Opposite trends of glycosides and alkaloids in Dendrobium nobile of different age based on UPLC‐Q/TOF‐MS combined with multivariate statistical analyses

Abstract Introduction Alkaloids and glycosides are the active ingredients of the herb Dendrobium nobile, which is used in traditional Chinese medicine. The pharmacological effects of alkaloids include neuroprotective effects and regulatory effects on glucose and lipid metabolism, while glycosides improve the immune system. The pharmacological activities of the above chemical components are significantly different. In practice, the stems of 3‐year‐old D. nobile are usually used as the main source of Dendrobii Caulis. However, it has not been reported whether this harvesting time is appropriate. Objective The aim of this study was to compare the chemical characteristics of D. nobile in different growth years (1–3 years). Methods In this study, ultra‐high‐performance liquid chromatography coupled with quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐Q/TOF‐MS) was employed to analyze the constituents of D. nobile. The relative abundance of each constituent was analyzed with multivariate statistical analyses to screen the characteristic constituents that contributed to the characterization and classification of D. nobile. Dendrobine, a component of D. nobile that is used for quality control according to the Chinese Pharmacopoeia, was assayed by gas chromatography. Results As a result, 34 characteristic constituents (VIP > 2) were identified or tentatively identified as alkaloids and glycosides based on MS/MS data. Moreover, the content of alkaloids decreased over time, whereas the content of glycosides showed the opposite trend. The absolute quantification of dendrobine was consistent with the metabolomics results. Conclusion Our findings provide valuable information to optimize the harvest period and a reference for the clinical application of D. nobile.

Caulis has the effects of nourishing Yin and clearing heat, and it is applicable to situations such as yin injury and depletion of body fluid, dry mouth and polydipsia, less food and retching, deficiency and heat after illness, and dark eye. 2 In the Chinese Pharmacopeia, D. nobile is listed as the primary medicinal source of Dendrobii Caulis. 3 Pharmacological research confirmed that D. nobile improves cognitive dysfunction, reduces gastric damage, has anti-tumor effects, and can be used to treat diabetes. 4 Due to its beneficial effects, D. nobile is also used as a tonic and functional food, such as Dendrobii Liquor. 5  To meet the demands, D. nobile was developed. The alkaloids and glycosides of D. nobile are considered as the principal biologically active components with extensive pharmacological effects. The alkaloids are used to protect neurons and regulate glucose and lipid metabolism, among others. 7 Dendromoniliside A, isolated from the stem of Dendrobium moniliforme, improves immune activity by stimulating the proliferation of B cells and inhibiting the proliferation of T cells in vitro. 8 Dendronobiloside A, isolated from the stem of D. nobile, was found to stimulate the proliferation of murine T and B lymphocytes in vitro. 9,10 The above compounds have different pharmacological effects. In the main producing region, the stems of D. nobile are usually harvested in the third year. However, little evidence is available to support this practice. To find out the best time to harvest, understanding the differences in metabolite profile between D. nobile grown for various years is necessary.
Fourteen alkaloids, including dendrobine, dendrobine-N-oxide, dobilonine, and dendroxine, were isolated from D. nobile in previous studies. 11,12 Among them, dendrobine is used as a quality marker for the quality control (QC) of D. nobile according to the Chinese Pharmacopeia. However, our previous study indicated that the content of dendrobine in D. nobile decreases over time. Moreover, it is unknown whether other alkaloids in D. nobile follow the same trend as dendrobine. In addition to alkaloids, some non-alkaloid chemical constituents, such as polysaccharides, sesquiterpenes, phenanthrene, bibenzyl, and fluorenones, also exist in D. nobile. 13 How the levels of these non-alkaloid chemical constituents in D. nobile change with age is also unclear. Therefore, it is unclear whether dendrobine should be used as a quality marker for the QC of D. nobile.
In the present study, ultra-high-performance liquid chromatogra-

| Sample collection
The fresh stems of 1-to 3-year-old D. nobile were harvested from Chishui city of Guizhou Province in China in October 2019. Samples were collected in triplicate for each feature. Details are listed in Table 1. 2.3 | Sample preparation to profile the metabolome of D. nobile by UPLC-Q/TOF-MS Clean fresh D. nobile stems were dried at 60 C in a drying oven. The dried stems were ground into fine powder (300 mesh), and then 75 mg was soaked in 1 mL of 70% (v/v) methanol, followed by ultrasonication (50 kHz, 400 W) for 30 min. Next, the liquid was centrifuged at 9705 g for 5 min, and the supernatant was used for UPLC-Q/TOF-MS analysis.

| Data processing
The raw data were processed by peak matching, peak alignment, ion fusion, and deconvolution using Agilent  2-hydroxydendrobine or 6-hydroxydendrobine, isomer of dendrobine, by searching our in-house library. However, the exact structure could not be confirmed due to a lack of reference standard or literature.  Figure 3B). As 2-and 6-hydroxydendrobine have been reported previously in D. nobile stems, we tentatively identify constituent #273-p as 2-or 6-hydroxydendrobine. As the structure of dendrobine had already been uniquely assigned to constituent #232-p, the link between constituent #418-p and dendrobine was broken. However, we still assume it is a dendrobine isomer based on its general structural features and our in-house library. In the same way, constituents (1) #34-p, N-isopentenyl-dendrobinium, nobiline, and N-methyl-dendrobinium, we also assumed they are dendrobine-type alkaloids.
In the negative mode of MS, 17 constituents with PLS-DA VIP scores of more than 2 were screened out. In the same way as above, using the exact molecular weight values, ion fragmentation mechanisms, and the information of reference compounds, constituents #44-n, #41-n, #171-n, and #47-n were uniquely identified as quinic acid, sucrose, violanthin, and protocatechualdehyde, respectively. By matching the exact molecular weights with members of our in-house library, constituents #121-n, #230-n, #251-n, #130-n, #163-n, #267-n, #280-n, and #150-n were identified as dendronobilosides or dendromonilisides ( Figure 4B). Their MS/MS fragment ions were also linked to parts of the predicted structures. Among those constituents, #280-n was uniquely identified as dendronobiloside A based on their formulae and fragmentation information obtained from the literature ( Figure 4A). 9 Others were only identified as isomers of their corresponding predicted structures because of the lack of corresponding reference compounds. Constituent #36-n was tentatively identified as one of the isomers of lactobionic acid. Regarding constituents #172-n, #249-n, #120-n, and #131-n, the exact molecular weight values matched nothing in our in-house library or any reference standards. Therefore, their structures remain unknown.

| Statistics of the structure-confirmed constituents with age-dependent expression and validation with dendrobine quantification
To find a marker compound to determine the age of D. nobile, the identification of one or multiple structure-confirmed constituents with an age-dependent expression pattern is important. Among all constituents with age-dependent expression patterns, #232-p, #339-p, #419-p, #278-p, and #207-p, which were detected in positive mode, were uniquely identified as dendrobine, dendroxine, N-isopentenyldendrobinium, nobiline, and N-methyl-dendrobinium, respectively; #44-n, #41-n, #171-n, #280-n, and #47-n, which were detected in negative mode, were uniquely identified as quinic acid, sucrose, violanthin, dendronobiloside A, and protocatechualdehyde, respectively. The statistical significance of the age-dependent expression of these structure-confirmed constituents was analyzed by one-way ANOVA followed by Tukey's test ( Figure 5). Constituents #339-p and #207-p passed the test between year 1 and year 2 and between year 1 and year 3; however, they failed to pass the test between year durations (0.37%) showed that the method was accurate, precise, stable, and reliable ( Figure 6C). In agreement with the MS data, the GC data confirmed that dendrobine content in D. nobile decreased with age. The difference of dendrobine content between two groups was statistically significant (P < 0.05, Figure 6D), confirming the MS results. rings. 14 This family is biosynthesized in various well-known medicinal plants, [15][16][17] and saponins are expressed in an age-dependent manner in Panax ginseng, Panax notoginseng, and Polygala tenuifolia. [18][19][20] Another famous glycoside family is formed by the flavonoid glycosides, which also exhibit a positively age-dependent expression pattern. 21  proposed an alternative biosynthesis pathway of dendrobine, in which indole alkaloid derivatives play an important role. 31 The shikimate pathway is upstream of the indole alkaloid pathway. 32 Meanwhile, shikimate is biosynthesized from 3-dehydroquinic acid, which is a major metabolite of quinic acid. 33 In the present study, we observed a decreasing trend of quinic acid during D. nobile growth. This agedependent decreasing expression pattern of quinic acid was not only observed in the present study but also in several other plant species. 34 Thus, taken together, the substrate-competitive effect from the generation of dendronobilosides and dendromonilisides and the substrate-reductive effect from the decreased synthesis of quinic acid and indole alkaloids may work together to contribute to the decreasing trend of dendrobine during D. nobile growth.
Dendrobine-type alkaloids share a sesquiterpene backbone that was verified to be synthesized mainly via the mevalonate pathway and the methyl-D-erythritol 4-phosphate pathway. 29 In the two pathways, 16  young leaves exhibit higher expression levels of the above genes than fully expanded leaves. 36 HMGR enzyme activity showed the same trend. 37 The above results are in agreement with the observation that the content of alkaloids in 1-year-old D. nobile stems is higher than were confirmed to be higher in the mature stage than in the young stage in Stevia rebaudiana, litchi, and strawberry. [39][40][41] Therefore, we deduced that the above enzymes, which are involved in the synthesis of alkaloids and glycosides, are among the major causes of the opposite trend observed for the two main compounds in D. nobile.

| CONCLUSION
In the present work, the metabolites in D. nobile stems were analyzed using UPLC-Q/TOF-MS with multivariate statistical analysis. The results reveal that trends of alkaloids and sesquiterpene glycosides in the stems of D. nobile in different growth years were the opposite.
The contents of alkaloids in stems were found to decrease with age, whereas the content of sesquiterpene glycosides follows an opposite trend. Furthermore, the absolute quantification of dendrobine confirmed the increasing trend of alkaloids. Combining our results with the pharmacological effects of the above chemical components, we recommend to use 1-year-old D. nobile for clinical treatment of neurological diseases, while 3-year-old D. nobile could be used to improve immunity. Finally, sesquiterpene glycosides might be qualityrelated constituents that could be used as quality markers in D. nobile.