Comparative Analysis of the Metabolites and Biological Activity of Cultivated and Wild Lignosus rhinocerotis

In this paper, Lignosus rhinocerotis (Cooke) Ryvarden (L. rhinocerotis) cultivated in rice medium (LRR) and in sawdust medium (LRS) was harvested. Then, in terms of the LRR, LRS, and wild L. rhinocerotis (LRW), the total flavonoid contents, total polyphenol contents, total polysaccharide contents, and metabolites were detected; antioxidants of their aqueous extracts and anti-inflammatory of their polysaccharides were performed. In addition, the possible mechanism of the polysaccharides of L. rhinocerotis inhibiting lung damage was elucidated. The results showed that 32 compounds were characterized in L. rhinocerotis, including flavonoids, terpenoids, lignans, and steroids and there were 20 compounds in cultivated and wild L. rhinocerotis; LRR has the highest total polyphenol and flavonoid contents, as well as ABTS and DPPH scavenging capacity. The total polysaccharide contents and the FRAP scavenging capacity of wild L. rhinocerotis were higher than those of cultivated L. rhinocerotis. The inhibition of polysaccharides of LRW (PLRW) on LPS-induced MRC-5 damage was stronger than that of the polysaccharides from cultivated L. rhinocerotis. The PLRW may alleviate lung damage by inhibiting the NLRP3 pathway and thereby suppressing the inflammatory response. In summary, both cultivated and wild L. rhinocerotis are abundant in bioactive components and have antioxidant and anti-inflammatory activities.


Introduction
Lignosus rhinocerotis (Cooke) Ryvarden, an ethnic medicinal mushroom of the Polyporaceae family, falls into the division Basidiomycota of fungi. L. rhinocerotis is geographically distributed only in tropical rainforests of South China, Thailand, Malaysia, Indonesia, Philippines, and Papua New. It has been extensively used in South China, Malaysia, and other Southeast Asian countries with significant therapeutic effects on cough and asthma [1,2]. The mature L. rhinocerotis consists of the sclerotium, stipe, and pileus. The sclerotium is the part of L. rhinocerotis with medicinal value. It is irregular hard mass with a diameter of 1-5 cm. The mature sclerotium is white, and it takes at least 3 months from mycelium to form sclerotium.
The cultivation methods mainly include solid culture and liquid culture. When the mycelium of L. rhinocerotis is cultured for one month, the mycelium is full of the medium and forms a layer of dense film on the surface. After three months of culture, mycelia begin to gather together to form a spherical sclerotium with a diameter of about 2 cm. At this time, sclerotium is small in size, light in color, and loose in texture. In about 6 months, sclerotium is dark and tight. A variety of active substances such as polysaccharide, polysaccharide-protein complex, protein, and protease can be extracted from sclerotium, which are anti-inflammation, antioxidative, antibacterial, and antivirus.
Mushrooms belonging to basidiomycetes are known for the presence of specific enzymes, called "ribotoxin-like proteins" whose prototype is Ageritin. This toxin possesses antifungal and antiviral activities and selective cytotoxicity against tumor cells [2]. Medicinal mushrooms produce a host of biochemicals with pharmacological effects on the process of growth, including ribotoxic proteins, bioactive peptides, polysaccharides, glycosides, terpenoids, alkaloids, steroids, and amino acids [3][4][5][6][7]. These bioactive substances are thought to have antitumor, antioxidant, anti-inflammatory, hypotensive, and hypocholesterolemic effects, as well as therapeutic effects on neurological diseases, liver system diseases, and respiratory system diseases [8][9][10][11][12]. In addition, the present reports have shown that the proteins and polysaccharides of L. rhinocerotis have antitumor, anti-inflammatory, and antioxidant effects [13][14][15][16]. However, the dramatic reduction in wild L. rhinocerotis has resulted in short supply and an artificial cultivation technique was implemented by Jamil et al. [17]. The phytochemical results showed that the total protein, total flavonoid, and total β-D glucan contents of cultivated L. rhinocerotis were higher than the wild. The mycelium could be harvested in artificially fertile living conditions, but the differences in the nutrients and the proportion of culture medium could account for its markedly different biomorphological properties, metabolites, and bioactivities. Few studies have been reported on the quality evaluation of cultivated L. rhinocerotis and whether cultivated L. rhinocerotis could be an alternative.
Pulmonary damage is an important part of the process of lung disease, and the overproduction of oxygen free radicals and inflammatory factors causes pulmonary damage, further leading to pulmonary fibrosis or lung cancer [18,19]. Therefore, inhibition of the effects of oxidation and pulmonary damage can effectively prevent lung disease. L. rhinocerotis plays a critical role in the treatment of lung diseases. Previous studies have reported that the hot water extract, cold water extract, and methanolic extract of L. rhinocerotis have good antioxidant capacity [20]. Muhamad et al. [1] reported that the hot water extract of L. rhinocerotis improves the symptoms of an OVA-induced airway inflammation model in mice.
In this study, the sclerotium of LRR and LRS was harvested. The phytochemical contents (total polyphenols, total flavonoids, and total polysaccharides) of LRR, LRS, and LRW were determined. We conducted a comparison of the metabolite differences between cultivated and wild L. rhinocerotis by UPLC-TOF-MS combined with principal component analysis (PCA) and hierarchical cluster analysis (HCA). In addition, the antioxidant ability of the aqueous extracts of L. rhinocerotis was detected by free radical scavenging experiments and the protective effect of the polysaccharides of L. rhinocerotis (PLR) on pulmonary injury was examined by in vitro experiments.

Extract Preparation.
The dry LRW, LRR, and LRS were powdered, and then, the powder suspended in 10 volumes of distilled water was ultrasound-assisted extracted for 1 h and filtered and the extraction operation was repeated once. The supernatants were combined, condensed and freeze dried, ground into powder, and stored at −20°C.
2.3.1. Polysaccharide Separation. Four volumes of 99.7% ethanol were added to the extraction solution, mixed, and rested at 4°C for 48 hours. The precipitates were collected by centrifugation, dissolved in distilled water, freeze dried, ground into powder, and stored at −20°C.

Total Polysaccharide, Phenolic, and Flavonoid Contents.
The sample solution was 25 mg of the dry aqueous extraction of LRW, LRR, and LRS dissolved in 1 mL of distilled water.

Total Polysaccharide Content (TPCC).
Based on the phenol-sulfuric method by Dubois et al. [21], all sample solutions were added to 9 volumes of 99.7% ethanol, mixed, and precipitated at 4°C for 48 h. The sediment was dissolved in distilled water after centrifugation at 4000 rpm for 10 min, and 500 μL of the solution was incubated with 500 μL of 5% phenol (w : w) and 2.5 mL of sulfuric acid (12 M) at 90°C for 15 min. The absorbance was measured at 490 nm with Ultraviolet-visible Spectrophotometer (AoYi-A390). The total polysaccharide content was expressed as μg glucose

Profile of the Metabolites Based on UPLC-TOF-MS.
Aqueous extraction was performed by an AB ExionLC UPLC system equipped with an AB Triple-TOF 6600 plus high-resolution mass spectrometer with an ACQUITY UPLC HSS T3 column (100 mm × 2:1 mm, 1.8 μm). Two microliters of sample solution were separated in the column at 45°C. Formic acid water (0.1%, mobile phase A) and formic acid-acetonitrile (0.1%, mobile phase B) were applied at a flow rate of 0.35 mL/min. The mobile-phase gradient elution procedure was as follows: Mass spectrum signal acquisition was performed in ESI+ and ESI− modes. The mass spectrometry conditions were set as follows: nebulizer gas and auxiliary gas pressure 55 psi, curtain gas pressure 55 psi, interface heater temperature 550°C, ion source temperature 550°C, and mass scan range 40~1000. Mobile phase A was the solution of aqueous containing 0.1% formic acid, and mobile phase B was the solution of acetonitrile containing 0.1% formic acid. The elution procedure (A : B (v/v) at time (min)) was as follows: (95 : 5) at 0 min, (95 : 5) at 2 min, (70 : 30) at 4 min, (50 : 50) at 8 min, (20 : 80) at 10 min, (0 : 100) at 14 min, (0 : 100) at 15 min, and (95 : 5) at 16 min, and the injection volume was 2 μL. The raw data were collected by UNIFI 1.8.1. software, and data processing was performed by Progenesis Q IV2.3 software (Nonlinear Dynamics, Newcastle, UK), including baseline filtering, peak identification, integration, retention time correction, peak alignment, and normalization (precursor tolerance: 5 ppm; product tolerance: 10 ppm; product ion threshold: 5%). The compounds were characterized based on their precise mass numbers, secondary fragmentation information, and isotopic distribution in The Human Metabolome Database (HMDB) and Lipidmaps (v2.3) as well as in the METLIN database. The data were extracted, the ion peaks with missing values ð0 valuesÞ > 50 % were removed from the group, and the 0 values were replaced by half of the minimum values and then scored according to the results of the compound characterization. The characterizations scored below 30 points were consid-ered inaccurate (full score 60) and deleted. It was necessary to convert the raw data, including retention time, m/z, and peak intensity, to the mass-retention time pair and peak intensity. All converted information was imported into SIMCA 14.1 (Umetrics, Sweden), and then, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were performed.
2.6. Antioxidant Activity 2.6.1. ABTS Assay. According to the method and modifying by Rajurkar and Hande [23]. Ten microliters of the sample solutions and 290 μL of ABTS+ radical solution mixed with 1 mL of potassium persulfate (0.7 mg/L) and 1 mL of ABTS (4 mg/L) at 25°C for 18 h in the dark were added to 96-well plates and kept at 25°C for 15 min. The absorbance was measured at 734 nm. The results are expressed as μg ascorbic acid equivalents per gram dry aqueous extraction (μg AAE/mg of DAE).

DPPH Assay. The method was modified by Pande and
Chanda [24]. Forty microliters of the sample solutions and 260 μL of DPPH+ radical solution (40 mg/L) were added to 96-well plates and kept at 25°C for 30 min. The absorbance was measured at 517 nm. The results were expressed as μg ascorbic acid equivalents per gram dry aqueous extraction (μg AAE/mg of DAE).

FRAP Assay.
Based on the phenol-sulfuric method by Rajurkar and Hande [23], the FRAP solution was mixed with 0.3 M sodium acetate solution, 10 mM TPTZ solution, and 20 mM ferric chloride solution at 10 : 1 : 1. Twenty microliters of the sample solution and 280 μL of the FRAP solution were added to 96-well plates and kept at 37°C for 10 min. The absorbance was measured at 593 nm. The results were expressed as μg ascorbic acid equivalents per gram dry aqueous extraction (μg AAE/mg of DAE).

Cell
Line. MRC-5, a human embryonic lung fibroblast line, was provided by Procell Life Science & Technology Co. Ltd. (Wuhan, China). Cells were cultured in complete growth medium: Minimum Essential Medium α ðMEM αÞ + 10% fetal bovine serum ðFBSÞ + 1% penicillinstreptomycin solution (P/S) and maintained in a humidified incubator at 37°C under an atmosphere of 5% CO 2 and 95% air. When the cells reached confluence, PBS was used to wash the cells twice and serum-free medium was used for further cell culture prior to subsequent experiments.
2.11. Western Blot. Rabbit anti-NLRP3 polyclonal antibody (diluted 1 : 1000) and rabbit anti-caspase-1 polyclonal antibody (diluted 1 : 1000) were used in these experiments. Rabbit anti-β-actin polyclonal antibody (diluted 1 : 1000) served as a loading control. As described in the literature, the proteins were separated by 10% (w/v) SDS-PAGE and transferred to polyvinyl fluoride (PVDF) membranes and incubated with primary antibodies overnight at 4°C, followed by horseradish peroxidase-(HRP-) conjugated goat anti-rabbit IgG (diluted 1 : 5000) at room temperature for 2 h. Blots were developed with enhanced chemiluminescence reagent and detected by the LAS4000 imager. Densitometric analysis of the blots was performed using Image-Pro Plus 6.0 software (Rockville, MD, USA).

Statistical
Analysis. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS for Windows, version 17). All of the data are expressed as the mean ± standard deviation (SD). Statistical differences between groups were analyzed using one-way analysis of variance (ANOVA) followed by a post hoc least-significant difference (LSD) test. p < 0:05 was considered to be statistically significant.

Proximate Analysis of the Physicochemical Content.
To explore the metabolite differences between cultivated and wild L. rhinocerotis, the physicochemical contents of LRR, LRS, and LRW were measured as shown in Table 1. The extraction rates of LRR, LRS, and LRW were 7.89%, 5.37%, and 1.86%, respectively. The TPCC of LRW was 73:15 ± 2:00 μg GE/mg of DAE, which was significantly higher than that of LRR and LRS. For TFC, LRR and LRW (4:69 ± 0:07 and 4:54 ± 0:16 μg QE/mg of DAE, respectively) were higher than LRS.

Metabolite Analysis.
The UPLC-TOF-MS technique with good efficiency and sensitivity is used to identify and characterize the biochemical compounds in food. In our study, UPLC-TOF-MS analysis led to the tentative characterization of 28, 22, and 25 compounds in LRR, LRS, and LRW, respectively. Twenty compounds were detected in LRR, LRS, and LRW, including flavonoids, terpenoids, alkaloids, and lignans. These compounds with their molecular formulas, retention times (RTs), ion modes, and errors in ppm are listed in Table 2. Figures 1(a) and 1(b) show the total ion chromatograms (both positive and negative ions) of the aqueous extracts of LRR, LRS, and LRW.

PLR Reduced the Expression of Cytokines after LPS-
Induced MRC-5 Injury. The expression of TNF-α and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The results showed that the release of TNF-α and IL-1β in the LPS group was significantly elevated compared to that in the control group (p < 0:01). Compared with the LPS group, the 160 μg/mL PLRW group and the 80 μg/mL PLRW group had significantly reduced expression (p < 0:01 ) and the other groups did not show significant difference ( Figure 3). Consequently, the levels of IL-6, IL-18, TNF-α, and IL-1β in response to the different concentrations of PLRW (40 and 160 μg/mL) were detected. Likewise, their expression in the LPS group was significantly higher than that in the control group (p < 0:01). Compared with the LPS group, the 160 μg/mL PLRW group had significantly decreased expression (p < 0:01), while the 40 μg/mL PLRW group did not show significant difference (Figure 3).

PLRW Inhibited the Activation of ROS, NLRP3, and
Caspase-1 after LPS-Induced MRC-5 Injury. Immunofluorescence assays were performed to measure the expression of ROS, NLRP3, and caspase-1. The results are shown in Figure 4. The levels of ROS, NLRP3, and caspase-1 in the LPS group were significantly increased compared to those in the control group (p < 0:01). Compared with the LPS group, the 160 μg/mL PLRW group had significantly reduced expression (p < 0:01), while the 40 μg/mL PLRW group did not show significant difference (p < 0:05).

PLRW Inhibited the Activation of NLRP3 and Pro-
Caspase-1 after LPS-Induced MRC-5 Injury. The Western blot results showing the expression of NLRP3 and procaspase-1 are in Figure 4. The levels of NLRP3 and procaspase-1 in the LPS group were significantly increased compared to those in the control group (p < 0:01). Compared with the LPS group, the 160 μg/mL PLRW group had signif-icantly reduced expression (p < 0:01), while the 40 μg/mL PLRW group did not show significant difference (p < 0:05).

Discussion
The bioactive compounds of medicinal mushrooms could be isolated from the mycelium, sclerotia and fruiting bodies during the growth process [25]. These biochemicals with pharmacological effects play a preventive and therapeutic role in disease. Polysaccharides are one of the therapeutic compounds of L. rhinocerotis, and numerous studies have shown that PLR is related to antitumor and immunomodulatory effects [26][27][28][29]. Phenolic compounds, as natural antioxidants, was able to scavenge free radicals, and the quantification of total phenolic content and total flavonoid content can be used as indicators to evaluate antioxidant capacity. Among the proximate analysis of physicochemical contents, the extraction rates of LRR (7.98%) was higher than those of LRS (5.37%) and LRW (1.86%) and the physicochemical content of cultivated LR was 4~5 times higher than wild L. rhinocerotis, which could be explained by the uncertain harvesting time and the harsh living conditions in the wild; conversely, the cultivated L. rhinocerotis was harvested under relatively fertile conditions. Per gram of dry aqueous extract, LRW had the highest polysaccharide content. LRS had the highest flavonoid and polyphenol contents. In short, both cultivated and wild L. rhinocerotis are abundant in bioactive compounds. The biochemical substances of medicinal mushrooms that have inhibitory and therapeutic effects on human diseases could be identified using mass spectrometry [30]. PCA and HCA were used to detect the metabolites within L. rhinocerotis. In the present study, we conducted multivariate analysis for the classification and characterization of biochemicals in the L. rhinocerotis samples. In the PCA and HCA score plots, the LRR samples, LRS samples, and LRW samples were separated, demonstrating that there were differences among LRR, LRS, and LRW. Modern pharmacology research has revealed the bioactivities and biochemicals of medicinal mushrooms [13,31]. At present, few studies have been reported on the metabolites of L. rhinocerotis. The results of the characterization of the metabolites of L. rhinocerotis showed that 13 flavonoids were isolated and identified, including 5 chalcones, 7 flavonoids, and 1 isoflavone. Cladrastin 7-O-glucoside (isoflavone glycosides) and lupinisoflavone M (isoflavones) were In each row, different letters (a, b, and c) mean significant differences (p < 0:05).

BioMed Research
International detected in LRR, isoliquiritin (flavonoid) was detected in LRR and LRS, and other flavonoids were detected in all of the samples. Lau et al. [32] used the UHPLC-ESI-MS/MS technique to analyze the metabolites in mycelium and culture broth under static liquid culture and shaking liquid culture conditions, and the main components were phenolic and fatty acids as well as lanostane triterpenoids. In this study, phenolic components, fatty acids, and lanostane triterpenes (ginsenoside III) were  BioMed Research International also detected. Moreover, 3-dehydroecdysone (insect ecdysone), 4β-hydroxywithanolide E (solanide), and 5dehydroepisterol (episterol) were found in all samples and the steroids were related to anti-inflammatory and antitumor activities [33]. Furthermore, (+)-eudesmin and dihydrodeoxy-8-epiaudiol, which are lignans found in LRR, LRS, and LRW, were used to suppress the growth of bacteria and inhibit the elongation of synapses [34,35].  The results were expressed as the mean ± standard deviation (n = 3); compared with the LPS group, * * p < 0:01 means statistically significant difference. Compared with the control group, ## p < 0:01 means statistically significant difference. 8 BioMed Research International The results were expressed as the mean ± standard deviation (n = 3); compared with the LPS group, * * p < 0:01 means statistically significant difference; * p < 0:05 means statistically difference. Compared with the control group, ## p < 0:01 means statistically significant difference. 9 BioMed Research International Most diseases are closely related to the excessive secretion of free radicals, and the excessive production of free radicals leads to oxidative damage, which exacerbates diseases and causes a series of pathologies; thus, the exploration of natural antioxidants is increasingly urgent [36]. The aqueous extracts of L. rhinocerotis have good antioxidant activity [37,38]. Antioxidant experiments, such as the ferric-reducing antioxidant power assay (FRAP), ABTS radical scavenging assay, and DPPH radical scavenging assay, were used to evaluate the antioxidant activity of various extracts [39]. For the ABTS and DPPH assays, the scavenging capacities of aqueous extracts of LRW and LRS were lower than that of LRR, which revealed that LRS has a stronger antioxidant activity. In the FRAP assay, LRW had the strongest FRAP scavenging capacity (7:37 ± 0:18 μg AAE/mg of DAE). This may be related to the high content of total polyphenols and flavonoids. The mechanisms of the inhibition of redox reactions by antioxidant ingredients are different, such as producing superoxide anions, facilitating metal ion chelation, and promoting free radical chain reactions.
The NLRP3 inflammatory vesicle complex is an important signaling molecule consisting of NLRP3, the junction protein ASC, and the effector pro-caspase-1, which can be activated by various pathogen-associated molecules [40]. ROS, one of the common inflammatory vesicle activators, are secreted at increased intracellular levels when stimulated by external factors, which can facilitate the separation of TRX from TXNIP and then the integration of TXNIP with NLRP3 inflammatory vesicles [41]. However, abnormal activation of the NLRP3 inflammatory vesicle complex plays an important role in lung injury. When cells are stimulated by adverse factors, ROS are activated and stimulates NLRP3 inflammatory vesicles, leading to the activation of inflammatory vesicle-mediated caspase-1 and initiates the downstream proinflammatory cytokines IL-6, IL-18, IL-1β, and TNF-α [42]. These cytokines are powerful promoters activating the cells to release more inflammatory factors, creating a cascade amplification effect, which in turn accelerates the process of tissue damage [43]. In this study, cytotoxicity and cytokine secretion were examined after LPS-induced MRC-5 cells were pretreated with PLR and PLR at a concentration of 20~320 μg/mL enhanced cell activity of LPS-induced MRC-5 cells. However, PLRR and PLRS did not show significant difference in the expression of IL-1β and TNF-α, while PLRW could significantly reduce their expression. PLRW shown good activity of antiinflammatory than PLRR and PLRS; this is might be associated with the higher polysaccharide content.

Conclusions
In this study, the differences between cultivated and wild L. rhinocerotis were elucidated. Both cultivated and wild L. rhinocerotis are abundant in bioactive components, and they both have antioxidant activities. This is the first time that metabolomics analysis has been applied to the study of L. rhinocerotis quality and the LRR, LRS, and LRW can be discriminated by untargeted metabolite profiling. In the antipulmonary injury experiments, the results suggested that wild L. rhinocerotis was more effective than cultivated. Moreover, PLRW plays an anti-inflammatory and antiapoptotic role in LPS-induced MRC-5 injury. PLRW may defend against cell damage by activating the NLRP3/caspase-1 pathway. In summary, both cultivated and wild L. rhinocerotis are abundant in bioactive components and have antioxidant and anti-inflammatory activities.