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Determination of biological activity of suillus granulatus mushroom extracts

In the last decade, in addition to the study of the nutritional composition of mushrooms, the study of biologically active compounds occupies an important place. However, there is a need to find new and lesser known mushrooms species that have biological activity and potential for application in industrial conditions. The aim of this research is to determine the biological activity of aqueous and ethanolic extracts of Suillus granulatus wild mushroom through the determination of the content of total carbohydrates, total, α and β-glucans, as bioactive compounds, as well as determination of cytotoxic activity. Total carbohydrate content was determined using spectroscopic method, total, α and β-glucans were analysed using specific kits, and MTT test (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used for cytotoxic activity. IR-ATR (infrared spectroscopy - attenuated total reflection) spectra of mushroom extracts were performed, too. Aqueous extracts had a higher content of total carbohydrates as well as glucan and had better cytotoxic activity against HeLa cells, while ethanolic extract of Suillus granulatus was characterized with better cytotoxic activity against HepG2 cells. Based on IR-ATR, the presence of different types of carbohydrates, glucans, proteins, phenols and flavonoids can be observed in aqueous and ethanolic, which is one of the reasons for the differences in their anticancer activity. The analysed extracts are an excellent basis for their further application in various products in order to obtain functional food with enriched biological value. Thus, using natural dietary supplements can significantly affect the positive changes in the health of consumers.


Introduction
Mushrooms are a food rich in proteins and carbohydrates, they have a low fat content, but a very high content of unsaturated fatty acids. Mushrooms are known to be a good source of almost all of the essential amino acids and vitamins, especially vitamins B 1 , B 2 , B 5 , C, and D [1,2]. Among and from the fruiting bodies of many fungi, mainly lectins and terpenes [7]. These compounds stimulate different cell populations such as macrophages, NK cells (lymphocyte type), neutrophils or lymphocytes and initiate cytokine synthesis [8]. Thus, some polysaccharides or polysaccharideprotein complexes of fungi can stimulate a nonspecific immune system and exert antitumor activity by stimulating host defense mechanisms [9].
The mechanism of immunomodulatory action is different, depending on the molecular weight of polysaccharides extracted from different types of fungi. Low molecular polysaccharides can enter the cells and thus have an enhanced effect on the immune system. When those with higher molecular weights cannot enter the cell, they bind to certain receptors on the cell membrane and thus manifest a response [10].
Polysaccharides achieve their anticancer effect indirectly by activating various defense immune reactions. Immunomodulatory properties of higher fungal polysaccharides include mitogenic activity, stimulation of pluripotent stem cells in haematopoiesis, activation of an alternative complement pathway, and activation of immune system cells such as macrophages, T helper cells (Th cells), and cytotoxic T cells (Tc cells) and B cells [11].
Suillus granulatus (L.) Roussel, known as the "weeping bolete", is an edible mushroom with a white, soon yellowish and non-staining flesh. It has a mild to slightly fragrant odour and tastes mild. Although this species is not one of the species most consumed as a delicacy, such as truffles or morels, it is widely harvested and consumed by the general population, particularly those who traditionally practice mushroom picking. This mushroom is characterized with a low fat content, and it is rich in plant fibers, carbohydrates and other compounds and because of that it is included in the category of functional foods [6].
Therefore, the aim of this research is to determine the in vitro cytotoxic activity of aqueous and ethanolic extract from Suillus granulatus, as well as determining the possibility of their use in the food industry for the production of functional food.

Collection, identification and drying of mushrooms
In this research, as a work material was used Suillus granulatus (L.) Roussel, edible mushroom collected from the territory of the Republic of North Macedonia (from Bistra Mountain near the village Sretkovo at an altitude of 1100 m, in a pine forest (Pinus), on a soil substrate). The determination was performed according to the key by Horak (2005) [12]. The collected fresh mushrooms were chopped into thin slices. The mushroom pieces were dried in a chamber dryer with hot air at a temperature of 40 ºC for 6 to 7 h, to a constant mass on a dry basis. Dried mushrooms were first ground to a fine powder and then extracted in two ways, with water and ethyl alcohol as extragens.

Preparation of aqueous extract
Aqueous extract was prepared by Sławińska et al. (2013) [13] and Ribeiro et al. (2015) [14] method. The measured mass of dried and finely powdered mushroom sample (10 g) was poured with about 200 mL of distilled water, and after that was extracted on a boiling water bath (BIOBASE, TB-1, Shandong, China) for 1 h. The extract was strained through filter paper, then rinsed once more with boiling water and the sample was filtered again. The resulting supernatant was combined and evaporated on a vacuum evaporator (Resona Technics Labo Rota 300 type B, The Netherlands). The samples are then further dried in a stream of warm air (40 ºC) to a constant mass.

Preparation of ethanolic extract
Ethanol extract was prepared by Vidović et al. (2011) [15] method. The measured mass of dried and finely powdered mushroom sample (10 g) was poured with 100 mL of 50% ethanol (Alkaloid, 1,006,278, N. Macedonia) and extract was covered for 40 min on the ultrasonic bath (VEVOR, 10 L 490 W Ultrasonic Cleaner JPS-40 A) at 45 °C. The sample was filtered through filter paper. The resulting supernatant extract was evaporated in a vacuum evaporator (Resona Technics Labo Rota 300 type B, The Netherlands) at 60 °C to constant mass. For each sample, the extraction procedure was done in triplicates.

Determination of total carbohydrates content
The content of total carbohydrates in the extracts was determined according to the modified method of Monsigny et al. (1988) in microplate [16]. A series of different concentrations of extracts was prepared, and the standard D-glucose was prepared, too, at a different concentration. 50 µL of extract was taken (50 µL of D-glucose was taken to create a standard curve), 15 µL of concentrated H 2 SO 4 and 30 µL of 5% phenol (5 mL of 100% phenol in 100 mL of distilled water) were added. All trials were performed in triplicate. The absorbance was read on a spectrophotometer (JEN-WAY 6305, United Kingdom) at a wavelength of 490 nm. The content of total carbohydrates was calculated on the basis of the calibration curve (absorbance depending on the concentration) of the standard D-glucose solution.
Eq values glucose/g dry matter in the tested mushroom extracts were obtained according to the following formula: mg eq. glucose/g d.m. = read conc. glucose (µg/mL)/ working conc. x 1000.

Determination of total, α and β-glucans
The content of total glucan and α-glucan in aqueous and ethanol extracts was determined using specific kits Mushroom and Yeast Beta-glucan Assay Procedure, K-YBGL 11, 2019 (Megazyme Co. Wicklow, Ireland). The β-glucan content was calculated as the difference between the total glucan content and the α-glucan content.

IR-ATR spectra of mushroom extracts
The spectra of the extracts were recorded on the FT-IR Perkin Elmer System 2000 using Specac Golden Gate ATR accessory. Diamond ATR single crystal was used for the purpose. ZnSe focusing lenses (a part of the accessory), restrained the measurements to 520 cm − 1 at the lower wavenumber side; the higher wavenumber side was at 4000 cm − 1 . The spectra were recorded using 4 cm − 1 resolution and 64 scans (both for the background -the N 2 /air, and the samples). The instrument was purged with 99.999% purity gaseous N 2 during the whole measurement process, to avoid the spectrum contamination with the bands due to air H 2 O and CO 2 . The recorded ATR spectra are presented as measured.
In vitrodetermination of cytotoxic activity. Two malignant cell lines were used to test cytotoxic activity: HeLa ATCCCCL−2 (ATCC, Poland) (cervical cancer) and HepG2 ATCCHB − 8065 (ATCC, Poland) (hepatocellular carcinoma). Cells were grown at 37 °C, in a CO 2 incubator (Model 199, LabX, USA) (5% CO 2 ), in nutrient medium DMEM (Dulbecco's Modified Eagle's Medium) (HyClone Logan, USA). HeLa and HepG2 were seeded on 96-well microtiter plates (Greiner 655,101, Germany), and then 6 concentrations of tested extracts were added. Final concentrations ranged from 0.01 to 3 mg/mL. The analysis was made according to the method of Kaišarević et al. (2007) [17]. At the end of the incubation time (24 and 72 h) a solution of MTT (MTT Assay Kit, ABCAM, UK) at a concentration of 0.5 mg/mL was added, which was prepared in the medium immediately before the addition. 100 µl of solution was added to each sample, so that the final concentration was 0.05 mg. After treatment, the cells were incubated for 3 h at 37 °C, whereby the activity of mitochondrial dehydrogenase in viable cells transformed MTT into purple-blue formazan crystals. After incubation, the medium was drained and the reaction was stopped by the addition of 100 µl of 0.4 M HCl in isopropanol (Merck, Germany), whereby the formazan crystals are dissolved.
After 10 min at room temperature, the plates were shaken on a shaker then the absorbance was measured at 690 nm using a spectrophotometer (JENWAY 6305, United Kingdom).
Based on the obtained results, the percentage of cytotoxicity (CI -cell inhibition) was calculated by the formula: CI(%)=(1-As/Ac)•100.
where As is absorbance of treated cells (sample) and Аc is control absorbance (untreated cells).
The efficiency of inhibition of tumor cell proliferation was quantitatively expressed as the IC 50 value.

Statistical analysis
The obtained results were statistically processed using the software package SPSS 20. The Independent Sample T-test was used to determine statistically significant differences (p = 0.05) between the values.

Content of total carbohydrates, total, α and β-glucans
The content of total carbohydrates in the analyzed extracts was higher in aqueous extracts compared to ethanolic extracts ( Table 1), which was probably due to the fact that carbohydrates are generally water-soluble compounds [18]. Moreover, aqueous extract from Suillus granulatus were characterized with statistically significant (p < 0.05) higher content of total, α and β-glucans compared to its ethanolic extracts.

IR-ATR spectra of mushroom extracts
The spectra of aqueous and ethanolic extract of Suillus granulatus are presented in Fig. 1. Spectra can be clearly divided in several general wavenumber regions. The first region between 3500 and 3000 cm − 1 is the one where -OH stretching vibrations from the glucane, phenols, water and protein side chains would appear.
One can easily recognize spikes on the broad ν(OH) band at 3350 and 3250 cm − 1 assigned to the ν(NH) A and B bands from the proteins, respectively (Fig. 1). The second region is the one between 3000 and 2800 cm − 1 , where stretching bands from the CH 2 groups of the protein side chain and lipids appear. Maxima around 2930, 2886 and 2852 cm − 1 speak on the behalf of these findings (Fig. 1). The third wavenumber region is between 1800 and 1200 cm − 1 . This region can be roughly subdivided in protein and sugar regions [24].
The protein region is comprised of Amide I band at ca. 1650 cm − 1 , Amide II band at 1550 cm − 1 and Amide III band 1400-1200 cm − 1 . However, this protein region is further burdened by the δ(H 2 O) band at ca. 1640 cm − 1 [25], and bands from phenolic and flavonoid compounds. The strong band appearing between 1658 and 1619 cm − 1 can be Polysaccharides are one of the most researched components of Suillis, Trametes and Phellinus species, which have shown a wide range of antimicrobial, antioxidant, anticancer, immunomodulatory and hepatoprotective effects [19].
Aqueous extraction is the only clinically proven method that allows the release of carbohydrates from chitin and their extraction. In this way, α and β-glucans are mostly concentrated, which are considered to be the main components when it comes to the medical activity of the fungus [20]. The β-glucans is thought to have the power to regulate the immune system, lower total cholesterol levels and LDL levels, and exhibit a number of other immunomodulatory effects [21].
Several studies have confirmed the activity of aqueous and ethanolic extracts from Suillus granulatus against different cancer cells, through different mechanisms: inhibition of cell growth by stopping their development cycle, stimulation of host immune response or by induction of apoptosis [22]. Some hydrosoluble compounds are thought to be responsible for these effects, such as β-D-glucans, β-Dglucans with heterosaccharide bonds of xylose, mannose, galactose, β-D-glucans with protein complexes (proteoglucans) and phenols, which indicate an immunomodulatory and therapeutic effect in humans and animals [22,23].  Figure 1 presents the main characteristics of the mid-IR spectra of the investigated mushrooms with the assignment of the bands Many studies show that proteins and polysaccharides are the major anticancer components in the extract from P. djamor, which show cytotoxic activity against HepG2 and MCF-7 cells, and polysaccharides from P. gilvus significantly inhibit melanoma growth [29,30], while proteins isolated from L. edodes are the main anticancer components that inhibit leukemia cells [31].
Besides, it is known that the anticancer activity of polysaccharides has a significant correlation with their structure, molecular mass, solubility, monosaccharide composition and extraction method. Hydrosoluble polysaccharides have also been shown to demonstrate higher immunomodulatory activity compared to insoluble ones. On the other hand, proteins show a strong cytotoxic effect against HeLa cells [32].
Also, the presence of double or triple helix, as a structural characteristic of biopolymers, or any structure, as well as the degree of degradation of sugar and non-sugar components (especially protein complexes or sulphur bonds), greatly affect the healing properties of fungi [2,33].

Cytotoxic activity of tested mushroom extracts
According to the data presented in Figs. 2 and 3 can be seen that both tested extracts showed moderate anticancer activity at different tested concentrations. Namely, in all samples, with concentration increasing, the cytotoxic effect of the examined cell types increased proportionally. Thus, the samples showed the highest activity at the highest tested concentration of 3 mg/mL. After 24 h of the treatment of HeLa cells can be noticed that aqueous extracts had better results (33.86 − 66.12% assigned to the ν(C = O) of the flavone ring, depending on the flavonoid compound present [26].
The aromatic rings are presented by the appearance of several ν(C = C) bands in the 1604-1392 cm − 1 wavenumber region. The weak band at 1463 cm − 1 can be attributed to δ(CH 2 ), while the strong band at 1398 cm − 1 may be attributed to the δ(C-OH) and/or ν(C = C) vibrations from the flavonoid and phenolic compounds [26,27]. The band at 1298 cm − 1 is the Amide III from the protein content [28].
Here it has to stress that the appearance of the band at 1398 cm − 1 might also be in connection to the ν s (COO − ) of the amino acids [25], however it never appears with such high intensity (compared to Amide I and II bands), as presented in this research.
The sugar region is mainly dependent on the vibrations of the glucans, which are the dominant polysaccharide in mushrooms. The bands at 1160 cm − 1 (appearing as a shoulder), 1078, 1048 and 908 cm − 1 originate from the presence of the β-D-glucane, while the bands at 1148, 1027 and 937 cm − 1 indicate the presence of α-D-glucane (Fig. 1) [24,25,27].
The Amide II band of the protein or the ν(C = C) of the flavone can be seen, where a strong and sharp band with a maximum at 1573 cm − 1 appears. That this band might be in a connection with the flavone content might be further proved with the band at 1398 cm − 1 , which appears to be the strongest in sample 1. Also, the glucan region is quite different, and it seems that the α-D-glucan is less present in the sample 1, than β-D-glucan, as observed through the intensities of the 937 cm − 1 band.

Fig. 2 Cytotoxic activity of aqueous and ethanolic extracts against HeLa cells after 24 and 72 h
In the case of HepG2 cells, after 72 h of incubation can be noticed that the ethanolic extract from Suillus granulatus showed a minimal reduction in the cytotoxic effect compared to the results after 24 h of incubation.
In accordance with the results for the percentage of cytotoxic activity are the IC 50 values of the analysed extracts ( Table 2). IC 50 values showed a statistically significant difference (p < 0.05) between aqueous and ethanolic extracts. Moreover, in the aqueous extract there was statistically significant difference (p < 0.05) between the incubation time of both tested cells, while in the ethanolic extract statistically significant difference (p < 0.05) showed the IC 50 values from HeLa cells between 24 and 72 h.
Glucans, phenols, flavonoids and a many other compounds, such as alcohols, esters, aldehydes, coumarins, etc., are thought to contribute to the cytotoxic activity of mushroom extracts [34].
In the MTT test should also be borne in mind that the results are largely dependent on mitochondrial activity, and if their activity is impaired by other factors, it may be shown that the cells are not alive, which would give false positive results [29]. Santos et al. (2013) [35] found that aqueous and ethanolic extract from Suillus luteus have an IC 50 > 400 µg/mL against tumor cells NCI-H460 and MFC-7, while methanol extract shows an IC 50 > 30.33 µg/mL and an IC 50 > 32.25 µg/mL, respectively. The values have been measured after 72 h of treatment, and the authors concluded that methanol extract had the best antimicrobial properties, but both aqueous and ethanol extracts showed good cytotoxic activity as a basis for their medical use. cytotoxic activity). A statistically significant difference (p < 0.05) was found between the anticancer activity of aqueous and ethanolic extracts.
Furthermore, after 24 h of the treatment of HepG2 cells can be seen that ethanolic extract from Suillus granulatus (36.94 − 66.67%) showed statistically significant (p < 0.05) better results compared to its aqueous extracts.
After 72 h of incubation can be observed that all tested extracts had better results compared to 24-hour incubation. Namely, in HeLa cells there was a proportional improvement of the obtained effects in all tested extracts. The effects from aqueous extract were statistically significant (p < 0.05) better than ethanol extracts (33.63 − 67.71% cytotoxic activity).

Conclusions
According to the results can be concluded that, both of aqueous and ethanolic extract of Suillus granulatus showed good biological activity. Thus, this mushroom can be classifying as functional food, because it's beneficial properties. Namely, aqueous extract had better anticancer activity against HeLa cells, while ethanolic extract of Suillus granulatus was characterized with better anticancer activity against HepG2 cells. Nevertheless, after 72 h of incubation, the examined extracts give better results, compared to the 24 h incubation.
Aqueous extract from Suillus granulatus were characterized with higher content of total, α and β-glucans compared to its ethanolic extracts. Aqueous extraction is the only clinically proven method that allows the release of carbohydrates from chitin and their extraction.
Based on IR-ATR, the presence of different types of carbohydrates, glucans, proteins, phenols and flavonoids can be observed in aqueous and ethanolic, which is one of the reasons for the differences in their cytotoxic activity.
Therefore the main conclusion from this research is that aqueous and ethanolic extracts of Suillus granulatus are suitable for use in the food industry as an excellent basis for the production of functional food.
Author contributions Monika Stojanova -mushroom collection, performing analysis and writing. Milena Pantic -conceptualization, final reviewing. Mitko Karadelev -mushroom collection. Vladimir Ivanovski -performing analysis. Miomir Niksic -conceptualization, final reviewing. All the authors discussed these results and contributed to the final manuscript.
Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
Data Availability All data analysed during this study are included in this published article.
On the other hand, Ünyayar et al. (2006) [37] concluded that methanol extract from Trametes versicolor showed a 45% ability to inhibit HeLa cells at a concentration of 10 µg/ mL. By reducing the extract concentration, the authors found a reduction of the effect up to 27% at a concentration of 1 µg/mL.
In their study, Knezević et al. (2018) [38] pointed out that the ethanolic extract from Trametes versicolor had an IC 50 value of 168.54 µg/mL against HeLa cells and an IC 50 > 200 µg/mL against LS174, A549 and MRC5 cells. The authors concluded that fungi of this species showed significant medical potential.
According to research by Lau et al. (2004) [39] the aqueous extract from Trametes versicolor had an IC 50 value of 269.3 µg/mL against NB-4 cells and an IC 50 of 147.3 µg/ mL against KL-60 cancer cells indicating high anticancer activity. Samchai et al. (2009) [40] in their study found that methanolic extract from Phellinus linteus has an IC 50 (µg/mL) of 17.36 against MFC 7, i.e. 19.14 against NCI-H187 cells. In the case of ethanolic extract, the IC 50 value (µg/mL) was determined to be 27.26 and 40.15, respectively.
Veljovic et al. (2017) [41] in their study, among other parameters, determined the antiproliferative effect of ethanolic mushroom extract from Ganoderma lucidum against HeLa cells, and found that this extracts showed a cytotoxic effect at a concentration of 500 µg/mL, i.e. IC 50 values range from 223.1 to > 500 µg/mL after 24 h and from 119.3 to 391.2 µg/mL after 48 h.
Aqueous and ethanolic extracts from G. lucidum have also been found to give significant results in preventing tumour proliferation in mice, which the presence of polysaccharides is thought to play a key role [42].
On the other hand, beside the cytotoxic activity, aqueous extracts of Suillus granulatus have high content of total phenols, and as a consequence good ability to capture DPPH radicals, ability to reduce iron ions and ability to chelate iron ions. Moreover, ethanolic extracts of these mushrooms have strong activity on the antioxidant test for reducing the presence of conjugated dienes, which is thought to be due to the higher content of flavonoids [43].