Evaluation of Multicomponent Changes of Schisandra chinensis Fruits with Different Drying Process by UPLC-QQQ-MS-Based Targeted Metabolomics Analysis

Schisandra chinensis fruit is a famous tonic traditional Chinese medicine and healthy food. The chemical components of dried fruits may vary with the initial processing methods of fresh fruits. Therefore, in this study, the influence of drying was evaluated based on targeted metabolomics analysis. The ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS) was applied in determination of multicomponent including lignans, organic acids, and oligosaccharides. The fresh fruits of Schisandra chinensis were processed by natural drying in the sun, hot air drying and vacuum drying at different temperatures, and vacuum freeze drying, respectively. The contents of lignans, organic acids, and oligosaccharides in different fruit samples were quantified and statistically analyzed by partial least squares discriminant analysis (PLS-DA). The variation of totally 10 lignans, 5 organic acids, and 9 oligosaccharides with different drying process were compared. The results showed that the drying time of natural drying method was much longer than the other methods using instrument. For hot air and vacuum drying samples, most of lignans, organic acids, and oligosaccharides gradually decreased with the increase of temperature. And the appearance of fruit drying at higher temperature was dark red. At the same processing temperature, vacuum drying takes longer time than hot air drying. In addition, organic acid and oligosaccharide contents of vacuum freeze drying treated samples were higher than the other processed samples significantly. According to the targeted metabolomics analysis, the lignans, organic acids, and oligosaccharides markers were screened out for holistic quality evaluation of Schisandra chinensis fruits. In comprehensive consideration of multicomponent, drying time, fruit appearance and storage, and production feasibility, hot air drying at 50°C is more suitable for the process of Schisandra chinensis. The result is expected to provide a scientific basis for the selection of proper method for drying process of fresh Schisandra chinensis fruit.


Introduction
As a kind of tonic traditional Chinese medicine and healthy food, Schisandra chinensis (Turcz.) Baill. fruits belong to the Magnoliophyta group [1]. It presented functions of astringency, invigorating qi while nourishing fluid and tonifying kidney, etc. [2,3], which was listed as top grade in Shennong's Herbal Classic with highly medicinal value. The primary processing of Chinese medicinal materials is the key step in their production, which is of great influence on their quality and pharmacological effects. The traditional primary processing of Schisandra chinensis fruits is drying in the sun. This drying pattern is greatly affected by weather and is difficult to standardize. With the development of drying technology, modern drying mode has been widely applied in Chinese medicinal material processing, such as hot air drying, vacuum drying, and vacuum freeze drying.
Schisandra chinensis fruits mainly contain lignans, organic acids, saccharides, and so on [4][5][6][7][8][9][10][11][12]. Lignan is used as characteristic active components in quality control generally, which has bioactivities such as antioxidation, hepatoprotective, and antitumor [13,14]. It is well known that organic acids and saccharides also presented important pharmacological effects [15][16][17]. Especially, oligosaccharides gained more attention in healthy caring field [18,19]. Therefore, simultaneous analysis of multiple components is more accurate and reliable in quality evaluation of Schisandra chinensis fruits. In this experiment, lignans, organic acids, and oligosaccharides were used as chemical markers to evaluate the influence of different drying methods on quality of Schisandra chinensis fruits, and furthermore, the optimized drying pattern was screened out.
In this study, UPLC-QQQ-MS-based targeted metabolomics technology was developed and applied to determine three main types of components of Schisandra chinensis fruits with different drying parameters. The lignans and organic acids were detected in positive and negative ionization mode of MS, respectively. In addition, the solid-phase methylation method was applied in analysis of oligosaccharides in Schisandra chinensis fruits for the first time. Under multiple reaction monitoring (MRM) mode, the accurate quantification of lignans, organic acids, and oligosaccharides was obtained. The contents of lignans, organic acids, and oligosaccharides were calculated and statistically analyzed. The influences of drying on variation of these three types components were investigated. And the Schisandra chinensis fruits with different drying were evaluated comprehensively. This study provided reference for selection of optimal drying conditions of Schisandra chinensis fruits, and the result was useful for improving quality of Schisandra chinensis fruits in production.

Materials and Instrument.
The fresh ripe fruits of Schisandra chinensis (Turcz.) Baill. were collected from Jingyu County, Baishan City, Jilin Province. After harvesting, the fruits should be dried immediately and according to different drying methods, respectively, as shown in Table 1. During natural, hot air, and vacuum dryings at different temperatures, the fruits were turned over every 12 h and drained the moisture. The samples in vacuum freeze drying should be prefreezed ahead at -80°C for 24 h. The water contents of dried fruits were less than 16%, and the drying time of each method is presented in Table 1.
After drying, the fruits were powdered and passed through 60 mesh sieve. Then, the powders were pretreated with different procedures to extract lignans, organic acids, and oligosaccharides for UPLC-QQQ-MS detection under optimized parameters, respectively.

Lignan Determination
2.3.1. Reference and Sample Solution Preparation. The 10 lignan references were accurately weighted and dissolved with 80% methanol-water to 1 mg/mL, individually. And then, reference solutions were mixed and prepared to stock solutions. After step by step dilutions, a series of mixed reference solutions with desired concentrations were obtained for quantification analysis.
The lignan extraction was according to Chinese Pharmacopoeia (2020 edition). The fruit powders were extracted with methanol by ultrasonication (250 W, 20 kHz) for 30 min at room temperature. The extraction was repeated five times and then diluted with methanol. Lastly, the extracts were passed through 0.22 μm microfilter for UPLC-QQQ-MS analysis.
Mass spectrometry conditions are as follows: positive ionization with scanning range m/z 150~1000 in MRM scan mode. Spray voltage is 3500 V. Sheath gas flow rate is 40 arb, and auxiliary gas flow rate is 12 arb. Ion transfer tube temperature is 333°C, and vaporizer temperature is 317°C. The preparation of organic acid extracts from Schisandra chinensis fruits was the same as "2.3.1 lignan extraction preparation." Mass spectrometry conditions are as follows: negative ionization with scanning range m/z 50~500 in MRM scan mode. Spray voltage is 3500 V. Sheath gas flow rate is 40 arb, and auxiliary gas flow rate is 10 arb. Ion transfer tube temperature is 320°C, and vaporizer temperature is 300°C.
2.5. Oligosaccharide Determination 2.5.1. Reference and Sample Extraction Preparation. The 9 oligosaccharide reference solutions were prepared using DMSO as solvent. 10 g Schisandra chinensis fruit powder was extracted with 200 mL water and stirred continuously in 70°C water bath for 5 hours. The supernatant was concentrated by reduced pressure, precipitated to 80% ethanol solution, and stood for 12 h at 4°C. After filtration and concentration, the oligosaccharide extracts were obtained and then freeze-dried. DMSO was added and Vortex oscillated to dissolve it for methylation.
The solid phase methylation column was filled with sodium hydroxide fine powder, dispersed in acetonitrile uniformly. After filling the column, pump DMSO at a flow rate of 200 μL/min to remove the residual acetonitrile. Meanwhile, the solid phase methylation column was cleaned with DMSO before and after methylation reaction. Take 900 μL reference or oligosaccharide extract DMSO solution mixed with 100 μL CH 3 I. The mixture was pumped into methylation column in 200 μL/min flow rate. And then, the methylation products in the column were rinsed out with 4 mL DMSO. Add 5 mL 5% acetic acid to the collected DMSO solution to stop methylation and then add the same volume of CH 2 Cl 2 for extraction with 3 times. The CH 2 Cl 2 extracts were combined and evaporated in water bath at 45°C. The residues were dissolved in 10 mL methanol and passed through 0.22 μm microfilter for UPLC-QQQ-MS quantification analysis.
Mass spectrometry conditions are as follows: positive ionization with scanning range m/z 50~2000 in MRM scan mode. Spray voltage is 3500 V. Sheath gas flow rate is 40 arb, and auxiliary gas flow rate is 10 arb. Ion transfer tube temperature is 340°C, and vaporizer temperature is 300°C.
2.6. Multivariate Statistical Analysis. UPLC-QQQ-MS data of lignans, organic acids, and oligosaccharides were processed using the Xcalibur software (Thermo Fisher Scientific Inc.). The contents of 10 lignans, 5 organic acids, and 9 oligosaccharides were imported into SIMCA-P software 13.0 for multivariate statistical analysis.

Methodology Validation of UPLC-QQQ-MS in
Determination of Lignans, Organic Acids, and Oligosaccharides. The structures of three types of components, lignans, organic acids, and oligosaccharides, are shown in Figure 1. The typical lignan, organic acid, and oligosaccharide solution was injected directly to mass spectrometer to optimize experimental parameters by syringe pump automatically. The positive ion mode was chosen for lignans and methylated oligosaccharides as higher signal intensity, while negative ion mode was in higher relative intensity for organic acid. The fragment ions and the corresponding collision energies were obtained by tuning automatically. After direct MS optimization, the pairs of quantitative and qualitative ions in MRM mode were selected in UPLC coupled to mass spectrometer analysis. The retention times of available references were determined. The validation of UPLC-MRM-MS quantification methodology is presented in Table 2. The linearities of references showed good relationship in linear range with correlation coefficients ðrÞ > 0:99. The precision was conducted by five repetitive detections in a day. The repeatability was investigated by three detections in each day of three consecutive days. The relative standard deviations (RSD) of precision and repeatability were less than 3.25%, respectively. The limits of detection (LOD) and quantification (LOQ) values demonstrated that this developed method was sensitive for quantifying lignan, organic acid, and oligosaccharide in dried Schisandra chinensis fruit extracts. The average recoveries of each reference at three different levels proved good accuracy of this UPLC-MRM-MS method.   Table 3.
The contents of schisandrol A in Schisandra chinensis fruits treated by different drying process methods were all more than 0.4%, which meets the standards of Chinese Pharmacopoeia (2020 edition). Among the 10 lignans, the contents of schisandrol A, gomisin J, schisandrol B, schizandrin A, schizandrin B, and schisandrin C were all higher in eight dried fruit samples. It may be due to these ingredients accumulate more in the fruit of Schisandra chinensis under the synthesis enzymes [1]. For hot air drying samples, the 10 lignans gradually decreased with the increase of temperature, and the highest content was presented in sample dried by hot air at 40°C. In vacuum drying with different temperatures, the changes of these 10 lignans were similar to those in hot air drying samples and the average contents were higher than that in hot air drying ones. It demonstrated that different temperatures presented little effect on the lignan contents changing under vacuum drying condition. From the sample of natural drying in the sun, the lignan contents were less than those in hot air drying and/or vacuum drying at 40°C and more than the higher temperature drying samples. The contents of lignans in Schisandra chinensis samples treated by different drying methods presented no significant difference.
Lignans are the most important active components in Schisandra chinensis, which play an important role in the digestive system, cardiovascular system, urinary system, and other fields [38]. In this experiment, lignans as representative component of Schisandra chinensis were determined. It is of certain significance to screen the optimized drying process methods of Schisandra chinensis. Table 3, the quinic acid, malic acid, citric acid, sorbic acid, and chlorogenic acid in Schisandra chinensis fruit samples were detected by validated UPLC-MRM-MS method in negative ion mode. The peaks were confirmed by retention time and ion pair with corresponding reference substances and were also quantified by regression equation. Among the five organic acids, the contents of malic acid and chlorogenic acid are higher, while the other three acids are relatively lower.

Variation of Organic Acids in Schisandra chinensis Fruits with Different Drying. As shown in
The average content of organic acids in Schisandra chinensis samples treated by hot air drying was higher than that treated by vacuum drying at different temperatures, respectively. With the increasing of drying temperature of fresh fruits, the organic acids decreased significantly. In addition,    Table  3: Contents of 10 lignans, 5 organic acids, and 9 oligosaccharides in Schisandra chinensis dried and fresh fruits (n = 10).

Contents
Dried fruits with different methods   In this experiment, the contents of organic acids in Schisandra chinensis fruits with different drying process methods were compared. The organic acid in Schisandra chinensis is high and is the material basis of "sour flavor." The organic acids in traditional Chinese medicine also have certain pharmacological effects. For example, citric acid can inhibit platelet aggregation [39], and quinic acid can be used in the treatment and diagnosis of tumors [6,40]. Therefore, it is of obvious significance to study the composition of organic acids in Schisandra chinensis. The results show that vacuum freeze drying or lower temperature drying is most suitable for retention of organic acids in Schisandra chinensis fruits.
3.4. Quantification of Oligosaccharides in Schisandra chinensis Fruits with Different Drying. It can be seen from Table 3 that the 9 oligosaccharides were extracted from Schisandra chinensis and methylated before UPLC-MRM-MS analysis. The contents of 9 oligosaccharides in the fruit samples treated by vacuum freeze drying were the highest, followed by the samples dried by hot air at 40°C. But the surface of vacuum freeze-dried fruits was sticky and is not conducive to storage of medicinal materials [1]. The average oligosaccharide content of Schisandra chinensis samples dried by hot air was higher than samples treated by vacuum drying at different temperatures. The total content of oligosaccharides in the natural drying group was the lowest.
Modern pharmacological studies show that the sugars in Schisandra chinensis have antifatigue, antioxidation, antiaging, liver protection, and other effects [7,8,[16][17][18]. Through the study and comparison, it was found that vacuum freeze drying is the most suitable drying method for oligosaccharides of Schisandra chinensis fruits.

Targeted Metabolomics Analysis of Multicomponents in
Schisandra chinensis Fruits with Different Drying. According to results of 10 lignans, 5 organic acids, and 9 oligosaccharides in Schisandra chinensis samples treated by different drying process methods, the targeted multicomponents were taken as statistical variable. The PLS-DA analysis was applied to investigate differences of all samples and evaluate the contribution of targeted multicomponents to fruits dried by different methods.
The PLS-DA scores plot using lignans (a), organic acids (b), and oligosaccharides () as variables are shown in Figure 2. Among the 8 groups of Schisandra chinensis samples treated by different drying process methods, the samples in the same group are relatively gathered. Different groups were distributed separately, indicating that there are significant differences between groups. It can also be seen that the vacuum freeze drying group is far away from the other groups significantly in Figures 2(b) and 2(c). It demonstrated that the contents of organic acids and oligosaccharides in vacuum freeze drying group were significantly different from that in other groups.
The contributions of each target components on difference of processed fruits were also calculated, and VIP value exceeded 1.0 was chosen as differential markers. The loading plot is shown in Figure 3, and each spot stood for a component. The spot was further away from the origin, and the contribution of this component to discrimination was greater. Therefore, organic acids and oligosaccharides were obtained as differential chemical markers. Meanwhile, the lignans were gathered around the origin. Among the three kinds of components, oligosaccharides presented greater contribution in distinction of drying methods.
The results showed that the drying time of natural drying method was much longer than the other methods using instrument. The shortest drying time was in hot air at 60°C group. But the appearance of fruits dried at higher temperature was dark red. At the same drying temperature, vacuum drying takes longer time than hot air drying. As under vacuum condition, moisture could not be released immediately. Therefore, the steam-pumping equipment is required. Lignans, organic acids, and oligosaccharides were screened out as chemical markers for holistic quality evaluation of Schisandra chinensis fruits. According to the contents of multicomponent, vacuum freeze drying was the best. But the vacuum freeze-dried method needs to prefreeze the samples resulting in an increase of total drying time.  Journal of Chemistry

Conclusions
In this study, the medicinal materials of Schisandra chinensis fruits were processed by different drying methods. Based on targeted metabolomics analysis, lignans, organic acids, and oligosaccharides in fruits samples were determined by UPLC-QQQ-MS. The content changes of these components with different drying methods were investigated. In comprehensive consideration of multicomponents, drying time, fruit appearance, storage of medicinal materials, and feasibility for production, hot air drying at 50°C is more suitable for the drying of Schisandra chinensis. This study provided a scientific basis for the selection of proper method for drying process of fresh Schisandra chinensis fruit.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest
The authors declare that they have no competing interests.