Abstract:
Background The multifaceted nature of phytotherapeutic products calls for methods able to provide a comprehensive and systems-level characterisation. Agricultural research suggests that copper chloride crystallisation (CCC) fingerprint analysis offers such possibilities. We therefore investigated the
applicability of CCC to phytopharmaceutical questions. In this pilot trial, we analysed plant extracts of the same genus (Viscum album L.), featuring three progressively subtler differences: 1) subspecies (subsp. album vs austriacum), 2) deciduous host trees (apple vs oak), and 3) blending procedures (machine vs hand).
Methods General experimental procedure and design
Extracts of mistletoe of two different subspecies, grown on three different host trees and subjected to two blending procedures, were used as samples. The extracts were mixed with CuCl2 solution and pipetted on glass plates. Evaporation of the solution under controlled conditions led to the formation of CCC fingerprints. These were analysed by computer image analysis and subsequent statistical evaluation. The experimental design was structured to assess the sensitivity (i.e. ability to statistically discriminate between samples) of CCC by testing three progressively finer differences in the extracts: 1) mistletoe subspecies, 2) mistletoe belonging to the same subspecies but growing on different deciduous trees, and 3) blending procedures. As a first step, the stability of the setup and the randomisation of the samples in the CCC chambers were investigated with 6 systematic control (hereafter SC) experiments. Subsequently, 12 Verum experiments were run. Samples were block-randomised and blinded throughout the experiments, with decoding performed during the statistical analysis phase.
Conclusion This article explores the potential applicability of CCC as a complementary method for characterising phytotherapeutic products, recognising their complex and multifaceted nature. As this methodology had not previously been applied in this field, our primary aim was to assess its sensitivity by testing its ability to detect differences between closely related phytotherapeutic samples.
We set up three sensitivity tests aiming to detect progressively finer differences between samples derived from plants of the same genus (i.e. Viscum album). Statistical differences were observed between the samples across the three tests, suggesting the differentiation ability of the method. Notably, the signals related to the three sensitivity tests were all extracted from the same set of experiments, hinting at the potential ability of the fingerprints to reflect multiple aspects of the products.