Cytotoxic Activities of Methanol Extract and Compounds of Porodaedalea pini Against Colorectal Cancer

: Porodaedalea pini is a medicinally important mushroom with antioxidant, cytotoxic, immunostimulating, antitumor, antiviral and immunomodulating activities. Therefore, in this study, P. pini methanol extract and isolated compounds from the methanol extract were tested for cytotoxic activities against DLD-1 (colorectal cancer) and CCD-18Co (human colon fibroblast cell line) by using Alamar Blue assay. Cytotoxic activity on DLD-1 was decreased in the order of P. pini methanol extract> 4-(3,4-dihydroxyphenyl)but-3-en-2-one ( 3 )> pinoresinol ( 2 )> ergosta-7,24(28)-dien-3 β -ol ( 1 ). P. pini methanol extract was determined to have the best cytotoxic activity with the lowest IC 50 value on DLD-1 (IC 50 : 25.33±0.29 µg/mL) and the highest IC 50 value on CCD-18Co (434.30±1.45 µg/mL). Within the scope of the findings, it is thought that P. pini mushroom can be used as a new and natural agent in the treatment of colorectal cancer.


INTRODUCTION
Cancer ranks second in deadly diseases after cardiovascular diseases worldwide. Approximately 10% of diagnosed cancers or cancer-related deaths each year consist of colorectal cancer (Dekker et al., 2019). Colorectal cancer is the second most common type of cancer diagnosed in women, while it is third cancer in men. The incidence and mortality in women are about 25% lower than in men. The worldwide incidence of colorectal cancer is estimated to increase to 2.5 million new cases by 2035(Arnold et al., 2017Dekker et al., 2019). Among the causes of colorectal cancer, various risk factors including diets are rich in animal fat, in vegetables and fruits, smoking, diabetes and obesity have been identified (Kelly et al., 2012;Perdue et al., 2014). Also, studies have reported that high consumption of red meat increases the risk of colorectal cancer, while adequate physical activity, especially outdoor activities, and the level of vitamin D in the bloodstream reduces the risk of colorectal cancer (Oba et al., 2006;Takachi et al., 2011;Surya et al., 2016). Worldwide, nutrition and diet are the most remarkable factors causing colorectal cancer. The literature studies revealed that many natural diet products can be beneficial in the prevention of cancer (Tao et al., 2018). Also, plants, fruits, vegetables, herbal teas, and mushrooms have been reported to have cytotoxic effects on colorectal cancer cells and due to the presence of dietary fibers and phytochemical compounds (Turati et al., 2015;Seidel et al., 2017).
In Chinese medicine, Phellinus genus has been reported to be used in the treatment of many diseases such as arthritis of the knee, stomachaches, tumors, inflammation, lymphatic and gastroenteric disorders and to promote longevity in Chinese medicine (Ganeshpurkar et al., 2010;Seephonkai et al., 2011). Bioactive compounds such as sesquiterpenes, triterpenes, steroids, pigments, and polysaccharides, responsible for estrogenic, antiviral, antioxidant, antitumor and anti-estrogenic activity have been purified from Phellinus species (Wu et al., 2010;Song et al., 2014;Wang et al., 2014;Pei et al., 2015). Porodaedalea pini, a member of Phellinus genus and Hymenochaetaceae family, is usually grown under the pine trees. It is known to lower blood sugar, increase immunity, and have anti-cancer properties (Ayer et al., 1996). Antioxidant, antifungal, anticholinesterase, anti-tyrosinase, cytotoxic activities, and inhibition effects on NO production in murine macrophages-RAW 264.7 cells by the compounds isolated from P. pini have been revealed by previous studies (Ayer et al., 1996;Wangun & Hertweck, 2007;Jang &Yang, 2011;. In recent years, the discovery of bioactive compounds from natural sources, and obtaining compounds and possible drug candidates with therapeutic properties have attracted more attention. In this context, mushrooms are considered as sources of natural bioactive compounds and are defined as promising therapeutic agents. The study was aimed to evaluate cytotoxic activities against DLD-1 (colorectal cancer) and CCD-18Co (human colon fibroblast cell line) of both P. pini methanol extract and ergosta-7,24(28)-dien-3β-ol, pinoresinol, and 4-(3,4dihydroxyphenyl)but-3-en-2-one compounds isolated from the methanol extract in our previous study  ).

Mushroom Material
Porodaedalea pini (Brot.) Murrill. was collected from the villages of Mentese district of Mugla in November-December 2014 and January 2015 from the area of Muğla, Turkey. The voucher specimen has been stored at the Research and Application Center for Mushrooms, Mugla Sitki Kocman University (Fungarium No AT-2446).

Spectral Measurements and Chemicals Used
For purification of the compounds, silica gel (Kieselgel 60, 70-230 mesh, Merck) was used for column chromatography; RP-18 F254S and silica gel 60 F254 plates for thin-layer chromatography (TLC). Separation and isolation of the compounds were performed by using C18 and GS-320 column-linked recycling HPLC (Japan Analytical Industry Co. Ltd.). UV-254nm light was used for detection of TLC spots and CeSO4 solution for visualization. Chemical structures of the isolated compounds were elucidated by using FT-IR, 1D-NMR, 2D-NMR spectroscopy techniques. Thermo Scientific one Nicolet IS10 FT-IR spectrometer was used to record the IR spectrum. The NMR spectrum was on Agilent-600-MHz instruments coupled with cooled cryoprobes probe for 1 H-and 13 C-NMR including HSQC, HMBC, and COSY. A 96-well microplate reader (MultiskanGo, Thermo Scientific Co., MA, USA) was used to analyze cytotoxic activity studies. Cytotoxic activity results were measured and calculated by using GraphPad Prism (GraphPad Software v5.0, USA).

Extraction and Isolation
For extraction of the aerial parts of P. pini (1900 g), powdered mushroom samples were macerated separately and respectively in solvents with increasing polarity: n-hexane, chloroform, acetone, and methanol for 24 h; and four times at room temperature. Solvents were vaporized by using a rotary evaporator. The methanol extract obtained was 49.70 g. The methanol extract was stored at +4 °C for further analysis. As a result of the chromatographic isolation of P. pini methanol extract, ergosta-7,24(28)-dien-3β-ol, pinoresinol, and 4-(3,4dihydroxyphenyl)but-3-en-2-one were obtained. Details about the isolation and characterization of the compounds can be seen in our previously published research (Deveci et al., 2019 a ).

Cell Viability Assay
1x10 4 cells were put into 96-well plate with a growth medium and incubated in 5% CO2 at 37 °C for 24 h until they attached to the bottom. Different concentrations (between 1 µg/mL and 1000 µg/mL) of the methanol extract and isolated compounds of P. pini were added to each well. Viability and proliferation of the cells were tested according to the previously described Alamar Blue assay (Karakurt & Adali, 2016). The results were measured at 570 nm and 610 nm by using a 96-well microplate reader. Doxorubicin was used as a positive control. The sigmoidal plot of the inhibition rate (%) versus the log concentration was used to calculate the IC50 values of P. pini methanol extract and the isolated compounds.

Statistical Analysis
All data on cytotoxic activity tests were the average of three parallel sample measurements. Data were recorded as mean ± S.E.M. Significant differences between means were determined by t-test, p <0.05 were regarded as significant.
From ancient times, people have described mushrooms as culinary wonders and valuable in folk medicine. In recent years, mushrooms have witnessed the intense interest of scientists due to their pharmaceutical potentials (Dimitrijevic et al., 2017). Until this time, the medicinal uses of mushrooms have been associated with their nephroprotective, antioxidant, antitumor, hypocholesterolemic, antidiabetic, immunomodulatory, anti-cancer, antiallergic, and antimicrobial properties. Literature studies have shown that Phellinus, Agaricus, Pleurotus, Clitocybe, Ganoderma, Antrodia, Cordyceps, Trametes, Calvatia, Xerocomus, Flammulina, Schizophyllum, Suillus, Inonotus, Funlia, Inocybe, Lactarius, Russula, Albatrellus, and Fomes mushroom species are effective against cancer. These mushroom species are defined as new generation biotherapeutics (Patel & Goyal, 2012). It is prominent that not only active isolated compounds but also extracts containing active compounds can be used as alternative agents with fewer side effects in cancer treatment and hence it is important to examine the cytotoxic effects of the mushroom species. According to the results obtained, it was determined that P. pini methanol extract has the best cytotoxic activity. In earlier studies, purification and bioactive properties of steroids, phenolics, terpenoids, and polysaccharides from P. pini have been reported Deveci et al., 2019 b ;Hong et al., 2013;Jang &Yang, 2011). Therefore, the higher cytotoxic activity of the methanol extract compared to other isolated compounds can be explained by the synergistic effect of various bioactive compounds contained in it. Among the isolated compounds, phenolic compounds indicated higher cytotoxic properties. It is known that phenolic compounds have anticancer activity associated with their high antioxidant properties or have direct cytotoxic effects on cancer cells (Ivanova et al., 2014). Also, the cytotoxic mechanism of action of phenolic compounds has been elucidated in previous studies as modulating carcinogen metabolism, altering gene expression levels, arresting the cell cycle, inducing apoptosis, and inhibiting various cell proliferation signaling pathways (Huang et al., 2010).

CONCLUSION
Chemotherapy, cytotoxic drugs, radiotherapy, and surgery are the main methods used in the treatment of colorectal cancer. Among these methods, the most effective method is surgery, and treatment is continued with chemotherapy or radiotherapy since there is a high possibility of recurrence of colorectal cancer after surgery. The negative side effects of chemotherapy compounded by the tendency of colorectal cancer reoccurrence, has made it imperative to search for new, natural, and effective agents with fewer side effects in its treatment. Cytotoxicity of P. pini methanol extract and ergosta-7,24(28)-dien-3β-ol (1), pinoresinol (2), and 4-(3,4-dihydroxyphenyl)but-3-en-2-one (3) isolated compounds were investigated on DLD-1 and CCD-18Co cells for the first time. The methanol extract of P. pini displayed significant cytotoxicity on DLD-1. As a result, this study recommends that P. pini mushroom, especially the methanol extract, can be used for further research that could lead to the development of new natural remedies in the treatment of colorectal cancer.

Declaration of Conflicting Interests and Ethics
The authors declare no conflict of interest. This research study complies with research publishing ethics. The scientific and legal responsibility for manuscripts published in IJSM belongs to the author(s).