Chemical Comparison of White Ginseng before and after Extrusion by UHPLC-Q-Orbitrap-MS/MS and Multivariate Statistical Analysis

Ultrahigh-performance liquid chromatography Quadrupole-Orbitrap tandem mass spectrometry (UHPLC-Q-Orbitrap-MS/MS) was used to compare the composition of ginsenosides in white ginseng (WG) and extruded white ginseng (EWG). A total of 45 saponins, including original neutral ginsenosides, malonyl-ginsenosides, and chemical transformation of ginsenosides, were successfully identified in both WG and EWG. Multivariate statistical analyses including supervised orthogonal partial least squared discrimination analysis (OPLS-DA) and hierarchical clustering analysis (HCA) were used to analyze components of white ginseng before and after extrusion. As a result, three ginsenosides (malonyl (M)-Rb1, M-Rb2, and M-Rc) were found to be increased in WG, while three ginsenosides (Rb2, Rc, and Rg1) were elevated in EWG. In the OPLS-DA S-plot, the different compositions of ginsenoside that were distinguished between WG and EWG were screened out. Experimental results indicate that the UHPLC-Q-Orbitrap-MS/MS is a useful tool to characterize variations of ginsenosides in WG and EWG.


Introduction
e root of ginseng (Panax ginseng C. A. Mey.) has been widely used as traditional medicine and functional foods in China and other Asian countries and regions for over 2,000 years [1]. It is found that ginseng contains lots of bioactive and pharmacological effects ingredients such as polysaccharides, saponin, amino acids, vitamins, protein, and phenolic compounds [2]. In China, WG (white ginseng) and RG (red ginseng) are two main types of ginseng that are widely used in Chinese herbal medicine and food markets. WG is produced by drying fresh ginseng in the sun, and RG is manufactured by steaming fresh ginseng at 95-100°C for 2-3 h and then drying. Although both white ginseng and red ginseng were processed with fresh ginseng, they were used for different purposes in the clinical application of TCM (Traditional Chinese Medicine) practice [3]. Meanwhile, ginseng raw materials and products were popularized worldwide as a natural healthy food along with the global trends of preference for natural products [4]. Modern studies showed that the bioactive and pharmacological effects components are mainly recognized as ginsenosides [5], which exhibit anti-inflammatory, antioxidant, antiapoptotic, anticancer, and immune-stimulant pharmacological activities [6][7][8][9][10][11][12][13][14][15].
Extrusion is a continuous process with high temperature, high pressure, and short time. Many chemical and physical reactions occur during the extrusion process, such as starch gelatinization, protein denaturation, and Millard reaction. Meanwhile, functional properties are also changed [16]. Numerous studies have reported that the extruding process could increase some bioactive and pharmacological effects of ginseng products, compared with unprocessed WG [17][18][19].
e physical characteristics and chemical composition of ginseng are changed during the extrusion process, which further alters the functional properties of ingredients [20][21][22][23][24]. Many researchers have extruded white ginseng and red ginseng samples conducted to improve the chemical and physical properties [8,16,18,19,[25][26][27]. e contents of crude saponin were higher in the extruded ginseng than that in unprocessed ginseng. In addition, the content of Rg2, Rh1, Rh2, and Rg3 in EWG was increased as the extrusion temperature was raised [28]. WG was more suitable for extrusion than RG because more significant increased antioxidant activity was obtained in extruded WG than that in RG [8]. Although studies on the physical, chemical, and functional properties of extrusion WG have been reported in the literature, the comparison of ginsenoside composition of WG before and after extrusion process has not yet been researched.
In the present study, we applied UHPLC-Q-Orbitrap-MS/ MS combined with multivariate statistical analysis approach to assess the ginsenoside compositions of WG and EWG. We also investigated the changing chemical structures of WG and EWG and the possible reasons.

Extrusion Process.
e WG root powder was extruded with a co-rotating intermeshing twin-screw extruder (Fumach Food Stuff Engineering & Technology Co., Hunan, China). e parameters of extrusion were as follows: the moisture content of 20%, screw speed of 200 rpm, the feed rate of 10 kg/h, and die diameter of 3.0 mm. e temperature profile from the feed section to the die exit was set to 140°C. e extrudate was directly dried in the oven at 60°C for 12 h.

Extraction of Ginsenosides.
e obtained powder was weighed (0.1 g) and extracted with 5 mL of 70% methanol in an ultrasonic water bath for 45 min, and the extract was filtered through a syringe filter (0.22 μm) and stored in a 4°C refrigerator for LC-MS analysis [29,30].

e Methods of UHPLC-Q-Orbitrap-MS/MS Analysis.
Chromatographic separation was performed on an Ultimate 3000 ultrahigh-performance liquid chromatography system ( ermo Fisher, San Jose, CA, USA) coupled with the Supelco C 18 column (3.0 × 50 mm, 2.7 μm; Sigma-Aldrich, USA). e column oven temperature was maintained at 35°C, and the mobile phases A and B were acetonitrile and water with 0.1% formic acid, respectively. e separation of experimental samples was programmed with the following gradient elution: the proportion of acetonitrile (A) was increased from 15% to 19% (0-5 min), 19-19% (5-10

Data Processing and Multivariate Analysis.
e SIEVE (version2.1, ermo Fisher, San Jose, CA, USA) software was used to process the raw data of samples, which could detect the mass, retention time, and intensity of the peaks in each TIC. e max retention time shift was set at 0.20 min, and the m/z width was 10 ppm to align the features. e base peak min intensity and background were set at 10 5 and 3, respectively. After being aligned, the intensity of each ion was normalized by the total ion intensity of each TIC. e resultant dataset, containing m/z value @ retention time, the normalized intensity, and the sample code, was used to perform the multivariate statistical analysis. en, the datasets were saved as .csv files and imported into SIMCA-P software 11.5 (Umetrics, Umea, Sweden) to conduct the multivariate statistical analysis, including orthogonal partial least squared discrimination analysis (OPLS-DA) and hierarchical clustering analysis (HCA). In the OPLS-DA model, ions with variable importance in projection VIP values larger than 1 were highlighted and were further filtered by t-test (SPSS19.0, Chicago, IL, USA). e components with p < 0.05 were considered significant and were selected as analytical markers.

UHPLC-Q-Orbitrap-MS/MS Analysis of White Ginseng and Extruded White Ginseng.
e ultrahigh-performance liquid chromatography combined high-resolution mass spectrometry has been proved as an effective analytical tool for ginsenoside analysis in complex extracts of Chinese herb medicine [31][32][33][34]. Figure 1 shows the total ion chromatogram (TIC) of the extracts of WG and EWG samples by UHPLC-Q-Orbitrap/MS in the negative ion mode. e ginsenoside compounds were effectively separated in 45 min by the established method. e intensities of several peaks were different before and after extrusion. Compared to WG, the total ion chromatogram of EWG showed obvious lower or higher intensity of some peaks from 25 to 30 min. ese demonstrated that the extrusion process changed the chemical composition of the ginseng sample. Meanwhile, the sample of ginseng contains water, and the determination of water content may be performed before extraction [35,36], which contribute to accurate determination of polysaccharide in future research. e Q-Orbitrap-MS was reliable and sensitive for measuring the exact mass values of the compounds from samples. Moreover, the retention time, accurate masses, and the characteristic fragment ions were compared with the standards to perform the compound identification.
Because the negative ion mode gave a much clearer fragmentation pattern for structure identification of ginsenoside compounds, we detected ginsenoside and confirmed the molecular weight by full-scan MS in negative ion mode. e [M-H] − and [M + HCOO] − ions of ginsenoside were formed in the negative ion mode. Compared to the theoretical value, all molecular ions were measured within the mass accuracy of 10 ppm. Table 1 shows the characteristic fragment ions of ginsenosides.
e aglycone type and the sequence of ginsenosides were confirmed by the tandem MS information. Figure 2 shows the tandem MS spectra of protopanaxadiol saponins (Rg1 and Rb1), malonyl saponins (mRb1), and oleanolic acid saponins (Ro). As shown in e experimental results show that the tandem mass spectrum fragmentation provided structural rich information for the elucidation of ginsenosides. By comparing the retention time, full scan, and the tandem MS spectra with the standard, ginsenoside structure could be identified. e components without standard could be tentatively assigned by comparing the data with the literature record. As a result of our analysis, a total of 45 ginsenosides were identified from WG and EWG. e identification information of ginsenosides is listed in Table 1.

Multivariate Statistical Analysis of White Ginseng and
Extruded White Ginseng. Some researchers obtained chemical profiling data using high-resolution mass spectrometry combined with multivariate statistical analysis [37][38][39][40][41]. e antioxidant activity of EWG was better than that of WG because of the chemical composition changes after extruding [16,18]. us, we used statistical methods to explore the differences in composition. e extract of WG and EWG was analyzed by the UHPLC-Q-Orbitrap-MS/MS. After data preprocessing, the dataset was generated and used to conduct multivariate statistical analysis.
Multivariate data analyses were performed to characterize the distinct composition of various chemicals from WG and EWG in detail. As shown in Figure 3(a), the score plot of OPLS-DA were effectively used todistinguishbetween WG and EWG samples. No overfitting was found because the permutation R 2 (0.993) and Q 2 (0.989) values on the right are higher than those on the left (Figure 3(b)). In the S-plot, the points of the chemical composition that importantly contributed to the variance between WG and EWG were plotted farther along the x-axis and y-axis (Figure 3(c)). In the comparison between WG and EWG, the increased components of WG were shown in the lower left quadrant of S-plot, while the upper right quadrant showed the increased components of EWG. e results showed that three ginsenosides (malonyl (M)-Rb1, M-Rb2, and M-Rc) and three ginsenosides (Rb2, Rc, and Rg1) were elevated in WG and EWG, respectively. In the OPLS-DA and S-plot, different compositions of the chemical were distinguished between WG and EWG.
In order to visualize the tendency of the variation of the chemical markers of WG and EWG, a heat map was constructed based on the relative intensity of each compound. As shown in Figure 4, color differences indicated the composition change in the WG group and the EWG group. Between the WG and EWG groups , the contents ofginsenoside-Rh2, -Rg3, -Rg1, -F1, -Rb3, -Ra1, -Rg7, -Rc, and Rd in EWG samples were significantly higher. However, the contents of malonylginsenoside-Rb1, -Rc, -Rb3, -Rd, -Rb2, -Rg1, and -Re, and ginsenoside-Re5 in all the WG samples were significantly higher.. is was illustrated in the heating trial, in which the concentration of ginsenosides was affected by the thermal processing condition and the degree of conversion between malonyl and neutral ginsenosides [37,42,43]. e results suggested that malonyl-ginsenoside was thermally unstable and especially susceptible to hydrolysis by extruding treatment. e malonyl residue was well preserved in WG because its dehydration was conducted in sunlight without extruding treatment.
In the case of crude saponin (some ginsenosides such as Rb1, Rg1, and Rc) and rare saponin (some ginsenosides such as Rh2 and Rg3) content, there was a slight increase after extrusion.
e malonyl-ginsenoside is prone to be transformed into ginsenoside due to heat and pressure treatment; the conversion efficiency of the ginsenosides was increased      II_Y01   II_Y03   II_Y04   II_Y05 II_Y06 II_Y07  II_Y09   II_Y08  II_Y10   II_Y02   II_J02   II_J01   II_J03  II_J04   II_J05   II_J07 II_J08  II_J09   II_J10   II_J06   1  by the extrusion process. e amount of several ginsenosides was increased by the extrusion process because the cell or tissues of ginseng underwent transformations, which allowed easier extraction of these components through the extrusion process. Meanwhile, this result was caused by the weakening of molecular bonds and increased water absorption at the high temperature, pressure, and shear forces involved in the extrusion process.

Conclusions
In this study, we perform the chemical comparison of white ginseng before and after extrusion by UHPLC-Q-Orbitrap-MS/ MS combined with multivariate statistical analysis. Multivariate statistical analyses were applied to differentiate the chemical components of WG and EWG, and 6 ginsenosides were found as the characteristic chemical markers of the analytical sample.
e experimental information will help to supervise the production of each type of processed ginseng product in the herbal medicine market or food industry. Finally, this study verified the application value of this method in the quality evaluation of WG and EWG. It is expected that the established method will be useful for evaluating processed products not only by ginseng samples but also other agricultural and food product processing such as tea, grape, meat, and so on.

Data Availability
e data used to support the results of this study are included within the article. Any further information is available from the authors upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.