Introduction

Cancer, diabetes, immune-system decline, cardiovascular diseases, and brain dysfunction are some of the degenerative diseases associated with reactive oxygen species (ROS) and aging [1]. Their linkage to free radicals is associated with the postulation that ROS-mediated damages to macromolecules and deoxyribonucleic acid (DNA) tend to accumulate with time and has been considered as a major form of endogenous damage that results to aging [2]. Mutagens such as hydroxyl radicals or hydrogen peroxide are also side-products of normal human body metabolism [3]. Humans have always embarked on the consumption of plant foods (fruits, red wines, fruits, and juices) and some mushrooms (to an extent) with the belief that they can confer them with some levels of protection against oxidative damages and diseases such as cancer [4]. This presumed protection was due to the ability of the antioxidants contained in these natural foods to scavenge the generated free radicals and keep their body healthy [5].

Before now, chemically-sourced antioxidants such as beta hydroxy acid (BHA) and butylated hydroxytoluene (BHT) have been commonly used in the stabilization of foods. However, these synthetic antioxidants were reported to be carcinogenic in experimental animals [6]. Consequently, efforts are geared towards finding alternative sources of natural antioxidants, especially from natural sources. In this respect, mushrooms have received much attention [7] considering that they have served as a major source of dietary nutrients and wholesome body benefits for ages due to their high phenolic contents, low fat and caloric contents, abundance in essential amino acids (leucine, cysteine, methionine) minerals (iron, zinc, manganese), and vitamins (vitamin C, folate, vitamin D, riboflavin, niacin, thiamin) [8]. Polyphenols are one of the non-essential dietary components with proven capabilities towards atherosclerosis and cancer inhibition [9]. The biological activity of polyphenols is related to their metal chelating, lipoxygenase inhibiting, and free radicals scavenging activities. Some of the previously identified polyphenols in the edible mushrooms include gallic acid, quercetin, pyrogallol, homogentisic acid, 5-sulfosalicylic acid, protocatechuic acid, chlorogenic acid, p-coumaric acid, o-coumaric acid, myricetin, resveratrol [10].

Several mushroom species contain several compounds with both medicinal and nutritional values. Mushrooms also contain other biologically active secondary metabolites such as polyketides, steroids, and terpenes [11] whose contents vary across mushroom species. Solvent polarity also contributes to the variation in the quantity of the extractable compounds from several mushrooms due to the differences in the solubility of the compounds in various compounds. Acetone and ethyl acetate have previously reported as effective solvents for the extraction of phytochemicals from biological extracts [12]. The justification for this study is based on the recent US breast cancer statistics report 2018, where breast cancer was projected as a leading cause of death around the world. According to the report, 266,120 new breast cancer cases are expected in the United States of America (USA) by 2018, while 63,960 new non-invasive cases are anticipated. Mortality-wise, approximately 40,920 breast cancer-related deaths are expected in the USA in 2018 [13]. These are challenging findings that require expedient actions towards diversifying the natural sources of anticancer agents. Hence, in this study, the anticancer activities of acetone and ethyl acetate extracts of white Hypsizygus tessellatus caps were investigated against 2 human breast cancer cell lines and Vero cells. The white variant of H. tessellatus (commonly called Bunapi shimeji) was selected for this study because there is no supportive evidence on its anticancer activity against any human breast cancer cell line despite its availability in the study area.

Materials and methods

Mushroom sample

The mushroom (white H. tessellatus), henceforth referred to as Bunapi shimeji, was identified based on literature evidence and supplied by a local supplier of biological materials (Orioner High-Tech Sdn. Bhd) in Kuantan, Pahang, Malaysia.

Chemicals and cell lines

Sodium carbonate anhydrous 99.99% purity trace metal basis (TMB), ferric chloride (> 99% purity), 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide, aluminum chloride (99.99% purity TMB), gallic acid (> 98% purity), Folin–Ciocalteu reagent, ascorbic acid (99% purity), potassium phosphate (monobasic and dibasic (99.95% purity)), quercetin (> 95% HPLC grade), potassium ferricyanide (99.5% REACH compliance), trichloroacetic acid (TCA > 99% purity CAS grade) Dulbecco’s modified eagles medium (DMEM), penicillin/streptomycin solution, fetal bovine serum (FBS), and phosphate buffered saline were bought from Merck-Darmstadt, Germany. MCF-10a, MDA-MB-231, and MCF-7 were procured from the American Type Culture Collection (ATCC- Manassas, VA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) kit was purchased from Solarbio-China.

Mushroom preparation and extraction

The caps of the mushrooms were first separated from the mushroom stems. The separated mushroom caps were dried to a regular weight in an incubator at 70 °C. Then, the dried caps were pulverized in a grinder, packaged in a dark plastic bag, and stored at 4 °C prior to usage. The pulverized mushroom caps (180 g) was weighed and added into two 500-mL flat bottom conical flasks. Then, 500 mL of either acetone or ethyl acetate (99%) was added to each flask. The flasks were sealed with aluminum foil and mounted on a shaker rotating at a speed of 250 revolutions per minute (rpm). The flasks were shaken on the shaker for 24 h at room temperature (25 ± 2.4 °C). After 24 h, the content of the flasks was filtered through several changes of Whatman no. 1 filter paper, then through a 0.45 µm syringe filter, concentrated in a rotary evaporator, and allowed to vaporize in a hood chamber. The dried extracts were collected in sterile universal bottles and preserved at − 20 °C prior to characterization.

Total phenolic content (TPC)

The extracts were characterized for TPC using the method described by Seonwook et al. [14]. Briefly, the extracts were first reconstituted in distilled water at the concentration of 1 mg/mL. Then, 1 mL of the reconstituted extract was added to 100-mL volumetric flasks. Then, 45 mL of distilled water was added to the flasks before adding Folin–Ciocalteu phenol reagent (1 mL). The flasks were thoroughly mixed and incubated with mild shaking for 3 min at ambient temperature. Then, 2% sodium carbonate solution (3 mL) was added to the flasks before further incubation for 2 h at ambient temperature with occasional shaking. After about 2 h of incubation, the absorbance of the solution was read in a spectrophotometer (Bibby Scientific Limited) at 760 nm. The analysis was performed in triplicate using gallic acid as a standard phenolic source. The results were presented as micrograms of gallic acid equivalents (µg GAE/mg) of the extract.

Total flavonoids content (TFC)

The flavonoids content of the extracts was determined using the method reported by Muhammad et al. [15]. In this method, 1 mL of 2% AlCl3 (prepared in absolute methanol) was incubated in a glass tube with 1 mL of the extract solutions (1 mg/mL) for 10 min at room temperature. After the incubation period, the absorbance of the solution was read at 415 nm in a spectrophotometer. Quercetin was used to prepare the standard flavonoid curve (R2 = 0.98) at a concentration range of 0.1–1.0 mg/mL. The results were presented as the mean ± standard deviation (SD) of triplicate measurements in micrograms of quercetin equivalent (µg QE/mg) of extracts.

LC–MS-QTOF analysis

The phytochemical composition of the extracts was determined using an AGILENT 6560 UHPLC-IM-QTOF-MS/MS system. For the analysis, the extract (5 mg) was first solubilized in methanol (1 mL) before injection into the system. The separation of the constituents was achieved using a mobile phase made up of 0.1% formic acid in water and acetonitrile. The sample was injected into the machine at a pre-set volume of 5 µL, maintaining a flow rate of 0.2 mL/min. The identification of the compounds was achieved by comparing the determined molecular weight to the theoretical molecular weights (MS/MS) from a standard database (NIST Mass Spectrometry Data Center) and calculating the mass error (ppm). This mass comparison and mass error calculation were automatically performed by the machine.

In vitro antioxidant activity

Three in vitro-based assay methods were deployed to ascertain the antioxidant activities of the extracts. These assays demonstrate the ability of different concentrations of the extracts to scavenge free hydrogen peroxide and DPPH radicals, as well as to reduce ferric ions to a ferrous state.

Free DPPH radicals scavenging

The free radicals scavenging activities of the extracts was studied at different concentrations against free DPPH radicals using a chemical method reported by Avni et al. [16]. In this method, DPPH solution (126 µM) was prepared in absolute methanol. Then, the extracts were prepared in distilled water at different concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL). Then, 1 mL of each concentration of the extracts was transferred to 10 mL glass test tubes, followed by adding 2 mL of the prepared DPPH solution. The tubes were incubated in a dark shelve for 30 min at ambient temperature. A control tube was prepared in a similar way using 1 mL of distilled water in the place of the extracts. The absorbance of the mixtures was determined at 517 nm. During the analysis, ascorbic acid was used as a standard radical scavenger. The scavenging activities of the extracts at different concentrations were determined using Eq. 1 and presented as both percentages (with respect to the initial level of DPPH radicals) and as IC50 values (required extract concentration to scavenge 50% of the initial DPPH radical concentration).

$$DPPH\,scavenging\;effect\;(\% )=\left[ {\frac{{{A_0} - {A_1}}}{{{A_0}}}} \right] \times 100$$
(1)

where A0 is the control absorbance, A1 is absorbance of test and standard.

Hydrogen peroxide radicals scavenging

The extracts were also evaluated for free hydrogen peroxide (H2O2) radicals scavenging using an in vitro method [17]. Prior to the analysis, a 40 mM H2O2 solution was prepared in phosphate buffer solution (50 mM, pH 7.4). The initial radical concentration of the H2O2 solution was determined against a phosphate buffer blank at 230 nm and recorded as A0. Then, 0.6 mL of different extract concentrations were mixed with the H2O2 solution (3.4 mL) in 10 mL glass test tubes. The tubes were incubated at ambient temperature for 10 min before determining their absorbances at 230 nm (recorded as A1). The scavenging of H2O2 by the extracts was calculated using Eq. 2 and reported both as percentages (with respect to the initial radical concentration) and as IC50 values. Ascorbic acid was used as a standard radical scavenger.

$$Hydrogen\;peroxide\;scavenged\,(\% )=\left[ {\frac{{{A_0} - {A_1}}}{{{A_0}}}} \right] \times 100$$
(2)

Ferric reducing antioxidant power (FRAP)

The extracts were also evaluated for metal-reducing activity using a chemical method as described by Maruthamuthu [18]. Here, different concentrations of the extracts (0.2–1.0 mg/mL) at a volume of 1 mL was added into test tubes containing 2.5 mL of 0.2 M sodium phosphate pH 6.6 buffer. Then, 2.5 mL of potassium ferricyanide solution (1% concentration) was introduced into the tubes. At this moment, the tubes were vortexed and incubated for 20 min at 50 °C. After the incubation, protein precipitation was ensured by adding 2.5 mL of 10% TCA into the tubes. The tubes were vigorously shaken and centrifuged for 10 min at 3000 rpm to obtain clear supernatants. The supernatants (2.5 mL) were carefully transferred to new tubes containing deionized water (2.5 mL), followed by the addition of 0.1% ferric chloride solution (0.5 mL) to facilitate a redox reaction between the antioxidants in the extracts and the unstable ferric ions. This redox reaction culminated in the formation of a colored complex upon the addition of TCA. The absorbance of this colored complex was determined in a spectrophotometer at 700 nm. Higher absorbances indicate stronger FRAP. Ascorbic acid served as a reference antioxidant source. The observed FRAP of the extracts was presented as the mean ± SD of triplicate absorbance measurements.

Cytotoxicity assay

MTT assay

The anticancer activity of the extracts was determined in vitro after the cultured cells had reached 70–80% confluence [19]. The extract stock solutions (50 mg/mL) were first prepared in dimethyl sulfoxide (DMSO). The required working concentrations (0.20–0.0125 µg/mL) of the extracts were prepared through a double dilution of the stock with supplemented DMEM. To perform the cytotoxicity assay, the cultured cells at the concentration of 5 × 103 cells/mL (counted using a hemocytometer) were first seeded into 96-well transparent microtiter plates and incubated for 24 h at 37 °C with 5% CO2 concentration. After 24 h, the old media in the wells were replaced with fresh media supplemented with different extract concentrations to final concentrations of 0.20, 0.10, 0.050, 0.025, and 0.0125 µg/mL. The media in the positive and negative control wells were replaced with doxorubicin-supplemented media and fresh DMEM supplemented with only FBS and P-S, respectively. All the treatment groups were prepared in three 96-well transparent microtiter plates. One set of the plates was incubated for 24 h, another set for 48 h, and the last set for 72 h. After end incubation period, thawed MTT solution (10 µL) was added all the well and further incubated for 4 h at 37 °C. After 4 h, 100 µL of 10% DMSO was used to solubilize the formed formazan crystals in each well. The absorbance of each well was read in a microplate reader at 570 nm, and from the absorbance readings, the cytotoxicity of the extracts on the cells was determined by calculating the percentage of the viable cells as a function of the untreated control using Eq. 3. The extracts concentration required to reduce the initial viable cells population by 50% (IC50 values) was determined from a concentration-cell death plot for each extract.

$$Cytotoxicity\,effect\,(\% )=\left[ {\frac{{OD\,of\,treated\,cell}}{{OD\,of\,control\,cell}}} \right] \times 100\%$$
(3)

Data analysis

All the data acquired from the study were analyzed using the non-paired Student t test package of Microsoft Excel® 2016 (Microsoft, Redmond, WA, USA) at a significance level of p < 0.05.

Results and discussion

LC–MS-QTOF analysis

The identified phytochemicals in the acetone and ethyl acetate extracts of Bunapi shimeji caps are presented in Tables 1 and 2, respectively. The base peak intensity (BPI) chromatograph and structure of some identified compounds in the acetone fraction are shown in Figs. 1 and 2 respectively, while the BPI and structure of some identified compounds in the ethyl acetate fraction are shown in Figs. 3 and 4, respectively. Some of the compounds identified in the acetone extract of Bunapi shimeji caps are xanthoxylin, forsythoside D, 2,4,6-trihydroxyacetophenone-2,4-di-O-β-d-glucopyranoside, feralolide, and p-cresol, while some compounds identified in the ethyl acetate extract of Bunapi shimeji caps include kukomine A, (3R,4R)-3,4-trans-7,2′-dihydroxy-4′-methoxy-4-[(3R)-2′,7-dihydroxy-4′-methoxy-isoflavan-5′-yl]-isoflavan, cyclocurcumin, and 6-gingerol. The ability of xanthoxylin in combination with a newly isolated ruthenium complex to induce S-phase arrest in HepG2 cells through a p53-independent pathway has been reported [20]. In addition, 6-gingerol has been reported to have cytotoxic effect against HCT15, L929, and Raw 264.7 in a concentration-dependent manner [21,22,23]. The antioxidant, anticancer, and anti-inflammatory activities of cyclocurcumin have been reported [24].

Table 1 List of LC–MS-QT of-identified compounds in the acetone fraction of Bunapi shimeji caps
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Table 2 List of LC-MS-QTof-identified compounds in the ethyl acetate fraction of Bunapi shimeji caps
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Fig. 1
figure 1

Base peak intensity plot of the LCMS-QTof-identified compounds in the acetone fraction of Bunapi shimeji caps

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Fig. 2
figure 2

Some of the LCMS-QTof-identified phytochemicals in the acetone fraction of Bunapi shimeji caps

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Fig. 3
figure 3

Base peak intensity plot of the LCMS-QTof-identified compounds in the ethyl acetate fraction of Bunapi shimeji caps

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Fig. 4
figure 4

Some of the LCMS-QTof-identified phytochemicals in the ethyl acetate fraction of Bunapi shimeji caps

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Total phenolics content (TPC) and total flavonoids content (TFC) in the extracts

The TPC and TFC of acetone and ethyl acetate extracts of Bunapi shimeji caps expressed as µg GAE/mg and µg QE/mg of the dry extract are presented in Table 3. The result showed that the TPC and TFC of the acetone fraction of Bunapi shimeji caps were significantly higher compared to those of ethyl acetate fractions. Flavonoids are a subgroup of phenolic compounds; therefore, the observed value of the TFC is expected to be lower than the TPC. A higher TPC and TFC was obtained from the acetone fraction due to the higher polarity of acetone compared to ethyl acetate. Several other factors such as the presence of phytochemicals with different side chains (including methoxy, hydroxyl, and methyl groups) in the extracts, the formation of glycosidic bond between molecules, and the degree of aromatic rings conjugation within the phytochemicals can interfere with the solubility of several phytochemicals in different solvents [11, 20]. Phenolic compounds are classified as either phenolic acids, simple phenolics, flavonoids, or hydroxycinnamic acid derivatives [11], these classes have different levels of biological activities.

Table 3 Total phenolic content (TPC) and total flavonoid content (TFC) of acetone and ethyl acetate fractions of Bunapi shimeji caps
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Previously, Addai et al. [24] and Dar et al. [25] reported solvent polarity-dependent differences in the TPC and TFC of some mushroom species. The solubility of phenolic compounds in different solvents is influenced by two factors: first is the level of the polarity of the solvent, and second is the number of OH− groups contained in the compound [26]. Compounds with a higher number of OH− groups are more soluble in polar solvents than those with less number of OH− groups [11]. The positive effect of dipole formation in the form of hydrogen bond formation in polar solvents on the solubility of solvating molecules has been reported by Visht and Chaturvedi [27]. Furthermore, different species of the same mushroom can contain different phytochemicals, however, their biological activities mainly depend on the type of phytochemicals they contain. Similarly, the TPC and TFC of extracts can vary due to the influence of environmental factors and genetic variations among. Hence, these results showed that Bunapi shimeji cap is a potential good natural source of phenolic compounds which has been shown to impact positively on human health owing to their antioxidant, anti-inflammatory, anticancer, and hypoglycemic properties [28].

In vitro antioxidant activity

A positive correlation has earlier been found between the antioxidant activity of extracts and their TPC. Phenolic compounds act as hydrogen donors, singlet oxygen quenchers, reducing agents, or as radical scavengers to bring about redox stability [5]. This study deployed three chemical-based assays (DPPH and H2O2 radicals scavenging and FRAP) to determine the antioxidant activities of the studied extracts [29]. The free radical-based assays (such as such as DPPH and H2O2 assays) manifest the presence of proton donors in a color formation or fade in original color [30], however, the metal-reducing-based methods (such as FRAP) manifest the ability of the extract to bring about a shift in the oxidation state of metals, such as the reduction of ferric ions to the ferrous state (Fe3+ to Fe2+).

Scavenging activity of DPPH and H2O2 radicals

One mechanism of lipid oxidation inhibition through antioxidants is its free radical scavenging as it can project the antioxidant activity of an extract within a short period [6]. Figures 5 and 6 illustrate the observed free DPPH and H2O2 radicals scavenging activities respectively, of acetone and ethyl acetate extracts of Bunapi shimeji compared to that of ascorbic acid. From the figures, the antioxidant activity of the acetone fraction was significantly higher against DPPH radicals (IC50 = 0.76 mg/mL) and H2O2 radicals (IC50 = 0.84 mg/mL) compared to that of the ethyl acetate fraction against DPPH radicals (IC50 = 1.10 mg/mL) and H2O2 radicals (IC50 = 1.26 mg/mL) (p < 0.05). A reduced scavenging activity was observed in ethyl acetate fraction probably due to the type of flavonoids extracted by ethyl acetate (lower polarity) compared to that of acetone. It should be noted that the abundance of flavonoids in extracts have a correlation with their antioxidant activities [31]. Another contributor to the antioxidant activity of extracts is the degree of hydrogen atom substitution by sugar on the C-3 position. This substitution has been reported to result in a slightly better antioxidant activity especially in compounds with one hydroxyl group [32]. The activities of extracts were also concentration dependent [12]. These findings agreed with previous studies where biological extracts have been reported to show a high antioxidant activity against DPPH and H2O2 [9, 32]. Siddhuraju and Becker reported the negative influence of glycosides presence in flavonoids on their antioxidant activity [33]. The scavenging activity of phenolics is reliant on the number of phenolic and hydroxyl groups they contain as these groups facilitate the transfer of protons to radicals, leading to the formation of phenoxide radicals and a consequent radical stabilization [34]. Cyclocurcumin, one of the identified phytochemicals in the extracts have been reported to have antioxidant activities [24].

Fig. 5
figure 5

DPPH radical scavenging activities of acetone and ethyl acetate fractions of Bunapi shimeji caps compared to standard ascorbic acid

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Fig. 6
figure 6

H2O2 radical scavenging activities of acetone and ethyl acetate fractions of Bunapi shimeji caps compared to standard ascorbic acid

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Ferric reducing antioxidant power (FRAP)

The ferric reducing power (FRAP) of the extracts is shown in Table 4. The FRAP of the extracts showed a similar concentration-dependent activity as observed with the radical scavenging assays. The table showed the acetone fraction to have more ferric reducing power compared to the ethyl acetate fractions. The reasons for the notable differences in the FRAP of the fractions are not far from the reasons for the differences in the radical scavenging activities that were earlier discussed. Hodzic et al. [35] and Schafer and Buettner [36] suggested FRAP as a good method for assessing the antioxidant activity of extracts. Although other factors such as solvent polarity can influence the biological activity of extracts, Moure et al. [37] further suggested that the FRAP of extracts can be due to their hydrogen ion donating ability.

Table 4 Ferric reducing antioxidant power (FRAP) of Bunapi shimeji caps extracted with acetone and ethyl acetate (absorbance@700 nm)
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Cytotoxicity assay

The concentrations of the extracts that inhibited the proliferation of the studied cell lines by 50% (IC50 values) are presented in Table 5. From the table, the acetone fraction was seen to be more cytotoxic against MCF-7 (IC50 range = 0.051–0.055 mg/mL) and MDA-MB-231 (IC50 range = 0.122–0.131 mg/mL) compared to MCF-10a (IC50 ≥ 250 mg/mL). Similarly, ethyl acetate fraction was more potent against MCF-7 (IC50 range = 0.075–0.096 mg/mL) and MDA-MB-231 (IC50 range = 0.161–0.164 mg/mL). The normal cells (MCF-10a) were significantly less sensitive to the extracts compared to cancerous cells (p < 0.05) likely due to the absence of specific proteins which were expressed by the cancerous cells but lacking in the normal cells. These proteins may contribute to the affinity of the active phytochemicals in the extracts towards binding on the cell surfaces to induce apoptosis [12, 38]. Similarly, the type of phenolic compound present in extracts contributes to their anticancer activities. Doxorubicin, a standard chemotherapeutic agent showed more antiproliferative activity on both cells (IC50 = 0.009–0.010 mg/mL against MDA-MB-231, 0.007–0.008 mg/mL against MCF-7, and 0.012–0.016 mg/mL against MCF-10a) compared to the extracts (p < 0.05).

Table 5 MTT-based anticancer activity of Bunapi shimeji caps extracted with acetone and ethyl acetate against MDA-MB-231, MCF-7, and MCF-10a
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In this study, a selective cytotoxicity was observed against the studied cell lines likely due to the type of protein markers expressed by the different cell lines [12]. The simple mechanism for the observed anti-inflammatory activities could be attributed to the induction of apoptosis by the polyphenol content of the extracts. The observed activity is mediated by the interaction of the hydroxyl groups contained in the phytochemicals with the polar receptor site of the mitochondrial cytochrome P450 enzyme. The attachment of these hydroxyl groups to the P450 enzyme could manifest in the intrinsic apoptotic pathway which is characterized by increased membrane permeability and its associated mitochondrial swelling, outer mitochondrial membrane rupture, and the discharge of proapoptotic factors from the intermembrane space [39].

Different classes of polyphenols have different mechanisms of inducing apoptosis and exerting antiproliferative effects. Flavonols inhibit cell proliferation by increasing the population of apoptotic cells and downregulating nuclear factor kB (NF-kB). They also induce apoptosis by increasing the expression of pro-apoptotic Bax proteins, Apaf-1, p53, cytosolic Cyt c, caspases-9 & -3, and cleavage of poly (ADP-ribose) polymerase (PARP). This cleavage of PARP results in the reduced expression of AKT, Bcl-2, Bcl-xL, and Mcl-1 which are anti-apoptotic proteins in different cancer cell lines [40]. Flavonols also induce the depolarization of, the mitochondrial membrane potential [41] and generally mediate apoptosis through a dose-dependent up-regulation of Bax protein expression, activation of caspase-3 and caspase-9, the release of Cyt c to the cytosol, and the increase of the mRNA levels of p53 as reported in mouse neuroblastoma cells [42]. Isoflavones induce apoptosis through the regulation of the protein kinase B survival signaling pathway as reported in breast and cancer cells [43]. They also regulate cell proliferation through the regulation of Bcl-2/Bax proteins expression and caspase-3 activation [44], Anthocyanins regulates cell proliferation by blocking the cell cycle at the G0/G1 phase. They also reduce the expression of antiapoptotic proteins (Bcl-2, xIAP, cIAP-1, and cIAP-2) [45]. Apoptosis due to anthocyanins manifests in the formation of apoptotic bodies, poly (ADP-ribose) polymerase proteolysis, and caspase-3 activation. Phenolic acids induce apoptosis through the inhibition of Bcl-2 activity, leading to the activation of caspase-3 and a subsequent release of Cyt c. These could represent are some of the possible mechanisms of the antiproliferative activities exerted by the extracts reported in this study [46].

Over the years, several reports on the bioactivity of several mushrooms have been documented, however, few have been taken to the next level of drug development likely due to the limited availability of the source mushrooms or due to the potentially high level of toxicity of such sources to human [11, 47]. Hence, this study was carried out on Bunapi shimeji, a widely available and non-toxic mushroom in Malaysia and South-East Asia which can be produced in great quantities with minimal labor and cost inputs. The antiproliferative activities of different mushroom extracts have been reported against different cancer cells [20, 23]. For instance, Kim et al. [48] reported the in vivo antiproliferative activity of L. deliciosus-derived hetero-polysaccharides, while Xu et al. [49] reported the ability of water extract of M. procera to inhibit the metastasis of 26-M3.1 cells. Previously, Kusavadee et al. [1] reported the antiproliferative activity of Sanghuangporus sp.1 extracts through a caspase-dependent pathway.

Conclusion

In this study, the in vitro anticancer activity of Bunapi shimeji caps extracted with acetone and ethyl acetate were studied against MCF-10a, MCF-7, and MDA-MB-231 cell lines. Both fractions of the extracts were biologically characterized before studying their anticancer activities. From the study, acetone extract showed more biological activities (antioxidant and anticancer) compared to ethyl acetate fraction (p < 0.05). Furthermore, MCF-7 cell line was more sensitive to both extracts based on the presented IC50 values compared to MDA-MB-231, while MCF-10a was generally less sensitive to both extracts, this might be due to the absence of some protein markers on the normal cells compared to the cancerous cells. Thus, the caps of Bunapi shimeji have the potential of being used as the good source of antioxidant and anticancer agents.