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Metabolomics analysis on different parts of acanthopanax senticosus based on UPLC-Q-TOF-MS

Abstract: Acanthopanax senticosus roots, rhizomes, or stems are the main site of ciwujia. Due to the limited resources of the roots, the leaves and pulp are sometimes used as health products or medication sites. In order to utilize the leaves and pulp, it is important to conduct the chemical comparison of the roots, leaves, and pulp of A. senticosus. In this study, Multivariate statistical analysis and UPLC-Q-TOF-MS/ MS was used to compare the chemical composition of the roots, leaves, pulp of A. senticosus, a total of 47 compounds were identified. The results suggested that the content of lignans, such as Eleutheroside D, Eleutheroside E, were so much higher in the roots than those in the leaves and pulp. The content of Eleutheroside B is similar between the roots and the pulp of A. senticosus and is so much higher in the roots, pulp than that in the leaves. This result laid a certain foundation for the pulp of A. senticosus to replace the roots as a medication site in some pharmacological effects. The leaves cannot replace the roots as a medication site due to the low content of Eleutheroside compounds. This study laid the foundation for the rational utilization of A. senticosus resources.

Metabolomics (or metabolic profiling) is an important part of systems biology that focuses on the identification of some small molecule (< 1500 Da) compounds in the metabolome (11)(12)(13)(14)(15)(16). Metabolomics has shown potential in the bioactivity evaluation and action mechanism of Traditional Chinese Medicine (TCM) as well as pharmaceutical research and development (17). Various studies have shown that metabolomics often uses the UHPLC-MS platform to analyze a series of metabolomics data efficiently to monitor the differences in various metabolites in body fluid (18)(19)(20).
This research intends to discuss the similarities and differences of the roots, leaves, pulp of A. senticosus using UPLC-HDMS, combined with MassLynx 4.0, Progenesis QI, PCA (principal components analysis), UNIFI 8.1. The results of this study will lay the foundation for the utilization of A. senticosus resources.

Plant materials
The roots, leaves, and pulp samples of A. senticosus were bought from the traditional Chinese medicinal material market in Bozhou (China), and the sample of Acanthopanax pulp is dried directly after picking. All samples are dried processed in the place of production. The information of all samples is as follows: The first batch, the second batch, and the third batch of samples originated from Jilin (China), and the numbers are 20170901, 20170902, 20170903, and the fourth, fifth, and sixth batches are from Heilongjiang (China), and the numbers are 20170904, 20170905, and 20170906. All samples were identified as A. senticosus by professor Qingmei Qiao from the College of Animal Medicine, Henan University of Animal Husbandry and Economy. All samples after drying in the place of production were dried at 40°C for 3 h in the laboratory and stored at room temperature before measurements.

Reagents
Acetonitrile and formic acid (LC/MS grade) were purchased from Thermo Fisher Co. (Waltham, MA, USA). Ultra-pure water was prepared using a Millipore Milli-Q purification system (Millipore Corp., Bedford, Mass., USA). All other reagents were of analytical grade.

Preparation of sample solutions
All dried roots, leaves, and pulp samples were broken into powder and filtered through a No. 65 sieve. In brief, 500 mg of per sample was weighed into a 10 mL tube, and 5 mL of methanol/water (1 : 1) was added. The sample powder was extracted for 20 min in an ultrasonic bath at 25°C. The supernatant solution was filtered through 0.22 μm PTFE membrane filter before injected into the UPLC system.
The injection volume was 5 μL. A dual ESI source was operated in positive ion mode (ESI + ). The mass range was set to acquire over m/z 50-2000 Da. The detailed MS conditions were as follows: source temperature of 130°C, desolvation temperature of 350°C, cone gas flow of 50 L/h, desolvation gas flow of 700 L/h, a capillary voltage of 2.5 kV, sample cone voltage of 21 V, extraction cone of 4 eV, collision energy of 15 eV. The leucine-enkephalin was continuously injected as a reference to ensure accuracy, which was detected during the run at a concentration of 500 pg/μL.

Data processing and multivariate statistical analysis
The mass chromatographic data acquisition and analyses of data were controlled by Waters MassLynx v4.0 software (Waters Corp.) and UNIFI v1.8.1 software (Waters Corp.). All chromatographic data were preprocessed and normalized. The MarkerLynx software and UNIFI software were used to calculate the ESI + raw data of all samples. A two-dimensional matrix consisting of data pairs of retention time (RT) and mass-to-charge ratio (m/z) was generated, and the mass values and intensities of peaks were exported to Excel for further chemometric analysis. Then, the data from Markerlynx were exported to SIMCA 13.0.3 software (Umetrics, Umeå, Sweden) for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA), which was used to identify different components among the three groups.

UPLC-HDMS analysis
MS data were obtained in positive ion mode. Most constituents exhibited their quasi-molecular ions [M + H] + ions in the positive ion mode. The based peak intensity (BPI) chromatograms of the roots, leaves, pulp were presented in Figure 1. Visual inspection of the spectra from the different parts of A. senticosus revealed obvious differences with each other. Accurate molecular data of each potential marker were determined up to four decimal places using UPLC-HDMS. Identification of compounds was achieved by comparison to mass spectral data in the available literature. The data were presented in Table 1.

Multivariate statistical analysis
Typically, PCA (Principal Components Analysis) can reduce the dimension of metabolites matrices based on their linear combinations of mutual features in the dataset. PCA is generally used for plotting the grouping and visualizing outliers based on natural interrelationships between samples (23). The peak list containing retention time, m/z, and ion intensity of each sample was imported into the SIMCA-P Software for multivariate statistical analysis, which was processed by PCA for discriminating all samples from different parts of A. senticosus. All metabolites were displayed as scores and loadings in a coordinate system of principal components resulting from data dimensionality reduction. The PCA scores plots of data were shown in Figure 2. UV scaling was used to perform PCA. R 2 = 0.749 and Q 2 = 0.559 suggested the model performed a relatively excellent prediction. Based on Figure 2, samples of roots are positioned on the right side and the top side (positive t [1] values and positive t [2] values) of the PCA score plot whereas the samples of leaves are located on the right side and the below side (positive t [1] values and negative t [2] values), the samples of pulp are located on the left side (negative t [1] values). These three classes can be separated clearly from each other.
In order to further explore the difference between different parts of A. senticosus, OPLS-DA (orthogonal partial least squares discriminant analysis) approach was applied to enhance the separation among the groups in PCA. Par scaling was used to perform OPLS-DA. As shown in Figure3A, applying the OPLS-DA study, samples were clearly separated in the scores plot. In the corresponding S-plot ( Figure 3B) ), MS data, and elemental composition of identified potential markers using UPLC-HDMS in positive ion mode (cont.).
in projection (VIP) > 1, the ion RT-m/z pairs were selected as potential chemical markers for the discrimination of the roots, leaves, and pulp.

Comparison of eleven chemical markers
According to Figure 3B, there were 275 chemical markers of VIP > 1 which were highlighted in the loading scatter plot of OPLS-DA. After using UNIFI 8.1 to identify the markers, eleven compounds were identified. The detailed information of the markers of VIP > 1 was summarized in Table 2. In order to provide a further comparison of the differences among roots, leaves, and pulp of A. senticosus at different maturation. The difference with p < 0.001 was considered significant. In order to further study the difference among roots, leaves, and pulp, the intensity of eleven chemical markers was analyzed by the one-way ANOVA test.
As shown in Figure 4, the marker levels varied considerably among roots, leaves, and pulp. The levels of Eleutheroside D, Eleutheroside E, liriodendrin in the roots showed particularly higher as compared to other parts of the leaves and pulp. The compounds can be used as the specific markers for the differentiation of roots from leaves and pulp. The levels of Indole-3-acrylic acid and 2-furoic acid were found remarkably higher in pulp than in other groups. The level of p-coumaric acid was higher in leaves and    pulp than that in roots. The intensity of syringin, the main active component of A. senticosus, was found higher in roots, pulp than that in leaves, it may lay a certain foundation for exerting the medicinal value of pulp. PCA and OPLS-DA score plots from UPLC-MS data showed a clear separation between the roots, leaves, and pulp of A. senticosus. A total of 47 compounds were identified in roots, leaves, pulp through a UNIFI 8.1 database. The roots contain main active phenylpropanoids, such as Eleutheroside B (syringin), Eleutheroside D, Eleutheroside E, while the leaves are basically free of phenylpropanoids. The pulp contains some phenylpropanoid, such as Eleutheroside B. The pulp also contains many acidic compounds, such as isoleucine, phenylalanine, 2-Furoic acid, coumaric acid, and so on.
Eleven compounds were identified as markers in this study. Those can be used as the specific markers for the differentiation of roots from leaves and pulp. Eleutheroside D and E are the different compounds of A. senticosus roots compared with leaves and pulp. The content of Eleutheroside D and E in the roots produced in the Northeast region is much higher than that in the leaves and pulp. The content of Eleutheroside E in the Eleutherococcus senticosus fruits produced in Bydgoszcz (Poland) is very little and almost nothing (24). The content of Eleutheroside B, also named syringin, is similar between the roots and the pulp, and the content of Eleutheroside B was found so much higher in the roots, pulp than that in the leaves of A. senticosus. But there is no Eleutheroside B in the fruits of E. senticosus in Bydgoszcz (Poland). It is suspected that the content of Eleutheroside B in the A. senticosus fruit is inconsistent due to the different origin. As stated in the introduction, the main active components of ciwujia injection, which is made by extracting the roots of A. senticosus, are Eleutheroside B, Eleutheroside E, Isazididine. Eleutheroside B and Eleutheroside E are the main effective ingredient for the treatment of cardiovascular diseases and neurological diseases, which is consistent with the various effects of clinical application of A. senticosus, such as angina, anti-fatigue, anti-cancer, etc. This may be one of the reasons why the roots can be the medication site of A. senticosus.
The content of Eleutheroside B is similar between the roots and the pulp. Thus we speculate that the pulp of A. senticosus may also have a certain pharmacological effect. The pulp of A. senticosus may be used as a substitute for the roots in some pharmacological effects, but it still needs further research. The content of Eleutheroside compounds in the leaves of A. senticosus is less than those in the roots and pulp. It is speculated that the leaves cannot replace the roots as the medication site.

CONCLUSIONS
We analyze the roots, leaves, and pulp of A. senticosus through UPLC-HDMS and multivariate statistical analysis. 47 components were identified in A. senticosus and they had been assigned to different parts. The results showed a clear separation between the roots, leaves, and pulp of A. senticosus. The content of Eleutheroside D and E is so much higher than those in the leaves and pulp. It may be one of the reasons why the roots can be the medication site of A. senticosus. The content of Eleutheroside B is similar between the roots and the pulp. This result laid a certain foundation for the pulp of A. senticosus to replace the roots as the medication site in some pharmacological effects, but it still needs further research. The leaves cannot replace the roots as the medication site due to the low content of Eleutheroside compounds. The study laid the foundation for the rational utilization of A. senticosus resources.