Anticancer and Antioxidant Activities in Ganoderma lucidum Wild Mushrooms in Poland, as Well as Their Phenolic and Triterpenoid Compounds

The goal of this study was to the assess anti-cancer and antioxidant properties of the Ganoderma lucidum fruiting body, and to identify bioactive compounds found in their extracts. Significant antiproliferative activity was observed against MCF-7, MCF-7/DX, LOVO, LOVO/DX, MDA-MB 231, SW 620, and NHDF cell lines. With IC50 values of 25.38 µg/mL and 47.90 µg/mL, respectively, the extract was most effective against MDA-MB 231 and SW 620 cell lines. The bioactive compounds were identified using an ACQUITY UPLC-PDA-MS system. The extracts contained 13 triterpenoids and 28 polyphenols from the flavonols, phenolic acids, flavones, flavan-3-ols, and stilbenes families. Ganoderic acid derivative was found to be the most abundant triterpenoid (162.4 mg/g DW), followed by ganoderic acid B (145.6 mg/g DW). Resveratrol was the most abundant phenolic in the extract (5155.7 mg/100 g DM). The findings could explain why G. lucidum extracts are used in folk medicine.


Introduction
Food's impact on the human body has received more attention in recent years. Lifestyle diseases are the leading cause of death in both Poland and other European countries. Cancer accounts for almost 27% of all deaths in the European Union [1]. Surgery is the most frequently used method of treating early-stage cancers [2], while later, chemotherapy and radiotherapy, hormone therapy, and immunotherapy are additionally used [3,4]. However, these methods have many side effects, such as high toxicity, pain, emotional stress, negative impact on fertility, and neurological sequelae [5]. In order to alleviate the negative effects and support the effectiveness of the therapies used, biologically active compounds found in medicinal plants can be used. Ganoderma lucidum (also known as "the fungus of immortality") is a traditional medicinal mushroom and one of nature's most effective adaptogens, acting as a regulator of biological activities. For over a thousand years, the Chinese have used G. lucidum for medicinal purposes. G. lucidum is still used as an adjuvant treatment for a variety of ailments in China as a traditional herbal medicine. Traditional herbal medicine is thought to have precise clinical effects by reducing inflammation and regulating immunity, as well as antiaging properties [6]. G. lucidum's pharmacological properties stem from its distinct chemical composition, which includes bioactive compounds such as terpenoids, polysaccharides, phenolics, steroids, proteins, glycopeptides, and fatty acids [7,8]. These substances have been linked to G. lucidum's antioxidant, antibacterial, anti-inflammatory, and anti-tyrosinase actions [9,10]. G. lucidum has also been used as an adjuvant in the treatment of a number of medical conditions, including insomnia, anorexia, chronic hepatitis, and hypertension [11]. Clinical studies show that the use of G. lucidum extracts in Table 1 and Figure 1 show the 28 compounds identified in the G. lucidum fruiting body methanolic extract.

Phenolic Acids and Derivatives
Peak 1 has an [M-H] molecular ion at m/z 827.0046 and was identified as tricaffeoylglucosyl-glucoside (Table 1) based on its product ions at m/z 665, 383, 341, 221, and 179, which is consistent with the data reported by Benayad et al. [22]. Peak 2 was identified as tricaffeoyl-glucosyl with an m/z of 665.0901. The fragment signals were found in the MS data at m/z 383, 341, 221, and 128 [22]. Peak 3 was identified as caffeoyl trihexoside because of its [M-H] ion at m/z 665.0853 and product ion at m/z 341 (caffeoyl hexose loss of two hexoses). Based on its UV-vis spectra and comparison with the corresponding authentic standard of protocatechuic acid, compound 4 with an m/z at 315.1666 was tentatively identified as protocatechuic acid hexoside. The MS data revealed a fragment signal at m/z 153 (hexose loss), which is typical of this compound. The observed fragmentation pathways, as well as the UV spectra and retention times of peaks 5 and 10, are consistent with authentic standards of 1-and 5-caffeoylquinic acids (

Quantitative Analysis of Polyphenols
The G. lucidum fruiting bodies had a total phenolic content of 13,991.10 mg/100 g dry weight (DW) ( Table 1). Stilbenes (5155.70 mg/100 g DW) were the most abundant group of compounds found in the samples, followed by flavones (total of 4572.51 mg/100 g DW). Resveratrol (compound 24) and apigenin (compound 28) were the most abundant compounds in the extract by content ( Figure 2 Based on the fragmentation pattern found in the literature, compounds 9 and 19 were tentatively identified as diosmetin pentosidine and chrysin-6-C-arabinoside-8-C-glucoside, respectively [26]. Peaks 27 and 28 were the precursors of apigenin ([M-H]¯ at m/z 269). Compound 27 was identified as an apigenin derivative (Table 1) (Table 1), based on previously published data [27].

Quantitative Analysis of Polyphenols
The G. lucidum fruiting bodies had a total phenolic content of 13,991.10 mg/100 g dry weight (DW) ( Table 1). Stilbenes (5155.70 mg/100 g DW) were the most abundant group of compounds found in the samples, followed by flavones (total of 4572.51 mg/100 g DW).
Resveratrol (compound 24) and apigenin (compound 28) were the most abundant compounds in the extract by content ( Figure 2). The phenolic acid content was the lowest, at 912.38 mg/100 g DW (Table 1). Apigenin was the dominant compound in the flavone group, accounting for approximately 88 percent of the group's content. The A-type procyanidin dimer accounted for approximately 41% of the flavan-3-ols, while the dominant compounds in the flavonols group were quercetin derivatives (about 59 percent). Caffeic acid derivatives predominated in the phenolic acid group, with 1-caffeoylquinic acid having the highest content (~55%).
Various concentrations of polyphenolics have previously been reported in the G. lucidum fruiting body. Due to differences in the determination method (application of the Folin-Ciocalteu method) or extraction technique, comparing those data with our results is difficult. Lin et al. [28] used CO2 extraction to obtain polyphenolic compounds similar to ours (4131-6376 mg/100 g DW), and Sheikh et al. [21] used the Folin-Ciocalteu method (4601-7143 mg/100 g). Kim et al. [20] obtained much lower levels of polyphenols (16.2 mg/100 g DW). Using the HPLC technique, they identified only ten compounds from the flavonol, phenolic acid, and flavone groups. Furthermore, Dong et al. [7] obtained much lower results (up to 468 mg/100 g DW) in mushrooms dried using various techniques. Yahia et al. [9] discovered nine phenolic compounds amounting to 48.47 mg/100 g DW using HPLC-ESI-MS.
So far, the health-promoting properties of Ganoderma fungi have been attributed primarily to the presence of triterpenoid compounds [29]. However, the high polyphenol The phenolic acid content was the lowest, at 912.38 mg/100 g DW (Table 1). Apigenin was the dominant compound in the flavone group, accounting for approximately 88 percent of the group's content. The A-type procyanidin dimer accounted for approximately 41% of the flavan-3-ols, while the dominant compounds in the flavonols group were quercetin derivatives (about 59 percent). Caffeic acid derivatives predominated in the phenolic acid group, with 1-caffeoylquinic acid having the highest content (~55%).
Various concentrations of polyphenolics have previously been reported in the G. lucidum fruiting body. Due to differences in the determination method (application of the Folin-Ciocalteu method) or extraction technique, comparing those data with our results is difficult. Lin et al. [28] used CO 2 extraction to obtain polyphenolic compounds similar to ours (4131-6376 mg/100 g DW), and Sheikh et al. [21] used the Folin-Ciocalteu method (4601-7143 mg/100 g). Kim et al. [20] obtained much lower levels of polyphenols (16.2 mg/100 g DW). Using the HPLC technique, they identified only ten compounds from the flavonol, phenolic acid, and flavone groups. Furthermore, Dong et al. [7] obtained much lower results (up to 468 mg/100 g DW) in mushrooms dried using various techniques. Yahia et al. [9] discovered nine phenolic compounds amounting to 48.47 mg/100 g DW using HPLC-ESI-MS.
So far, the health-promoting properties of Ganoderma fungi have been attributed primarily to the presence of triterpenoid compounds [29]. However, the high polyphenol content found in our study could indicate that this class of compounds plays an important role in shaping the therapeutic properties. Polyphenols, which have antioxidant and anti-inflammatory properties, have a wide range of health-promoting properties [30]. Table 2 and Figure 3 show the 13 triterpenoids identified in the G. lucidum fruiting bodies.  Table 2 and Figure 3 show the 13 triterpenoids identified in the G. lucidum fruiting bodies.   Table 2.

Characterization and Quantification of Triterpenoids
Analysis revealed the presence of typical Ganoderma species triterpenoid compounds. All triterpenoid compounds were identified using data from the literature.   Table 2.
Analysis revealed the presence of typical Ganoderma species triterpenoid compounds. All triterpenoid compounds were identified using data from the literature.
Peak 1 was identified as ganoderic acid C2 with [M-H] at m/z 517.3228 and a fragmentation ion at m/z 499 (Table 2) [29]. Peak 2 contained a pseudomolecular ion at m/z 529.2790 that fragmented at m/z 511 and was identified as ganoderic acid C6 [10]. Based on its [M-H] at m/z 459.2761, fragmentation ion at m/z 441, and literature data, compound 3 was identified as lucidenic acid N [29]. Peak 4 was identified as ganoderic acid G, with [M-H] at m/z 531.2991 and fragmentation ions at m/z 513 and 469 [10]. Based on literature data [8], peak 5 was identified as ganoderenic acid B, with its [M-H] at m/z 513.2840 and a fragmentation ion at m/z 495. Compound 6 was identified as ganoderic acid B, with [M-H] at m/z 515.2890 and fragmentation ion at m/z 497 [10]. Peak 7 contained a pseudomolecular ion with a mass of 529.2063, which fragmented at m/z 511 and was identified as a ganoderic acid derivative [10]. According to its fragmentation pathway and literature data, compound 8 was identified as lucidenic acid A [29]. Compound 9, identified as ganoderenic acid K, had a [M-H] of 571.2933 and a fragmentation ion of 553 [29]. Compound 10 was identified as ganoderic acid AM1 (Table 1) [29]. Peaks 11 and 12, with [M-H] at m/z 573.3075 and 569.2740, respectively, were identified as ganoderic acid K and F [8,29]. Compound 13 was identified as ganoderic acid A based on a ganoderic acid authentic standard and the literature [10,31].
The total triterpene concentration determined in the G. lucidum fruiting bodies was 769.1 mg/g of extracts DW. Ganoderic acid derivatives (total 478.9 mg/g DW) were the most abundant, followed by ganoderenic acid derivatives (total 308.0 mg/g DW) (Figure 4). The lucidenic acid derivative concentration was 36.8 mg/g DW (Table 1).  [10]. Peak 7 contained a pseudomolecular ion with a mass of 529.2063, which fragmented at m/z 511 and was identified as a ganoderic acid derivative [10]. According to its fragmentation pathway and literature data, compound 8 was identified as lucidenic acid A [29]. Compound 9, identified as ganoderenic acid K, had a [M-H]¯ of 571.2933 and a fragmentation ion of 553 [29]. Compound 10 was identified as ganoderic acid AM1 (Table 1) [29]. Peaks 11 and 12, with [M-H]¯ at m/z 573.3075 and 569.2740, respectively, were identified as ganoderic acid K and F [8,29]. Compound 13 was identified as ganoderic acid A based on a ganoderic acid authentic standard and the literature [10,31]. The total triterpene concentration determined in the G. lucidum fruiting bodies was 769.1 mg/g of extracts DW. Ganoderic acid derivatives (total 478.9 mg/g DW) were the most abundant, followed by ganoderenic acid derivatives (total 308.0 mg/g DW) ( Figure  4). The lucidenic acid derivative concentration was 36.8 mg/g DW (Table 1). These findings are similar to those reported by Lin et al. [28]. Total triterpenoid concentrations in their research on supercritical fluid extraction ranged from 196.03 to 643.06 mg/g DW. The concentration of triterpenoids in the study by Taofiq et al. [10] ranged between 280.46 and 531.21 mg/g, depending on the extraction technique. Bidegain et al. [32] obtained lower results. Triterpenoid compound concentrations in their study ranged between 37.4 and 47.6 mg/g, depending on the cultivation method.

Antioxidant Activity
Because of its high triterpenoid and polyphenolic concentrations, G. lucidum has significant antioxidant capacity according to the literature. The antioxidant activity of G. lucidum was assessed in this study using the DPPH and ABTS radical scavenging activity as well as the FRAP assay. DPPH, ABTS, and FRAP values were 51.30, 81.26, and 49.87 µmol of Trolox per 1 g DW, respectively, as shown in Table 3. Resveratrol and apigenin, as major components of the extract, as well as ascorbic acid were taken as positive controls. These findings are similar to those reported by Lin et al. [28]. Total triterpenoid concentrations in their research on supercritical fluid extraction ranged from 196.03 to 643.06 mg/g DW. The concentration of triterpenoids in the study by Taofiq et al. [10] ranged between 280.46 and 531.21 mg/g, depending on the extraction technique. Bidegain et al. [32] obtained lower results. Triterpenoid compound concentrations in their study ranged between 37.4 and 47.6 mg/g, depending on the cultivation method.

Antioxidant Activity
Because of its high triterpenoid and polyphenolic concentrations, G. lucidum has significant antioxidant capacity according to the literature. The antioxidant activity of G. lucidum was assessed in this study using the DPPH and ABTS radical scavenging activity as well as the FRAP assay. DPPH, ABTS, and FRAP values were 51.30, 81.26, and 49.87 µmol of Trolox per 1 g DW, respectively, as shown in Table 3. Resveratrol and apigenin, as major components of the extract, as well as ascorbic acid were taken as positive controls. Ascorbic acid was characterized by the highest value of antioxidant capacity in all the methods used. In the case of the DPPH method, the value of the antioxidant capacity of pure ascorbic acid was over 12 times higher than that of G. lucidum. For the ABTS and FRAP methods, the values were about 6 and 10 times higher. The results obtained for apigenin were comparable with those of G. lucidum extract. In the case of resveratrol, a higher value of antioxidant capacity was observed compared to the G. lucidum methanolic extract. The value was 1.9 times higher for the DPPH method, 11.7 times higher for ABTS, and over 32 times higher for FRAP.
The content of bioactive compounds, such as polyphenols and triterpenoids, influences antioxidant capacity. Dong et al. [7] discovered a strong correlation between DPPH, ABTS, and FRAP antioxidant capacities and polyphenolic and triterpenoid content in G. lucidum. Saltarelli et al. [38] also reported similar findings. The impact of bioactive compounds on antioxidant capacity formation has been discussed in the literature. Procyanidin polymers have a high antioxidant capacity, according to Rice-Evans et al. [39], due to the effects of hydroxylation and glycosylation on the B ring. In a study of the tocopherols found in sour jujube, Qiao et al. [40] discovered that some triterpenoids have more than 15 times the antioxidant activity of ascorbic acid, while others have a low antioxidant activity.

Antiproliferative Activity
The antiproliferative activity of G. lucidum was evaluated in ethyl acetate/hexane (1:1, v:v) mixture) after 72 h of incubation with MCF-7, MCF-7/DX, MDA-MB 231, LOVO, LOVO/DX, SW 620, and NHDF cell lines by the MTT assay [41]. The results obtained for breast and colon cancer cells and for normal cells are given in Figures 5-7, respectively. The IC 50 values are summarized in Table 4.    The results are the mean ± SD of three independent experiments. The significance of the differences was determined by Student's t-test. *** p < 0.001.  The results are the mean ± SD of three independent experiments. The significance of the differences was determined by Student's t-test. *** p < 0.001.
Here we observed that the extract obtained from G. lucidum exerted a significant antiproliferative effect on all tested cancer cell lines in a dose-dependent manner. Importantly, the extract did not exhibit cytotoxicity on normal cells up to 160 µg/mL, which indicates a specific antitumor activity.
The extract was most effective against MDA-MB 231 and SW 620 cell lines with the IC 50 values of 25.38 µg/mL and 47.90 µg/mL, respectively. In contrast, doxorubicin-resistant MCF-7/DX and LOVO/DX cell lines were found to be less sensitive to the extract at concentrations up to 160 µg/mL. In addition, the results showed that the extract was more effective against drug sensitive MCF-7 and LOVO cells compared to doxorubicin-resistant MCF-7/DX and LOVO/DX cells. The highest extract concentration, of 320 µg/mL, induced significant antiproliferative effects on all tested cancer cells line, achieving growth inhibition over 50% (from 53% up to 91%, depending on cancer cells). However, it should be noted that at this concentration, the extract also caused an important antiproliferative effect on normal cells (inhibition by 40%).
G. lucidum is a commonly used herbal medicine in many oriental countries [42] and has been studied by several research groups. Stojković et al. [43] evaluated the cytotoxic effect of two G. lucidum extracts from Serbia and China. They reported no cytotoxic effect of the Chinese extract on the HCT15 colon cancer cell line or MCF-7 cell line. It had a cytotoxic effect against MCF-7 cells with GI50 309.66 µg/mL but had no cytotoxic effect on HCT15 cells. In another study, an ethanolic extract containing phenolic compounds showed antiproliferative activity on HeLa (cervical carcinoma cell line), A549 (lung cancer cell line), EA.hy 926 (permanent human cell line derived by fusing human umbilical vein endothelial cells (HUVEC) with human lung adenocarcinoma epithelial cells (A549)), and colon LS174 (colon cancer cell line), as presented by Veljović et al. [44]. The authors found significant correlations between the antiproliferative effect and the total phenolic compounds/glucan content. The most abundant phenols in this extract were hesperetin and naringenin. In our study, a significant part of the antiproliferative activity of our extract towards cancer cells might be attributed to the high content of phenolic compounds: resveratrol had 5155.70 mg and apigenin had 4039.08 mg on 13,991.10 mg of total polyphenolic compounds per 100 g of the extract. Both phytochemicals are known for their potent anticancer activity, as well as chemopreventive and chemoprotective effects [45]. Our data confirm that resveratrol at low concentrations (up to 34 µg/mL) has significant antiproliferative activity on breast and colon cancer cell lines (data not shown).
In addition to phenolic compounds, other phytochemicals may also be responsible for cytotoxicity of the G. lucidum extracts towards cancer cells. Raj et al. [46] reported antiproliferative effects of polysaccharides and triterpenoids extracted from G. lucidum. In particular, triterpenes known as ganoderic acids are the important fractions responsible for the therapeutic efficacy of G. lucidum. Ganoderic acids are capable of inducing apoptosis and autophagy in various cancer cell types [47]. Our extract contains 13 different triterpenes in the amount of 769.1 mg per g of the extract, which may act synergically with phenolic compounds in anticancer effects. Naturally occurring compounds, in addition to their direct effect on cancer cells, often exhibit immunomodulatory effects, which may also induce an anti-neoplastic response [48]. Ganoderma has been shown to be a promising anticancer immunotherapy agent. Together with relatively low toxicity, the extract may be used in combination therapy or as a dietary supplement [49].

Extraction Procedure
Phenolic compounds were isolated from the lyophilized powder by extraction with 80% ethanol. The ratio of this solvent to the raw material was 3:1 (v/v). After filtration, the solution was concentrated with a vacuum pump and freeze-dried and then analyzed. A total of 0.1 g of sample was mixed with 5 mL of 30% UPLC-grade methanol, sonicated (20 min), centrifuged (19,000 g/10 min), and filtered (hydrophilic PTFE 0.20 µm membrane (Millex Samplicity Filter, Darmstadt, Germany)).

Determination of Antioxidant Activity
The DPPH, ABTS, and FRAP assays were performed in the same manner as previously described by Yen and Chen [51], Re et al. [52], and Benzie and Strain [53], respectively. All measurements were taken on a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). The standard curve was created by varying the concentrations of Trolox. As positive controls, resveratrol, apigenin, and ascorbic acid in concentrations of 1mg/mL were determined. The results are expressed in terms of µMol of Trolox equivalents (TE) per 1 g of extract (µmol TE/1 g).

Cell Lines and Culture Conditions
The study used seven cell lines: MCF-7, MCF-7/DX (doxorubicin-resistant subline of MCF-7 cells), and MDA-MB-231 from breast cancer; LOVO, LOVO/DX (doxorubicinresistant subline of LOVO/DX), and SW620 from colorectal cancer; and normal human dermal fibroblast from colorectal cancer (NHDF). The ATCC collection was used to obtain all cell lines. LOVO/DX and MCF-7/DX were created by cultivating parental counterparts in the presence of low concentrations of doxorubicin (DX). Cultures were grown in a humidified incubator with 5% CO 2 , 37 • C, and 95% humidity. The cell lines MCF-7/DX, MCF-7, MDA-MB-231, and NHDF were cultured in DMEM medium supplemented with 10% FBS, 2 mM L-glutamine, and 20 g/mL gentamicin sulfate. LOVO, LOVO/DX, and SW620 cell lines were grown in DMEM/F12 medium with the aforementioned supplements. At 37 • C, cells were passaged twice a week with TrypLE Express solution, then reduced and resuspended in culture medium.

Tested Compound
G. lucidum extract was dissolved in DMSO to make a stock solution of 64 mg/mL and stored at −20 • C. Before each experiment, a 100× dilution of the stock solution in a culture medium was used to make the working solution. The cells were subjected to serial dilutions of the tested extract (at final concentrations of 20, 40, 80, 160, and 320 g/mL). In the highest concentration of the extract, the final DMSO concentration in the cell culture did not exceed 0.5 percent.

MTT Assay
The MTT tests were carried out in accordance with the standard procedure outlined in the literature [54]. Using TrypLE solution, the cells were detached from the culture bottle surface (when confluency was greater than 70%), counted, and resuspended in full culture medium. The cells were then seeded in 96-well plates at 2.5 × 104 cells per well and incubated for 24 h. The cells were treated with various concentrations of the tested extract the next day and incubated for another 72 h. The medium was removed from the wells after incubation, and 50 µL of 1 mg/mL MTT solution in MEM was added to each well. Plates were placed in an incubator (at 37 • C) for 2 h. For 30 min in the dark, formazan crystals were solubilized in 100 µL of isopropanol. Finally, absorbance at 555 nm was determined using a Wallac 1420 Victor2 microplate reader (PerkinElmer, Waltham, MA, USA).
The extract's antiproliferative effect was compared to control cell cultures (without the extract) and expressed as E/E0 × 100 percent values. The IC 50 values were calculated based on the dose-response curves. The IC 50 is defined as the concentration of a tested extract that inhibits cell growth by 50%.

Statistical Analyses
The data of antiproliferative activity were expressed as the mean ± standard deviation of eight replicates from three independent experiments. The t-test was used to perform statistical analysis on the experimental data. GraphPad Prism 6 software was used for statistical analysis (InStat Software, San Diego, CA, USA). At the confidence level of p < 0.05, significant differences between each set of data were considered. For bioactive compounds and antioxidant activity, the data were expressed as the mean ± standard deviation of three replicates. Statistical analysis was conducted using Statistica 13.1 software (StatSoft, Kraków, Poland). Significant differences (p ≤ 0.05) between means were evaluated by one-way ANOVA and Duncan's test.

Conclusions
In this study, anticancer and antioxidant activities as well as bioactive compounds of the Ganoderma lucidum fruiting body were evaluated. The results demonstrate that the extract from G. lucidum is a rich source of bioactive components such as phenolic compounds and triterpenoids. Our study provides significant insight into the antiproliferative effects of phenolic compounds and triterpenoids contained in the extract of Ganoderma lucidum on breast cancer cell lines (MCF-7, MCF-7/DX, MDA-MB-231), a colorectal cancer cell line (SW 620), and colon cancer cell lines (LOVO, LOVO/DX). The anticancer activity of these compounds should be further investigated in terms of causing cancer cell death through various pathways that could help develop therapies against colon, colorectal, and breast cancer. This shows that G. lucidum extracts could be useful in the fight against diseases related to oxidative stress such as cancer.