Saponin composition comparison of black ginseng and white ginseng by liquid chromatography-mass spectrometry combined with multivariate statistical analysis

Abstract Black ginseng (BG) is one type of ginseng product, which is produced from fresh ginseng by steaming and drying several times. To characterize the differences in saponin composition of BG and white ginseng (WG), the ultra-high performance liquid chromatography Quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS) was used to analyze the ginseng samples. A total of 53 saponins were successfully identified, and the possible transformation pathways of several ginsenosides were described. Multivariate statistical analysis methods were used to perform the pattern recognition and further to select the marker compounds of samples. Twenty ginsenosides were considered to contribute most to the sample classification, six of which including Rg3, Rk1, Rh4, Rs3, Rs5, and Rk2 increased significantly in BG, while the other fourteen ginsenosides were greatly elevated in WG. The changes of ginsenoside in BG and WG were characterized by UHPLC-Q-Orbitrap-MS, which is of great significance for its quality control and effect evaluation. Graphical abstract


Introduction
Ginseng is the dry root and rhizome of Panax ginseng C. A. Meyer, a perennial herb of the Acanthopanax family, which has been widely used as traditional medicine and functional food in China for more than 4000 years (Chung et al. 2011). The processed products of ginseng mainly include white ginseng (WG), red ginseng (RG), and black ginseng (BG). WG is naturally dried from fresh ginseng; RG is processed through soaking, selection, steaming, drying, and other processes; and BG is ginseng with dark brown, which is processed by steaming and drying fresh ginseng multiple times.
Many studies have shown that the molecular weight of ginsenosides could be reduced by steaming and drying, which considerably improves the bioavailability of ginsenosides thereby improving their biological activity (Sun et al. 2011;Liu et al. 2012). For example, during the processing, a large number of rare ginsenosides, such as Rg3, Rg5, Rk1, and Rk3 were produced due to thermal cracking.
Since the composition of WG and BG saponins has not been compared, the UHPLC-Q-Orbitrap-MS was used to study their changes in different samples. Investigating the transformation that occurs during the steaming and drying, as well as exploring the possible transformation mechanism, is of great significance for the processing research, quality control, and effective and safe use of BG.

Results and discussion
2.1. UHPLC-Q-Orbitrap-MS analysis of ginseng sample Figure S1 shows the total ion current (TIC) of WG and BG extracts detected by UHPLC-Q-Orbitrap-MS in the negative ion mode. Ginsenoside compounds were effectively separated within 33 min by the established LC-MS method. The total ion chromatograms of WG and BG are significantly different at 14-28 min, which proved that nine times of steaming and drying process changed the chemical composition of ginseng.
The compound identification was conducted by matching the retention time, accurate m/z value, and tandem MS information to either the public database or our inhouse database record. The samples were analyzed by LC-MS under negative ion mode, therefore, the [M-H] À and [M þ HCOO] À signals are the main adduct forms of ginsenosides, and the mass accuracy was set to 10 ppm compared to the theoretical value. Tandem mass spectra provide a wealth of structural information for the identification of ginsenosides. The ginsenoside Re ion at RT_m/z 14.72 min_991.5516 was used as an example to illustrate the process of compound identification. As shown in Figure S2A, the base peak at m/z 991.5516 corresponded to [M þ HCOO]ion of ginsenoside Re. And its fragment information is shown in Figure S2B, the ion at 945.5413 represented the deprotonated ion of Re, while the ions at 783.4927, 637.4312, and 475.3755 were produced from the consecutive loss of glucose, rhamnose, and glucose residues from the ginsenoside ( Figure S2C). Using the above procedure, a total of 53 saponins have been identified from BG and WG. Table S1 lists the identification information of ginsenosides.

Multivariate statistical analysis of BG and WG
After data acquisition and data preprocessing, the resulted dataset was used to perform multivariate statistical analysis. As shown in Figure S3A, WG and BG samples were classed into two groups in the PCA score plot. The OPLS-DA model was established to screen out the compounds that most significantly contribute to the classification. The OPLS-DA score plot in Figure S3B exhibited the sample clusters, and the variants with the VIP value larger than 1 that located at both ends of the S-Plot ( Figure S3C) far away from the origin were highlighted as marker compound candidates which considered to contribute most to the sample classification. Then the student t-test was applied to perform the further filtration and the compound candidates with p < 0.05 were screened out as marker compounds enabling the differentiation between BG and WG samples. A total of 21 ginsenosides were identified and their detailed information was listed in Table S2.
In order to systematically evaluate the changes of chemical markers, heatmaps were generated and shown in Figure S4, which showed the varying levels of chemical marker intensity in different ginseng groups in an intuitive way. Red squares signified higher intensities, whereas blue ones represented lower levels. The intensity of ginsenosides Rg3, Rk1, Rh2 in the BG group was significantly higher than that in the WG group, while the content of other ginsenosides such as Rb1, Rg1, mRg1 showed the reverse trend, which indicated that the ginsenosides underwent significant changes during the processing of WG to BG.
According to previous findings, the chemical components of ginseng can be transformed into other forms through different chemical reactions during the steaming and drying process (Zheng et al. 2017;Yao et al. 2021). The chemical can be hydrolyzed, dehydrated, decarboxylated to form new compounds. For ginsenosides in this study, the structure changes mainly occurred at C-3, C-6, or C-20 for carbohydrate hydrolysis to generate the less polar ginsenosides, followed by dehydration at C-20, which is consistent with the previous reports that the steaming and drying process can increase the ratio of protopanaxadiol to protopanaxatriol ginsenoside in BG (Zheng et al. 2017;Metwaly et al. 2019).
As shown in Figure S5A, the PPD-type ginsenoside Rd is transformed into ginsenosides F2, Rg3, Rh2, and compound K by hydrolysis of sugar moiety at C-3 and C-20. The ginsenoside Rg3 is further undergone the dehydration reactions to produce Rg5 and Rk1, and then covered to Rh3 and Rk2, respectively, through hydrolyzation at C-3. Figure S5B exhibits that PPT-type ginsenosides Re and Rg1 are hydrolyzed into Rh1 and Rg2 at C-20, respectively. Ginsenosides Rg6, F4, Rk3, and Rh4 are further produced from Rg2 through dehydration and hydrolysis reaction at C-20 (Sun et al. 2011;Metwaly et al. 2019). It has been reported that the BG had better anti-tumor, immunomodulatory and anti-inflammatory effects than WG due to repeated steaming and drying process (Jang et al. 2016). Ginsenosides with one or two glycosyl residues, which are the highest proportion in steamed BG, such as Rg3, Rh2, and Rg5, showed promising anti-cancer activity in a variety of in vitro, in vivo, and clinical studies (Metwaly et al. 2019). The above transformation that occurred during the BG processing contributes to the chemical diversity between BG and WG, which further influences the biological activities of two ginseng products.

Conclusions
In this study, we used UHPLC-Q-Orbitrap-MS combined with multivariate statistical analysis to compare the saponin composition of BG and WG. Multivariate statistical analysis was used to display the sample classification. A total of 53 ginsenosides were tentatively identified, and 20 of them were considered to be characteristic markers of ginseng samples. This study verified the application of LC-MS to analyze the differences between different ginseng products, providing an experimental basis for traditional Chinese medicine manufacturers to produce the different processed ginseng products, as well as allowing for the explanation of effect differences of different ginseng samples.

Disclosure statement
No potential conflict of interest was reported by the authors.