In DMSO solvent, the DMSO control is showing more effect than the drug.
Hesperidin probably is a very complex molecule to dissolve because of the simultaneous presence of a hydrophilic and a lypophilic part. As Dr. Bangdula suggested you can try to use ethanol, but the final concentration of ethanol in cell culture medium should be lower than 0.01%to avoid a direct effect of EtOH, so may be that you get a re-precipitation of the drug. In that case you may try to complex hesperidin with FBS: first make a mother solution of hesperidin in FBS, dissolving it in the appropriate amount of FBS , warming it and put in a sonicating water bath and then take the appropriate aliquots for your required concentrations. FBS is made mostly of Fetuin A, that has a tendency to surround the poorly water soluble compounds (even inorganic and complete insoluble hydroxyapatite) with a layer and bring them in solution, the compound is then readily available by the cells and the micelles obtained are very stable (I used this method when I was treating cells with fatty acids with a long chain). Definitely DMSO at any concentration is not an option,because in a drug screening that I was doing, DMSO controls had always the highest readings of all the drugs tested, regardless of the concentration, it does interfere with too many cell functions.
Hesperidin probably is a very complex molecule to dissolve because of the simultaneous presence of a hydrophilic and a lypophilic part. As Dr. Bangdula suggested you can try to use ethanol, but the final concentration of ethanol in cell culture medium should be lower than 0.01%to avoid a direct effect of EtOH, so may be that you get a re-precipitation of the drug. In that case you may try to complex hesperidin with FBS: first make a mother solution of hesperidin in FBS, dissolving it in the appropriate amount of FBS , warming it and put in a sonicating water bath and then take the appropriate aliquots for your required concentrations. FBS is made mostly of Fetuin A, that has a tendency to surround the poorly water soluble compounds (even inorganic and complete insoluble hydroxyapatite) with a layer and bring them in solution, the compound is then readily available by the cells and the micelles obtained are very stable (I used this method when I was treating cells with fatty acids with a long chain). Definitely DMSO at any concentration is not an option,because in a drug screening that I was doing, DMSO controls had always the highest readings of all the drugs tested, regardless of the concentration, it does interfere with too many cell functions.
Hesperidin is a flavan-on glycoside (flavanone rutinoside) found in citrus fruits. Hesperidin is soluble in organic solvents DMSO and dimethyl formamide. Hesperidin is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, Hesperidin should first be dissolved in DMSO and then diluted with the aqueous buffer.
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