinese hamster cell 5 (XRCC5) and DNA polymerase delta catalytic subunit 1 (POLD1) showed reduced expression [1].

Xanthine oxidase inhibition assay: The inhibition of xanthine oxidase activity was measured according to the decrease in absorbance at 290 nm, indirectly determining superoxide formation by measuring uric acid production. The assay mixture consisted of 100 µl of the test compound (100, 200, and 300 µg/ml), 200 µl of xanthine substrate (final concentration 50 µM), hydroxylamine (final concentration 0.2 mM), 200 µl EDTA (0.1 mM) and 300 µl distilled water. The reaction was initiated by adding 200 µl xanthine oxidase (5.5 mU/ml) dissolved in phosphate buffer (0.2 M, pH 7.5). The assay mixture was incubated at 37°C for 30 min. Before measurement of uric acid production at 290 nm, the reaction was stopped by adding 0.1 ml of 0.5 M HCl. Absorbance was measured against a blank solution prepared by replacing xanthine oxidase with phosphate buffer. A control solution without the tested compound was prepared to measure total uric acid production (100% of uric acid production). The dose-effect curve for each test compound was linearized by regression analysis to derive IC50 values. Allopurinol was used as positive control [1].
DPPH radical scavenging assay: Ethanol solutions were prepared containing 100, 30, 10, 3, or 1 µg/ml of the tested compounds and 23.6 µg/ml of DPPH. After incubation of the mixture for 30 min at ambient temperature, the absorbance of the remaining DPPH was determined colorimetrically at 517 nm. The radical scavenging activity was expressed as the decrease of the absorbance of each sample versus DPPH standard solution. Percentage inhibition was calculated as [(OD control - OD sample) / OD control] × 100, where OD control was the initial absorbance and OD sample the value for added sample concentration. The mean inhibiting concentration IC50 was defined as the concentration (µg/ml) causing 50% loss of DPPH activity and was calculated using the Litchfield and Wilcoxon test [1].

Cytotoxicity assay (MTT assay): Human K562 cells were seeded in 96-well plates at 5×10⁴ cells/well and incubated for 24 h at 37°C. After treatment with various concentrations of Myricetin-3-o-galactoside (100, 200, 400, and 800 µg/ml), the cells were incubated for an additional 48 h at 37°C. After incubation, the medium was removed and cells in each well were incubated with 50 µl of MTT solution (5 mg/ml) for 4 h at 37°C. MTT solution was then discarded and 5 µl of dimethyl sulfoxide (DMSO) was added to dissolve insoluble formazan crystals. Optical density was measured at 540 nm. The cytotoxicity index (CI%) was calculated as (1 - T/C) × 100, where T and C represent the mean optical density of the treated group and vehicle control group, respectively [1].
Lipid peroxidation inhibitory activity assay: Cells were exposed to Myricetin-3-o-galactoside at various concentrations (110, 220, and 440 µg/ml in 0.1% DMSO) in the incubation medium for 2 h, followed by incubation with 30 µM H₂O₂ for 2 h. Cells were then washed with cold PBS and homogenized in ice-cold 1.15% KCl. Samples containing 100 µl of cell lysates were combined with 0.2 ml of 8.1% SDS, 1.5 ml of 20% acetic acid adjusted to pH 3.5, and 1.5 ml of 0.8% thiobarbituric acid. The mixture was brought to a final volume of 4 ml with distilled water and heated to 95°C for 120 min. After cooling to room temperature, 5 ml of a mixture of n-butanol and pyridine (15:1, v/v) was added and the mixture was shaken vigorously. After centrifugation at 3000 rpm for 10 min, the supernatant fraction was isolated and the absorbance was measured at 546 nm [1].
Comet assay (single-cell gel electrophoresis): Freshly withdrawn cells (60 µl) treated with 220 µg/ml of Myricetin-3-o-galactoside for 24 h (for genotoxicity study), or with the tested compound for 24 h followed by 30 µM H₂O₂ for 2 h (for antigenotoxic activity study), were added to 60 µl of 0.6% low melting agarose (LMA) in PBS and transferred onto degreased microscope slides previously dipped in 1% normal melting agarose (NMA) for the first layer. The agarose was allowed to set for 10 min at 4°C before addition of a final layer of LMA. After agarose solidification, slides were placed in lysing solution (2.5 M NaCl, 100 mM Na₂EDTA, 10 mM Tris-HCl, 1% Triton X-100, and 10% DMSO, pH 10) at 4°C overnight in the dark. Alkaline DNA-unwinding was carried out in a gel electrophoresis chamber containing freshly prepared buffer (1 mM Na₂EDTA, 300 mM NaOH, pH 13) for 40 min, and electrophoresis was performed in the same buffer for 30 min at 25 V and 300 mM. After electrophoresis, slides were washed in neutralization buffer (0.4 M Tris-HCl, pH 7.5). After staining with 50 µl ethidium bromide (20 µg/ml), observations were made under a fluorescence microscope equipped with an excitation filter BP 515-560 nm and a barrier filter LP 580 nm, using an image-analysis system. For each sample, 100 cells were analyzed, and the tail extent moment (TEM) was used to represent the data on antigenotoxic effects [1].
cDNA microarray for gene expression: K562 cells (about 5×10⁶) treated with 220 µg/ml of Myricetin-3-o-galactoside for 24 h were subcultured in 75 cm³ flasks and stressed by 50 µM H₂O₂ for 2 h. Cells were harvested 12 h after stress exposure, centrifuged (3000 rpm, 10 min at room temperature) and rinsed twice with PBS. Total RNAs were extracted from cell pellets using an RNeasy Mini Kit. Labeled cDNAs were prepared from total RNA by reverse transcription with incorporation of [α³³P] dCTP and oligo dT25 as a primer. Arrays were prehybridized and then hybridized with the labeled RNA probe for 72 h at 60°C. After washing, arrays were placed in a phosphorimager cassette for 48 h. Radioactive signal detection was performed using a phosphorimager. Data normalization was done by dividing all signals with intensity superior to background by the median of all signals [1].

Myricetin-3-o-galactoside showed concentration-dependent cytotoxicity on K562 cells with an IC50 of 220 µg/ml. At concentrations of 200 and 300 µg/ml in the xanthine oxidase assay, the compound exhibited increased xanthine oxidase activity, translating to a pro-oxidant effect at these doses [1].
In the Comet assay, Myricetin-3-o-galactoside (220 µg/ml for 24 h) was not genotoxic, showing no significant difference in tail extent moment (TEM = 3.79) compared to the negative control (TEM = 2.64) [1].

[1]. In vitro antioxidant and antigenotoxic potentials of myricetin-3-o-galactoside and myricetin-3-o-rhamnoside from Myrtus communis: modulation of expression of genes involved in cell defence system using cDNA microarray. Toxicol In Vitro. 2.

Myricetin-3-O-β-D-galactopyranoside is a glycosyloxyflavonoid formed by linking a β-D-galactose residue to the 3-position of myricetin. It is a metabolite. It is a β-D-galactopyranoside, belonging to the monosaccharide derivatives, pentahydroxyflavones, and glycosyloxyflavones. Functionally, it is related to β-D-galactose and myricetin. Myricetin-3-galactopyranoside has been reported to exist in tea (Camellia sinensis), clover (Trifolium pannonicum), and several other organisms with relevant data.

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Myricetin-3-o-galactoside was isolated from the leaves of Myrtus communis (Myrtaceae), collected from the National Park of Boukornine in the north east of Tunisia in November 2003. The aqueous extract of powdered leaves was partitioned successively with chloroform, ethyl acetate, and butanol. The compound was obtained from the ethyl acetate fraction (2 g) subjected to Sephadex LH-20 gel column chromatography with MeOH-H₂O (90:10) as eluent, followed by C-18 silica gel column chromatography eluted with a gradient of H₂O-MeOH (100:0 → 0:100), and finally purified by silica gel column chromatography to yield 2 mg of the compound [1].
The compound is a flavonoid glycoside with myricetin as the aglycone and galactose as the sugar moiety. The addition of the sugar moiety may improve the antioxidant activity of the aglycone part. The compound’s antimutagenic activity may be attributed to inhibition of microsomal enzyme activation or direct protection of DNA strands from electrophilic metabolites. The difference in antimutagenic activity between myricetin-3-o-galactoside and myricetin-3-o-rhamnoside is closely related to the glucidic group linked to the myricetin moiety, with the galactoside being more effective than the rhamnoside [1].

C21H20O13

480.3757

480.09

C, 52.51; H, 4.20; O, 43.30

15648-86-9

5491408

White to off-white solid powder

2.0±0.1 g/cm3

957.8±65.0 °C at 760 mmHg

198-201℃

334.9±27.8 °C

0.0±0.3 mmHg at 25°C

1.834

1.56

9

13

4

34

777

5

O1[C@]([H])([C@@]([H])([C@]([H])([C@]([H])([C@@]1([H])C([H])([H])O[H])O[H])O[H])O[H])OC1C(C2=C(C([H])=C(C([H])=C2OC=1C1C([H])=C(C(=C(C=1[H])O[H])O[H])O[H])O[H])O[H])=O

FOHXFLPXBUAOJM-MGMURXEASA-N

InChI=1S/C21H20O13/c22-5-12-15(28)17(30)18(31)21(33-12)34-20-16(29)13-8(24)3-7(23)4-11(13)32-19(20)6-1-9(25)14(27)10(26)2-6/h1-4,12,15,17-18,21-28,30-31H,5H2/t12-,15+,17+,18-,21+/m1/s1

5,7-dihydroxy-3-[(2S,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2-(3,4,5-trihydroxyphenyl)chromen-4-one

Myricetin 3-O-beta-D-galactopyranoside; Myricetin 3-galactoside; Myricetin 3-O-galactoside

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)