Metabolomics analysis of three Artemisia species in the Tibet autonomous region of China

The Artemisia species are widely distributed around the world, and have found important usage in traditional medicinal practice. This study was designed to investigate the metabolites of Tibetan Artemisia species and understand the metabolic pathways. The metabolites from three Artemisia species in Tibet, were analyzed using LC–MS/MS. The differential metabolites were classified and analyzed by principal component analysis (PCA), partial least squares analysis and hierarchical clustering. KEGG Pathway enrichment analysis was used to identify the key metabolic pathways involved in the differential metabolites of three Artemisia species. The metabolites of three Artemisia species were analyzed. Under the positive ion mode in LC–MS/MS, 262 distinct metabolites were differentially detected from Artemisia sieversiana and Artemisia annua, 312 differential metabolites were detected from Artemisia wellbyi and Artemisia sieversiana, 306 differential metabolites were screened from Artemisia wellbyi and Artemisia annua. With the negative ion mode, 106 differential metabolites were identified from Artemisia sieversiana and Artemisia annua, 131 differential metabolites were identified from Artemisia wellbyi and Artemisia sieversiana,133 differential metabolites were differentially detected from Artemisia wellbyi and Artemisia annua. The selected differential metabolites were mainly organic acids and their derivatives, ketones, phenols, alcohols and coumarins. Among these natural compounds, artemisinin, has the highest relative content in Artemisia annua. This is the first reported attempt to comparatively determine the types of the metabolites of the three widely distributed Artemisia species in Tibet. The information should help medicinal research and facilitate comprehensive development and utilization of Artemisia species in Tibet.


Background
Artemisia is a large genus of Anthemideae in the Compositae family. There are about 350 species in the world. The members of Artemisia are widely distributed in the temperate, frigid and subtropical regions of the northern hemisphere, with a few species distributed in the southern hemisphere [1]. It is well adapted in various environments and can survive in high altitude and extremely arid areas. Artemisia plants are mostly herbs, only a few are bushes or small shrubs, and most of them can be used as medicine and food for human consumption as well as animal feed [2,3]. There are 186 species and 44 varieties of Artemisia plants in China, which are distributed throughout the country and widely used [4] in traditional Chinese medicinal practice utilizing their properties of antibacterial, anti-inflammatory, and coagulant activity [5]. In addition, there are more than Open Access *Correspondence: luhao@nwsuaf.edu.cn † Xinyu Liu and Jinglong Wang contributed equally to this work. 1 College of Veterinary Medicine, Northwest A&F University, Yangling 712100, Shaanxi, China Full list of author information is available at the end of the article 30 Artemisia plants distributed in grassland and desert areas. They are highly resistant to the adverse conditions and have potential ecological and economic value [6,7]. They are important livestock feed, windbreak and sandstabilizing plants in pastoral areas [8,9].
Artemisia plant extracts contain polysaccharides, essential oils, organic acids, terpenes, flavonoids, with many of these components possessing the anti-inflammatory, immune-regulating, anti-tumor, anti-bacterial and anti-coagulant effects [10,11]. Artemisinin drugs extracted from this genus of Artemisia annua have been demonstrated to be the highly effective anti-malarial therapeutics. The anti-cholera drug "Artemisia wormwood" for liver and gallbladder diseases also belong to this genus.
Presently, the types of metabolites of Artemisia plants and the differences in metabolites among these plants are not clear. In this study, we selected three Artemisia plants for metabolomics analysis using LC-MC/MS methodology to determine the metabolites of these Artemisia plants and analyze the differences in metabolites in order to understand the constituents of the 3 species of Artemisia in Tibet. This study will provide new evidence for the potential medicinal use of the three Tibetan Artemisia species and lay the foundation for further exploration of the active constituents, their metabolic pathways, and pharmacological mechanisms of action.

Qualitative analysis of metabolites
The results are shown in the Additional file 1. In the negative ion mode, a total of 220 metabolites were identified from three Artemisia species. In the positive ion mode, a total of 535 metabolites were identified from three Artemisia species. The results showed that Artemisia plants contain polysaccharides, organic acids, flavonoids, terpenes, pigments, coumarin and other chemical components.

Principal component analysis (PCA)
PCA was used to distinguish the overall distribution trend between each two groups of samples (Fig. 1). As shown in Fig. 1A (a) and Fig. 1B (a), the samples of group D are all overlapped, and the correlation is good, while the Q group is mostly separated, and the degree of correlation is not as good as D.. There is no crossover between group D and group Q, which indicate that the difference between the two groups is relatively large, indicating that the metabolites between Artemisia sieversiana and Artemisia annua have a tendency to separate, and there are differences between groups. As shown in Fig. 1A (b) and Fig. 1B (b), the samples of group D are all overlapped, and the correlation is relatively good. There is no crossover between the D group and the Z group, which shows that the difference between the two groups is relatively large, indicating that the metabolites between Artemisia wellbyi and Artemisia sieversiana have a tendency to separate, and there are differences between groups. As shown in Fig. 1A (c) and Fig. 1B (c), the samples in group Q are all overlapped, and the correlation is better, while the Z group is mostly separated, and the correlation is not so good. There is no crossover between the Z group and the Q group, which shows that the difference between the two groups is relatively large, indicating that the metabolites between Artemisia wellbyi and Artemisia annua have a tendency to separate, and there are differences between groups.

Discriminant analysis of partial least squares (PLS-DA)
In the group (a) of Fig. 2A and Fig. 2B, the D group and the Q group are clearly separated, which shows that the metabolites between Artemisia sieversiana and Artemisia annua have a tendency to separate, which can explain the difference between the groups of Artemisia sieversiana and Artemisia annua is very large. In groups (b) of Fig. 2A and Fig. 2B, there is a clear separation between groups Z and D, demonstrating a trend towards separation of metabolites between Artemisia wellbyi and Artemisia sieversiana the inter-group differences between Artemisia wellbyi and Artemisia sieversiana are very large. The clear separation between groups Z and Q in groups (c) of Fig. 2A and Fig. 2B demonstrates the tendency for metabolites to segregate between Artemisia wellbyi and Artemisia annua and the inter-group differences between Artemisia wellbyi and Artemisia annua are observable.

Differential metabolites analysis
The Variable Importance in the Projection (VIP) value of the first principal component of the PLS-DA model was used. The VIP value represents the contribution rate of the metabolite difference in different groups; the difference multiple (Fold Change, FC) represents each metabolism. The ratio of the mean value of the repeated quantitative values of all metabolites in the comparison group; combined with the p value of t-test to find the differentially expressed metabolites, set the threshold value to VIP > 1.0, the multiple of difference FC > 1.2 or FC < 0.833 and p-value < 0.05, and the selected different metabolites are shown in Table 1. The information of the different metabolites selected from the 3 species of Artemisia plants is in Additional file 2. Scopoletin was a representative differential metabolite in Artemisia sieversiana and Biochanin A was a representative differential metabolite in Artemisia wellbyi.
Comparing group D with group Q, in the positive ion mode, a total of 535 metabolites are identified. Among the 535 metabolites, 262 are different. That is, there are 262 differential metabolites between Artemisia sieversiana and Artemisia annua. Total 149 of differential metabolites are up-regulated among the 262 differentially regulated metabolites. In the negative ion mode, a total of 220 metabolites are identified, and 106 of these 220 metabolites are different. Total 68 of differential metabolites are up-regulated among the 106 differentially regulated metabolites. Comparing group Z with group D, in the positive ion mode, a total of 535 metabolites are identified, and 312 of these 535 metabolites are different. That is, there are 312 differences between Artemisia wellbyi and Artemisia sieversiana metabolites, of which the total number of differential metabolites that are up-regulated is 163, and the total number of differential metabolites that are down-regulated is 149; in the negative ion mode, a total of 220 metabolites are identified, and 131 of these 220 metabolites are different, that is, there were 131 differential metabolites screened between Artemisia wellbyi and Artemisia sieversiana. The total number of differential metabolites was 69 up-regulated and 62 were down-regulated.
Comparing group Z with group Q, in the positive ion mode, a total of 535 metabolites are identified. Among these 535 metabolites, 306 are different. That is, there are 306 differential metabolites between Artemisia wellbyi the scores of the first and second ranked principal components respectively, the different coloured scatter points indicate samples from different experimental subgroups, and the ellipses are 95% confidence intervals (95% confidence ellipses cannot be shown when the number of biological replicates is less than 4). ("D" refer to Artemisia sieversiana. "Q" refer to Artemisia annua. "Z" refer to Artemisia wellbyi) and Artemisia annua. Among them, the total number of differential metabolites that are up-regulated is 148, and the total number of differential metabolites that are down-regulated is 158; in negative ion mode, a total of 220 metabolites are identified, and 133 of these 220 metabolites are different, namely Artemisia wellbyi. A total of 133 differential metabolites were screened from Artemisia annua, of which 59 were up-regulated and 74 were down-regulated.
Comparing the difference folds of the different metabolites in the samples of Artemisia sieversiana and Artemisia annua, as shown in Table 2 are the top 20 differentially expressed metabolic components in  Differentially present metabolites in the samples of Artemisia sieversiana and Artemisia wellbyi were compared, and the top 20 differential metabolites in terms of levels of presence are shown in Table 3. Compared to Artemisia sieversiana, Artemisia wellbyi showed a higher levels of 1-Naphthylamine, Isodehydrocostus lactone, Anastrozole, Pseudoivalin, Etodolac, Prostaglandin I2, Baicalin,Cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), Quercetin, Cyanidin 3-glucoside, Biochanin A, Telmisartan were different in content.

Volcano map of differential metabolites
The volcano chart can visually display the overall distribution of different metabolites, and the results are shown in Fig. 3. Figure 3A and 3B visually show the significantly different metabolites between the three Artemisia plants. The overall and visual display of the specific metabolites of each group and their differences can be used as a functional analysis of metabolic pathways. As shown in the Fig. 3, red is up-regulated, green is down-regulated, and gray is not occurring, that is, the metabolites is no difference.

Cluster analysis of differential metabolites
A hierarchical clustering analysis is performed on all the difference metabolites between the obtained comparison pairs, and the relative quantitative values of the difference metabolites are normalized and converted and clustered. As shown in Fig. 4.
Different colored areas in the figure represent differently clustered groups.Metabolites with similar expression patterns in the same group will be clustered together suggesting similar or identical biological processes. It can be seen intuitively from the positive ion pattern that the upper part of the Z group is red, and the upper part of the D and Q groups are blue, indicating that there are many different metabolites in the Artemisia wellbyi group that   are highly expressed, while the expression levels in Artemisia annua and Artemisia sieversiana group are relatively low.

Wayne analysis of different metabolites
In the positive ion mode, the number of different metabolites of different species identified by the multivariate statistical method is 125 (Fig. 5)  Positive ion mode (a) D vs Q differential metabolite volcano (b) Z vs D differential metabolite volcano (c) Z vs Q differential metabolite volcano diagram. B: Negative ion mode (a) D vs Q differential metabolite volcano diagram (b) Z vs D differential metabolite volcano diagram (c) Z vs Q differential metabolite volcano diagram. Negative ion mode (a) D vs Q differential metabolite volcano diagram (b) Z vs D differential metabolite volcano diagram (c) Z vs Q differential metabolite volcano diagram. ("D" refer to Artemisia sieversiana. "Q" refer to Artemisia annua. "Z" refer to Artemisia wellbyi) Fig. 4 Cluster Analysis of Differential Metabolites. Clustering heat map of total differential metabolites (the upper frame is the grouped heat map, the lower frame is the sample heat map; each frame is the first picture is the positive ion mode, the second picture is the negative ion mode). The vertical direction is the clustering of samples, and the horizontal direction is the clustering of metabolites. The shorter the cluster branches, the higher the similarity. The relationship between the clustering of metabolite content between groups and samples can be seen through horizontal comparison. ("D" refer to Artemisia sieversiana. "Q" refer to Artemisia annua. "Z" refer to Artemisia wellbyi)

KEGG pathway analysis of the metabolites
All the information on the metabolic pathways enriched by the differential metabolites detected in the 3 species of Artemisia is listed in Additional file 3. The significance analysis of KEGG can determine the main biological functions performed by the different metabolites. KEGG Pathway enrichment results of different metabolites are shown in Additional file 3. In the positive ion mode, 548 differential metabolites of D vs Q are annotated into metabolic pathways, 741 differential metabolites of Z vs D are annotated into metabolic pathways, and 631 differential metabolites of Z vs Q are annotated into metabolic pathways. In the metabolic pathway, the analysis showed that some metabolites can participate in multiple metabolic pathways, and multiple metabolic pathways are consistent among the comparison groups. In the negative ion mode, D vs Q has 392 differential metabolites annotated into the metabolic pathway, Z vs D has 532 differential metabolites annotated into the metabolic pathway, and Z vs Q has 510 differential metabolites are annotated into the metabolic pathway. The analysis showed that some metabolites can participate in multiple metabolic pathways, and multiple metabolic pathways are consistent among the comparison groups.

KEGG enrichment bubble chart
The enriched differentially expressed metabolites in KEGG pathway analysis also presented in bubble chart (only the results of top 20) are shown in Fig. 6A, 6B  (Fig. 6).
The differential metabolites of Artemisia sieversiana and Artemisia annua (D vs Q) are enriched in the Linoleic acid metabolism, Monoterpenoid biosynthesis and Lysine biosynthesis pathway. The differential metabolites of Artemisia wellbyi and Artemisia sieversiana (Z vs D) are enriched in the Styrene degradation and Isoquinoline alkaloid biosynthesis pathway. The differential metabolites of Artemisia wellbyi and Artemisia annua (Z vs Q) are enriched in the Styrene degradation and Monoterpenoid biosynthesis pathway.
The significant enrichment of these three species of Artemisia on these pathways is helpful to understand the metabolic pathways of Artemisia plants and their intermediate metabolites, which lays the foundation for their biological research.

Artemisinin content of three Artemisia plants
Based on the detection results of non-targeted metabolomics, we detected artemisinin from three different Artemisia plants, and through screening, we found that artemisinin is an important differential metabolite. Figure 7A is the secondary spectrum of artemisinin obtained from three Artemisia plants in non-targeted metabolomics.
Having found that artemisinin is an important differential metabolite, we then used high-phase liquid chromatography combined with mass spectrometry to target the artemisinin content in these three Artemisia plants. The standard curve was drawn according to the calculated regression equation: Y = 500.74237X + 1551.22512 (R = 0.99980). The concentration of artemisinin in Artemisia sieversiana is 3.545 ± 1.202 × 10 5 µɡ/ɡ. The concentration of artemisinin in Artemisia wellbyi is 4.799 ± 2.544 × 10 5 µɡ/ɡ. The concentration of artemisinin in Artemisia annua is 5.713 ± 0.385 × 10 7 µɡ/ɡ. Compared with Artemisia annua, the content of artemisinin in Artemisia wellbyi and Artemisia sieversiana was lower than that in Artemisia annua (Fig. 7B).

Discussion
In this study, the metabolomics of three representative species of Artemisia in blooming stage in Tibet were analyzed by metabolomic technology. The results of metabolite analysis showed that all three Artemisia plants contained fatty acids, glycerophospholipids, amino acids, sugars, nucleotides, phenolamines, organic acids, coumarins, catechins, vitamins, indole, and hydroxycinnamic acid. The metabolites of Artemisia annua are significantly different. Daidzin has a unique effect on breast cancer [12], prostate cancer [13], heart disease [14], cardiovascular disease [15] and other diseases [16]. Scopoletin has been shown to have anti-inflammatory effects [17], anti-tumor effects as well as analgesic effects [18][19][20][21][22]. We study the differential metabolites in Artemisia wellbyi. Quercetin has been found to have multiple biological activities, such as antioxidant [21], antiviral [22], and anti-inflammatory effects [23,24]. Baicalin has significant biological activity. It has antibacterial, diuretic, anti-inflammatory, cholesterol-lowering, anti-thrombosis, relief of asthma, detoxification, and hemostasis [25,26]. The pharmacological effects of these important metabolites are consistent with those recorded in the published literature [27,28].
We use LC-MS to target detection of artemisinin content in 3 species of Artemisia plants. This study found that artemisinin is present in the three representative Artemisia plants, Artemisia sieversiana, Artemisia wellbyi and Artemisia annua, collected from Tibet. Artemisia annua contains the highest concentration of artemisinin, with an average value of 57,130 µɡ/ɡ, the second is Artemisia wellbyi with an artemisinin content of 479.93 µɡ/ɡ, the last is Artemisia sieversiana, its content is 354.47 µɡ/ɡ. Xiang et al. [29] established a quick and easy UPLC-UV method for the detection of artemisinin, and tested the content of artemisinin in Artemisia annua from different producing areas, and found that the artemisinin content of Artemisia annua from Chongqing City was as high as 10,000.4 µɡ/ɡ. Cheng et al. [30] used UPLC-MS/MS detection to compare the artemisinin content of Artemisia annua from different sources, the results found that the origin of Artemisia annua with higher artemisinin content was Yunnan province, and the content was 3810.597 µɡ/ɡ, followed by Hainan province, with an average of 3702.952 µɡ/ɡ. By comparison, it is found that the artemisinin content of Artemisia annua in Tibet is the highest compared to other provinces, which indicates that as a traditional Tibetan plant of the genus Artemisia, Artemisia annua has properties of antibacterial, antitumor, antiviral, anti-inflammatory and these pharmacological properties may have important potential medicinal value.
Tibetan medicine is used for anti-inflammatory, visceral bleeding and so on [31]. Artemisia sieversiana is also a traditional herbal medicine used by Tibetan and Mongolian medicine. It mainly contains chemical components such as flavonoids, lignins, sesquiterpenes and volatile oils. The medicinal work "Compendium of Materia Medica" mentioned Artemisia selengensis and the x/y (the number of differential metabolites in the corresponding metabolic pathway/the total number of metabolites identified in the pathway). The larger the value, the higher the enrichment of differential metabolites in the pathway. The color of the dot represents the p-value of the hypergeometric test. The smaller the value, the greater the reliability of the test and the more statistically significant. The size of the dot represents the number of different metabolites in the corresponding pathway. The larger the dot, the more differential metabolites in the pathway. (If there is no enrichment result, there is no picture). ("D" refer to Artemisia sieversiana. "Q" refer to Artemisia annua. "Z" refer to Artemisia wellbyi) the dominant plants in Tibet's alpine desert grasslands and are also potentially important forage and medicinal plant resources. Moreover, they still play an important role in the ecological protection and economic development of Tibet's grassland. As a plant with both medicinal and edible value, Artemisia can also be developed as a functional food at the same time as a high-quality feed for livestock to improve vitality and disease resistance. In future, it is necessary to study the transcriptomics of the genes in these plants to understand their regulation in the synthesis of artemisinin in the three Artemisia plants and to transform them by genetic engineering technology to obtain high-yield artemisinin varieties, which can effectively solve the shortage of artemisinin sources.

Conclusions
This study is based on LC-MS/MS technology to qualitatively determine the differential metabolites of 3 species of Artemisia in Tibet. The types of differential metabolites screened out are mainly organic acids and their derivatives, ketones, phenols, alcohols and coumarins. Among them, artemisinin, as a representative differential metabolite, has the highest relative content in Artemisia annua. The content is 5.713 ± 0.385 × 10 7 µɡ/ɡ. The key metabolic pathways involved in the different metabolites analyzed by KEGG enrichment are Linoleic acid metabolism, Monoterpenoid biosynthesis and Isoquinoline alkaloid biosynthesis. This study profiled the differential metabolites of the three Artemisia plants in Tibet, provided new evidence for their medicinal research, and opened up new ideas for the comprehensive development and utilization of Artemisia plants in Tibet.

Plant material
Artemisia sieversiana, Artemisia wellbyi and Artemisia annua were collected in Jinbei, Caina Township, Qushui County, Lhasa City, Tibet Autonomous Region in July 2019. The wild samples in this experiment was permitted by Lhasa Forestry and Grassland Administration. Permission was not necessary for collecting these species, which have not been included in the list of national key protected plants. Te formal identifcation of the plant material was undertaken by Professor Zhaoyang Chang, College of Life Science, Northwest A&F university. The voucher specimens of Artemisia sieversiana, Artemisia wellbyi and Artemisia annua were deposited at Herbarium, Institute of Botany, Chinese Academy of Sciences (voucher number PE01890226,PE01890481 ,PE01997408). These plants were taken from each sampling site with a size of 10 m × 10 m, and 9 plants were sampled along the diagonal, for a total of 27 samples. All samples were dried, crushed, passed through a 40-mesh sieve (with an aperture of 0.425 mm), put into a paper bag, and stored in a desiccator at room temperature for later use. One g each of 27 samples were wrapped in tin foil, snap frozen in liquid nitrogen for storage, transported in dry ice to Beijing Tiangen Technology Co., Ltd. for analysis.

Experimental sample
The 27 plant samples were divided into 3 groups according to 3 different kinds of Artemisia plants, the first group "Artemisia sieversiana", was indicated by the letter "D"; the second group "Artemisia annua", was indicated by the letter "Q"; and the third group "Artemisia wellbyi", was marked by the letter "Z". The comparisons between the samples in the group are respectively denoted as D vs Q, Z vs Q, Z vs D, where D vs Q represents the metabolite comparison between "Artemisia sieversiana" and "Artemisia annua". There were 9 samples in each group, and 3 biological replicate experiments were performed respectively. Quality control samples (QC) were prepared by mixing equal amounts of three Artemisia extracts in three replicates and were treated and tested in the same way as the analytical samples, with one QC sample inserted in every 10 analytical samples tested during instrumental testing to investigate the stability and reproducibility of the entire analytical process.

Metabolite extraction
A 100 mg of liquid nitrogen ground tissue sample was placed in an EP tube, 500 μL of 80% methanol aqueous solution containing 0.1% formic acid was added, vortexed, left to stand in an ice bath for 5 min, and then centrifuged at 15,000 rpm, at 4 °C for 10 min. The supernatant (100µL) was diluted with mass spectrometry grade water to 53% methanol, and placed in a centrifuge tube at 15,000 g, 4 °C for 10 min. The supernatant was collected and injected into LC-MS for analysis. An equal volume of each sample was mixed as QC samples. The blank sample was replaced by aqueous 53% methanol solution containing 0.1% formic acid. The pretreatment process is the same as that of the experimental sample.