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

Authors: Qianqian He; Anjing Lu; Lin Qin; Qianru Zhang; Yanliu Lu; Zhou Yang; Daopeng Tan; Yuqi He
Journal: Journal of Analytical Methods in Chemistry
 (2022)

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## Abstract

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.


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## Body

## 1. Introduction

The genusDendrobium is one of the largest genera of the Orchidaceae, which contains 1500–2000 species [1]. Among them, Dendrobium officinale 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. officinale is widely cultivated in various regions of China, including Anhui, Zhejiang, Hunan, Fujian, Guangxi, Sichuan, and Yunnan provinces [3–5].D. officinale is a traditional Chinese medicine first 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. officinale are considered to be a drug of nourishing Yin, which comprises the efficacy of nourishing the stomach for promoting the production of fluid, 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. officinale has multiple promising bioactivities, including immunomodulation, antitumor, antioxidant, antifatigue, glycolipid regulation, hepatoprotection, etc [8–11]. According to current phytochemical investigations, more than 190 compounds have been isolated from D. officinale, including polysaccharides, alkaloids, amino acids, flavonoids, trace elements, and other nutrients, and its medicinal components are mainly composed of polysaccharides, bibenzyl, phenanthrene, flavonoids, and alkaloids [12–15]. 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.officinale 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. officinale varies greatly from different region and growing environment [18]. Fortunately, the manual cultivation of D. officinale has made a great breakthrough, and currently D. officinale 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. The components in Chinese herbal medicine are very complex. Thus, the quality of D. officinale cannot yet be accurately evaluated by its polysaccharides content alone. Therefore, the present study proposal explores the relationship between small molecular components and polysaccharides in D. officinale from greenhouse cultivation in different regions (AH, GX, GZ, and YN). To provide a theoretical basis for the collection, conservation, and utilization of germplasm resources of D. officinale in different regions.


## 2. Materials and Methods

### 2.1. Chemicals

LC-MS grade acetonitrile, methanol, formic acid, and water were purchased from Merck (Darmstadt, Germany), CNW Technologies GmbH (Duesseldorf, Germany), and Watsons (Hong Kong, China), respectively, for the LC-MS analyze. The reagents required for the polysaccharide assay including anhydrous ethanol, phenol, concentrated sulfuric acid, and glucose standards were purchased from Kelong Chemical Reagent Factory (Chengdu, China), Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), and Merck (Darmstadt, Germany), respectively.


### 2.2. Sample Preparation

A total of 71 samples ofD. officinale were collected from four regions, including D. officinale from Huoshan County of Anhui province (AH), D. officinale from Rong County of Guangxi province (GX), D. officinale from Danzhai County of Guizhou province (GZ) and D. officinale from Menglian County of Yunnan province (YN), in 2019 (see Table 1). The fresh stems of the 3-year-old samples were dried at 60°C, grounded into fine powder and stored at −80°C for the assay.Table 1
Information ofD. officinale samples.
No.
Collecting samples
Sample numbers
Average altitude (m)
Longitude (E)
Latitude (N)
1
AH
18
80
116°19′33″
31°23′20″
2
GX
17
97
110°38′20″
22°49′48″
3
GZ
18
895
107°65′76″
26°21′16″
4
YN
18
1116
99°42′55″
22°24′10″


### 2.3. Determination of Polysaccharides

The quantification of polysaccharides inD. officinale 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. The linear regression equation was showed as y = ax + b. And then, the D. officinale powder sample (0.06 g) was heated and refluxed in a tested round bottom flask 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. Then, the phenol-sulfuric 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. The absorbance of the reaction solution was measured at 488 nm and the polysaccharide content was calculated according to the glucose standard calibration curve.


### 2.4. Metabolite Extraction

Dried and crushedD. officinale 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. officinale by UPLC-Q-TOF/MS.


### 2.5. UPLC-Q-TOF/MS Conditions

Samples were evaluated using a Waters 1290 Infinity II UPLC liquid chromatograph system (Waters Corp., Milford, MA, USA) equipped with a binary pump, in-line degasser, autosampler, and thermostatically controlled column chamber. The separation of an aliquot of 1μL sample solutions was performed on a Waters CORTECS UPLC C18 (100 mm × 2.1 mm, 1.6 μm) maintained at 40°C. The mobile phase was solvent consisting of solvent A (0.1% formic acid in water, v/v) and solvent B (0.1% formic acid in acetonitrile, v/v) with a flow rate of 0.4 mL/min and a separation time of 10 min. Gradient elution conditions: 0–0.5 min, 5% B; 0.5–4 min, 40% B; 4–5 min, 75% B; 5–5.1 min, 95% B; 5.1–6.5 min, 95% B; 6.5–6.6 min, 5% B; 6.6–10 min, 5% B. The injection volume was 1 μL.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 flow rate, 10 L/min; nebulizer, 45 psig; shealth gas temperature, 350°C; sheath gas flow 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 Profinder analysis software.


### 2.6. 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 Profinder software (version 10.0). Fragmented peaks with false positives were excluded based on peak area, retention time, and molecular weight. The 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 differential compositions. The ggplot2, pheatmap, and other packages were applied in the R program (version 4.1.1) for other visualizations.p<0.05 was considered statistically significant.


## 2.1. Chemicals

LC-MS grade acetonitrile, methanol, formic acid, and water were purchased from Merck (Darmstadt, Germany), CNW Technologies GmbH (Duesseldorf, Germany), and Watsons (Hong Kong, China), respectively, for the LC-MS analyze. The reagents required for the polysaccharide assay including anhydrous ethanol, phenol, concentrated sulfuric acid, and glucose standards were purchased from Kelong Chemical Reagent Factory (Chengdu, China), Sinopharm Chemical Reagent Co., Ltd (Shanghai, China), and Merck (Darmstadt, Germany), respectively.


## 2.2. Sample Preparation

A total of 71 samples ofD. officinale were collected from four regions, including D. officinale from Huoshan County of Anhui province (AH), D. officinale from Rong County of Guangxi province (GX), D. officinale from Danzhai County of Guizhou province (GZ) and D. officinale from Menglian County of Yunnan province (YN), in 2019 (see Table 1). The fresh stems of the 3-year-old samples were dried at 60°C, grounded into fine powder and stored at −80°C for the assay.Table 1
Information ofD. officinale samples.
No.
Collecting samples
Sample numbers
Average altitude (m)
Longitude (E)
Latitude (N)
1
AH
18
80
116°19′33″
31°23′20″
2
GX
17
97
110°38′20″
22°49′48″
3
GZ
18
895
107°65′76″
26°21′16″
4
YN
18
1116
99°42′55″
22°24′10″


## 2.3. Determination of Polysaccharides

The quantification of polysaccharides inD. officinale 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. The linear regression equation was showed as y = ax + b. And then, the D. officinale powder sample (0.06 g) was heated and refluxed in a tested round bottom flask 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. Then, the phenol-sulfuric 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. The absorbance of the reaction solution was measured at 488 nm and the polysaccharide content was calculated according to the glucose standard calibration curve.


## 2.4. Metabolite Extraction

Dried and crushedD. officinale 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. officinale by UPLC-Q-TOF/MS.


## 2.5. UPLC-Q-TOF/MS Conditions

Samples were evaluated using a Waters 1290 Infinity II UPLC liquid chromatograph system (Waters Corp., Milford, MA, USA) equipped with a binary pump, in-line degasser, autosampler, and thermostatically controlled column chamber. The separation of an aliquot of 1μL sample solutions was performed on a Waters CORTECS UPLC C18 (100 mm × 2.1 mm, 1.6 μm) maintained at 40°C. The mobile phase was solvent consisting of solvent A (0.1% formic acid in water, v/v) and solvent B (0.1% formic acid in acetonitrile, v/v) with a flow rate of 0.4 mL/min and a separation time of 10 min. Gradient elution conditions: 0–0.5 min, 5% B; 0.5–4 min, 40% B; 4–5 min, 75% B; 5–5.1 min, 95% B; 5.1–6.5 min, 95% B; 6.5–6.6 min, 5% B; 6.6–10 min, 5% B. The injection volume was 1 μL.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 flow rate, 10 L/min; nebulizer, 45 psig; shealth gas temperature, 350°C; sheath gas flow 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 Profinder analysis software.


## 2.6. 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 Profinder software (version 10.0). Fragmented peaks with false positives were excluded based on peak area, retention time, and molecular weight. The 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 differential compositions. The ggplot2, pheatmap, and other packages were applied in the R program (version 4.1.1) for other visualizations.p<0.05 was considered statistically significant.


## 3. Results

### 3.1. Comparison of Polysaccharide Content inD. officinale in Different Regions

The polysaccharide contents of 71 samples collected from four regions were determined. The Ch.P. stipulates thatD. officinale polysaccharide content of not less than 25% is qualified. The results showed (see Figure 1) that the average polysaccharide contents in D. officinale 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. The polysaccharide content of D. officinale in GX was significantly higher than that of other places.Figure 1
The polysaccharide content assessment ofD. officinale from four regions. (a) Polysaccharide content of D. officinale from four regions. ∗ indicates significant difference compared to GX (p<0.05). (b) Polysaccharide qualification rate of D. officinale from four regions.
(a)
(b)


### 3.2. Secondary Metabolomics ofD. officinale in Different Regions

The metabolic information ofD. officinale from four regions was investigated by nontargeted metabolomics techniques. The 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 identified (see Table 2). In the BPC plots of typical samples from each region, there were differences 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 profile of the distribution of D. officinale samples from different regions could be observed.Figure 2
Profile analysis of secondary metabolomics ofD. officinale in different regions. The quality control and typical base peak chromatogram of D. officinale in positive ion mode (a, c) and negative ion mode (b, d). PCA score plot of D. officinale from four regions (e).
(a)
(b)
(c)
(d)
(e)


### 3.3. Discovery of Differential Metabolites ofD. officinale in Different Regions

To further reveal the differences in the chemical composition ofD. officinale in different regions, multivariate statistical analysis was used for the analysis. The PLS-DA model developed had good predictive power (R2X (cum) = 0.698, R2Y (cum) = 0.668) and confidence (Q2 (cum) = 0.504) (e.g., in Figure 3(a)). The results showed that YN and GX clustered separately by region, while the metabolite profiles of the two regions AH and GZ crossed obviously, indicating that the metabolites of D. officinale in the two regions have a similar expression. 32 differential 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 differential metabolites (e.g., in Figure 3(c)), and overall, the region with more differential metabolites was YN, followed by GZ, AH, and GX in that order.Figure 3
Discovery of differential components ofD. officinale from different regions. (a) PLS-DA of D. officinale from four regions. (b) VIP plot of D. officinale from four regions. (c) Heatmap of D. officinale from four regions.
(a)
(b)
(c)Table 2
Secondary metabolites ofD. officinale from four regions.
No.
Identification
RT (min)
Add ion
m/z
ppm
Molecular formula
1
Sucrose
0.587
[M-H]−
341.1092
0.7
C12H22O11
2
Succinylcarnitine
0.628
[M+H]+
262.1292
2.6
C11H19NO6
3
O-methylmalonyl-L-carnitine
0.755
[M+H]+
262.1291
2.4
C11H19NO6
4
Guanosine
0.807
[M-H]−
282.0838
−2.0
C10H13N5O5
5
O-glutaroyl-L-carnitine
0.898
[M+H]+
276.1453
4.0
C12H21NO6
6
2-Methyl-1H-indol-7-yl-β-D-mannopyranoside or isomer
1.416
[M+H]+
310.1289
1.4
C15H19NO6
7
Protocatechuic acid-3-O-glucoside
1.452
[M-H]-
315.0716
−1.7
C13H16O9
8
di-O-methylcrenatin
1.741
[M+H]+
347.1313
−6.9
C15H22O9
9
2,6-Dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-glucopyranoside
1.915
[M-H]−
447.1136
−1.8
C18H24O13
10
Benzyl-β-gentiobioside
1.992
[M-H]−
431.1549
−2.3
C19H28O11
11
Dendromoniliside C
2.251
[M-H]−
443.1912
−2.4
C21H32O10
12
2,6-Dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-xylopyranoside
2.306
[M-H]−
417.1031
−1.8
C17H22O12
13
Syringin
2.408
[M+Na]+
395.1310
−0.5
C17H24O9
14
Dihydrosyringin
2.480
[M+Na]+
397.1470
0.3
C17H26O9
15
Khaephuoside A
2.526
[M+Na]+
501.1575
−0.7
C20H30O13
16
Gastrodin
2.612
[M+Na]+
309.0946
0.3
C13H18O7
17
2-Methoxyphenyl-1-O-β-D-apiofuromosyl-(1⟶2)-β-D-glucopyranside
2.637
[M-H]−
417.1393
−2.4
C18H26O11
18
Vicenin 2
2.654
[M-H]−
593.1503
−1.5
C27H30O15
19
2-methylphenyl-O-β-D-xylopyranosyl-(1⟶6)-O-β-d-glucopyranoside
2.709
[M-H]−
401.1446
−1.7
C18H26O10
20
Vicenin 1
2.797
[M-H]−
563.1399
−1.3
C26H28O14
21
Schaftoside
2.858
[M-H]−
563.1398
−1.5
C26H28O14
22
Dihydromelilotoside
2.880
[M-H]−
327.1079
−1.9
C15H20O8
23
Isoschaftoside
2.951
[M-H]−
563.1398
−1.4
C26H28O14
24
Syringaresinol 4,4’-di-O-β-D-glucopyranoside
2.970
[M+Na]+
765.2566
−1.4
C34H46O18
25
Apigenin 6-C-β-D-xylopyranosyl-8-C-α-L-arabinopyranoside
3.012
[M-H]−
533.1292
−1.6
C25H26O13
26
Dendromoniliside E
3.026
[M+Na]+
621.2147
−1.1
C28H38O14
27
Shashenoside I
3.048
[M+Na]+
527.1734
−0.2
C22H32O13
28
Violanthin
3.084
[M-H]−
577.1555
−1.3
C27H30O14
29
Dendrobine
3.114
[M+H]+
264.1957
−0.4
C16H25NO2
30
Apigenin 6,8-di-C-arabinoside
3.106
[M-H]−
533.1291
−1.8
C25H26O13
31
Vicenin 3
3.111
[M-H]−
563.1394
−2.2
C26H28O14
32
Rutin
3.111
[M-H]−
609.1455
−1.1
C27H30O16
33
Apigenin-7-O-β-D-glucoside
3.161
[M-H]−
431.0972
−2.6
C21H20O10
34
Apigenin 6-C-α-L-arabinopyranosyl-8-C-β-D-xylopyranoside
3.238
[M-H]−
533.1291
−1.8
C25H26O13
35
Apigenin 8-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside
3.381
[M-H]−
563.1396
−1.8
C26H28O14
36
Apigenin 6-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside
3.425
[M-H]−
563.1399
−1.3
C26H28O14
37
Syringaresinolβ-D-glucoside
3.522
[M+Na]+
605.2197
−1.2
C28H38O13
38
N-p-cis-coumaroyltyramine
3.742
[M+H]+
284.1282
0.3
C17H17NO3
39
Paeonolide
3.767
[M-H]−
459.1501
−1.5
C20H28O12
40
N-trans-feruloyltyramine
3.930
[M+H]+
314.1387
−0.1
C18H19NO4
41
Dendrocandin E
3.894
[M-H]−
275.0917
−2.8
C15H16O5
42
icariol A2-4-O-β-D-glucopyranoside
4.007
[M+Na]+
621.2141
−2.0
C28H38O14
43
4,4’-dihydroxy-3,5-dimethoxybibenzyl
4.010
[M-H]−
273.1124
−3.1
C16H18O4
44
N-p-trans-coumaroyltyramine
4.012
[M+H]+
284.1280
−0.2
C17H17NO3
45
cis-N-Feruloyltyramine
4.128
[M+H]+
314.1385
−0.5
C18H19NO4
46
Dendrocandin C
4.175
[M-H]−
289.1075
−2.1
C16H18O5
47
3’,5,5’,7-tetrahydroxyflavanone
4.214
[M-H]−
287.0554
−2.6
C15H12O6
48
Bornyl-2-O-β-D-arabinofuranosyl(1⟶6)-β-D-glucopyranoside
4.379
[M-H]−
447.2228
−1.7
C21H36O10
49
Tristin
4.440
[M-H]−
259.0971
−1.8
C15H16O4
50
Naringenin
4.699
[M-H]−
271.0608
−1.6
C15H12O5
51
trans-Cinnamoyl-p-hydroxybenzenethylamine
5.005
[M+H]+
268.1333
0.5
C17H17NO2
52
Dendrocandin O
5.032
[M+H]+
455.1696
−1.0
C25H26O8
53
Trihydroxyoctadecaenoic acid
5.052
[M-H]−
329.2329
−1.4
C18H34O5
54
Dendrocandin M
5.109
[M+Na]+
493.1832
−0.2
C26H30O8
55
Dendrocandin K
5.123
[M-H]−
515.1702
−1.9
C30H28O8
56
Dendrocandin U
5.231
[M+H]+
469.1856
−0.3
C26H28O8
57
Dendrocandin N
5.264
[M+H]+
439.1751
−0.1
C25H26O7
58
Dendrocandin T
5.286
[M+Na]+
521.1779
−0.7
C27H30O9
59
Syringaresinol
5.324
[M+Na]+
441.1518
−0.5
C22H26O8
60
Dendrocandin R
5.377
[M-H]−
329.1386
−2.7
C19H22O5
61
Dengraol A
5.377
[M-H]−
499.1749
−2.6
C30H28O7
62
Dendrocandin H
5.377
[M-H]−
525.1179
−2.3
C30H22O9
63
3,4-Dihydroxy-5,4’-dimethoxybibenzyl
5.415
[M-H]−
273.1127
−2.0
C16H18O4
64
Erianin
5.434
[M+H]+
319.1540
0.0
C18H22O5
65
15-Methylhexadecaphytosphingosine or isomer
5.473
[M+H]+
304.2861
4.8
C17H37NO3
66
Dendrocandin G
5.476
[M-H]−
529.1856
−2.4
C31H30O8
67
Dendrocandin L
5.548
[M-H]−
509.1226
−3.2
C30H22O8
68
Physcion
5.567
[M+H]+
285.0759
0.4
C16H12O5
69
Dendrocandin J
5.548
[M-H]−
529.1854
−2.6
C31H30O8
70
2-Amino-octadecane-1,3,4-triol
5.589
[M+H]+
318.3010
2.4
C18H39NO3
71
Dendrocandin I
5.680
[M-H]−
543.1999
−4.6
C32H32O8
72
3-Methoxy-5-[2-(4-methoxyphenyl) ethyl] phenol
5.666
[M+H]+
259.1331
0.8
C16H18O3
73
Dendrocandin B
5.677
[M+H]+
483.2014
0.1
C27H30O8
74
2,3,4,7-Tetramethoxyphenanthrene
5.705
[M+H]+
299.1278
0.0
C18H18O4
75
Sphinganine
5.727
[M+H]+
302.3057
1.0
C18H39NO2
76
Dendrocandin F
5.823
[M-H]−
543.2010
−2.7
C32H32O8
77
Linolenic acid
5.986
[M+H]+
279.2321
0.8
C18H30O2
78
Linoleic acid
6.104
[M-H]−
279.2320
−3.4
C18H32O2
79
Aphanamixoid J or isomer
6.531
[M+H]+
609.2704
1.6
C34H40O10
80
Aphanamixoid J or isomer
6.614
[M+H]+
609.2722
4.5
C34H40O10
81
Aphanamixoid G or isomer
6.702
[M+H]+
593.2765
3.4
C34H40O9
82
Aphanamixoid G or isomer
6.818
[M+H]+
593.2747
0.4
C34H40O9
Note. A: amides; B: bibenzyls; D: disaccharide; F: flavonoids; O: organic nitrogenous compounds; P: phenolic glycosides.


### 3.4. Identification of Differential Compositions in D. officinale from Different Regions

Based on the analysis of the extracted ion chromatograms and the results of VIP >1, 22 compounds with clear chemical structures were tentative identified (see Figure4), including 1 amides (cis-N-feruloyltyramine: A1), 3 bibenzyls (dendromoniliside E; B1, dendrocandin U: B2, dendrocandin B: B3), 1 disaccharide (sucrose: D1), 9 flavonoids (vicenin 2: F1, vicenin 1: F2, schaftoside: F3, isoschaftoside: F4, apigenin 6,8-di-C-arabinoside: F5, vicenin 3: F6, rutin: F7, apigenin 8-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside: F8, apigenin 6-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside: F9), 3 organic nitrogenous compounds (succinylcarnitine: O1, O-methylmalonyl-L-carnitine: O2, O-glutaroyl-L-carnitine: O3), 5 phenolic glycosides (di-O-methylcrenatin: P1, 2,6-dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-glucopyranoside: P2, 2,6-dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-xylopyranoside: P3, 2-methylphenyl-O-β-D-xylopyranosyl-(1⟶6)-O-β-D-glucopyranoside: P4, paeonolide: P5).Figure 4
Chemical structures of differential compositions inD. officinale from four regions.In terms of the distribution of expression of these differential compositions (see Figure5), the highest expression of differential compositions was still YN, followed by GZ, AH, and GX in that order. Among flavonoids, the higher expression regions were YN and AH. It is also evident that this differential 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.Figure 5
Expression of differential compositions inD. officinale from four regions.


## 3.1. Comparison of Polysaccharide Content inD. officinale in Different Regions

The polysaccharide contents of 71 samples collected from four regions were determined. The Ch.P. stipulates thatD. officinale polysaccharide content of not less than 25% is qualified. The results showed (see Figure 1) that the average polysaccharide contents in D. officinale 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. The polysaccharide content of D. officinale in GX was significantly higher than that of other places.Figure 1
The polysaccharide content assessment ofD. officinale from four regions. (a) Polysaccharide content of D. officinale from four regions. ∗ indicates significant difference compared to GX (p<0.05). (b) Polysaccharide qualification rate of D. officinale from four regions.
(a)
(b)


## 3.2. Secondary Metabolomics ofD. officinale in Different Regions

The metabolic information ofD. officinale from four regions was investigated by nontargeted metabolomics techniques. The 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 identified (see Table 2). In the BPC plots of typical samples from each region, there were differences 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 profile of the distribution of D. officinale samples from different regions could be observed.Figure 2
Profile analysis of secondary metabolomics ofD. officinale in different regions. The quality control and typical base peak chromatogram of D. officinale in positive ion mode (a, c) and negative ion mode (b, d). PCA score plot of D. officinale from four regions (e).
(a)
(b)
(c)
(d)
(e)


## 3.3. Discovery of Differential Metabolites ofD. officinale in Different Regions

To further reveal the differences in the chemical composition ofD. officinale in different regions, multivariate statistical analysis was used for the analysis. The PLS-DA model developed had good predictive power (R2X (cum) = 0.698, R2Y (cum) = 0.668) and confidence (Q2 (cum) = 0.504) (e.g., in Figure 3(a)). The results showed that YN and GX clustered separately by region, while the metabolite profiles of the two regions AH and GZ crossed obviously, indicating that the metabolites of D. officinale in the two regions have a similar expression. 32 differential 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 differential metabolites (e.g., in Figure 3(c)), and overall, the region with more differential metabolites was YN, followed by GZ, AH, and GX in that order.Figure 3
Discovery of differential components ofD. officinale from different regions. (a) PLS-DA of D. officinale from four regions. (b) VIP plot of D. officinale from four regions. (c) Heatmap of D. officinale from four regions.
(a)
(b)
(c)Table 2
Secondary metabolites ofD. officinale from four regions.
No.
Identification
RT (min)
Add ion
m/z
ppm
Molecular formula
1
Sucrose
0.587
[M-H]−
341.1092
0.7
C12H22O11
2
Succinylcarnitine
0.628
[M+H]+
262.1292
2.6
C11H19NO6
3
O-methylmalonyl-L-carnitine
0.755
[M+H]+
262.1291
2.4
C11H19NO6
4
Guanosine
0.807
[M-H]−
282.0838
−2.0
C10H13N5O5
5
O-glutaroyl-L-carnitine
0.898
[M+H]+
276.1453
4.0
C12H21NO6
6
2-Methyl-1H-indol-7-yl-β-D-mannopyranoside or isomer
1.416
[M+H]+
310.1289
1.4
C15H19NO6
7
Protocatechuic acid-3-O-glucoside
1.452
[M-H]-
315.0716
−1.7
C13H16O9
8
di-O-methylcrenatin
1.741
[M+H]+
347.1313
−6.9
C15H22O9
9
2,6-Dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-glucopyranoside
1.915
[M-H]−
447.1136
−1.8
C18H24O13
10
Benzyl-β-gentiobioside
1.992
[M-H]−
431.1549
−2.3
C19H28O11
11
Dendromoniliside C
2.251
[M-H]−
443.1912
−2.4
C21H32O10
12
2,6-Dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-xylopyranoside
2.306
[M-H]−
417.1031
−1.8
C17H22O12
13
Syringin
2.408
[M+Na]+
395.1310
−0.5
C17H24O9
14
Dihydrosyringin
2.480
[M+Na]+
397.1470
0.3
C17H26O9
15
Khaephuoside A
2.526
[M+Na]+
501.1575
−0.7
C20H30O13
16
Gastrodin
2.612
[M+Na]+
309.0946
0.3
C13H18O7
17
2-Methoxyphenyl-1-O-β-D-apiofuromosyl-(1⟶2)-β-D-glucopyranside
2.637
[M-H]−
417.1393
−2.4
C18H26O11
18
Vicenin 2
2.654
[M-H]−
593.1503
−1.5
C27H30O15
19
2-methylphenyl-O-β-D-xylopyranosyl-(1⟶6)-O-β-d-glucopyranoside
2.709
[M-H]−
401.1446
−1.7
C18H26O10
20
Vicenin 1
2.797
[M-H]−
563.1399
−1.3
C26H28O14
21
Schaftoside
2.858
[M-H]−
563.1398
−1.5
C26H28O14
22
Dihydromelilotoside
2.880
[M-H]−
327.1079
−1.9
C15H20O8
23
Isoschaftoside
2.951
[M-H]−
563.1398
−1.4
C26H28O14
24
Syringaresinol 4,4’-di-O-β-D-glucopyranoside
2.970
[M+Na]+
765.2566
−1.4
C34H46O18
25
Apigenin 6-C-β-D-xylopyranosyl-8-C-α-L-arabinopyranoside
3.012
[M-H]−
533.1292
−1.6
C25H26O13
26
Dendromoniliside E
3.026
[M+Na]+
621.2147
−1.1
C28H38O14
27
Shashenoside I
3.048
[M+Na]+
527.1734
−0.2
C22H32O13
28
Violanthin
3.084
[M-H]−
577.1555
−1.3
C27H30O14
29
Dendrobine
3.114
[M+H]+
264.1957
−0.4
C16H25NO2
30
Apigenin 6,8-di-C-arabinoside
3.106
[M-H]−
533.1291
−1.8
C25H26O13
31
Vicenin 3
3.111
[M-H]−
563.1394
−2.2
C26H28O14
32
Rutin
3.111
[M-H]−
609.1455
−1.1
C27H30O16
33
Apigenin-7-O-β-D-glucoside
3.161
[M-H]−
431.0972
−2.6
C21H20O10
34
Apigenin 6-C-α-L-arabinopyranosyl-8-C-β-D-xylopyranoside
3.238
[M-H]−
533.1291
−1.8
C25H26O13
35
Apigenin 8-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside
3.381
[M-H]−
563.1396
−1.8
C26H28O14
36
Apigenin 6-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside
3.425
[M-H]−
563.1399
−1.3
C26H28O14
37
Syringaresinolβ-D-glucoside
3.522
[M+Na]+
605.2197
−1.2
C28H38O13
38
N-p-cis-coumaroyltyramine
3.742
[M+H]+
284.1282
0.3
C17H17NO3
39
Paeonolide
3.767
[M-H]−
459.1501
−1.5
C20H28O12
40
N-trans-feruloyltyramine
3.930
[M+H]+
314.1387
−0.1
C18H19NO4
41
Dendrocandin E
3.894
[M-H]−
275.0917
−2.8
C15H16O5
42
icariol A2-4-O-β-D-glucopyranoside
4.007
[M+Na]+
621.2141
−2.0
C28H38O14
43
4,4’-dihydroxy-3,5-dimethoxybibenzyl
4.010
[M-H]−
273.1124
−3.1
C16H18O4
44
N-p-trans-coumaroyltyramine
4.012
[M+H]+
284.1280
−0.2
C17H17NO3
45
cis-N-Feruloyltyramine
4.128
[M+H]+
314.1385
−0.5
C18H19NO4
46
Dendrocandin C
4.175
[M-H]−
289.1075
−2.1
C16H18O5
47
3’,5,5’,7-tetrahydroxyflavanone
4.214
[M-H]−
287.0554
−2.6
C15H12O6
48
Bornyl-2-O-β-D-arabinofuranosyl(1⟶6)-β-D-glucopyranoside
4.379
[M-H]−
447.2228
−1.7
C21H36O10
49
Tristin
4.440
[M-H]−
259.0971
−1.8
C15H16O4
50
Naringenin
4.699
[M-H]−
271.0608
−1.6
C15H12O5
51
trans-Cinnamoyl-p-hydroxybenzenethylamine
5.005
[M+H]+
268.1333
0.5
C17H17NO2
52
Dendrocandin O
5.032
[M+H]+
455.1696
−1.0
C25H26O8
53
Trihydroxyoctadecaenoic acid
5.052
[M-H]−
329.2329
−1.4
C18H34O5
54
Dendrocandin M
5.109
[M+Na]+
493.1832
−0.2
C26H30O8
55
Dendrocandin K
5.123
[M-H]−
515.1702
−1.9
C30H28O8
56
Dendrocandin U
5.231
[M+H]+
469.1856
−0.3
C26H28O8
57
Dendrocandin N
5.264
[M+H]+
439.1751
−0.1
C25H26O7
58
Dendrocandin T
5.286
[M+Na]+
521.1779
−0.7
C27H30O9
59
Syringaresinol
5.324
[M+Na]+
441.1518
−0.5
C22H26O8
60
Dendrocandin R
5.377
[M-H]−
329.1386
−2.7
C19H22O5
61
Dengraol A
5.377
[M-H]−
499.1749
−2.6
C30H28O7
62
Dendrocandin H
5.377
[M-H]−
525.1179
−2.3
C30H22O9
63
3,4-Dihydroxy-5,4’-dimethoxybibenzyl
5.415
[M-H]−
273.1127
−2.0
C16H18O4
64
Erianin
5.434
[M+H]+
319.1540
0.0
C18H22O5
65
15-Methylhexadecaphytosphingosine or isomer
5.473
[M+H]+
304.2861
4.8
C17H37NO3
66
Dendrocandin G
5.476
[M-H]−
529.1856
−2.4
C31H30O8
67
Dendrocandin L
5.548
[M-H]−
509.1226
−3.2
C30H22O8
68
Physcion
5.567
[M+H]+
285.0759
0.4
C16H12O5
69
Dendrocandin J
5.548
[M-H]−
529.1854
−2.6
C31H30O8
70
2-Amino-octadecane-1,3,4-triol
5.589
[M+H]+
318.3010
2.4
C18H39NO3
71
Dendrocandin I
5.680
[M-H]−
543.1999
−4.6
C32H32O8
72
3-Methoxy-5-[2-(4-methoxyphenyl) ethyl] phenol
5.666
[M+H]+
259.1331
0.8
C16H18O3
73
Dendrocandin B
5.677
[M+H]+
483.2014
0.1
C27H30O8
74
2,3,4,7-Tetramethoxyphenanthrene
5.705
[M+H]+
299.1278
0.0
C18H18O4
75
Sphinganine
5.727
[M+H]+
302.3057
1.0
C18H39NO2
76
Dendrocandin F
5.823
[M-H]−
543.2010
−2.7
C32H32O8
77
Linolenic acid
5.986
[M+H]+
279.2321
0.8
C18H30O2
78
Linoleic acid
6.104
[M-H]−
279.2320
−3.4
C18H32O2
79
Aphanamixoid J or isomer
6.531
[M+H]+
609.2704
1.6
C34H40O10
80
Aphanamixoid J or isomer
6.614
[M+H]+
609.2722
4.5
C34H40O10
81
Aphanamixoid G or isomer
6.702
[M+H]+
593.2765
3.4
C34H40O9
82
Aphanamixoid G or isomer
6.818
[M+H]+
593.2747
0.4
C34H40O9
Note. A: amides; B: bibenzyls; D: disaccharide; F: flavonoids; O: organic nitrogenous compounds; P: phenolic glycosides.


## 3.4. Identification of Differential Compositions in D. officinale from Different Regions

Based on the analysis of the extracted ion chromatograms and the results of VIP >1, 22 compounds with clear chemical structures were tentative identified (see Figure4), including 1 amides (cis-N-feruloyltyramine: A1), 3 bibenzyls (dendromoniliside E; B1, dendrocandin U: B2, dendrocandin B: B3), 1 disaccharide (sucrose: D1), 9 flavonoids (vicenin 2: F1, vicenin 1: F2, schaftoside: F3, isoschaftoside: F4, apigenin 6,8-di-C-arabinoside: F5, vicenin 3: F6, rutin: F7, apigenin 8-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside: F8, apigenin 6-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside: F9), 3 organic nitrogenous compounds (succinylcarnitine: O1, O-methylmalonyl-L-carnitine: O2, O-glutaroyl-L-carnitine: O3), 5 phenolic glycosides (di-O-methylcrenatin: P1, 2,6-dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-glucopyranoside: P2, 2,6-dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-xylopyranoside: P3, 2-methylphenyl-O-β-D-xylopyranosyl-(1⟶6)-O-β-D-glucopyranoside: P4, paeonolide: P5).Figure 4
Chemical structures of differential compositions inD. officinale from four regions.In terms of the distribution of expression of these differential compositions (see Figure5), the highest expression of differential compositions was still YN, followed by GZ, AH, and GX in that order. Among flavonoids, the higher expression regions were YN and AH. It is also evident that this differential 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.Figure 5
Expression of differential compositions inD. officinale from four regions.


## 4. Discussion

D. officinale, 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. officinale from different origins of greenhouse cultivation with relatively stable quality as the research object, and firstly analyzed their polysaccharide content according to the Ch.P. method. The 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]. This 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 thatD. officinale is rich in flavonoids, phenanthrenes, bibenzyl, and other small molecule chemical components in addition to polysaccharides [21–24]. Therefore, it is unsystematic and incomplete to use polysaccharide content alone as a criterion for the medicinal value and quality evaluation of D. officinale. Herein, we used metabolomic analysis, combined with heat map and hierarchical clustering analysis, to reveal the differences in small molecule chemical composition among D. officinale from four different origins. The results elucidated that 22 compounds, including 1 amides (Cis-N-Feruloyltyramine), 3 bibenzyls (Dendromoniliside E, Dendrocandin U, Dendrocandin B), 1 disaccharide (Sucrose), 9 flavonoids (Vicenin 1, Vicenin 2, Vicenin 3, Schaftoside, Isoschaftoside, Rutin, Apigenin 6,8-di-C-arabinoside, Apigenin 6-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside, apigenin 8-C-α-L-arabinopyranosyl-2”-O-β-D-glucopyranoside), 3 organic nitrogenous compounds (Succinylcarnitine, O-Methylmalonyl-L-carnitine, O-Glutaroyl-L-carnitine), 5 phenolic glycosides (Di-O-methylcrenatin, Paeonolide, 2,6-Dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-glucopyranoside, 2,6-Dihydroxybenzoic acid 2-O-β-D-apiofuranosyl-(1⟶2)-β-D-xylopyranoside, 2-Methylphenyl-O-β-D-xylopyranosyl-(1⟶6)-O-β-D-glucopyranoside) were the main differential chemical constituents in D. officinale from different origins. The growth environment of D. officinale cultivated in greenhouses is relatively controlled, and thus the differences in the chemical composition of D. officinale from these different regions are more from the influence of origin.Among the flavonoids, most of them were flavone-C-glycosides. In plants, flavonoids have a variety of biological functions, such as regulating cell growth, enhancing nutrient recycling, and resisting biotic and abiotic stresses [25,26]. A flavonoids-rich diet not only helps prevent some chronic diseases, but it also has biological activities such as anti-inflammatory, anticancer, cardiovascular protection, and blood lipid regulation [27–29]. It has been reported that Astragalus polysaccharides significantly improve the solubility, stability, and solubilizing effect of flavonoids [30]. Synergistic effects of polysaccharides with flavonoids have been shown, which may contribute to better pharmacological effects. However, almost all the differential flavonoids 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 qualified and unqualified polysaccharide contents. This also reinforces that the evaluation of the quality of D. officinale 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. The bibenzyls is one of the active ingredients inDendrobium genus. The main pharmacological activities identified in the compounds are anti-tumor, antidiabetic, antiplatelet aggregation, anti-inflammatory, etc [31–33]. It is expressed that the bibenzyls had a wide range of medicinal effects 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 specific 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, antineuroinflammatory, antiobesity and antioxidant, and other activities, mainly [35–38]. 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]. These studies also predicted that D. officinale containing different components is suitable for different disease treatment.


## 5. Conclusion

In summary, this study identified the differential compositions ofD. officinale cultivated in greenhouses in four regions of China (AH, GX, GZ, YN). The results showed that the polysaccharide content in D. officinale 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). There was also a significant difference in the small molecule chemical composition in D. officinale, where the content of flavonoids showed an opposite trend to that of polysaccharides. 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.



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*Source: 8026410-2022-11-04.xml*