Determination of design space and normal operating range of process parameters to produce purified Aesculus hippocastanum L. seed extract

Introduction. Aesculus hippocastanum L. seeds are a promising source of triterpene saponins – escin (β-escin) possessing venotonic, angioprotective, and anti-inflammatory activities. The production of β-escin substance in Russia is currently absent, which determines the relevance of developing a reproducible technology for obtaining purified horse chestnut seed extract with a high escin content.

Aim. To determine the optimal extraction parameters of triterpene saponins from Aesculus hippocastanum L. seeds using multivariate optimization, to develop an extract purification procedure, and to establish the Normal Operating Range of process parameters.

Materials and methods. Extraction process optimization was performed according to a Box – Behnken design. The effects of ethanol concentration, temperature, solid-to-solvent ratio, and extraction time on the yield of total triterpene saponins expressed as β-escin were investigated. The chemical composition of the extract was determined by HPLC-MS with ESI+/ESI− detection. The quantitative content of saponins in the extract was determined by spectrophotometry; β-escin content in the purified extract was determined by HPLC.

Results and discussion. A quadratic regression model of the extraction process was developed. The solid-to-solvent ratio and ethanol concentration were identified as the primary factors affecting saponin yield, while temperature within the studied range was found to be statistically non-significant. The optimal extraction conditions were: ethanol concentration – 49 %, temperature – 45 °C, solid-to-solvent ratio – 1 : 12, extraction time – 90 min; the predicted yield was 75.6 %. In accordance with ICH Q8(R2) principles, the Design Space (DS) (Y ≥ 65 %) and the Normal Operating Range (NOR) were established. Seven structural groups of escins were identified in the purified extract by HPLC-MS.

Conclusion. The Design Space and Normal Operating Range for the extraction process of triterpene saponins from Aesculus hippocastanum L. seeds were established within the ICH Q8(R2) / QbD framework, ensuring the production of purified extract with reproducible quality attributes.

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