Abstract

Background

   Dendrobium nobile and Dendrobium chrysotoxum are important species of
   the genus Dendrobium and have great economic and medicinal value.
   However, the medicinal properties of these two plants remain poorly
   understood. This study aimed to investigate the medical properties of
   D. nobile and D. chrysotoxum by conducting a comprehensive chemical
   profiling of the two plants. Additionally, active compounds and
   predictive targets for anti-hepatoma activity in D. chrysotoxum
   extracts were identified using Network Pharmacology.

Results

   Chemical profiling showed that altogether 65 phytochemicals were
   identified from D. nobile and D. chrysotoxum, with major classes as
   alkaloids, terpenoids, flavonoids, bibenzyls and phenanthrenes. About
   18 compounds were identified as the important differential metabolites
   in D. nobile and D. chrysotoxum. Furtherly, CCK-8 results showed that
   the extracts of stems and leaves of D. nobile and D. chrysotoxum could
   inhibit the growth of Huh-7 cells, and the anti-hepatoma activity of
   extracts were dose-dependent. Among the extracts, the extract of D.
   chrysotoxum showed significant anti-hepatoma activity. In order to find
   the potential mechanism of anti-hepatoma activity of D. chrysotoxum,
   five key compounds and nine key targets were obtained through
   constructing and analyzing the compound-target-pathway network. The
   five key compounds were chrysotobibenzyl, chrysotoxin, moscatilin,
   gigantol and chrysotoxene. Nine key targets, including GAPDH, EGFR,
   ESR1, HRAS, SRC, CCND1, HIF1A, ERBB2 and MTOR, could be considered as
   the core targets of the anti-hepatoma activity of D. chrysotoxum.

Conclusions

   In this study, the chemical composition difference and anti-hepatoma
   activity of stems and leaves of D. nobile and D. chrysotoxum were
   compared, and the potential anti-hepatoma mechanism of D. chrysotoxum
   was revealed in a multi-target and multi-pathway manner.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12906-023-04048-y.

   Keywords: D. nobile, D. chrysotoxum, Metabolomics, Network
   pharmacology, Hepatoma, Biomarkers

Introduction

   Hepatoma is the major cause of cancer associated mortality. In fact,
   since cancer treatment is often accompanied by side effects, there is a
   growing demand for innocuous and more effective anticancer drugs
   [[43]1]. Phytochemicals isolated from medicinal plants have been
   largely neglected in this context, although their pharmacological
   activities have been well investigated in the past, and they may have
   considerable medicinal potential [[44]2]. In particular, medicinal
   plants could be and more effective in the treatment of various
   diseases, including cancer [[45]3]. Various biomolecules presented in
   the plant extract such as alkaloids, terpenoids, polyphenols,
   polysaccharides, flavonoids, tannins, saponins, phenolics, amino acids,
   and proteins are natural sources of therapeutic drugs [[46]4–[47]8].

   With approximately 1,400 native species, Dendrobium is one of the
   largest families of orchids and is widely distributed around the world
   [[48]9]. Recent pharmacological studies have shown that Dendrobium has
   a wealth of medicinal effects, such as hepatoprotective,
   anti-inflammatory, anti-angiogenic and anti-oxidative properties
   [[49]9, [50]10]. The stems of D. nobile and D. chrysotoxum have great
   economic and medicinal value and are listed in the Chinese
   Pharmacopoeia of 2020 (Fig. [51]1A, B and C) [[52]11]. The chemical
   constituents of D. nobile and D. chrysotoxum are mainly alkaloids,
   terpenoids, polyphenols, flavonoids, phenanthrenes, bibenzyls and amino
   acids [[53]12–[54]14]. Recent pharmacological studies have shown that
   components of the stems of Dendrobium, possess a broad range of
   activities, encompassing hepatoprotective, anti-proliferative activity
   toward cancer cells, immunostimulating, anti-diabetic activity,
   cataractogenesis-inhibiting activity, neuroprotective activity,
   anti-inflammatory activity, anti-platelet aggregation activity and
   hemagglutininating activities and also exert beneficial actions on
   colonic health and alleviate symptoms of hyperthyroidism
   [[55]15–[56]17]. Because only the stems of D. nobile and D. chrysotoxum
   are permitted for use according to the Chinese Pharmacopoeia, their
   leaves are largely discarded [[57]18]. However, the leaves of these
   Dendrobium plants possess chemical constituents similar to those found
   in the stems [[58]19–[59]21].

Fig. 1.

   [60]Fig. 1
   [61]Open in a new tab

   Plants and metabolites. (A, B, C) Dendrobium species planting base in
   Wenshan, Yunnan

   Plant extracts act on certain molecular targets due to the synergistic
   effects of their chemical compounds and the fact that they could
   interact with many targets simultaneously [[62]3, [63]22]. In recent
   years, network pharmacological analysis has been effectively applied to
   predict the relationship between protein targets, active compounds and
   related diseases. This approach attempts to explore disease-related,
   multiple active compounds and targets, thus allowing to characterize
   the multi-target mechanism of bioactive compounds [[64]23, [65]24].
   This analysis can provide insights into the link between Traditional
   Chinese Medicine (TCM) bioactive compounds and diseases, possibly
   revealing the pharmacological properties of multiple biomolecules found
   in medicinal plants [[66]25].

   Screening and identification of active components from TCM is rather
   challenging due to the diversity and complexity of chemical components.
   Herein, comprehensive metabolite profiling of the D. nobile and D.
   chrysotoxum were conducted by LC-MS using an untargeted metabolomics
   approach. The anti-hepatoma activities of the extracts from the stems
   and leaves of D. Nobile and D. chrysotoxum were compared by CCK-8
   assay. A network pharmacology approach was applied to characterize the
   possible mechanism of action of D. chrysotoxum compounds on the human
   hepatoma Huh-7 cell line. Active compounds and potential targets of D.
   chrysotoxum, as well as related genes of hepatoma were obtained from
   the public databases, while the potential targets and signalling
   pathways were determined by protein-protein interaction (PPI), gene
   ontology (GO) and pathway enrichment analyses. Ultimately, the
   compound-target and target-pathway networks were constructed. This
   study compared the chemical composition and anti-hepatoma activity of
   D. nobile and D. chrysotoxum extracts, and revealed the potential
   anti-hepatoma action mechanism of D. chrysotoxum using a multi-target
   and multi-pathway approach.

Materials and methods

Reagents and materials

   Methanol (MeOH), acetonitrile and ethanol were obtained from Thermo
   Fisher Scientific (Waltham, MA, USA). 5-Fluorouracil was bought from
   Macklin Biochemica Co., Ltd.(Shanghai, China). CCK-8 kit was bought
   from Beyotime Biotechnology(Shanghai, China); Phosphate Buffered Saline
   (PBS) was bought from Biological Industries (The State of Israel); DMEM
   medium; Fetal bovine serum (FBS); Antibiotics; Trypsin were bought from
   Thermo Fisher Scientific (Massachusetts, US).

   D. nobile and D. chrysotoxum were harvested from Wenshan, Yunnan
   Province, China. The experiment was conducted using at 3-year-old
   wild-cultured D. nobile and D. chrysotoxum, which were collected in
   February.

   Human hepatocellular carcinoma (Huh7 cell line) was purchased from
   Hangzhou Qiannuo Biotechnology Co., Ltd (Yuanlong Commercial Building,
   Baiyang Street, Qiantang District, Hangzhou, China). Normal human
   hepatocytes (Lo2 cells line) was purchased from Shanghai Cell Bank,
   Chinese Academy of Sciences (No. 320 Yueyang Road, Xuhui District,
   Shanghai, China). ( Note:We used only Huh-7 and Lo2 (normal human
   hepatocytes) cell lines in our experiments, and no other human samples
   were used.)

Extraction procedure

   The stems and leaves of the D. nobile and D. chrysotoxum were cut into
   small segments completely dried at 50–60 ℃. Then crushed by a
   pulverizer and put into a sealed bag for storage. 10 g of crushed stems
   and leaves were extracted with 500 mL of 70% methanol aqueous solution
   with ultrasonic for 45 min respectively, the filtrate was collected,
   concentrated by a rotary evaporator, and freeze-dried [[67]26].

Liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis for
metabolite identification

Sample preparation for metabolomics

   The tissue samples were dried at 55℃ and ground into a fine powder.
   Approximately 0.01 g of root was homogenized with 10 mL of MeOH/H[2]O
   (70:30, v/v), centrifuged for 5 min and subjected to ultrasonic
   treatment for 20 min. The sample was then centrifuged at 10,000 rpm for
   10 min, and the resulting supernatant solution was passed through a
   0.22-µm pore-size membrane. The filtrate was transferred to sample
   vials for LC-MS/MS analysis [[68]26].

UPLC-Q-TOF-MS/MS analysis method

   Chromatographic separation was achieved using an Waters ACQUITY UPLC
   HSS T3 column (1.8 μm, 2.1 mm × 100.0 mm; Waters Corporation, Milford,
   MA, USA). The column temperature and flow rate were set at 40℃ and 0.4
   mL·min^− 1, respectively. The injection volume and detection wavelength
   of the chromatographic column were set at 1 µL and 254 nm,
   respectively. The mobile phase was composed of water (A) and
   acetonitrile (B), with a gradient elution program of 4% B (0–1 min),
   4–50% B (1–7 min), 50–70% B (7–8 min), 70–95% B (8–12 min), 95% B
   (12–15 min), 95 − 4% B (15–16 min) and 4% B (16–18 min).

   Mass spectrometric detection was performed in electrospray mode using a
   Xevo G2-XS Q-Tof mass spectrometer detector (Waters Corporation,
   Milford, MA, USA). Argon was used as the desolvation and collision gas.
   The full-scan data range was 50 − 1,200 Da, the source temperature was
   100 °C, the desolvation temperature was 400 °C and the scanning
   frequency was 1.000 s. The locking spray standard was 400 mg·mL^− 1
   with a collision energy of 6.000 V for the low collision energy scan
   and 30 to 70 V for the high collision energy scan of the mass
   spectrometer, and the UPLC system was controlled by MassLynx4.1
   software (Waters Corporation, Milford, MA, USA).

CCK-8 assay of plant extracts and cytotoxicity tests

   Human hepatoma Huh-7 cells were maintained routinely in DMEM
   supplemented with 10% FBS and 1% antibiotics in a humidified atmosphere
   of 5% CO[2]/50% air [[69]27]. The anti-hepatoma activity of extracts
   from the stems and leaves of the D. nobile and D. chrysotoxum were
   determined by CCK-8 assay. 100 µL cell suspension were cultured in a
   96-well plate for 24 h, then the medium was removed, and the cells were
   incubated with different concentrations of the extracts, the extracts
   were dissolved in DMEM medium containing 10% FBS and 1% antibiotics for
   48 h. 10 µL CCK-8 solution was added to each well for 2 h, the
   absorbance was measured at 450 nm using a microplate reader. The
   absorbance of blank group without cells was Abs blank. 5-Fluorouracil
   was used as a positive control [[70]28]. The cell inhibition rate of
   extract solution was calculated according to the following formula.
   graphic file with name M1.gif

   To evaluate the effect of D. nobile and D. chrysotoxum on the viability
   of Lo2 cells (normal human hepatocytes), cytotoxicity assays were
   performed using the Cell Counting Kit-8. We used the method of Xia et
   al. as a reference for experimental design, briefly, cells in the
   logarithmic growth phase were subjected to the cell passage cultivation
   and finally 100 µL of cell suspension (cell density of 1 × 10 ^5 /mL)
   was added to each well of the 96-well plate. The blank control was
   cell-free cell culture medium. The final concentrations of the
   experimental group were 0, 50, 100, 150, 200, 250, 500 µg/mL, and 6
   replicate wells were added for each concentration, and each group was
   repeated 3 times. For the control group, only 100 µL of cell culture
   medium was added. After incubation for 24 h, 10 µL of CCK-8 was added
   to each well and incubated for 2 h. The absorbance (OD) was measured at
   450 nm on an enzyme marker [[71]29].
   graphic file with name M2.gif

Network pharmacology analysis

Database construction and prediction of potential targets

   The SIMILES and InChIKey of the chemical components of aerial part of
   D. chrysotoxum were collected from the PubChem database. The GI
   absorption and bioavailability score of the compounds were calculated
   by SwissADME, and the compounds with GI absorption were high and
   bioavailability score > 0.3 were screened. The Swiss Target Prediction
   database was used to obtain the target corresponding of the components
   [[72]30]. Use “Liver cancer” as the key words, the liver cancer related
   genes were screened out by DisGeNET [[73]31], GeneCards [[74]32], OMIM
   [[75]33]. The obtained targets of the constituents and the disease
   targets of liver cancer were unified as UniProt ID through the UniProt
   database [[76]34].

Network construction

   Cytoscape software was used to construct the “drug components-target”
   network to explain the interactions between the core chemical
   components of D. chrysotoxum and potential targets in liver cancer
   therapy. In order to better analyze the protein-protein interaction,
   the PPI network of potential targets was constructed by using the
   STRING database using the common target of the liver cancer related
   genes and target corresponding of the components, the species was
   defined as Homo sapiens [[77]35]. The “drug components-target” network
   and PPI network was imported into Cytoscape software for topological
   attribute analysis, and the key nodes and degree values in the network
   map were analyzed to obtain the core components and targets of D.
   chrysotoxum.

Biological function and pathway analysis

   In order to illustrate the role of the core target in the gene function
   and signal pathway of the active ingredient in the treatment of liver
   cancer. David database was used to perform gene ontology (GO) function
   and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
   analysis to explore the mechanism of anti-hepatoma activity of D.
   chrysotoxum [[78]36–[79]39].

Data analysis

   We used the method of Xia et al. as a reference for data analysis, mass
   spectrometry data were collected using MassLynx V4.2 software (Waters
   Corporation, Milford, MA, USA) for automatic peak identification, peak
   matching, peak alignment, peak extraction, peak integration and
   normalization. The metabolites and their possible cleavage modes were
   identified using secondary mass spectrometry data, Unifi (Waters
   Corporation, Milford, MA, USA), online databases (SciFinder, Chemspider
   and PubMed) and data from previous studies. Unsupervised principal
   component analysis (PCA) and supervised orthogonal partial least
   squares discriminant analysis (OPLS-DA) were performed using SIMCA-P
   14.1 (Umetrics Corporation, Umea, Sweden) software [[80]29].

Results

Overview of the metabolomes of different tissues

   Comprehensive metabolite profiling of the D. nobile and D. chrysotoxum
   was conducted by LC-MS using an untargeted metabolomics approach. By
   observing the trend of ion peaks in BPI and the clustering of
   metabolite species between samples, it was indicated that there was a
   high similarity in the accumulation of metabolites between different
   tissues of the same Dendrobium species (Fig. [81]2A and B). In order to
   analyze the differences of accumulated metabolites between different
   samples, the identified metabolite types were assigned to different
   samples (Fig. [82]2B). In D. nobile, the major metabolite types were
   alkaloids (21 metabolites), terpenoids (8 metabolites), flavonoids (3
   metabolites) and phenanthrenes (3 metabolites); in D. chrysotoxum, the
   largest number of metabolite types belonged to bibenzyls (4
   metabolites), phenanthrenes (3 metabolites) and phenolic acids (2
   metabolites) (Figure [83]S1). Notably, As for the metabolites
   specifically enriched in D. nobile, alkaloids were abundant in stems
   but also were observed in leaves (Fig. [84]2B). In both plants, the
   predominant differential metabolites were alkaloids and bibenzyls.

Fig. 2.

   [85]Fig. 2
   [86]Open in a new tab

   Differentially accumulated metabolites among the four tissues of two
   Dendrobium species. (A)The base peak ion chromatogram (BPI) of
   different samples; (B) A heatmap of the abundance of metabolites in the
   four tissues of two Dendrobium species

Metabolomic multivariate statistical analysis of D. nobile and D. chrysotoxum

Metabolic profiling of D. nobile and D. chrysotoxum

   Using our in-house and public databases, we tentatively identified 65
   metabolites of negative and positive ion modes, and thus gave a
   comprehensive metabolome for the stems and leaves of D. nobile and D.
   chrysotoxum (Tables [87]S1, [88]S2, [89]S3 and [90]S4). The majority of
   metabolite types were alkaloids, terpenoids, bibenzyls, phenanthrenes,
   phenolic acids, flavonoids, carboxylic acids. Figure [91]3A presents
   two heat-maps corresponding to the relative contents of different
   metabolites in stems and leaves of different Dendrobium species. As
   shown in Fig. [92]3A, alkaloids had significantly greater metabolite
   diversity, especially in stems and leaves of D. nobile, such as
   dendrobine, N-isopentenyl-dendrobinium, N-isopentenyl-dendroxinium,
   N-methyldendrobinium, homocrepidine B and dendroxine. Different content
   of phenanthrene, as the main metabolite, was detected in the stems of
   the two Dendrobium species. Chemometric analysis was applied to
   determine metabolites abundance of D. nobile and D. chrysotoxum. The
   results of PCA and OPLS-DA analysis showed that D. nobile and D.
   chrysotoxum species were clearly separated, with significant
   differences in chemical profiles between the two species. In addition,
   the metabolites in the stems and leaves of D. nobile differed more
   markedly (Fig. [93]3B-C). The VIP value ≥ 1.0 derived from the OPLS-DA
   analysis revealed the differences in metabolite profiles between D.
   nobile and D. chrysotoxum (Fig. [94]4). Notably, 18 compounds were
   identified as important differential metabolites between D. nobile and
   D. chrysotoxum, including alkaloids, phenanthrenes and terpenoids.

Fig. 3.

   [95]Fig. 3
   [96]Open in a new tab

   The results of non-targeted metabolomics and chemometric analysis of D.
   nobile and D. chrysotoxum. (A) Heat map of metabolomics and
   chemometrics clustering of D. nobile and D. chrysotoxum; (B) Principal
   component analysis of D. nobile and D. chrysotoxum; (C) OPLS-DA
   analysis of D. nobile and D. chrysotoxum

   (D. nobile-S: D. nobile stem;D. nobile-L: D. nobile leaf; D.
   chrysotoxum-S: D. chrysotoxum stem; D. chrysotoxum-L: D. chrysotoxum
   leaf; No.1–5: Number of repetitions)

Fig. 4.

   [97]Fig. 4
   [98]Open in a new tab

   The values of VIP of the D. nobile and D. chrysotoxum

Evaluations of in vitro anti-hepatoma activity

   CCK-8 results showed that all four extracts could inhibit the growth of
   Huh-7 cells, and the anti-hepatoma activity of extracts were
   dose-dependent (Figure [99]S2). According to the Table [100]1, among
   the extracts, the extract of leaves of D. chrysotoxum showed the
   strongest anti-hepatoma activity, while the stems extract of D. nobile
   showed the weakest anti-hepatoma activity. In addition, the inhibitory
   activity of ethanolic extract of D. chrysanthum leaves against HeLa
   human cervical cancer cells was evaluated by in vitro and in vivo
   assays with an IC[50] value of 450 µg/mL [[101]18, [102]40], and this
   result was in excellent agreement with our experimental data. As shown
   in Fig. [103]S3, the Lo2 cells (normal human hepatocytes) survival rate
   was above 90% at all concentration gradients of Dendrobium samples. The
   results showed that all Dendrobium extracts with the same concentration
   are nontoxicity to normal cells.

Table 1.

   Anti-hepatoma activity of four extracts determined by CCK-8 (Each lower
   case indicates significant differences(P < 0.05))
   Sample              Inline graphic
                       Anti-hepatoma activity IC[50](µg/mL)
   5-Fluorouracil      30.04 ± 1.16^a
   D. Nobile-Stem      1315.67 ± 37.50^e
   D. Nobile-Leaf      815.00 ± 70.73^c
   D. chrysotoxum-Stem 1051.67 ± 48.09^d
   D. chrysotoxum-Leaf 493.50 ± 28.56^b
   [104]Open in a new tab

Network pharmacology analysis

PPI network analysis

   Because the stems and leaves of D. chrysotoxum have greater
   anti-hepatoma activity than that observed in D. nobile, the former
   species was selected. About 1526 liver cancer related targets were
   obtained by DisGeNET, GeneCards and OMIM. Finally, 112 potential
   targets for anti-hepatoma activity of D. chrysotoxum were obtained, the
   PPI network of the 112 potential targets were constructed by using the
   STRING database (Fig. [105]5A). The required interaction score was set
   to 0.400. Protein-protein interaction (PPI) network topological
   features, which have been widely used in bioinformatics to predict
   disease-related gene and drug targets [[106]41]. According to the
   topological attribute analysis of PPI network, GAPDH, EGFR, ESR1, HRAS,
   SRC, CCND1, HIF1A, ERBB2 and MTOR were the protein nodes with high
   degree and could be considered as the core-targets of the anti-hepatoma
   activity of D. chrysotoxum (Fig. [107]5B).

Fig. 5.

   [108]Fig. 5
   [109]Open in a new tab

   Construction of PPI network and identification of core targets. (A) The
   PPI network of potential targets was constructed. The darker the color,
   the higher the degree of the node; (B) According to the degree of the
   nodes, the top 9 targets were selected as the core targets. (C) The top
   12 items in the GO biological process, cellular component and molecular
   function; (D) The top 20 items in Kyoto Encyclopedia of Genes and
   Genomes (KEGG) signaling pathway. (Note: The PPI network contained 112
   nodes, 1192 edges, an average node degree of 21.3. Each node in the
   figure represents a protein, and the connections between the nodes
   represent the interaction between the proteins)

Pathway enrichment analysis

   DAVID is a biological information database that can provide systematic
   and comprehensive biological function annotation information for
   large-scale gene or protein lists, helping users to extract biological
   information from them [[110]42]. Biological function analysis of
   anti-hepatoma activity of chemical components were performed by David
   database. The GO enrichment results for the first 12 terms of
   biological process, cellular component and molecular function are shown
   in Fig. [111]5C. According to statistical analysis, the results of the
   first 20 KEGG pathways were shown in Fig. [112]5D, including prostate
   cancer, pathways in cancer, endocrine resistance, EGFR tyrosine kinase
   inhibitor resistance, central carbon metabolism in cancer, FoxO
   signaling pathway, etc.

Herbs-chemicals-targets-pathways-therapeutic effects network analysis

   22 components were identified by UPLC-Q-TOF-MS, 6 non-target proteins
   were eliminated and 454 targets were obtained by Pubchem, SwissADME and
   Swiss target prediction. Chrysotobibenzyl, chrysotoxin, moscatilin,
   gigantol and chrysotoxene were identified as core components by
   correlation analysis of “drug components-target”.

Discussion

   Plants are unique in the richness and diversity of their secondary
   metabolism, the literatures suggest that the number of metabolites
   produced in the plant kingdom exceeds 200,000 [[113]43]. More and more
   researchers are paying attention to the study of plant secondary
   metabolism and pharmacological efficacy [[114]44–[115]48]. Although
   there are hundreds of millions of phytochemicals, only a small number
   have been isolated and identified from plants [[116]49, [117]50]. The
   advancement of metabolomics in terms of techniques for measuring small
   molecule composition has enabled the rapid assay and quantification of
   numerous the endogenous metabolites of an organism [[118]51]. With the
   optimization of the analytics platform, such as gas or liquid
   chromatography mass spectrometry (GC-MS and LC-MS, respectively) and
   nuclear magnetic resonance spectroscopy (NMR), have enabled
   characterizing the dynamic of metabolites [[119]52–[120]54]. Based on
   this, UPLC-Q-TOF-MS was used to identify D. nobile and D. chrysotoxum
   comprehensive metabolome in different tissues. Research shows that the
   levels of compounds from different Dendrobium varied greatly. In D.
   nobile, the major metabolite types were alkaloids, terpenoids,
   flavonoids and phenanthrenes, however, in D. chrysotoxum, the largest
   number of metabolite types belonged to bibenzyls, phenanthrenes and
   phenolic acids, which was consistent with previous studies
   [[121]55–[122]57]. Notably, D. nobile stem is abundant and diversified
   in alkaloids, such as dendrobine, N-isopentenyl-dendrobinium and
   N-methyldendrobinium, in D. chrysotoxum, bibenzyls and phenanthrenes
   secondary metabolites were significantly accumulated, such as
   chrysotobibenzyl, chrysotoxin, moscatilin, and chrysotoxene. Therefore,
   these secondary metabolites can be used as quality markers for the two
   Dendrobium species, providing a reference for species identification
   and quality control of Dendrobium. Furthermore, chemometric analysis of
   the metabolites differences between the two species revealed that they
   are quite different in evolution [[123]58, [124]59]. Therefore,
   integrated metabolomics based on UPLC-Q-TOF-MS and chemometric analysis
   provides new insights for quality-oriented identification of chemical
   profiles of traditional chinese herbal medicines.

   Modern pharmacological studies have shown that D. nobile and D.
   chrysotoxum has had a strong anti-cancer effect [[125]15, [126]60]. For
   example, D. chrysotoxum natural products caused moderate growth delay
   in xenografted human hepatoma Bel7402 and melanoma A375 and induced
   significant vascular shutdown within 4 h of administering 100 mg/kg of
   the drug [[127]61]. In addition, D. nobile extracts down regulated the
   expression level of decoy receptor-3 and synergized with Fas ligand to
   bring about apoptotic cell death in pancreatic adenocarcinoma cells
   [[128]62]. However, the inhibition rates of the two dendrobium species
   on hepatocellular carcinoma cells were different. The study showed that
   the anti-hepatoma effect of D. chrysotoxum was significantly better
   than that of D. nobile, and it was worth noting that D. chrysotoxum
   leaves had the strong anti-hepatoma effect. Due to the wide variation
   in the types and contents of secondary metabolites of different
   Dendrobium species, the differences in their active ingredients may
   lead to the differences in their pharmacological activities.

   The result of KEGG enrichment indicated that the 112 potential targets
   were highly enriched in the EGFR tyrosine kinase inhibitor resistance
   and FoX O signaling pathway. It has been confirmed that EGFR targets
   have a significant impact on the proliferation and migration of liver
   cancer cells [[129]63, [130]64], which is consistent with the results
   of network pharmacological analysis in this study. In addition, FoX O
   signaling pathway related to a variety of tumors. The research of Yang
   et al. and Wang et al. indicated that FOXO1 is weakly expressed in
   liver cancer tissue, which results in abnormal cell proliferation and
   cell apoptosis [[131]65, [132]66]. The influence of D. chrysotoxum on
   hepatocellular carcinoma inhibition may depend on making an impact on
   EGFR tyrosine kinase inhibitor resistance pathway and FoX O signaling
   pathway.

   The stems of D. nobile and D. chrysotoxum had a long history of
   medicinal use, but the leaves are often discarded during the production
   of medicinal materials. And the leaves on the Dendrobium stems are
   sometimes peeled for aesthetic reasons. However, the metabolite
   fingerprint results here indicated that there were still a large number
   of bioactive compounds in the leaves of D. nobile and D. chrysotoxum.
   Previous pharmacological studies also have shown that have also shown
   that Dendrobium leaves play an important role in dermatologic
   disorders, metabolic syndromes, nervous system disorders, and
   musculoskeletal system disorders [[133]18]. Therefore, it is necessary
   to develop an approach for the secondary utilization of leaves.
   Moreover, comprehensive metabolomics results showed that leaves of D.
   nobile and D. chrysotoxum were rich in many bioactive components and
   had excellent pharmacological effects (Fig. [134]3; Table [135]1),
   indicating that the leaves should be preserved as much as possible in
   the process of crude drug production. However, in traditional
   processing of Dendrobiums, the leaves were removed after thoroughly
   washing. Therefore, Dendrobium leaves show potential for the research
   and development of pharmacological biomolecules.

Conclusions

   In this study, mass spectrometry-based metabolomic and multivariate
   statistical analysis were conducted to screen the differential
   metabolites in D. nobile and D. chrysotoxum. We screened differential
   ions in the positive-ion and negative-ion model of UPLC-Q-TOF-MS/MS
   using OPLS-DA and PCA. CCK-8 results showed that D. nobile and D.
   chrysotoxum extracts could inhibit the growth of Huh-7 cells, and the
   anti-hepatoma activity of extracts were dose-dependent. Network
   pharmacology analysis revealed chrysotobibenzyl, chrysotoxin,
   moscatilin, gigantol and chrysotoxene as relevant compounds for D.
   chrysotoxum anti-hepatoma activity. Our research provided a effective
   method for rapid screening and identification of the differential
   metabolites in different Dendrobium species, and provided candidate
   chemical markers for herb quality screening of D. nobile and D.
   chrysotoxum.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [136]Supplementary Material 1^ (1.5MB, doc)
   [137]12906_2023_4048_MOESM2_ESM.docx^ (23.4KB, docx)

   Supplementary Material 2: Review history.

Acknowledgements