Abstract

   graphic file with name ao9b04258_0006.jpg

   Endophytic fungi of medicinal plants have attracted wide attention due
   to their various active biochemical substances that are similar to
   those of the host plants and can be easily fermented and cultured. As a
   traditional medicine and food homologous plant in Xinjiang, Brassica
   rapa L. has a long history of applications. Recently, it has been shown
   that B. rapa L. has hypoglycemic, antimicrobial, immunomodulatory, and
   antioxidant properties. However, there are no studies on the function
   and diversity of enophytic fungi of B. rapa L. Four endophytic fungus
   (pr6, pr7, pr8, and pr10) strains were isolated from B. rapa L. in our
   laboratory. The metabolic extracts from pr10 have significant effects
   in terms of antitumor activity. In this study, in terms of types and
   contents, compared with those of the other three endophytic fungi, the
   dominant metabolites of pr10 were determined by comparative
   metabolomics analysis. The results of metabolomics analysis indicated
   that the metabolites of pr10 are rich in amino acids and sugar
   derivatives such as trehalose, whose ability to inhibit the A549 cell
   line has been proved. This study provides a theoretical basis for the
   development and utilization of B. rapa L. and its endophytic fungi to
   form antitumor agents.

1. Introduction

   As an important part of biological resources and biodiversity in
   nature, endophytic fungi include bacteria, fungi, actinomycetes, and
   algae; are ubiquitous in plants; and spend their whole life or a period
   of life cycle in host plant tissues, which could not cause disease
   symptoms in the host plant tissues.^[38]1,[39]2 Owing to the further
   study of the diversity and active metabolites of the endophytic
   fungi,^[40]3−[41]5 these have attracted global attention. The results
   showed that endophytic fungi have rich biodiversity and can positively
   regulate the growth and development of the host plants.^[42]6 In
   addition, endophytic fungi have important biological functions such as
   promoting the growth of the host plants and the defense ability against
   biotic and abiotic stresses.^[43]1,[44]7 Gond et al. extracted 18 kinds
   of endophytic fungi from the leaves of Nyctanthes arbortristsi, a
   famous medicinal plant in India, and 10 kinds from the stems. They
   conducted mycelium inhibition tests on eight pathogenic bacteria and
   eight pathogenic fungi, respectively, and found that the inhibition
   effect on bacteria was up to 75% and on pathogenic fungi was up to
   56.25%.^[45]1

   Importantly, endophytic fungi can produce the same or similar
   physiologically active biochemical substances, which have insecticidal,
   antimicrobial, antitumor, immunosuppression, antioxidant, and other
   biological activities.^[46]8,[47]9 Interestingly, different from the
   artificial chemical synthesis, such endophytic fungi can synthesize a
   variety of secondary metabolites and are easy to be fermented and
   cultured.^[48]7 Many studies have proved that endophytic fungi and
   their specific metabolites could enhance the defense response to both
   the abiotic and biotic stresses and effectively decrease the survival
   rate of tumor cells.^[49]10−[50]12 Strobel et al. isolated an
   endophytic fungus from the stem of Tripterygium wilfordii Hook.f.,
   which can produce a new cyclopeptide antibiotic with similar chemical
   properties to those of echinomycin. This cyclopeptide compound can
   inhibit human pathogenic fungi such as Candida albicans and Trichoderma
   and can be used in the treatment of fungal nail and skin
   diseases.^[51]11

   As a traditional medicine and food homology plant, Brassica rapa L. has
   a long history of consumption and is favored by Xinjiang Uygur and
   other ethnic minorities. It has been reported that B. rapa L. contains
   a large amount of flavonoids, sugars and glycosides, alkaloids,
   volatile oils, amino acids, and other biochemical components that are
   beneficial for human beings.^[52]13−[53]16 It has nonnegligible and
   significant value in inhibiting the mycelium growth of bacteria and
   fungi. Also, with diverse endogenous metabolites and endophytic fungi,
   such plants could enhance the immunity of human beings in various
   ways.^[54]17−[55]19 However, there are no research studies on the study
   of B. rapa L. enophytic fungi, especially on its unique metabolite
   fingerprint.

   In this study, we isolated and identified the endophytic fungi from B.
   rapa L. and determined their antibacterial and antitumor activities
   with their crude metabolic extracts. Ultimately, the strain pr10, whose
   metabolic extracts have effective antitumor properties, was isolated
   from four endophytic fungi. For the endophytic fungus with good
   antitumor effect in B. rapa L., comparative metabolomics was performed
   here to draw a metabolism map of pr10 to elucidate the antitumor
   mechanism, demonstrating a unique fingerprint of active metabolites
   synthesized by pr10 and B. rapa L. This study provides a theoretical
   basis for the development and utilization of B. rapa L. in forming
   antitumor drugs and the rational utilization of endophytic fungi.

2. Materials and Methods

2.1. Isolation of Endophytic Fungi and Preparation of Crude Extract

   The normal and nondamaged B. rapa L. was rinsed with tap water and 70%
   ethanol for 30 s, sterile water 3 times, sodium hypochlorite solution
   (2.5% effective Cl^–) for 3 min, and sterile water 4 times and dried
   under sterile conditions. Samples (5 g) were fully ground in a mortar
   containing a small amount of sterilized calcium carbonate and quartz
   sand mortar. Moreover, the ground samples were diluted 10 and 100 times
   with sterile water. The diluent was smeared on the Petri dishes
   containing potato dextrose agar (PDA) and cultured at 28 °C, away from
   light. The tip part of the newly formed mycelium was transferred to the
   new PDA medium, then purified, and cultured 5–7 times until the pure
   strain was obtained.

   Genomic DNA was extracted from the strains, according to the
   manufacturer’s protocols. The internal transcribed spacer (ITS) region
   of rDNA was amplified using primers ITS1F and ITF4. The sequences were
   compared with those available in GenBank via BLAST. Phylogenetic
   analysis was conducted using the neighbor-joining method in MEGA5.

   The endophytic strain was inoculated into the Erlenmeyer flask
   containing potato dextrose broth (PDB) medium and fermented at 28 °C
   for 15 days. Then, the culture medium was extracted by organic solvents
   ethyl acetate (EtAc) 3 times. After that, all extracts were evaporated
   in a rotary evaporator under reduced pressure.

2.2. Antitumor Activity Assay Determination (CCK8)

   Human alveolar adenocarcinoma cells (A549) in the logarithmic growth
   phase with good growth state were taken, the cell density was adjusted
   to 5 × 10^4 mL^–1 by Dulbecco’s modified Eagle’s medium (DMEM), and the
   cell suspension of 100 microcells per well was added into a 96-well
   plate. At the same time, the PBS blank group and normal cell control
   group were set for overnight culture at 37 °C (100 microcells were
   added into the holes around cell holes). Cells were treated
   respectively according to different groups and cell treatment settings.
   The extract (200 μg/mL), which is diluted by the medium, was added to
   the cells in the treatment group, PBS in the blank group, and no
   addition in the control group. Each group was cultured for 48 h in an
   incubator with 5% CO[2] and 37 °C. CCK8 (10 μL) was added to each well
   and cultured at 37 °C for 4 h. The absorbance OD[450] was determined by
   a microplate reader.

2.3. Sample Preparation and Extraction for Metabolomics Analysis

   The four endophytic fungi were expanded on PDA and cultured in PDB.
   Each fungus was fermented for 15 days at 180 rpm and 28 °C, and each
   fungus has six replicates. Mycelia and cell structure were broken by an
   ultrasonic instrument, then the organic products were extracted by
   ethyl acetate with an equal proportion 3 times, the organic products
   were rotation-dried at 45 °C, and the coarse metabolites of fungi were
   obtained by vacuum-drying in an oven. All of the samples were prepared
   for metabolomics analysis.

2.4. Gas Chromatography–Time-of-Flight Mass Spectrometry (GC-TOF-MS) Analysis

   GC-TOF-MS analysis was performed using an Agilent 7890 gas
   chromatograph coupled with a time-of-flight mass spectrometer. The
   system utilized a DB-5MS capillary column. An aliquot (1 μL) of the
   sample was injected in splitless mode. Helium was used as the carrier
   gas, the front inlet purge flow was 3 mL min^–1, and the gas flow rate
   through the column was 1 mL min^–1. The initial temperature was kept at
   50 °C for 1 min, then increased to 310 °C at a rate of 10 °C min^–1,
   and then kept for 8 min at 310 °C. The injection, transfer line, and
   ion source temperatures were 280, 280, and 250 °C, respectively. The
   energy was −70 eV in electron impact mode. The mass spectrometry data
   were acquired in full-scan mode with the m/z range of 50–500 at a rate
   of 12.5 spectra/s after a solvent delay of 6.33 min.

2.5. Raw Data Preprocessing

   The original data included eight quality control (QC) samples and 24
   experimental samples; ultimately, 296 peaks were extracted from the raw
   data profile. To better analyze the data, a series of preparations and
   arrangements were performed based on the original data. The noise was
   removed by filtering individual peaks. Also, deviation values were
   filtered based on the interquartile range and individual peaks were
   further filtered. Only the peak area data with a null value not more
   than 50% in a single group or a hollow value not more than 50% in all
   groups were retained. The missing value recoding in the original data
   was simulated by 1/2 of the minimum value. Moreover, the data were
   further normalized by an internal standard (IS). Finally, 275 peaks
   were preserved after preprocessing and all peaks in this study were
   searched and identified in the local database by its MS/MS information.

2.6. Statistical Analysis

   Metabolites have been used for hierarchical clustering analysis (HCA),
   principle component analysis (PCA), and partial least
   squares-discriminant analysis (OPLS-DA) by R ([56]www.r-project.org/)
   to study metabolite cultivar-specific accumulation, according to the
   normalized peak area of metabolites.^[57]20 The main analytical
   parameters of P-value and fold change were 0.05 and 2.0, respectively.

   To further determine the biological significance associated with
   antitumor activity, we used the Kyoto Encyclopedia of Genes and Genomes
   (KEGG) database to link differential metabolites to metabolic pathways
   in pr10 compared with other three endophytic fungi (pr6, pr7, and pr8).
   Enrichment P-values were computed from a hypergeometric distribution. A
   P-value of <0.05 was selected to reduce the false discovery rate.

3. Results

3.1. Antitumor Activity of Endophytic Fungal Metabolites

   Totally, four endophytic fungi were isolated and used to determine its
   antitumor activity. Antitumor activity of ethyl acetate extract of
   fungi (pr10, pr6, pr7, and pr8) against human alveolar adenocarcinoma
   cell line A549 was determined by CCK8 assays. The result shows that the
   200 μg/mL concentration extract produce more than 50% reduction in
   viability of A549 cells and its inhibition on other cell lines is also
   better than those of pr6, pr7, and pr8 ([58]Table [59]1
   ). However, pr6, pr7, and pr8 did not show significant inhibition of
   A549 cells and other cell lines. The phylogenesis suggests that pr10 is
   a member of Alternaria. It has the closest genetic relationship with
   Alternaria brassicae. The mycelium morphology also indicated that the
   hypha of pr10 has the characteristics of Alternaria such that the
   hyphae are septate and brown conidiophores are solitary or clustered
   ([60]Figure [61]1).

Table 1. Antitumor Activity of Extraction from Four Endophytic Fungi[62]^a.

   name A549 (mean±SD)      blank        control    inhibition ratio (%)
   pr10 0.584 ± 0.011** 0.206 ± 0.003 1.055 ± 0.012 55
   pr6  1.015 ± 0.013B* 0.206 ± 0.003 1.055 ± 0.012 5
   pr7   0.860 ± 0.011  0.206 ± 0.003 1.055 ± 0.012 23
   pr8   0.963 ± 0.011  0.206 ± 0.003 1.055 ± 0.012 11
   [63]Open in a new tab
   ^a

   **P < 0.01, *P < 0.05, blank represents the absorption values of pbs,
   and the control represents the value of A549 cell lines without
   treatment.

Figure 1.

   [64]Figure 1
   [65]Open in a new tab

   Phylogenetic identification and mycelial morphology of pr10. (A)
   Phylogenetic analysis of pr10 by its ITS gene sequence. pr10 and A.
   brassicae are used as a group. (B) Mycelial morphology of pr10 by an
   optical microscope (40×).

   To investigate the factor that causes the effective antitumor activity
   of pr10 that is different with other three endophytic fungi, six
   biological replicates of each endophytic fungus were harvested for
   metabolomics analysis. For this experiment, metabolites of such four
   endophytic fungi cultured for 15 days were extracted. Principle
   component analysis ([66]Figure [67]2
   A) was used to evaluate the biological variability among all samples
   and the metabolic differences among such four endophytic fungi. The
   result of PCA analysis indicated that four distinct regions with
   different colors were clustered, especially in sample pr10 that formed
   a red oval demonstrating that there is a significant metabolic
   difference between fungus pr10 and other three endophytic fungi. In
   addition, to verify the difference and variability of the overall
   metabolic profile, hierarchical clustering was performed for further
   analysis ([68]Figure [69]2B). The hierarchical clustering showed that
   four clades were clustered and each clade is made up of a specific
   fungus with its six replicates. Interestingly, the replicates of pr10
   formed a separate clade, demonstrating that there is a significant
   metabolic difference between pr10 and other three endophytic fungi.
   Therefore, with the antineoplastic activity of pr10, it could be
   concluded that the metabolic profile of pr10 has a potential antitumor
   effect.

Figure 2.

   [70]Figure 2
   [71]Open in a new tab

   Global analysis of the metabolic profile of endophytic fungi. (A)
   Principle component analysis indicating the distinct biological
   variation among all samples. The ellipses with different colors
   represent the replicates of each endophytic fungi (red, pr10; green,
   pr6; navy, pr7; and blue, pr8). Scatter colors and shapes represent
   experimental groupings of samples. All samples are within 95%
   confidence intervals (Hotelling’s T-squared ellipse). (B) Hierarchical
   clustering of the 24 samples used in this study showing two distinct
   clades: one comprised of pr10 exhibiting a significantly specific
   metabolic profile of pr10.

3.2. Discovery of Candidate Metabolites from All Metabolomics Profiles

   Based on the most effective antitumor activity of pr10 compared with
   those of other three endophytic fungi, the identified metabolites that
   matched the conditions (log 2 fold changed ≥ 2; FDR ≤ 0.05, and VIP >
   1.0) were defined as candidate metabolites ([72]Figure [73]3
   A–C). The volcano plot was used to excavate the candidate metabolites
   in each pairwise comparison group (D15-pr10 vs pr6, D15-pr10 vs pr7,
   and D15-pr10 vs pr8). Compared with the metabolic profile of pr6, 31
   metabolites could match the condition and be identified as candidate
   metabolites that have potential antitumor activity. Among such 31
   metabolites, 5 metabolites were unknown and 26 metabolites were
   identified against a local database with its MS/MS fragment information
   ([74]Figure [75]3A). After comparing pr7, 47 metabolites were increased
   in pr10 with various levels and 8 out of all identified metabolites
   were unknown. Finally, compared with pr8, 46 metabolites were in a
   dominant position and 9 out of all different metabolites were unknown.

Figure 3.

   [76]Figure 3
   [77]Open in a new tab

   Excavating of candidate metabolites in each pairwise comparison. (A–C)
   Volcano plot showed the metabolites match the condition (log 2 fold
   changed ≥ 2, FDR ≤ 0.05, and VIP > 1.0) in each pairwise comparison and
   dug out the candidate metabolites with various increased and decreased
   levels. Each point in the volcano diagram represents a metabolite, and
   the horizontal coordinate represents the multiple changes of the group
   of substances compared (log 2 fold change), and the vertical coordinate
   represents the P-value of Student’s t-test (−log[10]P-value). The red
   plot and blue plot represent the significantly increased and decreased
   metabolites, respectively. The metabolites labeled by the gray plot are
   not significant in a pairwise comparison. Moreover, the scatter size
   represents the VIP value of the OPLS-DA model; the larger the scatter
   point, the larger the VIP value. (a, c, e) OPLS-DA analysis of each
   pairwise comparison (D15-pr10 vs pr6, D15-pr10 vs pr7, and D15-pr10 vs
   pr8). As can be seen from the OPLS-DA score plot, the differences
   between each pairwise comparison group of samples are very significant
   and all samples are within 95% confidence intervals (Hotelling’s
   T-squared ellipse). (b, d, f) Permutation test of the OPLS-DA model for
   each pairwise comparison group. The vertical coordinate represents the
   value of R2Y or Q, and the green dot shows the value of R2Y obtained by
   the substitution test. The blue square shows the value of Q obtained by
   the substitution test, and the two dotted lines represent the
   regression lines of R2Y and Q, respectively.

   To clearly analyze the interference degree of the antitumor effect of
   pr10, the OPLS model was utilized. The OPLS score plot ([78]Figure
   [79]3
   a,c,e) at each point indicated a sample, and each clustering
   represented a corresponding metabolic pattern in six different groups.
   Obvious separation of all pairwise comparison groups (D15-pr10 vs pr6,
   D15-pr10 vs pr7, and D15-pr10 vs pr8) was observed, and the R2Y values
   of the permutation test of the OPLS-DA model for each pairwise
   comparison group were 0.75 (D15-pr10 vs pr6), 0.87 (D15-pr10 vs pr7),
   and 0.86 (D15-pr10 vs pr8) (P ≤ 0.05). The original model R2Y is very
   close to 1, indicating that the established model conforms to the real
   situation of sample data. The original model Q value is very close to
   1, indicating that if new samples are added to the model, approximate
   distribution will be obtained. In general, the original model can well
   explain the differences between the two groups of samples. The Q values
   of the random model of the permutation test are all smaller than the Q
   values of the original model. The regression line indicated that the Q
   values of the random model of permutation test are all smaller than the
   Q values of the original model. Moreover, in the random model, the
   gradually decreased Q value associated with the increase in the
   proportion of Y variable, indicating that the original model is robust
   and does not over fit. In addition, all these results of OPLS-DA
   analysis indicated high model reliability and significant difference in
   each pairwise comparison.

3.3. Macromaps of the Relative Content of Candidate Metabolites in Each
Pairwise Comparison Group

   To dig out the metabolites that caused the differences among these four
   endophytic fungi and identify unique and valuable compounds of fungus
   pr10, hierarchical clustering analysis was further performed based on
   the relative content of the metabolites in each pairwise comparisons.
   Also, the heatmaps indicated that all of the samples could be clustered
   together based on the significantly increased and decreased metabolites
   in each pairwise comparison ([80]Figure [81]4
   A–C). Moreover, the increased metabolites are less than the decreased
   metabolites in D15-pr10 vs pr6. However, the increased metabolites are
   significantly more than the decreased metabolites in D15-pr10 vs pr7.
   In addition, all of the metabolites display a stable character in each
   replicate of each endophytic fungi, demonstrating that it is meaningful
   to find some components having potential antitumor activities in the
   specific endophytic fungus pr10. All of the increased metabolites in
   pairwise comparison were isolated to perform further function analysis
   by pathway enrichment analysis.

Figure 4.

   [82]Figure 4
   [83]Open in a new tab

   Heatmap of hierarchical clustering analysis for each pairwise
   comparison group. In the figure, the horizontal coordinate represents
   different experimental groups (each column represent one replicate of
   metabolic profile of each endophytic fungus), the vertical coordinate
   represents different metabolites of this group, and the color blocks at
   different positions represent relative expressions of metabolites at
   corresponding positions. (A) D15-pr10 vs pr6, (B) D15-pr10 vs pr7, and
   (C) D15-pr10 vs pr8.

3.4. Pathway Enrichment Analysis of the Candidate Metabolites in Three
Pairwise Comparison Groups

   To further analyze the function of the candidate metabolites, and the
   significantly affected pathway that produces such key metabolites,
   pathway enrichment analysis was performed against the KEGG database.
   The enrichment results of the candidate metabolites from D15-pr10 vs
   pr6 indicated that amino acid metabolism is the main differential
   metabolic pathway, such as valine, leucine, and isoleucine
   biosynthesis; valine, leucine, and isoleucine degradation; and
   aminoacyl-tRNA biosynthesis; in addition, pantothenate and CoA
   biosynthesis and β-alanine metabolism are also significantly enhanced
   ([84]Figure [85]5
   ). For D15-pr10 vs pr7, the amino acid metabolism is still the most
   significantly different metabolism, such as valine, leucine, and
   isoleucine biosynthesis and valine, leucine, and isoleucine degradation
   ([86]Figure [87]6). Interestingly, the same as the results above, for
   the candidate metabolites from D15-pr10 vs pr8, the enrichment results
   indicated that amino acid metabolism is also the main significant
   metabolic pathway, such as arginine and proline metabolism; lysine
   biosynthesis; and valine, leucine, and isoleucine biosynthesis, which
   is higher in pr10 than that in other three endophytic fungi. β-Alanine
   metabolism is higher in the pr10 than in the other three endophytic
   fungi ([88]Figure [89]7). Such phenomenon indicated that the high-level
   expression of amino acid metabolism may lead the antitumor activity of
   pr10, which is more effective than other three endophytic fungi.

Figure 5.

   [90]Figure 5
   [91]Open in a new tab

   Pathway analysis of the candidate metabolites from D15-pr10 vs pr6. The
   bubble diagram demonstrated the results of metabolic pathway analysis
   based on the candidate metabolites from D15-pr10 vs pr6. Each bubble in
   the bubble diagram represents a metabolic pathway, and the abscissa
   coordinate and the bubble size represent the size of the influencing
   factors of this pathway in the topological analysis. The vertical
   coordinate and bubble color represent the P-value (−ln(P-value)) of
   enrichment analysis. Darker color means a smaller P-value and
   significant enrichment degree.

Figure 6.

   [92]Figure 6
   [93]Open in a new tab

   Pathway analysis of the candidate metabolites from D15-pr10 vs pr7. The
   results of pathway enrichment analysis of the candidate metabolites
   identified from D15-pr10 vs pr7 are shown in the bubble diagram, and
   each bubble means a metabolic pathway enriched in the pairwise
   comparison group.

Figure 7.

   [94]Figure 7
   [95]Open in a new tab

   Pathway analysis of the candidate metabolites from D15-pr10 vs pr8.
   Pathway analysis of the metabolites filtered in D15-pr10 vs pr8 by the
   conditions (log 2 fold change ≥ 1.0, P < 0.05, and VIP > 1.0).

   Besides this, the results of pathway enrichment analysis indicated that
   different levels of glucose metabolism were displayed in these three
   pairwise comparison groups, for example, pentose phosphate pathway,
   fructose and mannose metabolism, glycolysis or gluconeogenesis, and
   starch and sucrose metabolism. It has been reported that
   glycometabolism plays an important role in the antitumor activity and
   has potential antitumor activity.^[96]21 Other metabolites such as
   biochemical components produced by the biosynthesis of antibiotics
   ([97]Table [98]2
   ), which was enriched in all of these three pairwise comparisons and
   could function in defense response against biological stress, also have
   potential antitumor activities with their microorganism inhibiting
   property.

Table 2. Significance Level of Key Metabolic Pathways Filtered by the Venn
Diagram in the D15-pr6 vs pr10 Group.

   [99]3.4.
   [100]Open in a new tab

   Compared with those in the other three endophytic fungi pr6, pr7, and
   pr8, there are 13 metabolic pathways that were significantly different
   and highly expressed in pr10 ([101]Figure [102]8
   and [103]Tables [104]3–[105]5). All 13 metabolic pathways were
   variously enriched in these three pairwise comparison groups; for
   D15-pr10 vs pr6, only two pathways were significantly enriched (e.g.,
   valine, leucine, and isoleucine biosynthesis and β-alanine metabolism)
   and it had been reported that valine and leucine play an important role
   in a new antitumor drug that could improve the antitumor efficiency
   ([106]Table [107]3). For group pr10 vs pr7, four biosynthesis pathways
   were significantly enriched (P < 0.05) (e.g., glycine, serine, and
   threonine metabolism; valine, leucine, and isoleucine biosynthesis;
   β-alanine metabolism; and aminoacyl-tRNA biosynthesis) ([108]Table
   [109]4). Finally, in the pr8 vs pr10 group, except the metabolic
   pathways mentioned above, there were some other metabolic pathways such
   as the pentose phosphate pathway and lysine degradation pathway
   ([110]Table [111]5). Among all 13 pathways, the metabolites synthesized
   in starch and sucrose metabolism were most likely to have antitumor
   activity such as trehalose.^[112]22

Figure 8.

   Figure 8
   [113]Open in a new tab

   Key metabolic pathways enriched in all three pairwise comparison
   groups. Venn diagram analysis of the key metabolic pathways enriched in
   all three pairwise comparison groups (D15-pr10 vs pr6, D15-pr10 vs pr7,
   and D15-pr10 vs pr8). Each circle represents one pairwise comparison.
   Also, there are 13 metabolic pathways that were all enriched in such
   three groups.

Table 3. Significance Level of Key Metabolic Pathways Filtered by the Venn
Diagram in the D15-pr7 vs pr10 Group.

   [114]3.4.
   [115]Open in a new tab

Table 5. Significance of Indispensable Metabolites Filtered by the Upset
Diagram[116]^a.

     D15-pr8 vs pr10
     __________________________________________________________________

   D15-pr7 vs pr10
     __________________________________________________________________

   D15-pr6 vs pr10
     __________________________________________________________________

     VIP (VIP > 1.0) P-value (P < 0.05) VIP (VIP > 1.0) P-value (P < 0.05)
   VIP (VIP > 1.0) P-value (P < 0.05)
   pantothenic acid 1.25 0.03 1.32 0.03 1.18 0.03
   threonine 1 1.73 0.01 1.76 0.01 1.27 0.05
   analyte 132 1.81 0.00 1.91 0.00 1.76 0.00
   lysine 1.81 0.01 1.55 0.01 1.50 0.01
   gentiobiose 2 1.57 0.02 1.91 0.02 1.54 0.02
   phosphate 1.07 0.00 1.26 0.00 1.37 0.00
   phenylalanine 2 1.53 0.01 1.61 0.01 1.16 0.03
   tagatose 1 1.59 0.03 1.37 0.03 1.22 0.03
   analyte 197 1.56 0.00 1.85 0.00 1.34 0.00
   unknown 1.54 0.02 1.64 0.02 1.51 0.02
   tartaric acid 1.53 0.02 1.61 0.02 1.25 0.02
   analyte 173 1.09 0.00 1.19 0.01 1.08 0.00
   analyte 128 1.33 0.00 1.65 0.00 1.07 0.01
   cellobiose 1 1.50 0.01 1.37 0.01 1.15 0.01
   tyrosine 1 1.81 0.00 1.91 0.00 1.48 0.00
   unknown 1.80 0.02 1.90 0.02 1.76 0.02
   valine 1.26 0.01 1.86 0.01 1.43 0.01
   unknown 1.53 0.02 1.63 0.02 1.50 0.02
   unknown 1.53 0.04 1.61 0.04 1.51 0.04
   analyte 659 1.08 0.03 1.35 0.03 1.27 0.03
   [117]Open in a new tab
   ^a

   VIP, variable importance in the projection (VIP > 1.0); P < 0.05
   (Student’s t-test).

Table 4. Significance Level of Key Metabolic Pathways Filtered by the Venn
Diagram in the D15-pr8 vs pr10 Group[118]^a.

   [119]3.4.
   [120]Open in a new tab
   ^a

   Colors represent the significance of each metabolic pathway in the
   D15-pr6 vs pr10 group; red represents a highly significant difference.
   The metabolites involved in each pathway are shown in the Hits list.

3.5. Excavation of the Most Important and Specific Metabolites of pr10

   All of the increased metabolites in pairwise comparative groups were
   classified in the upset plot ([121]Figure [122]9
   ) to excavate the specific metabolites that are higher in pr10 than in
   other endophytic fungi. The count of the metabolites with such a
   property is 20, and in pr10, all such 20 metabolites are higher than
   their contents in the other three endophytic fungi. Moreover, there are
   various amounts of the metabolites involved in the intersection, which
   are shown in [123]Figure [124]10. All such 20 metabolites and their
   properties (VIP and P-value) are shown in [125]Table [126]5,
   demonstrating a significant difference in each pairwise comparison.
   However, among all 20 metabolites, 9 components were still not
   identified with a detailed MS/MS information, which need further
   identification and determination of their antitumor activities.

Figure 9.

   Figure 9
   [127]Open in a new tab

   Filtration of significantly increased metabolites of pr10. The upset
   diagram analysis show 20 metabolites, which were represented by red
   circles and recognized as indispensable components, that were increased
   in these three pairwise comparison groups (D15-pr6 vs pr10, D15-pr7 vs
   pr10, and D15-pr8 vs pr10). The black bars display the number of
   metabolites involved in each intersection.

Figure 10.

   Figure 10
   [128]Open in a new tab

   Relative content analysis of the indispensable metabolites in each
   pairwise comparison group. The heatmap displays the relative content of
   the indispensable metabolites in each pairwise comparative group. In
   the figure, the horizontal coordinate represents different pairwise
   comparative groups (D15-pr10 vs pr6, D15-pr10 vs pr7, and D15-pr10 vs
   pr8), the vertical coordinate displays the relative content of each
   component of all 20 indispensable metabolites, and colors represent the
   relative contents (log 2 fold change) of metabolites at their
   corresponding positions.

   Except for these 20 metabolites, there are other three metabolites that
   could function in the antitumor process such as trehalose, d-arabitol,
   and phenylalanine ([129]Figure [130]11
   ). For trehalose, the results indicated that, compared with that of
   pr7, the content of trehalose increased by 2.67-fold in pr10,
   demonstrating that pr10 could synthesize more trehalose in vivo
   ([131]Figure [132]11B). It has been reported that trehalose and its
   derivates could perform various functions in the antitumor
   process.^[133]23 For other metabolites, d-arabitol could effectively
   influence the proliferation of tumor cells and angiogenesis during
   tumor growth^[134]24 and the content of such valuable metabolites is
   30.65-fold more than its content in pr6. Many studies have reported
   that phenylalanine and its derivates could suppress the growth and
   survival rate of tumor cells in different ways.^[135]25 However, the
   content of phenylalanine is about 230 000-fold in pr10 more than that
   in the other three endophytic fungi. Moreover, all three metabolites
   have potential antitumor activities, contributing the antitumor
   activity of pr10 on A549 cell lines. Further analysis of the relative
   content of such 20 metabolites suggests that these metabolites could
   form three clades in the hierarchical clustering analysis. Also, in the
   upper cluster, four metabolites are identified (e.g., valine,
   phosphate, tagatose 1, and threonine 1). The contents of all of the
   metabolites in this cluster display a lower-level increase from about
   2.2- to 200-fold than their contents in the other three endophytic
   fungi ([136]Figure [137]10). In the bottom cluster, all of the
   metabolites were identified by MS/MS and such six metabolites (lysine,
   cellobiose, tyrosine, gentiobiose, tartaric acid, and phenylalanine)
   exhibit a relatively higher increase (about 8.0- to 220-fold increase)
   ([138]Figure [139]10). Moreover, it has been proved that phenylalanine
   and its derivates have potential antitumor activity.^[140]25 In the
   middle cluster, only one compound, pantothenic acid, was identified
   ([141]Figure [142]10). In pr10, the content of pantothenic acid is
   about 33 000-fold than its content in the other three endophytic fungi.
   In addition to this, the other six significantly increased metabolites
   were not identified (P < 0.05).

Figure 11.

   [143]Figure 11
   [144]Open in a new tab

   Relative contents of candidate metabolites in each endophytic fungi.
   Boxplot analysis displays the relative content of these three candidate
   metabolites, demonstrating a significant difference among these four
   endophytic fungi, and pr10 has a higher level of these three
   metabolites. The vertical coordinates represent the peak areas of
   metabolites, which means their relative content. A, d-arabitol; B,
   trehalose; C, phenylalanine; NS, not significant; *P <0.05; **P <0.01;
   ***P <0.001.

4. Discussion

   In the interaction process of the endophytic fungi and host plants, the
   host plants provide nutrition and shelter to the endophytic fungi and,
   in return, endophytic fungi could synthesize many effective and active
   biochemical components that could enhance the resistance against
   various biotic and abiotic stresses in the host plants.^[145]1 Although
   this has gained extensive attention all over the world, this is still
   in the initial stage of research in terms of the diversity and function
   of endophytic fungi, and such research studies could be a new addition
   to the available diversity of fungi.^[146]26 There is no research on
   the function and diversity of endophytic fungi of B. rapa L., a widely
   used plant with potential medicinal properties. It has been reported
   that B. rapa L. has abundant biochemical components such as flavonoids,
   phenolic acid, amino acids, carbohydrates, and vitamins; all of these
   metabolites could enhance the antioxidant ability and resistance of
   human beings in various aspects such as antitumor, immune regulation,
   biotic stress, and abiotic stress.^[147]17,[148]18,[149]27 In
   consideration of these, conducting research on the diversity and
   biological function of endophytic fungi of B. rapa L. is highly
   deserved.

   In this study, four endophytic fungi were isolated from B. rapa L. and
   further purified in vitro. It has been reported that endophytic fungus
   extracts from the medicinal plant Umea could effectively inhibit
   multidrug-resistant bacteria Escherichia coli, Pseudomonas aeruginosa,
   and Klebsiella pneumoniae and found that 60% of the crude extracts had
   antibacterial effects on these bacteria (Manila). In addition, Refaei
   et al. isolated eight strains of endophytic fungi from Dahua, among
   which three strains of crude extracts from fermentation broth had
   antibacterial effects on C. albicans.([150]5) To reveal its function
   and application value, its ability to mycelium inhibition in vitro was
   tested based on its crude extracts. The result indicated that the crude
   extracts could not effectively inhibit the growth of pathogenic
   bacteria (E. coli, Staphylococcus aureus, C. albicans, P. aeruginosa,
   and Enterococcus faecalis). In addition to the antibacterial ability of
   endophytic fungi, it has been reported that they have antitumor
   functions because of their uniquely synthesized metabolites.^[151]28 We
   further tested the antitumor ability of endophytic fungi with their
   crude extracts; the results indicated that among all of these four
   endophytic fungi the crude extracts of pr10 could effectively inhibit
   tumor cells by 54%; however, other three crude metabolites from pr6,
   pr7, and pr8 did not display significant antitumor effect. For the
   results of antitumor effect, it is likely due to the use of metabolized
   crude extract from pr10, including D-Arabito, Trehalose, and
   Phenylalanine. With effective antitumor metabolites, we performed a
   comparative metabolomics analysis to study the different metabolites
   among these four endophytic fungi from species to contents, especially
   the metabolites from pr10.

   For the metabolites in all endophytic fungi, there are 296 metabolites
   being acquired; and among all such acquired metabolites, 117
   metabolites were identified by their MS/MS fragments. The results
   indicated that these four endophytic fungi are rich in amino acids and
   sugars, which are beneficial for the health of human beings. Also, in
   this study, by comparative metabolomics analysis, we found that the
   metabolic profile of pr10 is greatly different than those of other
   endophytic fungi ([152]Figure [153]4
   ), which is also demonstrated by the OPLS-DA analysis ([154]Figure
   [155]4). This phenomenon showed that in the metabolite levels from the
   content to the diversity of metabolites, pr10 displays a more specific
   property than other endophytic fungi. Compared with those in pr6,
   contents of 31 metabolites are higher in pr10, such as d-arabitol that
   has been researched to found that it possesses antitumor activity by
   decreasing the survival rate of tumor cells.^[156]24 For pr7 vs pr10,
   in total, 47 metabolites were identified and filtered by their contents
   and found that these were synthesized by pr10 in a higher level
   ([157]Figures [158]9 and [159]4). Also, there is an attractive
   metabolite, trehalose, and it has been reported that brartemicin, a
   derivate of trehalose, has a good ability to inhibit the invasion of
   26-L5 cells in colon cancer.^[160]23 The advantage is that trehalose
   could inhibit A549 tumor cells.^[161]29 In addition, the abundance of
   trehalose in pr10 explains the effective antitumor activity of pr10 on
   A549. Anything else, there are other 20 metabolites that were
   specifically synthesized at a high level in pr10. Among all of these 20
   metabolites, except unknown compounds, carbohydrate compounds and amino
   acids and their derivates are the main compounds. Also, it has been
   reported that amino acids such as phenylalanine and its derivates, for
   example, l-phenylalanine dipeptide derivatives, are effective in
   treating cancer and have a preferable inhibitory effect on prostate
   cancer cell line PC3 and K562 cells in vitro.^[162]25 Carbohydrates and
   their derivates such as fucose, arabinose, mannose, galactose, and
   glucose could act on McF-7 tumor cells of breast cancer, promote the
   proliferation of spleen cells, and stimulate the immune activity, thus
   inhibiting the growth of tumor cells.^[163]24 Moreover, in addition to
   direct antitumor activity, the metabolism of amino acids and sugar
   derivatives provides small molecular nutrients that are more readily
   absorbed by humans. The intake of these amino acids and
   carbohydrate-derived nutrients will also enhance human immunity,
   leading to an effective antitumor capacity.^[164]30 Due to the
   limitations of the current MS database, the unknown compounds, which
   were the main candidate metabolites in pr10, needed to be further
   studied in terms of metabolite identification. Moreover, such unknown
   metabolites have potential direct and indirect antitumor activities,
   which are helpful to make more rational use of endophytic fungi. Based
   on these above-mentioned advantages, it could be concluded that B. rapa
   L. with its specific endophytic fungi and metabolites synthesized by
   endophytic fungi could enhance the resistance of human beings in terms
   of tumors and other biotic and abiotic stresses.

   In addition to the antitumor activity mentioned above, B. rapa L. has
   many other valuable characteristics such as antioxidant function and
   free-radical scavenging.^[165]21 It is the first time that comparative
   metabolomics was performed to systematically elucidate the antitumor
   mechanism of endophytic fungi of B. rapa L. The results manifested that
   B. rapa L. has unique kinds of endophytic fungi and such endophytic
   fungi could directly inhibit the tumor cells with their various
   biochemical metabolites. This research provides a theoretical basis and
   metabolic fingerprint database, at the biochemical metabolic level, for
   better use of B. rapa L. and its endophytic fungi to develop antitumor
   agents and give direction for digging out the potential value of
   antitumor medicinal plants at the biometabolic level in the future.

5. Conclusions

   In view of the fact that previous experiments in vitro have proved that
   pr10 has antitumor properties there are no reports on the metabolomics
   of B. rapa L. endophytic fungi. Therefore, in this experiment, the
   method of comparative metabolomics was used to analyze the metabolomics
   of four strains of B. rapa L. endophytic fungi and the data were
   searched in the database. The metabolites of four strains of fungi were
   compared statistically, and the metabolism map of pr10 was drawn. The
   unique metabolites of endophytic fungus pr10 are rich in amino acids
   and sugar derivatives such as phenylalanine, d-arabitol, cellobiose,
   and trehalose. These metabolites have potential antitumor activity,
   especially trehalose whose antitumor activity on A549 has been
   reported.

   This work was supported by the National Natural Science Foundation of
   China (81460673, 81960169, 81760169) and the Projects supported by the
   Natural Science Foundation of Xinjiang Uygur Autonomous Region
   (2019D01C219, 2017D01C234)

   The authors declare no competing financial interest.

References