Development of approaches to standardization of Juglans nigra L. leaves by HPLC

Introduction. Juglans nigra L., family Juglandaceae), is a deciduous tree, which is one of the most promising types of medicinal plant raw materials. Due to the high content of biologically active compounds, Juglans nigra L. leaves are of interest as a new type of medicinal plant raw materials. In addition to the leading group of biologically active compounds (naphthoquinones), that the raw materials contain other chemical compounds, including flavonoids (quercitrin, myricitrin), which can make a significant contribution to the anti-microbial and tonic activity of the specified pharmaceuticals. The urgency of the problem is due to the fact that, despite the content of a large number of biologically active compounds and high pharmacological activity, the Juglans nigra L. leaves are not included in the State Pharmacopoeia of the Russian Federation. Consequently, there is a need for standardization of Juglans nigra L. leaves, including the development of quantitative determination meth- ods using modern methods of analysis. The article presents the results of studies on the development of quantitative analysis of myricitrin and quercitrinin, Juglans nigra L. leaves in order to include medicinal plant raw materials of Juglans nigra L. leaves in the State Pharmacopoeia of the Russian Federation. The study was carried out within the framework of the complex Research and development work topics of SamSMU "Chemical-pharmaceutical, biotechnological, pharmacological and organizational-economic research on the development, analysis and use of pharmaceutical substances and drugs" (No. АААА-А19-119051490148-7 dated 05.14.2019). The purpose of the study. Development of methods for a quantitative analysis of biologically active compounds of Juglans nigra L. leaves, namely myricitrin and quercitrin. Material and methods. The objects of the study were samples of Juglans nigra L. leaves harvested in March-April 2018-2020 on the territory of the Botanical Garden of Samara University (Samara), received by the team of the Department of Pharmacognosy with Botany and Fundamentals of Phytotherapy of SamSMU reference standard of myricitrin and quercitrin with a purity of at least 98%. High-performance liquid chromatography (HPLC) was used as methods for analyzing the extracts, which was carried out using a Milichrom-6 chromatograph (NPAO "Nauchpribor") under the following conditions of reverse phase chromatography in an isocratic mode: steel column "KAKH-6-80-4" (No2; 2 mm x 80 mm; Separon-C18 7 microns), mobile phase-acetonitrile: 1% solution of acetic acid in water in a ratio of 2:8, 100 μL/min, eluent volume - 1500 μL. The substances were detected at a wavelength of 360 nm. The volume of injected samples for extraction from the bark of black walnut, as well as solutions of quercitrin and myricitrin 4 μl. The myricitrin and quercitrin content were calculated using the external standard method. The suitability of the chromatographic system was evaluated in accordance with the OFS.1.2.1.2.0001.15 "Chromatography". Results. Using the method of column chromatography, the dominant flavonoids - myricitrin (myricetin - 3-O-α-L-rhamnopyranoside) and quercitrin (quercetin-3-O-α-L-rhamnopyranoside), were identified on the basis of UV, NMR, mass spectra, as well as the results of acid hydrolysis, were isolated from the water-ethanolic extractionof Juglans nigra L. leaves. Methods for the quantitative determination of myricitrin and quercitrin in Juglans nigra L. leaves by microcolumn HPLC under isocratic conditions (mobile phase - acetonitrile : 1% solution of acetic acid in water in a ratio of 2:8) have been developed. The error of a single determination of the content of myricitrin in the leaves of Juglans nigra L. with a confidence probability of 95% was ±4.91 %. The content of myricitrin varied (2.58±0.15)% to (2.67±0.13)%. In addition, the quercitrincontent varied from (1.36±0.12)% to (1.45±0.15)%. The error of a single determination of the quercitrin content in Juglans nigra L. leaves with a confidence probability of 95% was ±5.18 %. Conclusion. Results of the conducted studies have shown satisfactory metrological characteristics of the HPLC method for determining the quantitative content of myricitrin and quercitrin proposed for standardization of raw materials of Juglans nigra L.

Juglans nigra L, Juglans nigra L, leaves, quercitrin, myricitrin, high-performance liquid chromatography, standardization