Effectiveness of an innovative prototype Subtracted Diversity Array (SDA) for fingerprinting commercial Chinese herbal medicines
Creators: name: Niu, Lin
Publisher: name: RMIT University
Year: 2024

description: Chinese herbal medicines (CHM) are widely used in China and exported worldwide. However, the presence of adulterants and substitutes in the market is a recurring problem, and may result in completely different or weaker pharmacological actions compared with their authentic counterparts or even adverse reactions. Therefore, the identification or authentication of CHM is not only important to ensure the therapeutic potency of dried commercial CHM but also to protect public health and safety.<br><br>Since genomic DNA was used directly for target preparation for the subtracted diversity array (SDA), the quality and purity of the DNA is crucial for this technique. A developed CTAB method was used in this study based on a comparison with three previously published methods and a modified CTAB method. Furthermore, DNA isolated from four dried commercial CHM samples representing different species were used to prepare targets in a separate experiment. These species were successfully discriminated in a preliminary fingerprinting.<br><br>Seven dried commercial CHM were correctly clustered using a developed SDA based on comparison of the signals of the features on hybridizations. The hierarchical clustering generated from the fingerprinting of dried materials conformed to the predicted taxonomical relationships of these species in the APG II classification system (2003). Furthermore, in a separate experiment Panax ginseng (Chinese ginseng) and P. quinquefolius (American ginseng), two species that were not used in the creation of the initial genomic DNA pool, were discriminated. Also, a small level of substitution (10% P. quinquefolius DNA in pure P. ginseng DNA) was differentiated from these two Panax species, indicating that the developed DNA microarray-based technique may be suitable for the fingerprinting of dried CHM samples at the species level.<br><br>To establish the identity of the polymorphic sequences, and to calculate the redundancy of the probes for the SDA, 152 clones spotted as features on the SDA were randomly selected and sequenced. 106 individual sequences (70.6%) possessed "significant matches" in the SwissProt® and SpTrEMBL® databases. In total, 151 DNA sequences were divided into five categories according to the descriptions of the database entries. Forty DNA sequences (26.7%) were matched to "retroelements", 35 (23.3%) were matched to "genes", 42 (28.0%) were matched to "putative uncharacterized protein", 12 (8.0%) were classified as "unknown" due to poor match, and the remaining 21 DNA sequences (14.0%) obtained "no hits" in the existing databases. Also, 81 unique sequences were subsequently identified, giving a redundancy of 1.9 fold (53%) for the SDA probe set.<br><br>Further work is required using the SDA to differentiate a greater range of closely related species and samples from the same species derived from different locations and processed and/or stored under different conditions in order to evaluate the utility of this method for commercial application in CHM quality control. To do this, refinements to increase the sensitivity and discrimination level of the SDA may be necessary.<br>