Endophytic Streptomyces sp. LRE541 isolated from Lilium davidii var. Unicolor Cotton as a Cell Factory for Antimicrobials and Anticarcinogens with Inducing Apoptosis and Cell Cycle Arrest Properties

Background: Endophytic actinomycetes, as emerging sources of bioactive metabolites, play a vital role in pharmaceutical development. Recent reports demonstrated that endophytic Streptomyces isolates could yield compounds with potent anticancer and antimicrobial properties that may be developed into chemotherapeutic drugs. Our study displayed that Streptomyces sp. LRE541 obtained from the root tissues of Lilium davidii var. unicolor Cotton, could be a potential source of anticarcinogens and antimicrobials. Results: Isolate LRE541 was characterized and identied as belonging to the genus Streptomyces based on the 16S rDNA sequence analysis, with highest sequence similarity to Streptomyces tauricus JCM4837 T (98.81%). It produced extensively branched red substrate and vivid pink aerial hyphae that changed into amaranth, with elliptic spores sessile to the aerial mycelia. The secondary metabolites (EtOAc extract) produced by isolate LRE541 exhibited signicant anticancer activities with IC 50 values of 0.021, 0.2904, 1.484, 4.861, 6.986, 8.106, 10.87, 12.98, and 16.94 μg/mL against cancer cells RKO, 7901, HepG2, CAL-27, MCF-7, K562, Hela, SW1190 and A549, respectively, evaluated by the MTT assay. In contrast, the EtOAc extract showed less cytotoxicity activity against the normal human pulmonary artery endothelial cell (HPAEC) with an IC 50 value of > 20 μg/mL than that of the cancer cells. To further explore the mechanism underlying the decrease in viability of cancer cells following the EtOAc extract treatment, cell apoptosis and cell cycle arrest assays were performed using two cancer cell lines, RKO and 7901. The result demonstrated that the EtOAc extract inhibited cell proliferation of RKO and 7901 cells by causing cell cycle arrest both at the S phase and inducing apoptosis in a dose ‐ dependent manner. Moreover, the EtOAc extract of isolate LRE541 with the concentrations within 100 μg/mL also possessed the antagonistic activities against E. coli ATCC 25922, MRSA ATCC 25923, P. aeruginosa and C. albicans ATCC 66415, and the antagonistic potent against the tested pathogens all displayed a dose-dependent manner. The UHPLC-MS/MS analysis of the EtOAc extract revealed that the presence of antitumor, potential antitumor and antimicrobial compounds could account for the potent antineoplasmic and antagonistic properties of the extract. Conclusion: This study provides the potential therapeutic applications of the bioactive metabolites from Streptomyces sp. LRE541 as novel antimicrobial and anticancer agents. cytotoxicity 7-isoprenylindole-3-carboxylic acid A549 50


Introduction
Although major progress has been achieved in the eld of cancer therapy for the past few decades, cancer remains a serious public health threat (1,2). Chemotherapy is one of the common therapeutic approaches for controlling cancers. Unfortunately, most patients eventually relapse and develop drug resistance (3,4). Furthermore, patients, especially those undergoing cancer chemotherapy, joint replacement surgery and organ transplantation, starve for effective antibiotics to prevent postoperative infection (5). On this account a continuous supply of novel drugs with high effectiveness and safety is urgently needed. Besides, druginduced apoptosis of malignant cells is a promising antitumor strategy with emerging evidence supporting its e cacy against various cancer types (6)(7)(8), thus, extensive and intensive studies on the underlying anti-tumor mechanism of the drugs are also required.
Streptomyces, with a vast distribution and innate capability of producing diverse bioactive secondary metabolites, has served as an important source of novel antibiotic candidates for decades (9)(10)(11). Nowadays, bioprospecting for Streptomyces sp. from untapped or unique ecosystems may be an effective way to meet the everlasting demand for novel drugs and other biomolecules, which have been preferred attributing to their potent therapeutic applications and desired pharmacokinetic properties for clinical uses and served as precursors of drug semi-synthesis or the template of drug chemical synthesis (12)(13)(14). Over the past decade, endophytic Streptomyces spp. from medicinal plants in various ecotopes, as relatively unexploited fascinating sources of novel natural products, have been explored extensively and gained some remarkable results. For example, reports covering new endophytic Streptomyces species and their novel secondary metabolites along with antimicrobial and antioxidant activities have sprung up (14)(15)(16)(17). Moreover, anticancer and cytotoxic compounds have also been discovered in endophytic Streptomyces spp., although such reports are sporadic compared to marine actinomycetes; however, their anticancer effects or cytotoxic activities are comparable to those of their marine counterparts, even stronger (18,19). In addition, it is widely accepted that medicinal plants are rich sources of precious bioactive compounds, and increasing evidence indicates that endophytic actinomycetes may participate in the metabolic pathways of their host plants, and obtain some genetic information to yield bioactive compounds similar to their host plants (5,20). These ndings suggest that endophytic Streptomyces sp. of medicinal plants may be a good choice for anticarcinogens and antimicrobials.
Lilium davidii var. unicolor Cotton, a famous health-care edible medicinal plant, rich in amino acid, vitamins, glycosides, alkaloids and polysaccharides, possesses antioxidant activities (21). Accordingly, the actinomycetes from the plants may develop adaptive strategies and yield chemically unique secondary metabolites. However, there is no report concerning the antimicrobial and anticancer activities of Streptomyces sp. from Lilium davidii var. unicolor Cotton in vitro. Given the immense potential of the secondary metabolites of endophytic Streptomyces sp. for pharmaceutical applications, we isolated and characterized a Streptomyces isolate from the root tissues of Lilium davidii var. unicolor Cotton, and investigated the antimicrobial and antitumor effects of the secondary metabolites of the isolate, as well as the inductive effects on apoptosis and cell cycle arrests of tumor cells.

Phenotypic characteristics of Isolate LRE541
The isolate LRE541 obtained from the root tissues of Lilium davidii var. unicolor Cotton is Gram-positive and aerobic. The cultural characteristics of LRE541 on various media were shown in Table 1. It grew well on all the tested media (ISP2 ~ ISP7 and Gao's No. 1) with varying colors of aerial and substrate myceliums on different medium. Compared with ISPs, the diffusible pigment was only produced on Gao's No. 1 medium. As presented under the scanning electron microscopy in Fig. 2, isolate LRE541 produced elliptic spores sessile to the aerial hyphae, which extensively branched and grew in segments with verrucous protrusions. As shown in Table 2, the growth of LRE541 was observed at the temperature range of 18-37 ℃ (optimum at 23 ℃ ), and pH range of 4-12; however, at NaCl concentration above 6% (w/v), no growth was observed. Isolate LRE541 was positive for cellulose utilization but negative for both methyl red test and H 2 S production. In the extracellular enzyme activity tests, the isolate demonstrated to produce various enzymes such as urease, catalase, amylase, protease, and lipase. Furthermore, it was found that LRE541 had broad utilizations of carbon and nitrogen sources ( Table 2). These phenotypic properties of LRE541 were congruent with the genus Streptomyces as depicted by E. A. Barka et al. (22) in that they are proli c aerobic Gram-positive bacteria possessing extensively branched vegetative form and aerial hyphae and produce various water-soluble pigments.   Fig. 3, compared to HPAEC, the cell viability of RKO and 7901 cells were dramatically decreased when the concentration of the EtOAc extract was within 10 µg/mL, and below 30% when reached 10 µg/mL.

Induction of apoptosis in 7901 and RKO cells
Inducing apoptosis and necrosis of tumor cells is the main mechanism of chemotherapy drugs inhibiting tumors, and it is also one of the main indicators for evaluating the e cacy of chemotherapy drugs (23,24). Herein, we quantitatively detected the cell death type triggered by the EtOAc extract in 7901 and RKO cells using the annexin V-FITC and PI double staining, which were presented in Fig. 4a. After the cells were processed with the EtOAc extract (2 µg/mL) for 48 h, FITC-positive cells accounted for ~ 50% and ~ 40% of the total cells in 7901 and RKO cells, respectively, suggesting that apoptosis was a major mechanism of the cytotoxicity of the EtOAc extract whether in 7901 or RKO cell, and the EtOAc extract induced apoptotic cell death in a dose-dependent manner in both two cells (Fig. 4b). However, the apoptosis patterns of the RKO and 7901 cells were distinctly diverse. As demonstrated in Fig. 4c, for the 7901 cell, the proportion of early apoptotic cells was higher than that of late apoptotic cells at low concentration of the EtOAc extract; however, the number of early apoptotic cells gradually decreased as the concentration increased, while the number of late apoptotic cells sharply increased with the increased concentration of EtOAc extract. In contrast, for the RKO cell, the late apoptotic cells were dominant at rst and displayed a dose-dependant manner, while the number of early apoptotic cells slightly increased with the increasing of concentration.
The EtOAc extract inhibits the cell cycles of 7901 and RKO cells Flow cytometric analysis of DNA showed a dose-dependent accumulation of cells in the S phase of the cell cycle both in 7901 and RKO cells, with a concomitant decrease in the proportion of cells in the G 0 /G 1 phase when treated with a concentration gradient of the EtOAc extract for 48 h, indicating the EtOAc extract blocked the cell cycle of 7901 and RKO in S phase ( Fig. 5a and 5b).

Antimicrobial activity of the EtOAc extract
The antimicrobial potential of EtOAc extract from Streptomyces sp. LRE541 was investigated against four human pathogens. As presented in Fig. 6, the EtOAc extract showed antagonistic activities against all of the tested microorganisms, and the inhibition zone diameters of the four human pathogens displayed a dose-dependent manner. However, the antimicrobial potent of the EtOAc extract against the four pathogens varied. For example, when the concentration of the EtOAc extract was 100 µg/mL, the maximum activity was found against E. coli ATCC 25922 (inhibition zone of 10.94 ± 0.45361 mm diameter), followed by MRSA ATCC 25923 (10.7567 ± 0.92716 mm), P. aeruginosa (10.4533 ± 0.47154 mm) and C. albicans ATCC 66415 (7.665 ± 0.78421 mm).

Chemical Pro ling of the EtOAc extract Using UHPLC-MS/MS Analysis
To examine the compounds that may be responsible for its antineoplastic and antimicrobial properties, the EtOAc extract of isolate LRE541 was subjected to ultra-high performance liquid chromatographytandem mass spectrometry (UHPLC-MS/MS) analysis (Additional le 1: Fig. S1). Based on the UHPLC-MS/MS analysis, we successfully detected thirty-nine antitumor compounds, ten potential antitneoplastic compounds, sixteen antimicrobial compounds and thirty compounds without speci c literatures to support their biological activities, but with a relative ratio above 1% in the EtOAc extract. The detailed information of the ninety-ve chemical compounds, including retention time, molecular formula, molecular weight and relative ratio, are listed in Table 4 and Additional le 1: Table S1, respectively, and their chemical structures are presented in Fig. 7 and Additional le 1: Fig. S2.

Discussion
To date, despite cancer treatment has made great advancement and innovation based on the development of basic science, genetic engineering technology and big data analysis technology, cancer remains one of the most refractory diseases nowadays, and the number of patients has increased year by year, causing a heavy burden on families and society (2,25,26). Moreover, the frequently occurrence of multidrug resistant pathogenic microorganisms is another challenge for researchers (40). Therefore, the discovery and development of safer and novel drugs based on natural products has become one of the research hotspots (27)(28)(29). While as an emerging source of novel natural products, the endophytic actinomycete has attracted increasing attention to academics (30)(31)(32). Our study obtained an endophytic isolate, designated as LRE541 from the root tissues of Lilium davidii var. unicolor Cotton, and found that the secondary metabolites of LRE541 possess potent antitumor and antimicrobial potential by inhibiting a variety of malignancies and pathogens growth. and lipase, which are industrially important. In addition, LRE541 could tolerate high pH value up to 12.0, a salinity of 6% NaCl, and temperature up to 37 °C. In sum, these physiological traits are consistent with the characteristics of streptomycetes that they are proli c and possess high adaptive capability for surviving in many unique niches (33)(34)(35), what′s more, re ecting the physiological exibility of Streptomyces isolate to adverse environmental conditions (36,37). Furthermore, the availability of a broad spectrum of carbon and nitrogen sources plays a vital role in the production of diverse secondary metabolites by Streptomyces sp. (38). Here, isolate LRE541 also exhibited the capability to utilize a wide range of carbon and nitrogen sources. This data provided an overview of the metabolite pro le of LRE541, potentially serving as references for future research concerning fermentation optimization for higher yield of the desirable bioactive metabolites.
In view of the remarkable physiological capabilities above mentioned, the secondary metabolites (EtOAc  (50). Taken together, these results suggested that the endophytic isolate LRE541 colonizing the root tissues of Lilium davidii var. unicolor Cotton, could produce secondary metabolites with antitumor activities in vitro, but exhibit different anticancer potent against different cancer cells. This may be related to the speci c molecular genetic background of different cancer cells, but further research will be needed.
It has been con rmed that apoptosis and necrosis are two patterns of cell death (51,52). Compared to necrosis, an abnormal form of cell death, cell apoptosis regulated by various intra and extracellular signals and governed by several genes, plays a signi cant role in stress responses, control of normal cell proliferation and development of an organism (53,54). While tumorigenesis is closely related to antiapoptotic pathways (6). Thus, druginduced apoptosis of malignant cells is an e cient strategy in cancer therapy (6,55). Our data presented that the EtOAc extract from the endophytic Streptomyces sp. LRE541 validly inhibited the cell viabilities of RKO and 7901 cells predominantly through the induction of apoptosis in a dose-dependant manner. Apparently, the apoptosis patterns between the two cells were greatly diverse, which suggested distinct mechanisms of the secondary metabolites actions occurring in the two cancer cells. Furthermore, previous studies have shown that cell cycle is likewise intimately associated with the tumorigenesis. Pathological or physiological apoptotic stimuli would greatly affect cell cycle progression, and disorder of cell cycle regulators is a common property of human cancer, which signi es that regulation of cell cycle progression in cancer cells is taken for an available method in the treatment of human malignancies (56,57). This study, the EtOAc extract dramatically inhibited the cell proliferation of RKO and 7901 in a dose-dependent manner through inducing S phase arrest of cell cycle and apoptosis in vitro. Several studies had discovered that chemotherapeutic drugs originating from marine actinomycetes could cause cancer cell cycle arrest as well. Echinosporin and 7-deoxyechinosporin produced by Streptomyces albogriseolus A2002 were exhibited to block the cell cycle of HCT-15, K562 and tsFT210 cells mainly at the G 0 /G 1 phase and promote apoptosis in these cells (58). Proximicins obtained from two rare actinomycetes Verrucosispora sp. MG-37 and Verrucosispora maris AB-18-032 isolated from marine sediments were found to block human gastric adenocarcinoma AGS cells at G 0 /G 1 phase. Likewise, thiocoraline produced by the marine actinomycete Micromonospora sp. L-13-ACM2-092 caused an arrest in G 1 phase of the cell cycle of human colon cancer cell lines (18). Collectively, chemotherapeutics with greater therapeutic e ciency and fewer side effects are of utmost desirability, and drug induced cancer cell death mode plays an important role in chemotherapy.
In the current study, the antagonistic activity of EtOAc extract associated with isolate LRE541 was Similarly, the antagonism of endophytic Streptomyces sp. in this study has been discovered in other reports as well. For example, the endophytic Streptomyces sp. T3SB005 from root tissues of Thymus roseus exhibited antagonistic activities against three human pathogens (59); the culture ltrate of Streptomyces sp. HUST012 from Dracaena cochinchinensis Lour. was found against E. coli ATCC 25922 and MRSA ATCC 25923 with an inhibition zone of 18.9 mm (5). These ndings suggested that endophytic streptomycetes may be exciting sources of bioactive compounds.
The antineoplastic and antimicrobial capacities of the EtOAc extract from isolate LRE541 suggest that the presence of antitumor and antimicrobial agents in the mixture of the extract may account for it. And we pursued this via ultra-high performance liquid chromatography-tandem mass spectrometry method.
Based on the UHPLC-MS/MS analysis, the EtOAc extract was detected to contain thirty-nine compounds testi ed to exhibit various antitumor activities and cytotoxicity, besides, sixteen compounds proved to be antagonistic against diverse microorganisms. For example, ferulic acid, relative ratio of 15.3965% in the ESI (−) mode, as a new broblast growth factor receptor 1 (FGFR1) inhibitor, could inhibit the melanoma growth and angiogenesis using a melanoma model in vivo (60); similarly, 4-Hydroxybenzylalcohol, a promising anti-angiogenic agent, suppressed the vascularization and growth of newly developing CT26.WT tumors in vivo (61); while formononetin and catechin suppressed MCF-7 proliferation through blocking the cell cycle arrest or inducing apoptosis, respectively (62,63); citral in combination with doxorubicin, and glycocholic acid with epirubicin, can dramatically increase the chemosensitivity of doxorubicin in human lymphoma Ramos cells, epirubicin in Caco-2 cells, respectively (64,65). Sorbic acid and Lauric acid with relative ratios of 14.9% and 1.2% in the ESI (+)/(−) modes, respectively, have broadspectrum antagonistic activities against most of the Gram-positive, Gram-negative pathogens and fungi (66,67). Cefradine and Pleuromutilin are common broad-spectrum, high effective and low toxic antibiotics. Moreover, trans-Cinnamaldehyde and Methyl cinnamate due to their antimicrobial activities have been used as preservatives in the food industry (68,69). As a whole, these chemical constitutes detected by UHPLC-MS/MS may be directly responsible for the antineoplastic and antimicrobial properties of the EtOAc extract.
It is reported that oxidative stress and chronic in ammation can develop into cancerous lesions (70,71).
Here, several natural phenolic compounds (rosmarinic acid, 4-Hydroxyphenylacetic acid and eugenol) and avonoids compounds (eriodictyol, daidzein, glycitein) detected from the EtOAc extract, have been reported to possess excellent antioxidant activity (72)(73)(74)(75)(76). (-)-Caryophyllene oxide and cynaropicrin present in the extract had been demonstrated to have potent anti-in ammatory effect (77,78). A NADPH oxidase inhibitor, 7-Methylguanosine (79), and cNIIIB nucleotidase inhibitor, apocynin (80), were all detected in the extract. Taken together, the above compounds may play an indirectly role in the antitumor activity of the EtOAc extract through antioxidant or anti-in ammatory effects, although further experimental evidence is necessary to testify their antineoplastic activity. Moreover, considering the potent antineoplastic activity of the EtOAc extract in the present study, the antitumor activity of the thirty compounds with relative ratio above 1% deserved to be further research. in Qilihe District, Lanzhou City, Gansu Province, China. The plant roots were dug out carefully to ensure its integrity, then kept in aseptic plastic bags at 4 ℃ and processed within 24 h after collection. After washed in running water, the surfaces of the roots were sterilized by sequential immersion in 0.1% Tween 20 for 5 min, following by 75% alcohol for 5 min, a solution of 2% sodium hypochlorite for 5 min, and 10% sodium bicarbonate solution for 5 min. Samples were washed in sterile distilled water at least three times to remove surface sterilization agents. Meanwhile, an aliquot (0.2 mL) of the last washing water was spread on agar plates, and incubated at 28 ℃ for 7 days to con rm surface sterilization. The surface-sterilized roots were then aseptically sectioned by commercial blender and spread onto the isolation media Gao's No. 1 medium supplemented with cycloheximide (25 mg/mL) and nystatin (10 mg/mL), followed by incubation at 23 ℃ for up to 2 weeks. Isolate LRE541 was puri ed with new Gao's No. 1

16S rDNA sequencing and phylogenetic analysis
The genomic DNA (gDNA) of isolate LRE541 was extracted as described by Orsini et al. (83) with minor adjustment. The universal bacterial primers targeted 16S rDNA, 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1525 R (5′-AAGGAGGTGATCCAGCCGCA-3′), were used for polymerase chain reaction (PCR) ampli cation following the manufacture's protocol (Takara, Japan) with optimized adjustment. The checked PCR products were directly subjected to cycle sequencing using an ABI3100 automated sequencer (Beijing Sangon Biotech, Beijing, China). The sequenced 16S rDNA of isolate LRE541 was matched with the nearest gene sequences of Streptomyces spp. retrieved from a public database using the EzBioCloud tool with Clustal W program. The phylogenetic tree was constructed by using the neighbor-joining algorithms (84) and p-distance model with bootstrap analysis of 1,000 replicates (85) in the MEGA version 7.0 package.
The 16S rDNA sequence of isolate LRE541 has been submitted to the GenBank nucleotide sequence databases under accession no. MK138546.

Fermentation and Extraction of isolate LRE541
Isolate LRE541 cultured on a slant agar medium was inoculated into 500 mL Erlenmeyer asks each containing 100 mL of the seed medium consisting of 15 g/L soluble starch, 10 g/L soybean powder, 1 g/L NaCl, 5 g/L glucose, 5 g/L tryptone and 5 g/L CaCO 3 (pH 7.3). The asks were cultivated on a rotary shaker (150 rpm) at 28 °C for 3 days. Then the seed culture (11 mL) were transferred into 1,000-mL Erlenmeyer asks each containing 500 mL of Gao′s liquid medium and incubated at 28 ℃, 150 rev min − 1 for 9 d. After the fermentation process, the biomass was discarded by centrifugation at 10000 g for 20 min while the supernatant was harvested and extracted three times with an equal volume of ethyl acetate. Then the ethyl acetate fractions were concentrated at 40 ℃ in a rotary vacuum distillation apparatus, and dissolved in DMSO (1 mg/mL) for the investigation of antitumor and antimicrobial activities.
In Vitro Cytotoxic Assay

Cell cultures
The antitumor activity of the EtOAc extract was examined against a wide variety of cell lines, including nine human cancer cell lines (HepG-2, SW-1190, CAL-27, 7901, RKO, MCF-7, Hela, K562, A549) and one normal human pulmonary artery endothelial cell (HPAEC), which were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were incubated in RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine and 100 units/mL streptomycinpenicillin, then maintained in a humidi ed atmosphere of 5% CO 2 at 37 °C.

Cytotoxicity assay
The cell survival was evaluated using the MTT assay (42). In short, the cells were seeded at a density of 1 × 10 4 cells/well in 96-well plates for 24 h, then the medium was replaced with fresh medium containing different concentrations of ethyl acetate extract (EtOAc extract) for 48 h. Cells treated with DMSO alone were set as controls. Later, 10 µL MTT (5 mg/mL) reagent was added to each well and incubated for an additional 4 h at 37 °C. Absorbance (490 nm) of the medium was measured using a microplate reader (Thermo Scienti c Multiskan GO, Finland).
Cell apoptosis analysis RKO and 7901 cells were seeded in 6-well plates for 24 h, then incubated with EtOAc extract of various concentrations (0, 0.5, 1, and 2 µg/mL) for 48 h. Then, both of the cells were collected and washed with PBS three times. Afterwards, cell samples were stained with uorescein 5-isothiocyanate (FITC)conjugated annexin V and PI following the manufacturer′s instructions (Zoman Biotech, Beijing, China). Data were obtained and analyzed using a FACS-Canto ow cytometer (BD Biosciences, San Jose, CA, USA) with FlowJo software.

Cell cycle analysis
Cell cycle analysis was also performed by ow cytometry (86,87). In brief, RKO and 7901 cells were

Evaluation of antimicrobial activities
The antimicrobial activities of the secondary metabolites (EtOAc extract) from strain LRE541 were investigated against four human pathogens, including Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 25923 representing Gram-positive bacteria, Escherichia coli ATCC 25922 representing Gram-negative bacteria, a clinical isolated Pseudomonas aeruginosa strain representing freshly pathogenic multi-resistant bacterial strain and Candida albicans ATCC 66415 representing yeast-like fungi. The EtOAc extract was prepared in ten concentration gradients of 10-100 ug/mL for antimicrobial activity assays by the agar well diffusion method (40), and the antimicrobial activity was assessed by measuring the diameter (mm) of inhibition zone (59). Each test was conducted in sextuplicate, and the DMSO (0.5%) was used as the negative control.

Metabolite pro les by UHPLC-MS/MS Analysis
The EtOAc extract of strain LRE541 was subjected to a Vanquish UHPLC system equipped with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher) for metabolite pro le analyse. The processed samples were injected onto a Hyperil Gold column (100 × 2.1 mm, 1.9 µm) using a 16-min linear gradient at a ow rate of 0.2 mL/min. The eluents for the positive polarity mode were eluent A (0.1% formic acid in Water) and eluent B (Methanol), and for the negative polarity mode were eluent A The raw data from UHPLC -ESI -Q -TOF -MS and MS/MS were analyzed using the Compound Discoverer 3.1 (CD3.1, Thermo Fisher) to perform peak alignment, peak picking, and quantitation for each metabolite. The normalized data was used to predict the molecular formula based on additive ions, molecular ion peaks and fragment ions. And then peaks were matched with the mzCloud (https://www.mzcloud.org/) mzVault and MassList database to obtained the accurate qualitative and relative quantitative results.

Statistical Analysis
Data are expressed as the means ± SD for at least three independent experiments. SPSS software was applied to perform the statistical analysis, and the statistical differences between two groups were assessed by Student's t test. P < 0.05 was used as the criterion for statistical signi cance.

Declarations
Availability of data and materials All data generated or analyzed during this study are included in this paper (and its additional le).