Study on the Source and Characteristics of Evodia rutaecarpa Based on Chemical Pattern Recognition

In this study, the appearance and texture of E. rutaecarpa were linked with the chemical constituents to explore methods of classification of E. rutaecarpa . The Chemometrics such as Hierarchical cluster analysis (HCA), principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models were used for analysis. According to the models, samples of E. rutaecarpa were divided into three categories based on their source: Evodia, Stone Tiger and Sparse Evodia. The Evodia category could be subdivided into two categories, one representing large fruits with a greater degree of cracking and the other representing large fruits with little or no cracking. The method provided by this study combines chemometrics with HPLC fingerprints, which can provide a basis and reference for the identification of E. rutaecarpa and enables establishment of a grade standard.


Materials and reagents
The 31 batches of E. rutaecarpa samples were collected from eight different provinces (Zhejiang, Jiangxi, Guangxi, Guizhou, Sichuan, Hunan, Hubei, and Shanxi) Table S1 and the physical charcters of Evodia were differentiated by chemometrics is shown in Table S2. Images of E. rutaecarpa samples from different sources are shown in Figure S1.

Sample preparation
Samples of E. rutaecarpa from various batches were collected, finely powdered, and passed through a 50-mesh sieve. Accurately weighed samples (0.2 g) of each powder were added to 80% methanol (10 mL), soaked for 1 h, and then sonicated for 40 min.
A mixed standard solution was obtained by dissolving accurately weighed samples of dehydroevodiamine, evodiamine and rutacarpine in methanol.

HPLC methodological evaluation
Six successive injections of the same E. rutaecarpa sample solution were subjected to chromatography as described in section 1.3. The relative retention time and relative peak area of each common peak were calculated by using evodiamine (peak number 10) as the reference peak. The RSD for the relative peak retention time was < 2.0% and the RSD for the relative peak area was < 3.0%, indicating good precision.
Six samples of E. rutaecarpa were prepared in parallel from the same group of E. rutaecarpa samples according to the method in section 1.2. The relative retention time and relative peak area of each common peak were calculated by using evodiamine (peak number 10) as the reference peak. The RSD for the relative peak retention time was < 1.6% and the RSD for the relative peak area was < 2.67%, indicating good repeatability.
Samples of the same Evodia solution, prepared according to section 2.2, were analyzed at 0, 2, 4, 8, 12, and 24 h. The relative retention time and relative peak area of each common peak were calculated by using evodiamine (peak number 10) as the reference peak. The RSD for the relative peak retention time was < 2.11% and the RSD for the relative peak area was < 2.98%, indicating that the sample was stable for 24 h.

Software requirements
LabSolutions/LCsolution workstation data management software (SHIMADZU, Japan) was used to collect chromatographic data on samples of E. rutaecarpa, including peak area, retention time and  Tables   Table S1. Description of E. rutaecarpa samples.      Table 1. VIP (variable importance for the project) plot).