Veremchuk O.A., Moiseyev D.V.
Validation of the technique of flavonoids quantitative determination in heather shoots
Education establishment "Vitebsk State Order of Peoples’ Friendship Medical University"
The objective of the present research work was to develop and validate the technique for quantitative determination of flavonoids on dry material in heather shoots by liquid chromatography method.
The chromatographic separation was performed on a Zorbax SB C8 column (4,6T250 mm, 5 µm). The mobile phase comprised phosphate buffer solution pH=3,0 and acetonitrile (80:20 v/v).
Validation of the developed technique of flavonoids quantitative determination in heather shoots was performed with the following characteristics taken into account: specificity, linearity, accuracy, precision (repeatability and intermediate precision) and robustness. The detector response (peak area for isoquercitrin) was linear for concentrations ranging from 50 to 2000 µg/ml and correlation coefficient was 0,9995. The accuracy of the developed technique was checked by carrying out the recovery experiment by using the method of «standard addition». The value of percent recovery was found to be 102,1±1,3%. The technique is precise because the values of relative standard deviations proved to be less than 5,0%. Changes in the extraction time (60±5 min), mobile phase composition (±1%) and rate (1,0±0,1 ml/min) had no significant effect on the detector response, thus the technique may be considered robust. The flavonoids content on isoqercitrin and dry material in heather shoots made up 36,8±1,3 mg/g.
Thus the developed and validated technique for quantitative determination of flavonoids in heather shoots is specific, linear, accurate, precise and robust within the range of application, and can be used for routine quality control of heather shoots.
Key words: flavonoids, isoquercitrin, liquid chromatography, heather.
1. TKP 451-2012 (02041), BY. Proizvodstvo lekarstvennykh sredstv. Spetsifikatsii: metody ispytanii i kriterii priemlemosti dlia lekarstvennogo rastitel'nogo syr'ia, produktov iz lekarstvennogo rastitel'nogo syr'ia i lekarstvennykh sredstv rastitel'nogo proiskhozhdeniia [Production of pharmaceuticals. Specifications: test methods and acceptance criteria for medicinal plant materials, products of medicinal plants and herbal medicines]. Vved. 29.11.12. Minsk, RB: Departament farmatsevticheskoi promyshlennosti Ministerstva zdravookhraneniia Respubliki Belarus'; 2012. 17 р.
2. Monschein M, Neira JI, Kunert O, Bucar F. Phytochemistry of Heather (Calluna vulgaris (L.) Hull.) and its altitudinal alteration. Phytochem Rev. 2010;9:205-15.
3. Jalal MAF, Read DJ, Halsamt E. Phenolic composition and its seasonal variation in Calluna vulgaris. Phytochemistry. 1982;21(6):1397-1401.
4. Orhan I, Küpeli E, Terzioğlu S, Yesilada E. Bioassay-guided isolation of kaempferol-3-O-β-d-galactoside with anti-inflammatory and antinociceptive activity from the aerial part of Calluna vulgaris L. J Ethnopharmacol. 2007 Oct;114(1):32-7.
5. Joshi UJ, Gadge AS, D’Mello P, Sinha R, Srivastava S, Govil G. Anti-inflammatory, antioxidant and anticancer activity of Quercetin and its analogues. International Journal of Research in Pharmaceutical and Biomedical Sciences. 2011;2(4):1756-66.
6. Tarakhovsky YuS, Kim YuA, Abdrasilov BS, Muzafarov EN. Flavonoidy: biokhimiia, biofizika, meditsina [Flavonoids: biochemistry, biophysics, medicine]. Pushchino, RF: Sуnchrobook; 2013. 310 р.
7. Gasparotto AJ, Gasparotto FM, Boffo MA, Lourenço EL, Stefanello MÉ, Salvador MJ, da Silva-Santos JE, Marques MC, Kassuya CA. Diuretic and potassium-sparing effect of isoquercitrin-an active flavonoids of Tropaeolum majus L. J Ethnopharmacol. 2011 Mar;134(2):210-5.
8. Jäger AK, Saaby L. Flavonoids and CNS. Molecules. 2011 Feb;16(2):1471-85.
9. Mandel SA, Weinreb O, Amit T, Youdim MB. Molecular mechanisms of the neuroprotective/neurorescue action of multi-target green tea polyphenols. Front Biosci (Schol Ed). 2012 Jan;4:581-98.
10. Saaby LH, Rasmussen B, Jäger AK. MAO-A inhibitory activity of quercetin from Calluna vulgaris (L.) Hull. Journal of Ethnopharmacology. 2009;121(1):178-81.
11. Orlova OA. Podbor optimal'nykh uslovii ekstraktsii iz vereska obyknovennogo [Selection of the optimal extraction conditions of ling]. V: Aktual'nye voprosy sovremennoi meditsiny i farmatsii: materialy 65-i itogovoi nauch.-prakt. konf. studentov i molodykh uchenykh, 24-25 apr. 2013 goda. Vitebsk, RB; 2013. р. 398-399.
12. Godovalnikov GV, red. Gosudarstvennaia farmakopeia Respubliki Belarus' [State Pharmacopoeia of the Republic of Belarus]. V 3 t. T. 1. Obshchie metody kontrolia kachestva lekarstvennykh sredstv. Minsk, RB: Minskii gosudarstvennyi PTK poligrafii; 2006. 656 р.