Anticancer, antithrombotic, antityrosinase, and anti‐α‐glucosidase activities of selected wild and commercial mushrooms from Pakistan

Abstract Mushrooms have been accepted as nutraceutical foods because of their high nutritional and functional values. They have also gained interest due to their medicinal properties, economic importance, and organoleptic merit. In this study, wild Ganoderma lucidum and four commercial mushrooms, that is, Pleurotus ostreatus, Volvariella volvacea, Hericium erinaceus, and Lentinus edodes from Pakistan were screened for their biological activities such as anticancer, antityrosinase, anti‐α‐glucosidase, and antithrombotic activities from their methanol, ethanol, and water extracts. Enzyme inhibition assay showed that selected mushrooms are potent inhibitors with %age inhibition ranging from 19.00% to 80.91%, and 32.85% to 83.38% for tyrosinase and α‐glucosidase, respectively. The best tyrosinase inhibition was shown by P. ostreatus whereas L. edodes was found best as α‐glucosidase inhibitor. These mushrooms were tested against cancer cell lines including HT‐29 colon and H‐1299 lungs carcinoma cell lines. G. lucidum showed 29% and 24% viability of cells against HT‐29 and H‐1299 cell lines, respectively. This antiproliferative effect was in dose‐dependent manner, and the maximum inhibition was observed at 200 μg/ml. Mushrooms extracts were also found effective against clot lysis. The percentage of clot lysis was in the range of 27%–29%. The research would provide knowledge to the people of Pakistan about the importance of locally available commercial mushrooms and wild mushrooms for health improvement and prevention against different kinds of diseases.

According to the International Agency for Research on Cancer (IARC), 148,041 people were reported to have cancer in 2012 among which death cases were about 101,113 in Pakistan (Sarwar & Saqib, 2017). The global increase of cancer incidence has been estimated by GLOBOCAN, an international agency for cancer research and it reported 12.7 and 8.2 million deaths in 2008 and 2012, respectively, and 14.1 million new cases in 2012, 64% of which belonged to developed countries due to cancer causing behavioral life style especially smoking (Ferlay et al., 2010(Ferlay et al., , 2015. Currently available anticancer drugs are not target specific and pose several side effects and some complications in clinical management which encourage the urgent need for novel, effective, and nontoxic therapeutic approaches. According to the World Health Organization (WHO), cardiovascular diseases (CVDs) (acute myocardial infarction and peripheral arterial thrombosis) are the causes of approximately 30% of deaths worldwide (Palomo, Fuentes, Padro, & Badimon, 2012). At this time, available thrombolytic agents are tissue plasminogen activator (t-PA), streptokinase (SK), and urokinase (UK) that might cause serious bleeding complications along with reinfarction and reoccolution. Recently, a number of bioactive compounds from natural sources have been explored and identified as inhibitors to various cancerous cells and are safe alternative to treat cardiovascular and diabetes (Patel & Goyal, 2012). The search for new anticancer agents resulted in the isolation and purification of number of bioactive compounds from various mushroom species that were shown to have antitumor activity (Borchers, Krishnamurthy, Keen, Meyers, & Gershwin, 2008).
This study was aimed at the exploration of the potential of selected mushroom against cancer and cardiovascular diseases.

| Sample collection and preparation
The wild locally grown Ganoderma lucidum (Fr.) P. Karst., was isolated from the stem of Salmalia malabarica plant collected from Jinah garden,

| Extraction of selected mushrooms
The selected mushrooms were extracted in methanol, ethanol, and water according to the method given by Gangadevi, Yogeswari, Kamalraj, Rani, and Muthumary (2008) with slight modification.
Briefly, dried mushroom powder 20 g was extracted with 200 ml of methanol, ethanol (80%), and water using an orbital shaker (Gallenkamp, UK) for 8 hr at room temperature. The extracts were separated from solid residue by filtering through Whatman No. 1 filter paper. The extract was evaporated in rotary evaporator (EYELA, N-N Series; Rikakikai Co. Ltd. Japan) to yield the residue and stored at 4°C for subsequent analysis.

| Anticancer potential of selected mushrooms
The in vitro cell proliferation assay was conducted as described by Jeff et al. (2013). The number of living cells at the end of incubation period was determined by colorimetric assay based on the tetrazolium salt MTT. In this assay, the tested samples were compared with control (without sample). All the experiments were performed in triplicate, and cell proliferation under each condition was expressed as a percentage of the control, which was set at 100%. All in vitro results were expressed as the proliferation ratio of tumor cells calculated as follows: where A and B are the average numbers of viable tumor cells for the control and samples, respectively, (Jeff et al., 2013).

| α-Glucosidase inhibition activity
The α-glucosidase inhibition activity was performed according to the slightly modified method of Kwon, Apostolidis, and Shetty (2008) and Dong, Li, Zhu, Liu, and Huang (2012). Total volume of the reaction mixture of 100 μl contained 70 μl 50 mm phosphate buffer saline, pH 6.8, 10 μl (0.5 mm) test compound, followed by the addition of 10 μl (0.057 units) enzyme. The contents were mixed, preincubated for 10 min at 37°C, and preread at 400 nm. The reaction was initiated by the addition of 10 μl of 0.5 mm substrate (p-nitrophenyl glucopyranoside). Acarbose was used as a positive control. After 30 min of incubation at 37°C, absorbance was taken at 400 nm using microplate reader (BioTek-USA).
The percent inhibition was calculated by the following equation: IC 50 values (concentration at which there is 50% in enzyme catalyzed reaction) of compounds were calculated using EZ-Fit Enzyme Kinetics Software (Perrella Scientific Inc. Amherst, USA).

| Tyrosinase inhibition activity
The antityrosinase effect of mushrooms was determined by calculating the hydroxylation of L-tyrosine to L-DOPA. Inhibition assay was conducted in 96-well microplates, a spectrophotometer reader was used to determine the absorbance at 490 nm. Kojic acid was used as a positive control. (Momtaz et al., 2008).

| Thrombolytic activities of selected mushrooms extracts and fractions
Clot lysis activity was checked using different mushrooms extracts and fractions (Prasad et al., 2006). The streptokinase was used as a positive control for in vitro clot lysis. In commercially available lyophilized streptokinase (SK) vial, 5 ml phosphate buffered saline (PBS) was added and assorted properly. This suspension was used as a stock from which proper dilutions were made to examine the anticlot activity.

| Sample preparation
Each extract (10 mg) was suspended in 1 ml dimethylsulfoxide (1%), and the suspension was shaken vigorously on a vortex.
Blood samples of different healthy volunteers were collected from different hospitals and laboratories of Faisalabad, Pakistan.
Venous blood was drawn from healthy human volunteers without a history of oral contraceptive or anticoagulant therapy and irrespective of gender. Blood (500 μl) was transferred to the previously weighed microcentrifuge tubes. W c = w m-w e

| Addition of mushrooms extracts and fractions
Each mushroom extract (100 μl) was added in tubes, where streptokinase and distilled water were applied as positive and negative controls, respectively. All the microcentrifuge-tubes were again incubated at 37°C for 90 min. for clot lysis. Then, tubes were inverted and left overnight. Microcentrifuge tubes were taken out of the incubator and the fluid obtained after lysis along with the applied agents (extract, streptokinase, and distilled water) was removed carefully. Tubes were weighed to calculate the weight of clot after lysis. The weights of clots were determined by taking difference between weights of clot after lysis (W1) and weight of empty tubes (We).
Then, percentage of clot lysis activity of different mushrooms extracts was determined by the difference between weight of clots before (Wb) and after lysis (W1) dividing by weight of clot before lysis and multiplied by 100.

| Effect of concentrations, incubation time, and amount of sample on clot lysis
Mushrooms extracts of different concentrations (1%, 0.3%, and 0.6%) and incubated at different time intervals (30, 60 and 90 min), and 30, 60, and 100 μl of each concentration (1%, 0.6%, and 0.3%) of mushroom extract were used to determine the effect on thrombolysis.

| Statistical analysis
The results obtained were presented by means ± standard deviation.

| Anticancer potential of studied mushrooms
Cancer diseases are one of the main causes of death worldwide (Liu, Wang, Zhao, & Wang, 2013). The discovery of new molecules from natural origin is a global trend currently for the less toxicity of natural products (Wang et al., 2012). A number of bioactive compounds from natural resources had been investigated, identified, and isolated as inhibitor to various cancer cell lines (Ma, Chen, Dong, & Lu, 2013). In this study, the anticancer activity of water extracts of selected mushrooms was subjected to in vitro cytotoxicity assay in certain cancer cell lines including HT-29 colon and H-1299 lungs carcinoma cell lines. It was found that higher the concentration, the lower was the cell viability percentage (Thetsrimuang, Khammuang, Chiablaem, Srisomsap, & Sarnthima, 2011). The antiproliferative effect was in dose-dependent manner, and the maximum inhibition was observed at the concentration of 200 μg/ml. The inhibitory activities of the water extracts on these cell lines are shown in Figure 1a,b.
All the mushrooms exhibited inhibition against HT-29 cell lines.  Clot lysis (%) = It is concluded that the anticancer potential of G. lucidum might be due to high percentage of fiber contents that is 54% in our study.
As dietary fibers are the carbohydrates in the diet that are not hydrolyzed by enzymes in either the stomach or small intestine, therefore they have importance for the management of different ailments. The anticancer potential of wild G. lucidum might be due to this attribute in addition to its antioxidant potential.

| α-glucosidase and tyrosinase inhibition activities of selected mushrooms
In the synthesis of melanin pigments, tyrosinases are responsible for coloring hairs, skin, and eyes and also for the treatments of some dermatological hyper pigmentation illness connected with overproductions of melanin (Chen, Ying, Li, & Yua, 2012  It is concluded from the above experiment that wild mushroom showed best tyrosinase and α-glucosidase inhibition activities.
Among the selected commercial mushrooms, locally cultivated P. ostreatus was the best tyrosinase and α-glucosidase inhibitor as compared to the exotic commercial mushrooms.

| Thrombolytic activity of mushrooms extracts and fractions
According to the World Health Organization (WHO), cardiovascular diseases (CVDs) (acute myocardial infarction, cerebrovascular disease, and peripheral arterial thrombosis) are the causes of approximately 30% of deaths worldwide (Palomo et al., 2012). In normal, body process coagulation and fibrinolysis processes are controlled properly. The dysfunction of fibrinolysis process or myocardial or cerebral infarction is a serious consequence of the thrombus formed in blood, and thus, blockage of blood vessels due to blood clot (fibrin clot) results in vascular disorders such as deep-vein thrombosis, stroke, myocardial infarction, and pulmonary embolism (Choi et al., 2014). Thrombolytic agents are used to dissolve the already formed clots in the blood vessels (Ansari, Siddiqui, & Singh, 2012). Currently available thrombolytic agents are streptokinase (SK), tissue plasminogen activator (t-PA), and urokinase (UK). They might cause serious bleeding complications along with reinfection and reoccolution and therefore secure and effective thrombolytic agents that can lyse a blood clot are desirable (Prasad et al., 2006).
TA B L E 1 α-glucosidase and tyrosinase inhibition activities of selected mushrooms water extracts (%DW)

| Effect of concentration, volume and time of incubation of selected mushrooms on thrombolysis
The percentage values of clot lysis of mushrooms extracts were di-  (Table 3). There was an increase in absorbance with increasing incubation time. The average percentage lysis values of natural extracts were close to synthetic compounds.
Up to our knowledge, no study has been conducted on thrombolytic potential of these selected mushrooms. This study showed that L. edodes and H. erinaceus extracts have significant thrombolytic activity as compared to the local cultivated mushrooms P. ostreatus.
Wild G. lucidum also showed moderate thrombolytic potential.

ACK N OWLED G M ENT
The authors are highly thankful to Higher Education Commission (HEC), Islamabad, Government of Pakistan for financial support for this work.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.