An UPLC-Q-TOF/MS-Based Analysis of the Differential Composition of Dendrobium officinale in Different Regions

Dendrobium officinale (D. officinale) is a valuable traditional Chinese herbal medicine with high commercial value. In Chinese Pharmacopoeia (Ch.P., 2020 edition), the quality of D. officinale is mainly evaluated by its polysaccharide content. However, varying growth and production conditions, such as cultivation environment, origin, harvesting process, or processing methods, resulting in highly variable yields, quality, and composition. The aim of this study was to investigate whether the content of secondary metabolites in D. officinale from different origins is consistent with the polysaccharide content. The results showed that the polysaccharide content and pass rate were ranked as GX > AH > GZ > YN. Based on the nontargeted metabolomics approach, we searched for differential components in 22 different regions of D. officinale, including amides, bibenzyls, disaccharide, flavonoids, organic nitrogenous compounds, and phenolic glycosides. The overall expression was opposite to the polysaccharide, and the most expressed was YN, followed by GZ, AH, and GX. These results indicated that the current quality standard for evaluating the quality of D. officinale by polysaccharide content alone is imperfect, and small molecule compounds need to be included as quality markers.


Introduction
Te genus Dendrobium is one of the largest genera of the Orchidaceae, which contains 1500-2000 species [1]. Among them, Dendrobium ofcinale Kimura et Migo is the major source of Dendrobii caulis, a traditional Chinese medicine, which is widely distributed worldwide, such as the United States, Australia and Japan [2]. Especially, D. ofcinale is widely cultivated in various regions of China, including Anhui, Zhejiang, Hunan, Fujian, Guangxi, Sichuan, and Yunnan provinces [3][4][5].
D. ofcinale is a traditional Chinese medicine frst recorded in the "Shen Nong's Herbal Classic" and used alone or as a prescription [6]. According to the practitioner of Chinese medicine's consensuses, the fresh or dried stems of D. ofcinale are considered to be a drug of nourishing Yin, which comprises the efcacy of nourishing the stomach for promoting the production of fuid, nourishing Yin and clearing heat, brightening the eyes, strengthening the waist and tonifying lung and kidney [7]. In addition, modern pharmacological studies have revealed that D. ofcinale has multiple promising bioactivities, including immunomodulation, antitumor, antioxidant, antifatigue, glycolipid regulation, hepatoprotection, etc [8][9][10][11]. According to current phytochemical investigations, more than 190 compounds have been isolated from D. ofcinale, including polysaccharides, alkaloids, amino acids, favonoids, trace elements, and other nutrients, and its medicinal components are mainly composed of polysaccharides, bibenzyl, phenanthrene, favonoids, and alkaloids [12][13][14][15]. Among them, polysaccharide was selected as the only quality marker in the current edition of the Ch.P., the relationship between polysaccharide and other small molecular components content is not yet known.
Due to the overexploitation and depletion of wild plant resources, D. ofcinale has become one of the rarest and most endangered Chinese herbal medicines in China, step by step [16,17]. In addition, with the current quality standards, the quality of D. ofcinale varies greatly from diferent region and growing environment [18]. Fortunately, the manual cultivation of D. ofcinale has made a great breakthrough, and currently D. ofcinale was mainly grown in greenhouses with the advantages of fast growth, high yield, and stable production, and became the most important source of the traditional Chinese medicine Dendrobii caulis. Te components in Chinese herbal medicine are very complex. Tus, the quality of D. ofcinale cannot yet be accurately evaluated by its polysaccharides content alone. Terefore, the present study proposal explores the relationship between small molecular components and polysaccharides in D. ofcinale from greenhouse cultivation in diferent regions (AH, GX, GZ, and YN). To provide a theoretical basis for the collection, conservation, and utilization of germplasm resources of D. ofcinale in diferent regions.

Sample Preparation.
A total of 71 samples of D. ofcinale were collected from four regions, including D. ofcinale from Huoshan County of Anhui province (AH), D. ofcinale from Rong County of Guangxi province (GX), D. ofcinale from Danzhai County of Guizhou province (GZ) and D. ofcinale from Menglian County of Yunnan province (YN), in 2019 (see Table 1). Te fresh stems of the 3-year-old samples were dried at 60°C, grounded into fne powder and stored at −80°C for the assay.

Determination of Polysaccharides.
Te quantifcation of polysaccharides in D. ofcinale follows the guidance of the Ch.P. (2020 edition) [6]. Firstly, a calibration curve was prepared by each UV absorbances(y) against concentrations (x, μg/mL) of glucose standard. Te linear regression equation was showed as y � ax + b. And then, the D. ofcinale powder sample (0.06 g) was heated and refuxed in a tested round bottom fask for 2 h. After cooling, it was precipitated with anhydrous ethanol for 1 h, then washed with 80% ethanol and the precipitate was dissolved with hot water to obtain the sample to be tested. Ten, the phenolsulfuric acid reaction was performed: 5% phenol solution (1.0 mL) and concentrated sulfuric acid (5.0 mL) were added sequentially to the sample to be tested (1.0 mL), rapidly shaken, heated in boiling water for 20 min, immediately removed and ice bathed for 5 min. Te absorbance of the reaction solution was measured at 488 nm and the polysaccharide content was calculated according to the glucose standard calibration curve.

Metabolite Extraction.
Dried and crushed D. ofcinale powder (75 mg) was weighed precisely, dispersed in 1 mL of 70% methanol, extracted by ultrasonication (400 W, 50 kHz) for 30 min, cooled, centrifuged at 12000 rpm for 5 min, and the supernatant was taken to analyze the secondary metabolites of D. ofcinale by UPLC-Q-TOF/MS.
Mass spectrometry was performed using an Agilent Q-TOF/MS system (Agilent, MA, USA), with separate acquisitions in positive and negative ion mode. Ion source parameters: mass scan ranged m/z 50 to 1200; gas temperature, 350°C; dry gas fow rate, 10 L/min; nebulizer, 45 psig; shealth gas temperature, 350°C; sheath gas fow rate, 11 L/min; vcap voltage, 4000 V; nozzle voltage, 1000 V; fragmentor, 175 V. Secondary mass spectrometry information was acquired using Auto MS/MS mode with CE of 20, 30, and 40 V, respectively. Data acquisition and processing were performed by Agilent Mass Hunter Profnder analysis software.

Data Processing and Analysis of Secondary Metabolites.
Peak matching, peak alignment, ion fusion, and deconvolution were performed on the raw data using Agilent Mass Hunter Profnder software (version 10.0). Fragmented peaks with false positives were excluded based on peak area, retention time, and molecular weight. Te analysis was performed by unsupervised pattern recognition principal component analysis (PCA) and projections on the latent structure-discrimination analysis (PLS-DA) models of SIMCA 14.1 software, with variable importance in the projection (VIP) value greater than 3 was used as a threshold to screen diferential compositions. Te ggplot2, pheatmap, and other packages were applied in the R program (version 4.1.1) for other visualizations. p < 0.05 was considered statistically signifcant.

Comparison of Polysaccharide Content in D. ofcinale in Diferent
Regions. Te polysaccharide contents of 71 samples collected from four regions were determined. Te Ch.P. stipulates that D. ofcinale polysaccharide content of not less than 25% is qualifed. Te results showed (see Figure 1) that the average polysaccharide contents in D. ofcinale of the four regions AH, GX, GZ, and YN were 33.51%, 40.11%, 28.39%, and 26.26%, and their passing rates were 83%, 94%, 72%, and 50%, respectively. Te polysaccharide content of D. ofcinale in GX was signifcantly higher than that of other places.

Secondary Metabolomics of D. ofcinale in Diferent
Regions. Te metabolic information of D. ofcinale from four regions was investigated by nontargeted metabolomics techniques. Te base peak chromatogram (BPC) of quality control samples showed retention times mainly within one to eight min in positive ion (e.g., in Figure 2(a)) and negative ion (e.g., in Figure 2(b)) modes. A total of 82 metabolites were identifed (see Table 2). In the BPC plots of typical samples from each region, there were diferences in metabolic composition between them in both positive ion (e.g., in Figure 2(c)) and negative ion (e.g., in Figure 2(d)) modes. In the PCA plot (e.g., in Figure 2(e)), the overall profle of the distribution of D. ofcinale samples from diferent regions could be observed.

Discovery of Diferential Metabolites of D. ofcinale in Diferent Regions.
To further reveal the diferences in the chemical composition of D. ofcinale in diferent regions, multivariate statistical analysis was used for the analysis. Te PLS-DA model developed had good predictive power (R2X (cum) � 0.698, R2Y (cum) � 0.668) and confdence (Q2 (cum) � 0.504) (e.g., in Figure 3(a)). Te results showed that YN and GX clustered separately by region, while the metabolite profles of the two regions AH and GZ crossed obviously, indicating that the metabolites of D. ofcinale in the two regions have a similar expression. 32 diferential metabolites were screened according to the variable importance for the projection (VIP) value > 1 (e.g., in Figure 3(b)). Further cluster heatmap analysis was done for these diferential metabolites (e.g., in Figure 3(c)), and overall, the region with more diferential metabolites was YN, followed by GZ, AH, and GX in that order.      In terms of the distribution of expression of these differential compositions (see Figure 5), the highest expression of diferential compositions was still YN, followed by GZ, AH, and GX in that order. Among favonoids, the higher expression regions were YN and AH. It is also evident that this diferential composition was almost absent in GX samples. Among the phenolic glycosides, the higher expression regions were YN and GX. In the organic nitrogenous compounds and amides, the highest expression region was YN. In the bibenzyls and disaccharide, the higher expression regions was GZ.

Discussion
D. ofcinale, as a widely used valuable Chinese herbal medicine, adopts polysaccharides as its main active ingredient and quality control standard in the Ch.P. In the present study, we selected D. ofcinale from diferent origins of greenhouse cultivation with relatively stable quality as the research object, and frstly analyzed their polysaccharide content according to the Ch.P. method. Te results showed that in terms of polysaccharide content, the order was GX > AH > GZ > YN. It has been observed that the production of polysaccharides in wolfberry and lingonberries decreases gradually, with the increase in altitude and decrease in temperature [19,20]. Tis has similarity with our results, in that GX (average altitude 97 m) and AH (average altitude 80 m) were at lower altitude and they both had high polysaccharide content, followed by GZ (average altitude 895 m), YN (average altitude 1116 m), respectively. Previous literature reported that D. ofcinale is rich in favonoids, phenanthrenes, bibenzyl, and other small molecule chemical components in addition to polysaccharides [21][22][23][24]. Terefore, it is unsystematic and incomplete to use polysaccharide content alone as a criterion for the medicinal value and quality evaluation of D. ofcinale. Herein, we used metabolomic analysis, combined with heat map and hierarchical clustering analysis, to reveal the diferences in small Among the favonoids, most of them were favone-Cglycosides. In plants, favonoids have a variety of biological functions, such as regulating cell growth, enhancing nutrient recycling, and resisting biotic and abiotic stresses [25,26]. A favonoids-rich diet not only helps prevent some chronic diseases, but it also has biological activities such as antiinfammatory, anticancer, cardiovascular protection, and blood lipid regulation [27][28][29]. It has been reported that Astragalus polysaccharides signifcantly improve the  Journal of Analytical Methods in Chemistry solubility, stability, and solubilizing efect of favonoids [30]. Synergistic efects of polysaccharides with favonoids have been shown, which may contribute to better pharmacological efects. However, almost all the diferential favonoids were higher in the YN sample with the lowest polysaccharide content, while the opposite result was observed in the GX sample with the highest polysaccharide content. And, it was corroborated in the YN samples from the samples with qualifed and unqualifed polysaccharide contents. Tis also reinforces that the evaluation of the quality of D. ofcinale by polysaccharide content alone is imperfect, and the content of small molecule components needs to be examined systematically. Bibenzyls and disaccharide were mainly highly expressed in GZ. Te bibenzyls is one of the active ingredients in Dendrobium genus. Te main pharmacological activities identifed in the compounds are antitumor, antidiabetic, antiplatelet aggregation, anti-infammatory, etc [31][32][33]. It is expressed that the bibenzyls had a wide range of medicinal efects in Dendrobium genus. Sucrose, a disaccharide, is the main product of photosynthesis and can act as a signaling molecule for a wide range of plant growth processes [34]. It has specifc functions in plant metabolism, growth, and development. Phenolic glycosides, amides, and organic nitrogenous compounds were the main sign component of YN. Of these, phenolic glycosides are widely distributed in plants and are phenylalanine and tyrosine metabolites with antimalarial, antineuroinfammatory, antiobesity and antioxidant, and other activities, mainly [35][36][37][38]. It has lower biological activity compared to the corresponding glycosides, some of which may later be used as nutritional agents or adjuvants [35]. Some studies have shown that the accumulation of total phenols may be positively correlated with altitude gradient [39]. It has been observed that succinylcarnitine (O1) correlates with total cholesterol or LDL, activated partial thromboplastin time [40]. Tese studies also predicted that D. ofcinale containing diferent components is suitable for diferent disease treatment.

Conclusion
In summary, this study identifed the diferential compositions of D. ofcinale cultivated in greenhouses in four regions of China (AH, GX, GZ, YN). Te results showed that the polysaccharide content in D. ofcinale was strongly related to its growing altitude, with the highest average polysaccharide content in the GX sample (altitude: 97 m) and the lowest average polysaccharide content in the YN sample (altitude: 1116 m). Tere was also a signifcant difference in the small molecule chemical composition in D. ofcinale, where the content of favonoids showed an opposite trend to that of polysaccharides. Tese results indicated that the current quality standard for evaluating the quality of D. ofcinale by polysaccharide content alone is imperfect, and small molecule compounds need to be included as quality markers.

Data Availability
Te original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Conflicts of Interest
Te authors declare that there are no conficts of interest.