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Widely Targeted Metabolomics Analysis to Reveal Transformation Mechanism of Cistanche Deserticola Active Compounds During Steaming and Drying Processes

Widely Targeted Metabolomics Analysis to Reveal Transformation Mechanism of Cistanche Deserticola... ORIGINAL RESEARCH published: 14 October 2021 doi: 10.3389/fnut.2021.742511 Widely Targeted Metabolomics Analysis to Reveal Transformation Mechanism of Cistanche Deserticola Active Compounds During Steaming and Drying Processes Ziping Ai, Yue Zhang, Xingyi Li, Wenling Sun and Yanhong Liu* College of Engineering, China Agricultural University, Beijing, China Edited by: Cistanche deserticola is one of the most precious plants, traditionally as Chinese Yasmina Sultanbawa, medicine, and has recently been used in pharmaceutical and healthy food The University of Queensland, Australia industries. Steaming and drying are two important steps in the processing of Reviewed by: Cistanche deserticola. Unfortunately, a comprehensive understanding of the chemical Bárbara Socas-Rodríguez, composition changes of Cistanche deserticola during thermal processing is limited. Institute of Food Science Research In this study, ultra-performance liquid chromatography-tandem mass spectrometry (CIAL), Spain Mohamed Fawzy Ramadan (UHPLC-MS/MS)-based widely targeted metabolomics analysis was used to investigate Hassanien, the transformation mechanism of Cistanche deserticola active compounds during Zagazig University, Egypt steaming and drying processes. A total of 776 metabolites were identified in Cistanche *Correspondence: Yanhong Liu deserticola during thermal processing, among which, 77 metabolites were differentially liuyanhong@cau.edu.cn regulated (p < 0.05) wherein 39 were upregulated (UR) and 38 were downregulated (DR). Forty-seven (17 UR, 30 DR) and 30 (22 UR, 8 DR) differential metabolites were Specialty section: This article was submitted to identified during steaming and drying, respectively. The most variation of the chemicals Nutrition and Food Science was observed during the process of steaming. Metabolic pathway analysis indicated Technology, that phenylpropanoid, flavonoid biosynthesis, and alanine metabolism were observed a section of the journal Frontiers in Nutrition during steaming, while glycine, serine, and threonine metabolism, thiamine metabolism, Received: 16 July 2021 and unsaturated fatty acid biosynthesis were observed during drying. The possible Accepted: 10 September 2021 mechanisms of the chemical alterations during thermal processing were also provided by Published: 14 October 2021 the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Furthermore, Citation: Ai Z, Zhang Y, Li X, Sun W and Liu Y the blackening of the appearance of Cistanche deserticola mainly occurred in the (2021) Widely Targeted Metabolomics steaming stage rather than the drying stage, which is associated with the metabolism of Analysis to Reveal Transformation the amino acids. All results indicated that the formation of active compounds during the Mechanism of Cistanche Deserticola Active Compounds During Steaming processing of Cistanche deserticola mainly occurred in the steaming stage. and Drying Processes. Front. Nutr. 8:742511. Keywords: cistanche deserticola, widely targeted metabolomics, steaming, drying, active compounds, formation doi: 10.3389/fnut.2021.742511 mechanism Frontiers in Nutrition | www.frontiersin.org 1 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola INTRODUCTION specific metabolite groups, rather than all the components in the sample. Non-targeted metabolomics technology can qualitatively Cistanche deserticola, belonging to the orobanchaceae family, determine the metabolites based on existing databases, with is one of the most famous tonic medicines and is mainly high coverage of compounds, however, low accuracy. The key distributed in the tropical and subtropical regions of the world, metabolites must be confirmed by standard products (14). Widely such as China, Iran, India, Mongolia, and so on (1–3). Cistanche targeted metabolomics is a new technology that integrates the deserticola serves as one of the most commonly utilized herbal advantages of non-target and targeted metabolites detection medicines for the treatments of kidney deficiency, impotence, technologies to achieve wide coverage, high throughput, female infertility, morbid leucorrhea, profuse metrorrhagia, and and sensitivity (15). Consequently, this technology has been senile (4, 5). Modern pharmacological research showed that widely used in the study of ingredient changes in different Cistanche deserticola has the effects of improving immunity, anti- materials during processing, such as active ingredients in fatigue, anti-aging, and enhancing learning and memorization functional foods by different processing methods (16), flavonoids ability (6). Owing to these health benefits, Cistanche deserticola and phenylpropanoids compounds in Chinese water chestnut tea made from its stem tubers has been developed as a processed with different methods (17), rice yellowing mechanism nourishing supplement and is being increasingly favored by during yellowing process (18), and the formation mechanism consumers. The active ingredients of Cistanche deserticola of characteristic non-volatile chemical constitutes during oolong have been shown to be responsible for its medicinal functions tea manufacturing process (19). Therefore, it is theoretically (7). Some active ingredients in Cistanche deserticola, such as feasible to use widely targeted metabolite technology to study phenylpropanoids (for example phenylethanoid glycosides), the mechanism of the conversion of active ingredients during the flavonoids, polysaccharides, oligosaccharides, iridoids, and processing of Cistanche deserticola. lignans have been reported in the previous studies (6, 8). Thus, the objectives of the present study were to (1) Due to the perishable and seasonal features, the all-year- provide useful information on the chemical changes in round supply of fresh Cistanche deserticola is unavailable, Cistanche deserticola during steaming and drying processes by accordingly, the processed Cistanche deserticola becomes the using ultraperformance liquid chromatography-tandem mass main consumption form. The quality of Cistanche deserticola spectrometry (UHPLC-MS/MS) combined with a widely targeted is dependent on many factors, such as climate, habitats, hosts, metabolomic approach; (2) identify the differential metabolites harvest time, processing technology, and position on the plant, and their regulation rules, and reveal the possible conversion among which processing technology is particularly important (4). pathways in Cistanche deserticola during processing. This study Steaming and drying are two important steps in the processing of is, therefore, expected to provide a theoretical reference for the Cistanche deserticola. Usually, the harvested Cistanche deserticola formation mechanism of high-quality Cistanche deserticola. rhizome was steamed in a steaming boiler at 93 C for 30 min and then dried at 60 C until the moisture content of 10% MATERIALS AND METHODS on a wet basis (w.b.) (9). Previous studies have shown that steaming can promote the accumulation of active ingredients Materials and Chemicals in Cistanche deserticola, such as phenylethanoid glycosides, Raw materials: Fresh Cistanche deserticola samples were obtained soluble sugars, and polysaccharides, accompanied by blackening from the Hetian region in Xinjiang Province of China. The of appearance color (9–11). However, most of the previous samples were carefully selected with the same size (average studies focused on certain specific compounds, very rare research length, diameter, and weight were 11.7 ± 1.1 cm, 7.0 ± 1.1 cm, are about the changes in all the chemical compounds and the and 360 ± 8.9 g, respectively). The samples were stored at room metabolite conversion mechanism during processing. Therefore, temperature in a dark environment with an initial moisture it is necessary to clarify the metabolite changes of Cistanche content of about 78.56% ± 3.47%. Prior to the experiments, deserticola in different processing stages. Cistanche deserticola samples were washed with tap water to Metabolomics is usually applied to qualitative and quantitative remove the dust on the surface. Excess water on its surface was analysis of all small molecules (namely, targeted and non- removed by blotting paper. targeted compounds) detected in the sample (12). Analysis of Chemicals: Methanol, acetonitrile, and formic acid were changes in various chemical components during food processing liquid-chromatography mass spectrometry grade (LC-MS) and helps to deepen the understanding of the mechanism of chemical purchased from Merck (Sigma Aldrich, MO, USA). The other component transformation in food processing (12). In recent analytical standards presented a purity higher than 98% (Sigma years, metabolomics has also been applied to the study of Aldrich, MO, USA). Cistanche deserticola for the discrimination of different parts (11) and different Cistanche deserticola species (13). The detection Experimental Design methods of metabolites in these studies were mostly based on Previous studies have shown that the chemical compounds targeted and non-targeted metabolomics. Among them, targeted distribute unevenly in the longitudinal direction of Cistanche metabolomics is based on standard products, with high data deserticola (1). Therefore, to obtain the same initial contents of accuracy and reliability, however, limited coverage of metabolites. chemical compounds in each sample, in the present research Targeted metabolomics is an important part of metabolomics all selected Cistanche deserticola was cut into three equal parts research, it is the targeted and specific detection and analysis for for the fresh group (A), steamed without drying group (B), Frontiers in Nutrition | www.frontiersin.org 2 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola and dried after steaming group (C), respectively, by longitudinal ESI-QTRAP-MS/MS Conditions segmentation with the longitudinal symmetry axis as the A triple quadrupole (QQQ)-linear ion trap mass spectrometer center (20). (QTRAP, API 6500 QTRAP UHPLC-MS/MS) + QQQ For group B, the samples were successively steamed for spectrometer equipped with an ESI turbo ion-spray interface 8 min according to preliminary experiments. A pulsed vacuum (Sciex Technologies, Framingham, MA, USA) was applied for steaming equipment (self-developed by China Agricultural MS analysis. The analytical conditions were as follows: ion University, Beijing, China) was used for steaming treatment of spray voltage: +5,500 V (positive ion mode)/−4,500 V (negative fresh Cistanche deserticolas. Steamed samples were dried in a ion mode), curtain gas: 35 psi, source temperature: 400 C, vacuum freeze-dryer (LGJ-25C, Si Huan Scientific Instrument ion source gas 1: 60 psi, ion source gas 2: 60 psi, declustering Factory Co., Beijing, China). The heating plate and cold trap potential: ±100 V. QQQ scans were acquired as multiple reaction temperature were 30 and −60 C, respectively. For group C, monitoring (MRM) experiments with collision gas (nitrogen) set the samples were successively steamed using pulsed vacuum to 5 psi. equipment for 8 min and dried in the hot air impingement Qualitative and Quantitative Analysis of dryer (self-developed by China Agricultural University, Beijing, China) until the final moisture content of 10% (w.b.). The airflow Metabolites rate and temperature were set at 6 m/s and 60 C, respectively, Qualitative and quantitative analyses of metabolites were referring to the research results of Zou et al. (11). All samples performed according to the methods by Liu et al. (18). Primary were stored at−20 C no more than 7 days before further analysis. and secondary mass spectrometry data were qualitatively analyzed based on the self-built human metabolome database (MWDB) (Metware Biotechnology Co., Ltd. Wuhan, China) Determination of Appearance Color of and the public database. Meanwhile, to ensure the accuracy Cistanche Deserticola of the qualitative analysis of some substances, interferences + + The appearance color of the Cistanche deserticola before and after from repeated signals of Na , NH , K and ions, and each thermal processing was measured using a colorimeter (SMY- repetitive signals of fragment ions derived from other relatively 2000SF, Shengming Yang Co., Beijing, China), and the blackness large molecules and isotope signals were removed during identification. Metabolite structural analysis was performed with was characterized by L value. reference to the public databases (Mass Bank, KNApSAcK, HMDB, MoTo DB, and METLIN). Sample Preparation and Extraction Metabolite quantification was carried out using the MRM The metabolite extraction was carried out according to the mode of the QQQ mass spectrometry. In the MRM mode, method reported previously by Chen et al. (21) with some the precursor ions (parent ions) of the target substances and minor modifications. In brief, the dried samples were crushed excluded ions corresponding to other substances with different using a mixer mill (MM 400, Retsch Company, Haan, Germany) molecular weights were screened first using the quadrupole rod with a zirconia bead for 2 min at 60 Hz. Then 50 mg powder to initially eliminate interference. The precursor ions then break (sifted through a 65 mesh sieve) of each sample was precisely through the collision chamber to form many fragment irons weighed, transferred to an Eppendorf tube, and extracted with after ionization, which were filtered by QQQ to select single- 1 ml methanol/water mixture (v:v = 3:1). After 30 s vortex, the fragment ions with the desired characteristics while eliminating mixture was homogenized twice at 35 Hz for 4 min, sonicated interference from non-target ions. Finally, after obtaining the for 15 min in an ice-water bath, and then shaken overnight metabolite mass spectrometry data of different samples, the ◦ ◦ at 4 C. After centrifugation at 12,000 rpm for 15 min at 4 C, mass spectrum peaks of all substances were integrated, and the supernatant was collected and filtered through a 0.22-μm the mass spectra peaks of the same metabolite in different membrane, then the obtained extract was transferred to 2-ml samples were integrated and corrected using Multi Quant glass vials and store at −80 C until the UHPLC-MS/MS analysis. version 3.0.2 (ABSCIEX, Concord, Ontario, Canada). The corresponding relative metabolite contents were represented as chromatographic peak area integrals. Metabolites Analysis by UHPLC-MS UHPLC Conditions Data Processing and Analysis The UHPLC separation was carried out using an EXIONLC The metabolic data were processed using orthogonal partial system (Sciex Technologies, Framingham, MA, USA). The least squares-discriminant analysis (OPLS-DA) and hierarchical analytical conditions were as follows: column: Waters ACQUITY cluster analysis (HCA). OPLS-DA was used to discriminate UHPLC HSS T3 C18 (1.8μm, 2.1 × 100 mm); solvent system: each group; it is more sensitive than other statistical methods mobile phase A (0.1% formic acid in water) and mobile phase B to variables with low correlations (17). The OPLS-DA models (acetonitrile containing). The gradient program: 98% A/2% B at were validated through a permutation analysis (200 times). 0 min, 50% A/50% B at 10 min, 5% A/95% B at 11 min, 98% A/2% The model was considered stable when the model parameters 2 2 B at 13.1 min, and 98% A/2% B at 15 min. Flow rate: 0.40 ml/min; (R and Q ) were both close to 1. The variable importance column temperature: 40 C; injection volume: 2 μl; automatic projection (VIP) values of metabolites were calculated. Any injection temperature: 4 C. metabolite with VIP values greater than 1.0 and p-values Frontiers in Nutrition | www.frontiersin.org 3 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola less than 0.05 were selected as biomarkers for each paired carboxylic acids and derivatives, 14 organic acids and derivatives, comparison between different thermal processing stages of 12 phytohormone, and 28 other chemicals. Among them, the Cistanche deserticola. The screening of different metabolites largest group was amino acid and derivatives, the relative was visualized in the form of the volcano plot. Metabolites content of which accounted for 30.26% of the total metabolite accumulation among different samples was analyzed by using composition. In addition, 10 kinds of phenylethanoid glycosides, the R package (www.rproject.org/). The Venn diagram was such as echinacoside and verbascoside, were detected and built according to the program web-based smart diagram R classified in the phenylpropanoids group. (https://cloud.smartdraw.com/). The commercial databases, such The accumulation pattern of metabolites among different as Kyoto Encyclopedia of Genes and Genomes (KEGG) (https: treatment groups was analyzed by HCA. As shown in Figure 3, //www.kegg.jp/kegg/), Pub Chem (https://pubchem.ncbi.nlm. 107 identified metabolites of Cistanche deserticola were clustered nih.gov/), the Small Molecule Pathway Database (SMPDB) in heat maps based on Euclidean distance arithmetic. Metabolites (https://smpdb.ca/), and HMDB (https://hmdb.ca/), were used identified at different thermal processing stages were gathered for enrichment analysis of differential metabolites and finding into three clusters according to the dendrogram. The brighter metabolic pathways. color indicates the higher content of a particular metabolite in the respective sample. The heat map of HCA showed larger di?erences in abundance between the fresh and steamed samples RESULTS AND DISCUSSION than those between steamed and dried samples, indicating that metabolites in Cistanche deserticola may have different Appearance Color Changes of Cistanche transformations during the steaming and drying stage, and the Deserticola During Thermal Processing types and quantities of metabolites involved in the steaming The difference in appearance color of Cistanche deserticola process are more than those in the drying process. between the fresh, steamed and dried samples are representatively displayed in Figure 1. From fresh to dried sample with the going of the processing stage, the appearance Differential Metabolite Analysis of color of the samples changed from yellow-brown to dark Cistanche Deserticola at Different Thermal black, and the darkness in color became more and more Processing Stages obvious (the corresponding L value was reduced from 50.26 For a better understanding of the impact of each processing on to 24.90). Obviously, the appearance changes of Cistanche the metabolites of Cistanche deserticolas, the OPLS-DA scatter deserticola mainly occurred in the steaming process. The scores of pairwise comparison groups are shown in Figure 4A, Maillard reaction, during which sugars react with amino acids showing that the fresh, steamed, and dried after steaming under thermal conditions (22), would be greatly responsible Cistanche deserticolas were significantly different. Moreover, for the dark-colored appearance of processed rhizomes of 2 2 the R Y and Q (as shown in Supplementary Figure 2) with Cistanche deserticola. Previous research studies have shown high-test values indicated that this model was highly reliable that precursors were converted into colorants and generated without overfitting. substances with dark color in the Maillard reaction (23). Similar To screen the expression level of metabolites between the findings were also observed in previous studies for steaming fresh, steamed, and dried after steaming Cistanche deserticola, of Polygonum multiflorum (24) and rhizomes of Polygonatum the analysis of volcano plot was further applied among all 776 cyrtonema (25). The darkness of the steamed samples was further metabolites identified according to the fold-change, combined deepened after drying. This phenomenon was probably due with VIP values to screen the differentially expressed metabolites. to the occurrence of a decrease in the pigment concentration Significant differential metabolites were selected according to during the drying process. the criterion that a fold change score of ≥2 or ≤0.5 with a VIP ≥ 1. The screening results are illustrated in Figure 4B. In Overview of the Metabolites in Raw and the volcanic map, each point represents a metabolite and the Thermal Processed Cistanche Deserticola color of the scattered dots represents the final screening result. Samples Red represents metabolites that are significantly upregulated The total ion chromatogram (TIC) of quality control (QC) (UR), green represents those significantly downregulated (DR), sample (a mixture of all the samples investigated) and a multi- and gray represents those insignificantly different. As shown in peak detection plot of chemicals in the MRM mode of the same Figure 4B, 47 metabolites in the fresh vs. steamed group (17 sample are illustrated in Supplementary Figure 1. Different UR and 30 DR), 30 metabolites in steamed vs. dried group (22 colored peaks represented different components in the sample. UR and 8 DR), and 65 metabolites in the fresh vs. dried group As shown in Figure 2, a total amount of 776 metabolites (29 UR and 36 DR) were selected to be significantly differential. were identified in the current study (Supplementary Table 1) The number of significantly different metabolites in the fresh in the fresh Cistanche deserticola samples, which were divided vs. steamed group was higher than those in the steamed vs. into 15 classes, including 40 amino acid and derivatives, 33 dried group, indicating that the influence on metabolites in the phenylpropanoids, 23 flavonoids, 68 flavone, 67 terpenes, 67 steaming process is higher than that of the drying process. The phenols, 87 alkaloids, 13 carbohydrates, 28 nucleotide and differential metabolites produced during thermal processing of derivatives, 5 alcohols and polyols, 3 purine nucleosides, 15 Cistanche deserticola were further classified and compared. These Frontiers in Nutrition | www.frontiersin.org 4 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola FIGURE 1 | Appearance color characteristics during Cistanche deserticola thermal processing. (A, B, C) represent samples at the thermal processing stage before steaming, after steaming without drying, and after steaming and drying, respectively. FIGURE 2 | Classification and composition of the 776 metabolites of Cistanche deserticola. differentially expressed metabolites were classified into 21 classes, amino acids and their derivatives (such as N6-Acetyl-L-lysine, 1- mainly amino acids and their derivatives, flavonoids and their Methy-L-histidine, and L-Phenylalanine) were significantly UR. derivatives, phenylpropanoids, alkaloids, terpenes, phenols, and However, in the steamed vs. dried group, the expression trends of nucleotide and their derivatives (Table 1). In fresh vs. steamed these types of differential metabolites were opposite. Some amino group, it can be found that flavonoids (such as isoquercitrin, and their derivatives (such as N, N-Dimethylglycine), nucleotide troxerutin, cyanidin, and fisetin), phenylpropanoids (such as and their derivatives (such as 2 -Deoxyuridine; Deoxyuridine) chlorogenic acid and 3-(3,4-Dihydroxy-5-methoxy)-2-propenoic were significantly DR, while most of the phenols (such as acid), and nucleotide and their derivatives (uracil and beta- methyl gallate and 4 -Prenyloxyresveratrol), flavonoids (such as Nicotinamide mononucleotide) were significantly DR, while isoquercitrin and cyanidin), phenylpropanoids (verbascoside), Frontiers in Nutrition | www.frontiersin.org 5 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola FIGURE 3 | Heat map of the hierarchical clustering analysis of differential chemicals among samples of fresh (A), steamed (B), and dried after steaming (C). and terpenes (such as terpinolene and furanodiene) were to promote hydrolysis, redox, isomerization, substitution, and significantly UR. other thermophysical and chemical reactions of metabolites These results showed that the chemical composition of (26). In this study, it was found that the metabolites, Cistanche deserticola has undergone conversion during thermal such as flavonoids and phenylpropanoids, were significantly processing, which is mainly reflected in the conversion accumulated in the steamed Cistanche deserticola compared of flavonoids, phenylpropanoids, and amino acids, and the to their corresponding fresh one, indicating that some key conversion mechanism of these components is different in physiological and metabolic activities leading to the synthesis of different processing stages. The use of a high temperature flavonoids and phenylpropanoids might be activated under high during the steaming and drying processes was previously found temperature and humidity. This result can also be supported Frontiers in Nutrition | www.frontiersin.org 6 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola FIGURE 4 | Differential metabolite analysis in all Cistanche deserticola samples processed at different stages. (A) the score plots of the differential metabolites generated from OPLS-DA; (B) the volcano plot of the differential metabolite in different samples; (C) the Veen diagram of the differential metabolite in different samples. A, fresh; B, steamed without drying; C, dried after steaming. OPLS-DA, orthogonal partial least squares discriminant analysis. by the report from Peng et al. (10) who found that the heat-sensitive components during the long-term drying process. content of PhGs (belonging to phenylpropanoids) increased after Previous studies have shown that flavonoid glycosides can be steaming. However, the accumulation of these components in decomposed into sugar bodies and flavonoid aglycones under the dried sample after steaming showed a significant decrease, thermal conditions, and flavonoids loss during the drying which may be attributed to the thermal degradation of these process was synthetically affected by temperature and drying Frontiers in Nutrition | www.frontiersin.org 7 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola time (26, 27). The upregulation of amino acids and their used to comprehend the impact of thermal processing stages on derivatives (N6-Acetyl-L-lysine, 1-Methy-L-histidine, and L- Cistanche deserticola composition. Phenylalanine) is attributed to the high-temperature-promoting Based on the KEGG annotation and enrichment analysis, protein degradation during steaming processing. In addition, four metabolic pathways (phenylpropanoid biosynthesis, it was also observed that some other amino acids and their flavonoid biosynthesis, alanine metabolism, and glycine, derivatives of N, N-Dimethylglycine, L- Kynurenine, glycine, serine, and threonine metabolism) were chosen as key serine, and threonine were DR. The decrease in the content metabolites to characterize the conversion of the main of these amino acids might be associated with thermal-induced active components of Cistanche deserticola during thermal Maillard reaction during which reducing sugars react with amino processing (Figures 5b1,b2). The current study indicated acids to generate 5-HMF, contributing to the production of that phenylpropanoids and flavonoids were accumulated but black appearance in Cistanche deserticola (22). The results of amino acids were degraded in steamed Cistanche deserticola Section appearance color changes of cistanche deserticola during compared to fresh and dried samples. The phenylpropanoid thermal processing further verified this hypothesis. Therefore, biosynthetic pathway is upstream of biosynthetic pathway of the blackening of Cistanche deserticola during steaming was flavonoid. Similar conclusions were published by Liu et al. (18) probably related to the metabolism of amino acids. who reported that the accumulation level of phenylpropanoids Venn diagram was used to differentiate the common and in the process of rice yellowing has increased significantly, exclusive metabolites of Cistanche deserticola during different compared with normal rice. Phenylpropanoids are derived from thermal processing stages. As shown in Figure 4C, both cinnamic acid, and their precursor is phenylalanine, which can be common and unique metabolites exist between the different synthesized by activating the activity of phenylalanine ammonia- comparison groups. Twenty-one common metabolites were lyase (PAL) when heated (28). Previous studies reported that the observed between the fresh and steamed group, while only 5 phenylpropanoid pathway led to the biosynthesis of coumarins, and 10 metabolites were found common between the fresh and flavones, isoflavones, and flavanols, which are the important dried group, steamed and dried group, respectively. Thus, a total weapons for plant defense (29), and to prevent cell death of 23 and 17 exclusive metabolites (p < 0.05) were observed caused by the strong heat stress in the steaming process, the in Cistanche deserticola during the thermal processing stage of phenylpropanoid pathway may be enhanced due to the biological steaming and drying, respectively. This result further confirmed stress caused by high temperature (30, 31). Flavonoids are the that steaming was particularly critical for the conversion of main secondary metabolites derived from phenylpropanoids metabolites during Cistanche deserticola processing. (32), and their accumulation could protect plants from oxidative damage by scavenging-free radicals (33). Compared to the fresh and dried Cistanche deserticola, the higher biosynthesis of Enrichment Analysis and KEGG Pathway flavonoids in the steamed Cistanche deserticola may be associated Impact Analysis of Differential Metabolites with enhanced heat stress during the steaming process providing The differential metabolites (p < 0.05) in fresh and processed protection against reactive oxygen species (ROS) (34, 35). As samples were mapped to the KEGG, HMDB, and PubChem shown in Figures 5b3,b4, amino acid metabolism played an online databases, which contain knowledge of the molecular important role in the thermal processing of Cistanche deserticola. interaction, reaction, and relation networks, and the Content changes of alanine, glycine, serine, and threonine after enrichment results and detailed metabolic pathways are steaming found in medicinal herbs have been used to indicate shown in Supplementary Table 2 and Figure 5. As shown in the occurrence of the Maillard reaction (36). Nevertheless, due to Figures 5a1,a2, pathway impact revealed the enrichment of the complicated process of the Cistanche deserticola steaming, a phenylpropanoid biosynthesis, flavonoid biosynthesis, alanine comprehensive evaluation of the Cistanche deserticola steaming, metabolism, riboflavin metabolism, taurine and hypotaurine such as blackening in appearance, active compounds, and metabolism, and nicotinate and nicotinamide metabolism during metabolic biomarkers, should be further investigated. steaming of Cistanche deserticola. Whereas, during the drying process after steaming, the metabolic pathways of the differential metabolites mainly contained glycine, serine and threonine CONCLUSIONS metabolism, thiamine metabolism, pyrimidine metabolism, and unsaturated fatty acids biosynthesis. Furthermore, some In the present study, UHPLC-MS/MS-based widely targeted metabolic pathways between these two pairwise comparisons metabolomics approach was employed to study the formation overlapped, such as nicotinate and nicotinamide metabolism, mechanism of active compounds at different thermal processing phenylpropanoid biosynthesis, and flavonoid biosynthesis, but stages of Cistanche deserticola. The current results revealed their enrichment levels were very different in two pairwise that the biosynthesis of some key metabolites, such as comparisons. These results suggested that the conversion phenylpropanoids and flavonoids, was significantly enhanced pathways of metabolites between the steaming and drying during the steaming process. The expression level of amino processes of Cistanche deserticola were different, and the acids in steamed Cistanche deserticola was enhanced, indicating differences in metabolic pathways could explain the differences the transformation between primary and secondary metabolites. in the presence of differentially exclusive metabolites during In addition, the blackening of the appearance of Cistanche thermal processing. These biochemical alterations might be deserticola mainly occurred in the steaming stage rather Frontiers in Nutrition | www.frontiersin.org 8 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola TABLE 1 | List of significantly different metabolites up/downregulated in Cistanche deserticola under different thermal processing stages. KEGG-ID Molecular Metabolite name Class VIP P-value Fold change Regulation mass Thermal processing stage: steaming 101.05 1- Phytohormone 2.00 0.01 0.07 Downregulated Aminocyclopropanecarboxylic acid C05623 464.09 Isoquercitrin Flavonoids 2.01 0.01 0.12 Downregulated C06802 645.25 Acarbose Alkaloids 1.79 0.04 0.15 Downregulated C10526 286.12 (–)-Sativan Flavonoids 1.84 0.04 0.15 Downregulated 742.23 Troxeruti Flavonoids 2.13 0.00 0.16 Downregulated C16959 216.15 Furanodiene Sesquiterpenoids 1.98 0.01 0.17 Downregulated C08493 145.05 Indole-3-carboxaldehyde Phytohormone 2.12 0.00 0.19 Downregulated C12634 610.15 Kaempferol3-O-beta- Flavonoids 2.04 0.00 0.19 Downregulated sophoroside C05905 286.05 Cyanidin Flavonoids 2.06 0.00 0.20 Downregulated C09372 367.11 (+)-Bicuculline Alkaloids 1.95 0.01 0.24 Downregulated C00106 112.03 Uracil Nucleotide and derivatives 1.86 0.03 0.26 Downregulated C00852 354.10 Chlorogenic acid Phenylpropanoids 1.93 0.02 0.27 Downregulated C00455 334.06 beta-Nicotinamide Nucleotide and derivatives 1.94 0.02 0.29 Downregulated mononucleotide C01965 290.14 Trimethoprim Phenol ethers 1.82 0.04 0.29 Downregulated C10414 268.07 Dalbergin Coumarins 1.82 0.03 0.39 Downregulated C12312 133.05 Indolin-2-one Alkaloids 1.89 0.02 0.39 Downregulated C00345 276.02 6-Phosphogluconic acid Organooxygen 1.93 0.02 0.40 Downregulated compounds C12298 198.16 Citronellyl acetate Monoterpenoids 2.12 0.00 0.41 Downregulated C01118 219.07 O-Succinyl-L-homoserine 1.86 0.03 0.41 Downregulated C10851 175.08 Calystegine B2 Alkaloids 1.86 0.03 0.44 Downregulated C01378 288.06 Fisetin Flavonoids 1.88 0.02 0.49 Downregulated C06575 134.11 P-Cymene Monoterpenoids 1.77 0.05 0.54 Downregulated C05123 125.99 2-Hydroxyethanesulfonate Organic acids 1.96 0.01 0.56 Downregulated C18326 234.14 N-p-Coumaroyl putrescine Phenolamides 1.77 0.04 0.56 Downregulated C05610 208.07 Trans-3,5-Dimethoxy-4- Phenylpropanoids 2.07 0.00 0.58 Downregulated hydroxy cinnamaldehydee C05619 210.05 3-(3,4-Dihydroxy-5- Cinnamic acids and 1.83 0.03 0.59 Downregulated methoxy)-2 propenoic derivatives acid C07650 263.07 Gemcitabine Pyrimidine nucleosides 1.88 0.03 0.62 Downregulated C02107 150.02 D-tartaric acid Organic acids and 1.76 0.05 0.65 Downregulated derivatives C09922 386.10 Cleomiscosin A Coumarins 1.88 0.02 0.74 Downregulated C00568 137.05 P-Aminobenzoate Benzoic acid derivatives 1.95 0.01 0.84 Downregulated C02727 188.12 N6-Acetyl-L-lysine Amino acid and 2.07 0.00 1.51 Upregulated derivatives C17756 151.06 Leukoaminochrome Indoles and derivatives 1.76 0.04 1.56 Upregulated C11045 294.12 Aspartame Carboxylic acids and 1.85 0.03 1.62 Upregulated derivatives C05138 332.24 17a-Hydroxypregnenolone Steroidsand steroid 1.89 0.02 1.76 Upregulated derivatives C00255 376.14 Riboflavine Vitamins 2.11 0.00 2.05 Upregulated C10372 272.10 9-Methoxy-alpha-lapachone Quinones 2.05 0.00 2.47 Upregulated C10875 412.12 Podophyllotoxinone Lignans 1.91 0.02 2.51 Upregulated (Continued) Frontiers in Nutrition | www.frontiersin.org 9 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola TABLE 1 | Continued KEGG-ID Molecular Metabolite name Class VIP P-value Fold change Regulation mass C01152 169.09 1-Methy-L-histidine Amino acids 1.88 0.02 2.75 Upregulated C09274 310.20 Tabernanthine Alkaloids 1.95 0.01 2.82 Upregulated C00079 165.08 L-Phenylalanine Amino acid and 1.93 0.02 3.00 Upregulated derivatives C05198 251.10 5’-Deoxyadenosine Nucleotide and derivatives 1.84 0.03 4.84 Upregulated C08431 251.10 Cordycepin Nucleotide and derivatives 1.84 0.03 4.84 Upregulated C00153 122.05 Nicotinamide Alkaloids 2.01 0.01 7.72 Upregulated C02353 329.05 Adenosine 2’,3’-cyclic Purine nucleotides 2.07 0.00 57.29 Upregulated phosphate C00942 345.05 Guanosine 3’,5’-cyclic Nucleotide and derivatives 1.78 0.04 58.52 Upregulated monophosphate C10190 372.12 Tangeretin Flavonoids 1.97 0.01 195.63 Upregulated 374.28 Ginkgolic acid C17:1 Phenols 2.10 0.00 1737.4 Upregulated Thermal processing stage: drying C05243 299.15 N-Methylcoclaurine Alkaloids 1.87 0.04 0.17 Downregulated C01026 103.06 N,N-Dimethylglycine Amino acid and 2.03 0.01 0.25 Downregulated derivatives C09202 376.15 Tripdiolide Diterpenoids 2.20 0.00 0.48 Downregulated C09868 150.10 (R)-Menthofuran Prenol lipids 1.89 0.04 0.48 Downregulated C05380 180.05 Nicotinurate Carboxylic acids and 2.15 0.00 0.56 Downregulated derivatives C17496 350.25 10-Gingerol Phenols 2.09 0.01 0.57 Downregulated C02666 178.06 Coniferylaldehyde Phenylpropanoids 2.03 0.01 0.59 Downregulated C00526 228.07 2’-Deoxyuridine Nucleotide and derivatives 1.94 0.04 0.64 Downregulated C10501 624.21 Verbascoside Phenylpropanoids 1.92 0.04 1.16 Upregulated C00576 101.08 Betaine aldehyde Organonitrogen 1.99 0.03 1.32 Upregulated compounds 184.04 Methyl gallate Phenols 1.88 0.04 1.35 Upregulated C08316 338.32 Erucic acid Fatty Acyls 2.11 0.001 1.53 Upregulated C10283 312.14 4’-Prenyloxyresveratrol Phenols 2.07 0.01 1.62 Upregulated C05905 286.05 Cyanidin Flavonoids 2.21 0.00 1.81 Upregulated C17497 194.09 Zingerone Phenols 1.96 0.03 1.89 Upregulated C00153 122.05 Nicotinamide Alkaloids 2.13 0.01 1.89 Upregulated C00881 227.09 Deoxycytidine Nucleotide and derivatives 1.95 0.04 2.06 Upregulated 174.10 6(1H)-Azulenone, Miscellaneous 2.09 0.01 2.25 Upregulated 2,3-dihydro-1,4-dimethyl C06075 136.13 Terpinolene Monoterpenoids 2.01 0.02 2.32 Upregulated 804.38 Rebaudioside B Diterpenoids 2.03 0.02 2.43 Upregulated C07650 263.07 Gemcitabine Pyrimidine nucleosides 1.90 0.04 2.73 Upregulated C00328 208.08 L-Kynurenine Amino acid and 2.00 0.03 2.77 Upregulated derivatives C10333 367.16 Isatidine Alkaloids 1.92 0.04 2.83 Upregulated C09770 334.07 Cedeodarin Flavonoids 2.06 0.02 3.06 Upregulated C13202 472.39 DL-alpha-Tocopherylacetate Phenols 1.86 0.04 3.44 Upregulated C04294 143.04 4-Methyl-5-thiazoleethanol Azoles 2.04 0.01 4.06 Upregulated C10640 372.16 Kadsurin A Lignans 2.13 0.01 4.61 Upregulated C16959 216.15 Furanodiene Sesquiterpenoids 1.87 0.04 7.61 Upregulated C16968 366.11 Neoglycyrol Coumarins 2.21 0.00 12.69 Upregulated C05623 464.10 Isoquercitrin Flavonoids 1.85 0.04 21.52 Upregulated VIP, variable importance projection. Frontiers in Nutrition | www.frontiersin.org 10 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola FIGURE 5 | Metabolic enrichment pathway analysis in two comparative groups (a1,a2) and important KEGG pathway maps (b1,b4). (a1,a2) represent the enrichment analysis of different metabolites in the steaming and drying processes, respectively; (b1,b4) respectively represent phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, alanine metabolism pathway, glycine, serine and threonine metabolism pathway. A, fresh; B, steamed without drying; C, dried after steaming. KEGG, Kyoto Encyclopedia of Genes and Genomes. Frontiers in Nutrition | www.frontiersin.org 11 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola than the drying stage, this characteristic is associated with AUTHOR CONTRIBUTIONS the amino acids’ metabolism pathway. However, the levels ZA conducted experimental design, performed the experiments, of the above metabolites decreased significantly during the generated the data, and wrote this manuscript. YZ performed drying process, suggesting the formation of active compounds the metabolomics analysis. XL provided the statistical analysis. mainly occurred in the steaming stage during the thermal WS conducted data processing and investigation. Funding processing of Cistanche deserticola. To the best of our acquisition, overall framework, and writing-reviewing were knowledge, this is the first time that the widely targeted completed by YL. All authors contributed to the article and metabolomic method was used to reveal that the mechanism approved the submitted version. of active compounds changes during the thermal processing and their crucial contribution to the Cistanche deserticola blackening. 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Widely Targeted Metabolomics Analysis to Reveal Transformation Mechanism of Cistanche Deserticola Active Compounds During Steaming and Drying Processes

Frontiers in Nutrition , Volume 8 – Oct 14, 2021

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Abstract

ORIGINAL RESEARCH published: 14 October 2021 doi: 10.3389/fnut.2021.742511 Widely Targeted Metabolomics Analysis to Reveal Transformation Mechanism of Cistanche Deserticola Active Compounds During Steaming and Drying Processes Ziping Ai, Yue Zhang, Xingyi Li, Wenling Sun and Yanhong Liu* College of Engineering, China Agricultural University, Beijing, China Edited by: Cistanche deserticola is one of the most precious plants, traditionally as Chinese Yasmina Sultanbawa, medicine, and has recently been used in pharmaceutical and healthy food The University of Queensland, Australia industries. Steaming and drying are two important steps in the processing of Reviewed by: Cistanche deserticola. Unfortunately, a comprehensive understanding of the chemical Bárbara Socas-Rodríguez, composition changes of Cistanche deserticola during thermal processing is limited. Institute of Food Science Research In this study, ultra-performance liquid chromatography-tandem mass spectrometry (CIAL), Spain Mohamed Fawzy Ramadan (UHPLC-MS/MS)-based widely targeted metabolomics analysis was used to investigate Hassanien, the transformation mechanism of Cistanche deserticola active compounds during Zagazig University, Egypt steaming and drying processes. A total of 776 metabolites were identified in Cistanche *Correspondence: Yanhong Liu deserticola during thermal processing, among which, 77 metabolites were differentially liuyanhong@cau.edu.cn regulated (p < 0.05) wherein 39 were upregulated (UR) and 38 were downregulated (DR). Forty-seven (17 UR, 30 DR) and 30 (22 UR, 8 DR) differential metabolites were Specialty section: This article was submitted to identified during steaming and drying, respectively. The most variation of the chemicals Nutrition and Food Science was observed during the process of steaming. Metabolic pathway analysis indicated Technology, that phenylpropanoid, flavonoid biosynthesis, and alanine metabolism were observed a section of the journal Frontiers in Nutrition during steaming, while glycine, serine, and threonine metabolism, thiamine metabolism, Received: 16 July 2021 and unsaturated fatty acid biosynthesis were observed during drying. The possible Accepted: 10 September 2021 mechanisms of the chemical alterations during thermal processing were also provided by Published: 14 October 2021 the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Furthermore, Citation: Ai Z, Zhang Y, Li X, Sun W and Liu Y the blackening of the appearance of Cistanche deserticola mainly occurred in the (2021) Widely Targeted Metabolomics steaming stage rather than the drying stage, which is associated with the metabolism of Analysis to Reveal Transformation the amino acids. All results indicated that the formation of active compounds during the Mechanism of Cistanche Deserticola Active Compounds During Steaming processing of Cistanche deserticola mainly occurred in the steaming stage. and Drying Processes. Front. Nutr. 8:742511. Keywords: cistanche deserticola, widely targeted metabolomics, steaming, drying, active compounds, formation doi: 10.3389/fnut.2021.742511 mechanism Frontiers in Nutrition | www.frontiersin.org 1 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola INTRODUCTION specific metabolite groups, rather than all the components in the sample. Non-targeted metabolomics technology can qualitatively Cistanche deserticola, belonging to the orobanchaceae family, determine the metabolites based on existing databases, with is one of the most famous tonic medicines and is mainly high coverage of compounds, however, low accuracy. The key distributed in the tropical and subtropical regions of the world, metabolites must be confirmed by standard products (14). Widely such as China, Iran, India, Mongolia, and so on (1–3). Cistanche targeted metabolomics is a new technology that integrates the deserticola serves as one of the most commonly utilized herbal advantages of non-target and targeted metabolites detection medicines for the treatments of kidney deficiency, impotence, technologies to achieve wide coverage, high throughput, female infertility, morbid leucorrhea, profuse metrorrhagia, and and sensitivity (15). Consequently, this technology has been senile (4, 5). Modern pharmacological research showed that widely used in the study of ingredient changes in different Cistanche deserticola has the effects of improving immunity, anti- materials during processing, such as active ingredients in fatigue, anti-aging, and enhancing learning and memorization functional foods by different processing methods (16), flavonoids ability (6). Owing to these health benefits, Cistanche deserticola and phenylpropanoids compounds in Chinese water chestnut tea made from its stem tubers has been developed as a processed with different methods (17), rice yellowing mechanism nourishing supplement and is being increasingly favored by during yellowing process (18), and the formation mechanism consumers. The active ingredients of Cistanche deserticola of characteristic non-volatile chemical constitutes during oolong have been shown to be responsible for its medicinal functions tea manufacturing process (19). Therefore, it is theoretically (7). Some active ingredients in Cistanche deserticola, such as feasible to use widely targeted metabolite technology to study phenylpropanoids (for example phenylethanoid glycosides), the mechanism of the conversion of active ingredients during the flavonoids, polysaccharides, oligosaccharides, iridoids, and processing of Cistanche deserticola. lignans have been reported in the previous studies (6, 8). Thus, the objectives of the present study were to (1) Due to the perishable and seasonal features, the all-year- provide useful information on the chemical changes in round supply of fresh Cistanche deserticola is unavailable, Cistanche deserticola during steaming and drying processes by accordingly, the processed Cistanche deserticola becomes the using ultraperformance liquid chromatography-tandem mass main consumption form. The quality of Cistanche deserticola spectrometry (UHPLC-MS/MS) combined with a widely targeted is dependent on many factors, such as climate, habitats, hosts, metabolomic approach; (2) identify the differential metabolites harvest time, processing technology, and position on the plant, and their regulation rules, and reveal the possible conversion among which processing technology is particularly important (4). pathways in Cistanche deserticola during processing. This study Steaming and drying are two important steps in the processing of is, therefore, expected to provide a theoretical reference for the Cistanche deserticola. Usually, the harvested Cistanche deserticola formation mechanism of high-quality Cistanche deserticola. rhizome was steamed in a steaming boiler at 93 C for 30 min and then dried at 60 C until the moisture content of 10% MATERIALS AND METHODS on a wet basis (w.b.) (9). Previous studies have shown that steaming can promote the accumulation of active ingredients Materials and Chemicals in Cistanche deserticola, such as phenylethanoid glycosides, Raw materials: Fresh Cistanche deserticola samples were obtained soluble sugars, and polysaccharides, accompanied by blackening from the Hetian region in Xinjiang Province of China. The of appearance color (9–11). However, most of the previous samples were carefully selected with the same size (average studies focused on certain specific compounds, very rare research length, diameter, and weight were 11.7 ± 1.1 cm, 7.0 ± 1.1 cm, are about the changes in all the chemical compounds and the and 360 ± 8.9 g, respectively). The samples were stored at room metabolite conversion mechanism during processing. Therefore, temperature in a dark environment with an initial moisture it is necessary to clarify the metabolite changes of Cistanche content of about 78.56% ± 3.47%. Prior to the experiments, deserticola in different processing stages. Cistanche deserticola samples were washed with tap water to Metabolomics is usually applied to qualitative and quantitative remove the dust on the surface. Excess water on its surface was analysis of all small molecules (namely, targeted and non- removed by blotting paper. targeted compounds) detected in the sample (12). Analysis of Chemicals: Methanol, acetonitrile, and formic acid were changes in various chemical components during food processing liquid-chromatography mass spectrometry grade (LC-MS) and helps to deepen the understanding of the mechanism of chemical purchased from Merck (Sigma Aldrich, MO, USA). The other component transformation in food processing (12). In recent analytical standards presented a purity higher than 98% (Sigma years, metabolomics has also been applied to the study of Aldrich, MO, USA). Cistanche deserticola for the discrimination of different parts (11) and different Cistanche deserticola species (13). The detection Experimental Design methods of metabolites in these studies were mostly based on Previous studies have shown that the chemical compounds targeted and non-targeted metabolomics. Among them, targeted distribute unevenly in the longitudinal direction of Cistanche metabolomics is based on standard products, with high data deserticola (1). Therefore, to obtain the same initial contents of accuracy and reliability, however, limited coverage of metabolites. chemical compounds in each sample, in the present research Targeted metabolomics is an important part of metabolomics all selected Cistanche deserticola was cut into three equal parts research, it is the targeted and specific detection and analysis for for the fresh group (A), steamed without drying group (B), Frontiers in Nutrition | www.frontiersin.org 2 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola and dried after steaming group (C), respectively, by longitudinal ESI-QTRAP-MS/MS Conditions segmentation with the longitudinal symmetry axis as the A triple quadrupole (QQQ)-linear ion trap mass spectrometer center (20). (QTRAP, API 6500 QTRAP UHPLC-MS/MS) + QQQ For group B, the samples were successively steamed for spectrometer equipped with an ESI turbo ion-spray interface 8 min according to preliminary experiments. A pulsed vacuum (Sciex Technologies, Framingham, MA, USA) was applied for steaming equipment (self-developed by China Agricultural MS analysis. The analytical conditions were as follows: ion University, Beijing, China) was used for steaming treatment of spray voltage: +5,500 V (positive ion mode)/−4,500 V (negative fresh Cistanche deserticolas. Steamed samples were dried in a ion mode), curtain gas: 35 psi, source temperature: 400 C, vacuum freeze-dryer (LGJ-25C, Si Huan Scientific Instrument ion source gas 1: 60 psi, ion source gas 2: 60 psi, declustering Factory Co., Beijing, China). The heating plate and cold trap potential: ±100 V. QQQ scans were acquired as multiple reaction temperature were 30 and −60 C, respectively. For group C, monitoring (MRM) experiments with collision gas (nitrogen) set the samples were successively steamed using pulsed vacuum to 5 psi. equipment for 8 min and dried in the hot air impingement Qualitative and Quantitative Analysis of dryer (self-developed by China Agricultural University, Beijing, China) until the final moisture content of 10% (w.b.). The airflow Metabolites rate and temperature were set at 6 m/s and 60 C, respectively, Qualitative and quantitative analyses of metabolites were referring to the research results of Zou et al. (11). All samples performed according to the methods by Liu et al. (18). Primary were stored at−20 C no more than 7 days before further analysis. and secondary mass spectrometry data were qualitatively analyzed based on the self-built human metabolome database (MWDB) (Metware Biotechnology Co., Ltd. Wuhan, China) Determination of Appearance Color of and the public database. Meanwhile, to ensure the accuracy Cistanche Deserticola of the qualitative analysis of some substances, interferences + + The appearance color of the Cistanche deserticola before and after from repeated signals of Na , NH , K and ions, and each thermal processing was measured using a colorimeter (SMY- repetitive signals of fragment ions derived from other relatively 2000SF, Shengming Yang Co., Beijing, China), and the blackness large molecules and isotope signals were removed during identification. Metabolite structural analysis was performed with was characterized by L value. reference to the public databases (Mass Bank, KNApSAcK, HMDB, MoTo DB, and METLIN). Sample Preparation and Extraction Metabolite quantification was carried out using the MRM The metabolite extraction was carried out according to the mode of the QQQ mass spectrometry. In the MRM mode, method reported previously by Chen et al. (21) with some the precursor ions (parent ions) of the target substances and minor modifications. In brief, the dried samples were crushed excluded ions corresponding to other substances with different using a mixer mill (MM 400, Retsch Company, Haan, Germany) molecular weights were screened first using the quadrupole rod with a zirconia bead for 2 min at 60 Hz. Then 50 mg powder to initially eliminate interference. The precursor ions then break (sifted through a 65 mesh sieve) of each sample was precisely through the collision chamber to form many fragment irons weighed, transferred to an Eppendorf tube, and extracted with after ionization, which were filtered by QQQ to select single- 1 ml methanol/water mixture (v:v = 3:1). After 30 s vortex, the fragment ions with the desired characteristics while eliminating mixture was homogenized twice at 35 Hz for 4 min, sonicated interference from non-target ions. Finally, after obtaining the for 15 min in an ice-water bath, and then shaken overnight metabolite mass spectrometry data of different samples, the ◦ ◦ at 4 C. After centrifugation at 12,000 rpm for 15 min at 4 C, mass spectrum peaks of all substances were integrated, and the supernatant was collected and filtered through a 0.22-μm the mass spectra peaks of the same metabolite in different membrane, then the obtained extract was transferred to 2-ml samples were integrated and corrected using Multi Quant glass vials and store at −80 C until the UHPLC-MS/MS analysis. version 3.0.2 (ABSCIEX, Concord, Ontario, Canada). The corresponding relative metabolite contents were represented as chromatographic peak area integrals. Metabolites Analysis by UHPLC-MS UHPLC Conditions Data Processing and Analysis The UHPLC separation was carried out using an EXIONLC The metabolic data were processed using orthogonal partial system (Sciex Technologies, Framingham, MA, USA). The least squares-discriminant analysis (OPLS-DA) and hierarchical analytical conditions were as follows: column: Waters ACQUITY cluster analysis (HCA). OPLS-DA was used to discriminate UHPLC HSS T3 C18 (1.8μm, 2.1 × 100 mm); solvent system: each group; it is more sensitive than other statistical methods mobile phase A (0.1% formic acid in water) and mobile phase B to variables with low correlations (17). The OPLS-DA models (acetonitrile containing). The gradient program: 98% A/2% B at were validated through a permutation analysis (200 times). 0 min, 50% A/50% B at 10 min, 5% A/95% B at 11 min, 98% A/2% The model was considered stable when the model parameters 2 2 B at 13.1 min, and 98% A/2% B at 15 min. Flow rate: 0.40 ml/min; (R and Q ) were both close to 1. The variable importance column temperature: 40 C; injection volume: 2 μl; automatic projection (VIP) values of metabolites were calculated. Any injection temperature: 4 C. metabolite with VIP values greater than 1.0 and p-values Frontiers in Nutrition | www.frontiersin.org 3 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola less than 0.05 were selected as biomarkers for each paired carboxylic acids and derivatives, 14 organic acids and derivatives, comparison between different thermal processing stages of 12 phytohormone, and 28 other chemicals. Among them, the Cistanche deserticola. The screening of different metabolites largest group was amino acid and derivatives, the relative was visualized in the form of the volcano plot. Metabolites content of which accounted for 30.26% of the total metabolite accumulation among different samples was analyzed by using composition. In addition, 10 kinds of phenylethanoid glycosides, the R package (www.rproject.org/). The Venn diagram was such as echinacoside and verbascoside, were detected and built according to the program web-based smart diagram R classified in the phenylpropanoids group. (https://cloud.smartdraw.com/). The commercial databases, such The accumulation pattern of metabolites among different as Kyoto Encyclopedia of Genes and Genomes (KEGG) (https: treatment groups was analyzed by HCA. As shown in Figure 3, //www.kegg.jp/kegg/), Pub Chem (https://pubchem.ncbi.nlm. 107 identified metabolites of Cistanche deserticola were clustered nih.gov/), the Small Molecule Pathway Database (SMPDB) in heat maps based on Euclidean distance arithmetic. Metabolites (https://smpdb.ca/), and HMDB (https://hmdb.ca/), were used identified at different thermal processing stages were gathered for enrichment analysis of differential metabolites and finding into three clusters according to the dendrogram. The brighter metabolic pathways. color indicates the higher content of a particular metabolite in the respective sample. The heat map of HCA showed larger di?erences in abundance between the fresh and steamed samples RESULTS AND DISCUSSION than those between steamed and dried samples, indicating that metabolites in Cistanche deserticola may have different Appearance Color Changes of Cistanche transformations during the steaming and drying stage, and the Deserticola During Thermal Processing types and quantities of metabolites involved in the steaming The difference in appearance color of Cistanche deserticola process are more than those in the drying process. between the fresh, steamed and dried samples are representatively displayed in Figure 1. From fresh to dried sample with the going of the processing stage, the appearance Differential Metabolite Analysis of color of the samples changed from yellow-brown to dark Cistanche Deserticola at Different Thermal black, and the darkness in color became more and more Processing Stages obvious (the corresponding L value was reduced from 50.26 For a better understanding of the impact of each processing on to 24.90). Obviously, the appearance changes of Cistanche the metabolites of Cistanche deserticolas, the OPLS-DA scatter deserticola mainly occurred in the steaming process. The scores of pairwise comparison groups are shown in Figure 4A, Maillard reaction, during which sugars react with amino acids showing that the fresh, steamed, and dried after steaming under thermal conditions (22), would be greatly responsible Cistanche deserticolas were significantly different. Moreover, for the dark-colored appearance of processed rhizomes of 2 2 the R Y and Q (as shown in Supplementary Figure 2) with Cistanche deserticola. Previous research studies have shown high-test values indicated that this model was highly reliable that precursors were converted into colorants and generated without overfitting. substances with dark color in the Maillard reaction (23). Similar To screen the expression level of metabolites between the findings were also observed in previous studies for steaming fresh, steamed, and dried after steaming Cistanche deserticola, of Polygonum multiflorum (24) and rhizomes of Polygonatum the analysis of volcano plot was further applied among all 776 cyrtonema (25). The darkness of the steamed samples was further metabolites identified according to the fold-change, combined deepened after drying. This phenomenon was probably due with VIP values to screen the differentially expressed metabolites. to the occurrence of a decrease in the pigment concentration Significant differential metabolites were selected according to during the drying process. the criterion that a fold change score of ≥2 or ≤0.5 with a VIP ≥ 1. The screening results are illustrated in Figure 4B. In Overview of the Metabolites in Raw and the volcanic map, each point represents a metabolite and the Thermal Processed Cistanche Deserticola color of the scattered dots represents the final screening result. Samples Red represents metabolites that are significantly upregulated The total ion chromatogram (TIC) of quality control (QC) (UR), green represents those significantly downregulated (DR), sample (a mixture of all the samples investigated) and a multi- and gray represents those insignificantly different. As shown in peak detection plot of chemicals in the MRM mode of the same Figure 4B, 47 metabolites in the fresh vs. steamed group (17 sample are illustrated in Supplementary Figure 1. Different UR and 30 DR), 30 metabolites in steamed vs. dried group (22 colored peaks represented different components in the sample. UR and 8 DR), and 65 metabolites in the fresh vs. dried group As shown in Figure 2, a total amount of 776 metabolites (29 UR and 36 DR) were selected to be significantly differential. were identified in the current study (Supplementary Table 1) The number of significantly different metabolites in the fresh in the fresh Cistanche deserticola samples, which were divided vs. steamed group was higher than those in the steamed vs. into 15 classes, including 40 amino acid and derivatives, 33 dried group, indicating that the influence on metabolites in the phenylpropanoids, 23 flavonoids, 68 flavone, 67 terpenes, 67 steaming process is higher than that of the drying process. The phenols, 87 alkaloids, 13 carbohydrates, 28 nucleotide and differential metabolites produced during thermal processing of derivatives, 5 alcohols and polyols, 3 purine nucleosides, 15 Cistanche deserticola were further classified and compared. These Frontiers in Nutrition | www.frontiersin.org 4 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola FIGURE 1 | Appearance color characteristics during Cistanche deserticola thermal processing. (A, B, C) represent samples at the thermal processing stage before steaming, after steaming without drying, and after steaming and drying, respectively. FIGURE 2 | Classification and composition of the 776 metabolites of Cistanche deserticola. differentially expressed metabolites were classified into 21 classes, amino acids and their derivatives (such as N6-Acetyl-L-lysine, 1- mainly amino acids and their derivatives, flavonoids and their Methy-L-histidine, and L-Phenylalanine) were significantly UR. derivatives, phenylpropanoids, alkaloids, terpenes, phenols, and However, in the steamed vs. dried group, the expression trends of nucleotide and their derivatives (Table 1). In fresh vs. steamed these types of differential metabolites were opposite. Some amino group, it can be found that flavonoids (such as isoquercitrin, and their derivatives (such as N, N-Dimethylglycine), nucleotide troxerutin, cyanidin, and fisetin), phenylpropanoids (such as and their derivatives (such as 2 -Deoxyuridine; Deoxyuridine) chlorogenic acid and 3-(3,4-Dihydroxy-5-methoxy)-2-propenoic were significantly DR, while most of the phenols (such as acid), and nucleotide and their derivatives (uracil and beta- methyl gallate and 4 -Prenyloxyresveratrol), flavonoids (such as Nicotinamide mononucleotide) were significantly DR, while isoquercitrin and cyanidin), phenylpropanoids (verbascoside), Frontiers in Nutrition | www.frontiersin.org 5 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola FIGURE 3 | Heat map of the hierarchical clustering analysis of differential chemicals among samples of fresh (A), steamed (B), and dried after steaming (C). and terpenes (such as terpinolene and furanodiene) were to promote hydrolysis, redox, isomerization, substitution, and significantly UR. other thermophysical and chemical reactions of metabolites These results showed that the chemical composition of (26). In this study, it was found that the metabolites, Cistanche deserticola has undergone conversion during thermal such as flavonoids and phenylpropanoids, were significantly processing, which is mainly reflected in the conversion accumulated in the steamed Cistanche deserticola compared of flavonoids, phenylpropanoids, and amino acids, and the to their corresponding fresh one, indicating that some key conversion mechanism of these components is different in physiological and metabolic activities leading to the synthesis of different processing stages. The use of a high temperature flavonoids and phenylpropanoids might be activated under high during the steaming and drying processes was previously found temperature and humidity. This result can also be supported Frontiers in Nutrition | www.frontiersin.org 6 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola FIGURE 4 | Differential metabolite analysis in all Cistanche deserticola samples processed at different stages. (A) the score plots of the differential metabolites generated from OPLS-DA; (B) the volcano plot of the differential metabolite in different samples; (C) the Veen diagram of the differential metabolite in different samples. A, fresh; B, steamed without drying; C, dried after steaming. OPLS-DA, orthogonal partial least squares discriminant analysis. by the report from Peng et al. (10) who found that the heat-sensitive components during the long-term drying process. content of PhGs (belonging to phenylpropanoids) increased after Previous studies have shown that flavonoid glycosides can be steaming. However, the accumulation of these components in decomposed into sugar bodies and flavonoid aglycones under the dried sample after steaming showed a significant decrease, thermal conditions, and flavonoids loss during the drying which may be attributed to the thermal degradation of these process was synthetically affected by temperature and drying Frontiers in Nutrition | www.frontiersin.org 7 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola time (26, 27). The upregulation of amino acids and their used to comprehend the impact of thermal processing stages on derivatives (N6-Acetyl-L-lysine, 1-Methy-L-histidine, and L- Cistanche deserticola composition. Phenylalanine) is attributed to the high-temperature-promoting Based on the KEGG annotation and enrichment analysis, protein degradation during steaming processing. In addition, four metabolic pathways (phenylpropanoid biosynthesis, it was also observed that some other amino acids and their flavonoid biosynthesis, alanine metabolism, and glycine, derivatives of N, N-Dimethylglycine, L- Kynurenine, glycine, serine, and threonine metabolism) were chosen as key serine, and threonine were DR. The decrease in the content metabolites to characterize the conversion of the main of these amino acids might be associated with thermal-induced active components of Cistanche deserticola during thermal Maillard reaction during which reducing sugars react with amino processing (Figures 5b1,b2). The current study indicated acids to generate 5-HMF, contributing to the production of that phenylpropanoids and flavonoids were accumulated but black appearance in Cistanche deserticola (22). The results of amino acids were degraded in steamed Cistanche deserticola Section appearance color changes of cistanche deserticola during compared to fresh and dried samples. The phenylpropanoid thermal processing further verified this hypothesis. Therefore, biosynthetic pathway is upstream of biosynthetic pathway of the blackening of Cistanche deserticola during steaming was flavonoid. Similar conclusions were published by Liu et al. (18) probably related to the metabolism of amino acids. who reported that the accumulation level of phenylpropanoids Venn diagram was used to differentiate the common and in the process of rice yellowing has increased significantly, exclusive metabolites of Cistanche deserticola during different compared with normal rice. Phenylpropanoids are derived from thermal processing stages. As shown in Figure 4C, both cinnamic acid, and their precursor is phenylalanine, which can be common and unique metabolites exist between the different synthesized by activating the activity of phenylalanine ammonia- comparison groups. Twenty-one common metabolites were lyase (PAL) when heated (28). Previous studies reported that the observed between the fresh and steamed group, while only 5 phenylpropanoid pathway led to the biosynthesis of coumarins, and 10 metabolites were found common between the fresh and flavones, isoflavones, and flavanols, which are the important dried group, steamed and dried group, respectively. Thus, a total weapons for plant defense (29), and to prevent cell death of 23 and 17 exclusive metabolites (p < 0.05) were observed caused by the strong heat stress in the steaming process, the in Cistanche deserticola during the thermal processing stage of phenylpropanoid pathway may be enhanced due to the biological steaming and drying, respectively. This result further confirmed stress caused by high temperature (30, 31). Flavonoids are the that steaming was particularly critical for the conversion of main secondary metabolites derived from phenylpropanoids metabolites during Cistanche deserticola processing. (32), and their accumulation could protect plants from oxidative damage by scavenging-free radicals (33). Compared to the fresh and dried Cistanche deserticola, the higher biosynthesis of Enrichment Analysis and KEGG Pathway flavonoids in the steamed Cistanche deserticola may be associated Impact Analysis of Differential Metabolites with enhanced heat stress during the steaming process providing The differential metabolites (p < 0.05) in fresh and processed protection against reactive oxygen species (ROS) (34, 35). As samples were mapped to the KEGG, HMDB, and PubChem shown in Figures 5b3,b4, amino acid metabolism played an online databases, which contain knowledge of the molecular important role in the thermal processing of Cistanche deserticola. interaction, reaction, and relation networks, and the Content changes of alanine, glycine, serine, and threonine after enrichment results and detailed metabolic pathways are steaming found in medicinal herbs have been used to indicate shown in Supplementary Table 2 and Figure 5. As shown in the occurrence of the Maillard reaction (36). Nevertheless, due to Figures 5a1,a2, pathway impact revealed the enrichment of the complicated process of the Cistanche deserticola steaming, a phenylpropanoid biosynthesis, flavonoid biosynthesis, alanine comprehensive evaluation of the Cistanche deserticola steaming, metabolism, riboflavin metabolism, taurine and hypotaurine such as blackening in appearance, active compounds, and metabolism, and nicotinate and nicotinamide metabolism during metabolic biomarkers, should be further investigated. steaming of Cistanche deserticola. Whereas, during the drying process after steaming, the metabolic pathways of the differential metabolites mainly contained glycine, serine and threonine CONCLUSIONS metabolism, thiamine metabolism, pyrimidine metabolism, and unsaturated fatty acids biosynthesis. Furthermore, some In the present study, UHPLC-MS/MS-based widely targeted metabolic pathways between these two pairwise comparisons metabolomics approach was employed to study the formation overlapped, such as nicotinate and nicotinamide metabolism, mechanism of active compounds at different thermal processing phenylpropanoid biosynthesis, and flavonoid biosynthesis, but stages of Cistanche deserticola. The current results revealed their enrichment levels were very different in two pairwise that the biosynthesis of some key metabolites, such as comparisons. These results suggested that the conversion phenylpropanoids and flavonoids, was significantly enhanced pathways of metabolites between the steaming and drying during the steaming process. The expression level of amino processes of Cistanche deserticola were different, and the acids in steamed Cistanche deserticola was enhanced, indicating differences in metabolic pathways could explain the differences the transformation between primary and secondary metabolites. in the presence of differentially exclusive metabolites during In addition, the blackening of the appearance of Cistanche thermal processing. These biochemical alterations might be deserticola mainly occurred in the steaming stage rather Frontiers in Nutrition | www.frontiersin.org 8 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola TABLE 1 | List of significantly different metabolites up/downregulated in Cistanche deserticola under different thermal processing stages. KEGG-ID Molecular Metabolite name Class VIP P-value Fold change Regulation mass Thermal processing stage: steaming 101.05 1- Phytohormone 2.00 0.01 0.07 Downregulated Aminocyclopropanecarboxylic acid C05623 464.09 Isoquercitrin Flavonoids 2.01 0.01 0.12 Downregulated C06802 645.25 Acarbose Alkaloids 1.79 0.04 0.15 Downregulated C10526 286.12 (–)-Sativan Flavonoids 1.84 0.04 0.15 Downregulated 742.23 Troxeruti Flavonoids 2.13 0.00 0.16 Downregulated C16959 216.15 Furanodiene Sesquiterpenoids 1.98 0.01 0.17 Downregulated C08493 145.05 Indole-3-carboxaldehyde Phytohormone 2.12 0.00 0.19 Downregulated C12634 610.15 Kaempferol3-O-beta- Flavonoids 2.04 0.00 0.19 Downregulated sophoroside C05905 286.05 Cyanidin Flavonoids 2.06 0.00 0.20 Downregulated C09372 367.11 (+)-Bicuculline Alkaloids 1.95 0.01 0.24 Downregulated C00106 112.03 Uracil Nucleotide and derivatives 1.86 0.03 0.26 Downregulated C00852 354.10 Chlorogenic acid Phenylpropanoids 1.93 0.02 0.27 Downregulated C00455 334.06 beta-Nicotinamide Nucleotide and derivatives 1.94 0.02 0.29 Downregulated mononucleotide C01965 290.14 Trimethoprim Phenol ethers 1.82 0.04 0.29 Downregulated C10414 268.07 Dalbergin Coumarins 1.82 0.03 0.39 Downregulated C12312 133.05 Indolin-2-one Alkaloids 1.89 0.02 0.39 Downregulated C00345 276.02 6-Phosphogluconic acid Organooxygen 1.93 0.02 0.40 Downregulated compounds C12298 198.16 Citronellyl acetate Monoterpenoids 2.12 0.00 0.41 Downregulated C01118 219.07 O-Succinyl-L-homoserine 1.86 0.03 0.41 Downregulated C10851 175.08 Calystegine B2 Alkaloids 1.86 0.03 0.44 Downregulated C01378 288.06 Fisetin Flavonoids 1.88 0.02 0.49 Downregulated C06575 134.11 P-Cymene Monoterpenoids 1.77 0.05 0.54 Downregulated C05123 125.99 2-Hydroxyethanesulfonate Organic acids 1.96 0.01 0.56 Downregulated C18326 234.14 N-p-Coumaroyl putrescine Phenolamides 1.77 0.04 0.56 Downregulated C05610 208.07 Trans-3,5-Dimethoxy-4- Phenylpropanoids 2.07 0.00 0.58 Downregulated hydroxy cinnamaldehydee C05619 210.05 3-(3,4-Dihydroxy-5- Cinnamic acids and 1.83 0.03 0.59 Downregulated methoxy)-2 propenoic derivatives acid C07650 263.07 Gemcitabine Pyrimidine nucleosides 1.88 0.03 0.62 Downregulated C02107 150.02 D-tartaric acid Organic acids and 1.76 0.05 0.65 Downregulated derivatives C09922 386.10 Cleomiscosin A Coumarins 1.88 0.02 0.74 Downregulated C00568 137.05 P-Aminobenzoate Benzoic acid derivatives 1.95 0.01 0.84 Downregulated C02727 188.12 N6-Acetyl-L-lysine Amino acid and 2.07 0.00 1.51 Upregulated derivatives C17756 151.06 Leukoaminochrome Indoles and derivatives 1.76 0.04 1.56 Upregulated C11045 294.12 Aspartame Carboxylic acids and 1.85 0.03 1.62 Upregulated derivatives C05138 332.24 17a-Hydroxypregnenolone Steroidsand steroid 1.89 0.02 1.76 Upregulated derivatives C00255 376.14 Riboflavine Vitamins 2.11 0.00 2.05 Upregulated C10372 272.10 9-Methoxy-alpha-lapachone Quinones 2.05 0.00 2.47 Upregulated C10875 412.12 Podophyllotoxinone Lignans 1.91 0.02 2.51 Upregulated (Continued) Frontiers in Nutrition | www.frontiersin.org 9 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola TABLE 1 | Continued KEGG-ID Molecular Metabolite name Class VIP P-value Fold change Regulation mass C01152 169.09 1-Methy-L-histidine Amino acids 1.88 0.02 2.75 Upregulated C09274 310.20 Tabernanthine Alkaloids 1.95 0.01 2.82 Upregulated C00079 165.08 L-Phenylalanine Amino acid and 1.93 0.02 3.00 Upregulated derivatives C05198 251.10 5’-Deoxyadenosine Nucleotide and derivatives 1.84 0.03 4.84 Upregulated C08431 251.10 Cordycepin Nucleotide and derivatives 1.84 0.03 4.84 Upregulated C00153 122.05 Nicotinamide Alkaloids 2.01 0.01 7.72 Upregulated C02353 329.05 Adenosine 2’,3’-cyclic Purine nucleotides 2.07 0.00 57.29 Upregulated phosphate C00942 345.05 Guanosine 3’,5’-cyclic Nucleotide and derivatives 1.78 0.04 58.52 Upregulated monophosphate C10190 372.12 Tangeretin Flavonoids 1.97 0.01 195.63 Upregulated 374.28 Ginkgolic acid C17:1 Phenols 2.10 0.00 1737.4 Upregulated Thermal processing stage: drying C05243 299.15 N-Methylcoclaurine Alkaloids 1.87 0.04 0.17 Downregulated C01026 103.06 N,N-Dimethylglycine Amino acid and 2.03 0.01 0.25 Downregulated derivatives C09202 376.15 Tripdiolide Diterpenoids 2.20 0.00 0.48 Downregulated C09868 150.10 (R)-Menthofuran Prenol lipids 1.89 0.04 0.48 Downregulated C05380 180.05 Nicotinurate Carboxylic acids and 2.15 0.00 0.56 Downregulated derivatives C17496 350.25 10-Gingerol Phenols 2.09 0.01 0.57 Downregulated C02666 178.06 Coniferylaldehyde Phenylpropanoids 2.03 0.01 0.59 Downregulated C00526 228.07 2’-Deoxyuridine Nucleotide and derivatives 1.94 0.04 0.64 Downregulated C10501 624.21 Verbascoside Phenylpropanoids 1.92 0.04 1.16 Upregulated C00576 101.08 Betaine aldehyde Organonitrogen 1.99 0.03 1.32 Upregulated compounds 184.04 Methyl gallate Phenols 1.88 0.04 1.35 Upregulated C08316 338.32 Erucic acid Fatty Acyls 2.11 0.001 1.53 Upregulated C10283 312.14 4’-Prenyloxyresveratrol Phenols 2.07 0.01 1.62 Upregulated C05905 286.05 Cyanidin Flavonoids 2.21 0.00 1.81 Upregulated C17497 194.09 Zingerone Phenols 1.96 0.03 1.89 Upregulated C00153 122.05 Nicotinamide Alkaloids 2.13 0.01 1.89 Upregulated C00881 227.09 Deoxycytidine Nucleotide and derivatives 1.95 0.04 2.06 Upregulated 174.10 6(1H)-Azulenone, Miscellaneous 2.09 0.01 2.25 Upregulated 2,3-dihydro-1,4-dimethyl C06075 136.13 Terpinolene Monoterpenoids 2.01 0.02 2.32 Upregulated 804.38 Rebaudioside B Diterpenoids 2.03 0.02 2.43 Upregulated C07650 263.07 Gemcitabine Pyrimidine nucleosides 1.90 0.04 2.73 Upregulated C00328 208.08 L-Kynurenine Amino acid and 2.00 0.03 2.77 Upregulated derivatives C10333 367.16 Isatidine Alkaloids 1.92 0.04 2.83 Upregulated C09770 334.07 Cedeodarin Flavonoids 2.06 0.02 3.06 Upregulated C13202 472.39 DL-alpha-Tocopherylacetate Phenols 1.86 0.04 3.44 Upregulated C04294 143.04 4-Methyl-5-thiazoleethanol Azoles 2.04 0.01 4.06 Upregulated C10640 372.16 Kadsurin A Lignans 2.13 0.01 4.61 Upregulated C16959 216.15 Furanodiene Sesquiterpenoids 1.87 0.04 7.61 Upregulated C16968 366.11 Neoglycyrol Coumarins 2.21 0.00 12.69 Upregulated C05623 464.10 Isoquercitrin Flavonoids 1.85 0.04 21.52 Upregulated VIP, variable importance projection. Frontiers in Nutrition | www.frontiersin.org 10 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola FIGURE 5 | Metabolic enrichment pathway analysis in two comparative groups (a1,a2) and important KEGG pathway maps (b1,b4). (a1,a2) represent the enrichment analysis of different metabolites in the steaming and drying processes, respectively; (b1,b4) respectively represent phenylpropanoid biosynthesis pathway, flavonoid biosynthesis pathway, alanine metabolism pathway, glycine, serine and threonine metabolism pathway. A, fresh; B, steamed without drying; C, dried after steaming. KEGG, Kyoto Encyclopedia of Genes and Genomes. Frontiers in Nutrition | www.frontiersin.org 11 October 2021 | Volume 8 | Article 742511 Ai et al. Transformation Mechanism of Cistanche Deserticola than the drying stage, this characteristic is associated with AUTHOR CONTRIBUTIONS the amino acids’ metabolism pathway. However, the levels ZA conducted experimental design, performed the experiments, of the above metabolites decreased significantly during the generated the data, and wrote this manuscript. YZ performed drying process, suggesting the formation of active compounds the metabolomics analysis. XL provided the statistical analysis. mainly occurred in the steaming stage during the thermal WS conducted data processing and investigation. Funding processing of Cistanche deserticola. To the best of our acquisition, overall framework, and writing-reviewing were knowledge, this is the first time that the widely targeted completed by YL. All authors contributed to the article and metabolomic method was used to reveal that the mechanism approved the submitted version. of active compounds changes during the thermal processing and their crucial contribution to the Cistanche deserticola blackening. However, further investigation is needed for a better FUNDING understanding of the relationship between the biosynthesis of This work was financially supported by the Department active compounds and the blackening of the appearance during of Science and Technology of Guangdong Province thermal processing. (No. 2018B020241003). DATA AVAILABILITY STATEMENT SUPPLEMENTARY MATERIAL The original contributions presented in the study The Supplementary Material for this article can be found are included in the article/Supplementary Material, online at: https://www.frontiersin.org/articles/10.3389/fnut.2021. further inquiries can be directed to the 742511/full#supplementary-material corresponding author/s. REFERENCES 11. Zou P, Song Y, Lei W, Li J, Tu P, Jiang Y. Application of 1 H NMR-based metabolomics for discrimination of different parts and development of a 1. Wang X, Wang J, Guan H, Xu R, Luo X, Su M, et al. Comparison new processing workflow for Cistanche deserticola. Acta Pharm Sin B. 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