Development and validation of a method for quantitative determination of saponins in Aesculus hippocastanum L. flowers

Introduction. Saponins are an extensive class of natural glycosides that perform important functions in plant life processes. Horse chestnut ( Aesculus hippocastanum L.), a plant of the genus Aesculus L., family Hippocastanaceae Burnett, is a promising medicinal plant. In official medicine, chestnut seeds are used as a venotonic and capillary-protective agent due to the complex of triterpene saponins contained in them, the main active ingredient of which is the glycoside β-escin. In folk medicine, flowers, leaves, bark, buds and capsules of chestnut are also used. Horse chestnut flowers contain a wide range of biologically active substances (BAS), which makes them a promising type of medicinal plant raw materials (MPRM) with a pronounced therapeutic potential. However, the group of triterpene saponins in their composition remains insufficiently studied. Aim. To develop and validate a spectrophotometric method for determining saponins in horse chestnut flowers, as well as to test it on raw materials from various growing regions to scientifically substantiate raw material quality standards. Materials and methods. The study involved air-dried horse chestnut flowers harvested in the Petrozavodsk urban district, Leningrad, Moscow, Voronezh, and Volgograd regions, and Stavropol Krai during the mass flowering period (May — June) in 2024. To quantify the total saponin content as β-escin, a spectrophotometric method was adapted. This method involves oxidation with concentrated sulfuric acid, accompanied by dehydration, which leads to the formation of colored quinoid structures. Optical density was measured using an SF-2000 spectrophotometer in the wavelength range of 250—450 nm. The method was validated in accordance with the general pharmacopoeial article (PA) "Validation of analytical methods" of the State Pharmacopoeia of the Russian Federation, 15th edition. Results and discussion. A method for quantifying the amount of triterpene saponins in terms of β-escin in horse chestnut flowers by spectrophotometry has been developed and validated. The obtained validation results allow us to recommend the method for the quantitative determination of saponins in horse chestnut flowers. It was found that optimal extraction conditions include the use of 70 % ethanol at a raw material : extractant ratio of 1 : 100 and an extraction time of 90 minutes. The variability of accumulation of this group of BAS in raw materials (from 1.27 to 2.95 %) was determined depending on the combination of ecological and geographical factors of the growing region. The highest saponin content was found in raw materials from the Petrozavodsk City District (2.95 %) and the Volgograd Region (2.76 %). Conclusion. The conducted research, being a key stage in the systematic study of horse chestnut flowers, expands the prospects for using this raw material as a source of triterpene saponins. Based on the results obtained, it is possible to recommend a unified criterion for assessing the quality of this MPRM for the future PA project "Aesculus hippocastanum flores" in terms of the "Quantification" of the amount of triterpene saponins in terms of β-escin — at least 1 %.