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HPLC method for determination of eleutherosides E and B in dry extract of Siberian Ginseng
Section 11. Chemistry
Bobok Maxim Nikolaevich, First Moscow State Medical University n. a. I. M. Sechenov,
Postgraduate student, Laboratory of bioactive compounds Institute of Pharmacy E-mail: maximbobok@outlook.com Pavlova Ludmila Anatolievna E-mail: l-a-pavlova@yandex.ru Smirnov Valeriy Valerievich, Department of pharmaceutical and toxicological chemistry
E-mail: vall@mail.mipt.ru
HPLC method for determination of eleutherosides E and B in dry extract of Siberian Ginseng
Abstract: The article presents a modern method for quantitative determination of eleutherosides B and E in dry extract of Siberian Ginseng (Eleutherococcus senticosus (Rupr. Et Maxim.)). The dry extract of Siberian Ginseng contains eleutherosides B (syringin) and E (syringaresinol-4',4-O-bis-ß-D-glucoside) as the most pharmacologically active compounds. In connection with the above, it is important to develop and validate a method for determining eleutherosides B and E.
Quantification of eleutherosides B and E by HPLC were determined. Chromatography method in parallel with the standard samples eleutherosides B and E was performed. Validation method is presented by the following characteristics: specificity, linearity and limit of quantification.
Keywords: eleutherosides, chromatography, quantitative determination, validation method.
Introduction. Liquid extract of Siberian Ginseng is used in physical and mental fatigue, neurasthenia and psychasthenia, functional exhaustion of the nervous system which is associated with reduced capacity for work, in vegetative neurosis and conditions after surgery. Due to the presence of eleutherosides in the extract, Siberian Ginseng increases physical and mental work capacity, resistance to adverse environmental factors, improves metabolism, shows a small stimulatory gonadotropic and hypoglycemic effect.
The composition of the dry extract of Eleutherococcus identified several eleutherosides different structure — A, B, B^ C, D, E, F. They belong to different classes of biologically active compounds: eleutheroside A — a steroid; eleutheroside B — a derivative phenyl-acrylic acid; eleutheroside B1 — isofraxidin-7-glucoside; eleuthero-sides D and E — lignans.
Thepurpose of the study is the development of method of quantitative determination eleutherosides B and E in the dry extract of Siberian Ginseng and its validation.
Experimental
Method of sample preparation. Standard solutions of eleutherosides were prepared by dissolving 5 mg. (accurately weighed) of standard samples (Cromodex, USA) in a volumetric flask of 25 ml. in deionized water and methanol (30/70). Were shaken until dissolved and diluted to volume with the same solvent. The concentration of the resulting solutions was 200 mcg/ml. Further, by diluting the obtained 200 mg/ml solution concentrations of 80 mcg/ml, 40 mcg/ml, 20 mcg/ml, 8 mcg/ml were prepared.
Solution of the dry extract prepared by dissolving an accurate sample of the extract (41.0 mg.) in 1.5 ml. of the same solvent. Further, the solution was stirred for 15 minutes then the resulting solution was filtered through a filter with a pore diameter of 9 microns.
Conditions of chromatography. Quantitative determination on HPLC Agilent 1200 with MS 6120 detector, Agilent Technologies, USA was performed. The data were processed using the software Chem Station (ver. B.03.01-SR1), Agilent Technologies, USA. Mobile phase: 0.1 % formic acid in water/acetonitrile (2:98), was pre-filtered and degassed on device for filtering under vacuum. Speed of stream the mobile phase: 1 ml/min. Stationary phase: chromatographic column Agilent Eclipce XDB-C18 4.6x50 mm; 1.8 microns at 23 °C. Injected sample volume: 10 ^l. Time of chromatography: 2 min. Detecting: MS detector ionization type: ESI + APCI (at atmospheric pressure electrospray); scan mode: SIM (by the characteristic peak of m/z 785.3 for eleutheroside E and 416.10 for eleutheroside B). Retention time: 1.2 for eleutheroside E and 1.4 for eleutheroside B.
Results and Discussion
Validation. Validation of bioanalytical method based on the guidelines prepared by the FDA (Guidance for Industry: Bioanalytical method validation. U. S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evolution and Research (CDER). U. S. Government Printing Office: Washington, DC, 2001) and EMA (Guideline on validation of bioanalytical methods (draft). European Medicines Agency. Committee for medicinal products for human use: London, 2009) was performed.
Specificity. 6 samples of pure solvents were analyzed and the sample of standard solution of eleutherosides in a concentration range from 8 mg/ml to 80 mcg/ml. In the chromatograms of the samples of the pure solvents were not observed with a retention time of peaks corresponding to retention time of eleutherosides.
Linearity. 4 samples of solutions of eleutherosides B and E with concentrations: 8 mcg/ml, 20 mcg/ml, 40 mcg/ml, 80 mcg/ml were analyzed. According to the obtained values calibration curve
Section 11. Chemistry
for eleutheroside B (r 2 > 0.995) was constructed, as is shown on Fig. 1 together with the calibration curve equation.
For eleutheroside E (r 2 > 0.991), as is shown on Fig. 2, together with the calibration curve equation.
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Fig. 1. Calibration graph depending of peak area of concentration eleutheroside B
Fig. 2. Calibration graph depending of peak area of concentration eleutheroside E
1 1.2 1.4 1.6 1.8
Fig. 3. The chromatogram of the extract obtained in the chromatographic conditions for eleutheroside B
Fig. 4. The chromatogram of the extract obtained in the chromatographic conditions for eleutheroside E
Herbicide activity of acetylenic aminoalcohols and their ammonium bases
Limit of quantification. According to the methodology on the basis oflinearity the limit of quantification (LOQ) was determined. LOQwas 8 mcg/ml.
Determination of eleutherosides B and E in the dry extract. Chromatogram of the extract obtained in the chromatographic conditions for eleutheroside B and E are shown in Fig. 3 and 4 respectively.
The studies revealed that the content eleutheroside B is 0.08 %, eleutheroside E is 0.06 %, thus the total content eleutherosides B and E is — 0.14 %.
Conclusion
Thus we have developed and confirmed method of quantitative determination eleuterosides B and E in a dry extract of
Eleutherococcus according to parameters: specificity, linearity and LOQ. Based on studies it can be concluded that in the quantitative content eleutheroside B is 0.08 %, the eleutheroside E is 0.06 % and this fraction equals 0.14 %.
Summary
The method of determination eleuterosides B and E in the dry extract of Eleutherococcus by HPLC was developed. Represented validation techniques according to parameters: specificity, linearity, LOQ.
Quantitative content of eleutherosides in dry extract of Eleutherococcus: eleutheroside B is 0.08 %, eleutheroside E is 0.06 % and total content eleutherosides B and E is 0.14 %.
References:
1. Watson D. G. Pharmaceutical Analysis: A Textbook for Pharmacy Students and Pharmaceutical Chemists, 2nd Revised edition. -Churchill Livingstone, 2005. - 398 c.
2. Zhang Q., Liu Y., Li J., Xu Q., Li X., Yang X., Yao D., Sun J., Cui G., Ying H. Simultaneous determination of Eleutheroside B and Eleutheroside E in rat plasma by high performance liquid chromatography-electrospray ionization mass spectrometry and its application in a pharmacokinetic study//J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. - 2013, Feb 15. - 917-918: 84-92.
3. Zhang L., Sun Y. Semipreparative separation and determination of eleutheroside E in Acanthopanax giraldii Harms by high-performance liquid chromatography//J. Chromatogr. Sci. - 2005, May-Jun. - 43(5): 249-252.
Turgunov Erxan, Associate Professor of the Faculty of Chemistry of the National University of Uzbekistan E-mail: erxon1955@yandex.ru Suleymanova Gulchehra Gaybullaevna, Tashkent Pediatric Medical Institute, assistant of the Department of Biology, Inorganic and Organic Chemistry, PhD
E-mail: gulchehra@mail.ru Sobirova Diloram Kabulovna, Tashkent Automobile and Road Institute, senior lecturer in Chemistry, PhD E-mail: Sobirova_66@umail.uz
Hudoyberdieva Zarifa, 4th year student of the Faculty of Chemistry of the National University of Uzbekistan E-mail: Zarifochka.15@gmail.com Kolyadin Vladimir Grigorevich, Associate Professor of the Faculty of Chemistry of the National University of Uzbekistan E-mail: erxon1955@yandex.ru Fazullaeva Mariyam, PhD, Department of Chemistry of the National University of Uzbekistan E-mail: marifai@mail.ru
Herbicide activity of acetylenic aminoalcohols and their ammonium bases
Abstract: Studied herbicide action of some synthesized acetylenic amino alcohols, hydrochloride, various chlor- and bro-moniy compounds based on them, as well as the selectivity of their actions in relation to such important crops as cotton and corn. It was found that the pretreatment of seeds and weeds studied drugs, depending on their chemical structure and dose exhibit various herbicide activity against annual dicotyledonous (shirica) and annual cereals (hen's proso) weeds.
Keywords: Mannich reaction, Favorskiy reaction, Acetylenic amino alcohols, acetylenic hydrochlorides of amino alcohols, chloroniy salts of acetylenic amino alcohols, Bromoniy salts of acetylenic amino alcohols, herbicide activity, cotton, corn, shirica, hen's proso.
