Authors:  ¹Vivekanandan Kalaiselvan, M. Pharm, Ph. D., ²Ahmed Sultana, M. Pharm., ²Muthusamy Kalaivani, M. Pharm, ²Santhanam Prasath, M. Pharm., ²Aiyalu Rajasekaran, M. Pharm, Ph.D

¹Indian Pharmacopoeia Commission, Sector 23, Raj Nagar, Ghaziabad, UP-201002, India

² Kovai Medical Center and Hospital (KMCH) – College of Pharmacy, Coimbatore 35, Tamilnadu, India

ABSTRACT
Mushrooms are fungi; some of them have been used as food by humans for millennia. Edible mushrooms are recognized as a source of antioxidants and as a potential chemotherapy treatment for cancer. The present study was designed to investigate the antioxidant, immunomodulatory, and anticancer properties of aqueous extracts of the edible mushroom Pleurotus florida. In vivo immunomodulatory activity was determined in male Balb/c mice following a Cyclophosphamide-induced challenge (10 mg/kg). Anticancer potential of P florida extracts was evaluated using the Ehrlich ascites carcinoma cell line. Positive modulation of antioxidant enzymes such as catalase, superoxide dismutase, and glutathione peroxidase was observed in the P florida treated group compared with the control group. Pleurotus florida (500 mg/kg) significantly increased white blood cell counts. In addition the tumor-reducing effect of the extract at a higher dose (1000 mg/kg) approached that of Cisplatin (4 mg/kg). Following histopathology, mice treated with a combination of P florida and cyclophosphamide developed no ductal adenoma in liver samples whereas in the cyclophosphamide only group, ductal carcinomas were evident. Further studies of the isolated active principle from P florida are warranted, to explore the full mechanisms of anticancer activity.

Introduction
Reactive oxygen species possess a wide range of pathologic effects, including cellular degeneration, DNA damage, and carcinogenesis.¹ Neutralization of free radical activity by natural antioxidant substances is generally considered a safe and effective therapy at mitigating these risks. Components of the immune system can be damaged by reactive oxygen species thus leading to immunodeficiency.² Immunomodulators used in the treatment of cancer can act by inducing stimulatory cytokines (such as interferon-alpha, tumor necrosis factor, and interleukin 12) and by activating T cells and antigen-presenting dendritic cells.³ The US National Cancer Institute has intensified its examination of natural products such as plants, marine organisms, and selected classes of microorganisms as sources for discovering new drugs. Many available and clinically useful anticancer drugs include natural plant products or the derivatives of natural products, including such examples as paclitaxel from the plant Taxus brevifolia and vincristine from Catharanthus roseus.⁴ Because of adverse effects from synthetic drugs and owing to a decline in the number of new molecular entities generated by the pharmaceutical industry, further novel anticancer agents are being sought from natural sources.^5,6 Natural products continue to offer a wide range of compounds with diverse structures and activities for use in modern cancer therapy.

Several clinically well-established drugs, including penicillin, griseofulvin, ergot alkaloids, and cyclosporine, are of fungal origin. Mushrooms are known to contain major therapeutic substances.^7,8 About 700 species of higher mushrooms have been found to possess significant pharmacologic properties.⁹ Pleurotus florida is cultivated for edible purposes in many countries, including India. It is valued for its nutritional content and for its high medicinal value among mushrooms cultivated worldwide.¹⁰ Pleurotus species have demonstrated antioxidant, anti-inflammatory, and anticancer properties.¹¹ Different extracts of this species have exhibited activity against various chronic diseases, including hypertension and hypercholesterolemia.^12-15 These beneficial medicinal effects of Pleurotus species were discovered independently in different countries. Awareness of the medicinal properties of these fungi comes not only from Asia but also through the folklore of central Europe, South America, and Africa.

Superoxide is generated during oxidation and is sequentially reduced to hydrogen peroxide and hydroxyl radicals. These free radical species damage DNA thereby producing cancer-initiating mutations. Immunomodulators and antioxidant compounds may have a vital role in the treatment of cancer by improving the immunologic response activity of white blood cells to enable recognition and destruction of cancer cells. A mushroom with antioxidant and immunomodulatory potential could well be a novel anticancer agent. The present study was undertaken to evaluate the antioxidant, immunomodulatory, and anticancer potential of P florida using a preclinical model.

Materials and Methods

All chemicals were purchased from S. D. Fine Chemicals, Ltd, Mumbai, India. An Ehrlich ascites carcinoma (EAC) cell line was obtained from Amla Cancer Research Institute, Thrissur, Kerala, India.

Animals

Male Balb/c mice were procured from the National Institute of Nutrition, Hyderabad, India. All animal investigations were approved by the institutional animal ethics committee (registration No. 685/02/a/CPCSEA).

Cultivation and Extraction

Pleurotus florida spawns were obtained from Tamil Nadu Agricultural University, Coimbatore, India. Cultivation and extraction were performed according to the method by Tan.¹⁶ The spawns were cultivated in polythene covers. Paddy straw (brown) was collected, cut into small pieces, and sterilized. Eight to 10 holes (each 2 cm diameter) were punctured in the polythene covers. Sterilized paddy straw was first packed to a depth of 5.1 mm (2 in), and spawns were spread over the straw. The polythene was then tied at the top of the mouth. The spawns were maintained at 25°C, and water was supplied daily at the top of the covers in which the mushrooms were grown. All spawns were kept in a dark shed. After 13 days, small buds appeared; after 25 days, fully grown mushrooms were collected and processed for the study. The mushrooms were dried in the shade and pulverized. One hundred grams of powder was mixed with sufficient water and was kept for 7 days in a process of cold maceration. Care was taken to avoid bacterial and fungal growth by adding sodium benzoate as a preservative, and the whole process was performed under aseptic conditions. After 7 days, the content was filtered. The filtrate was lyophilized and was then suspended in 5% gum acacia for pharmacologic studies.

Evaluation of In Vivo Immunomodulatory Effect

Pathogen-free male Balb/c mice were selected and divided into various groups. Group 1 received saline intraperitoneally (IP); group 2 received cyclophosphamide IP (10 mg/kg of body weight); and groups 3, 4, and 5 received aqueous extracts of P florida extract orally (100, 250, and 500 mg/kg, respectively). Group 6 received an aqueous extract of P florida extract (500 mg/kg) combined with cyclophosphamide IP (10 mg/kg). On day 8, blood samples were collected from the orbital vein and washed in Hank solution (containing calcium chloride, hydrated magnesium sulfate, potassium chloride, monopotassium phosphate, sodium bicarbonate, sodium chloride, sodium phosphate dibasic dihydrate, and d-glucose). Blood specimens were analyzed to determine white blood cell (WBC) counts. Cells were counted in a Neubauer chamber.

Evaluation of In Vivo Antioxidant Property

Animals were euthanized after completion of the immunomodulatory study, and their livers were dissected out. Liver tissue was homogenized and prepared for further analysis. The homogenate was centrifuged, and aliquots were used for the estimation of catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPX) activity.

Catalase activity was estimated using the method described by Sinha.¹⁷ One milliliter of phosphate buffer and 50 µL of aliquot were considered a blank. One hundred microliters of hydrogen peroxide was added to each sample, and change in optical density was recorded at 650 nm in a spectrophotometer for 30 seconds at 5-second intervals. One unit of catalase activity represents the amount of enzyme required to decompose 1 µmol of hydrogen peroxide per minute.

Superoxide dismutase activity was measured using the method by Misra and Fridovich.¹⁸ The assay is based on the oxidation of epinephrine adenochrome transition by the enzyme. Carbonate buffer (0.75 mL) and 50-µL aliquots of sample were used as blanks. Samples were immediately reacted with epinephrine (10 µL), and change in SOD of each sample was recorded at 480 nm for 2 minutes at 15-second intervals. Auto-oxidation of epinephrine was also recorded. The SOD activity was calculated from the auto-oxidation of epinephrine. One unit of SOD represents the amount of enzyme required to produce 50% inhibition of epinephrine auto-oxidation.

Glutathione peroxidase activity was measured using the method by Paglia and Valentine.¹⁹ Aliquots were reacted with hydrogen peroxide, and change in optical density per minute for each sample was recorded at 340 nm for 2 minutes at 15-second intervals. The glutathione peroxidase activity was calculated from the extinction coefficient of nicotinamide adenine dinucleotide phosphate (NADPH). One unit of enzyme activity represents the amount of enzyme required to use 1 nmol of NADPH per minute at 37ºC.

Histopathologic Examination

Histopathologic studies were performed among liver samples obtained from control animals and from treated animals. These examinations were conducted on day 8 after completion of the immunomodulatory studies.

Evaluation of In Vivo Anticancer Activity

Anticancer potential was assayed using the ascites tumor model developed by Ajith and Janardhanan.²⁰ Male Balb/c mice (weight, 20-25 g) were used for the study. The animals were divided into 5 groups, with 6 animals in each group. Animals were injected subcutaneously with EAC cells (106 cells per 0.1 mL) on the right hind limb. Aqueous extracts of P florida were administered intraperitoneally at doses of 250, 500, and 1000 mg/kg after 24 hours of tumor inoculation, and drug administration was continued for 10 days. Cisplatin was used as the standard, and the group injected with EAC cells alone served as a positive control. Tumor development for animals in each group was measured using a Vernier caliper, and tumor volume (V) was calculated using the following formula: V = (4/3) r₁ r₂, where r₁ and r₂ are the radii of the tumors).²¹

Statistical Analysis

All data is expressed as the mean with standard deviation in brackets (SD). The groups were compared by 1-way analysis of variance (ANOVA) using post hoc Dunnett test. P < .05 was considered significant.

Results

Preparation of P florida Extract

Different stages of growth of P florida during cultivation are shown in Figure 1. The mushrooms were dried, powdered, and extracted in water. Extract was lyophilized, producing a yield of 9.5% wt/wt.

Figure 1: Different stages of growth of Pleurotus florida during cultivation

A. Spawns packedin sterilized paddy straw
B. After 12 days, appearance of small buds
C, D, E, and F: Mushroom growth on 13tt, 14th, 15th, and 16th day respectively
G: Mushroom fully matures on day 17th day

 

Effects of P florida on Immunologic Variables

Administration of aqueous extract of P florida increased total WBC counts in healthy Balb/c mice. The mean maximal WBC count in animals treated with P florida (500 mg) was 9.40 cells/µL on day 8 after drug administration; this corresponds to a 49% increase over the controls (P < 01) (Table 1). The mean maximal WBC count in animals treated with the combination of P florida and cyclophosphamide was 4.55 cells/µL, corresponding to a 9% increase over the cyclophosphamide-only treated group (P < 001). There was no appreciable change in differential count or hemoglobin content after administration of P florida.

Effects of P florida on Organ Weight

The effects of P florida on organ weight are summarized in Table 2. Livers were heavier after administration of P florida. The size and weight of kidneys also increased significantly after administration of combined P florida (500 mg/kg) and cyclophosphamide (10 mg). Overall, 22%, 15%, and 12% increases were found in the weights of livers, kidneys, and spleens, respectively, with administration of P florida compared with controls. The weight of the spleen increased slightly with administration of P florida.

Antioxidant Variables

Catalase, SOD, and GPX activity in granulation tissue was significantly increased in mice treated with aqueous extracts of P florida (250 and 500 mg/kg) compared with the cyclophosphamide-treated group (P < .001) (Table 3). Enzyme activity was measured as international units per milligram of protein.

Figure 2: Effect of Pleurotus florida on histopathological studies

A: Control- No cells were destroyed in the liver portion of the control Balb/c mice on 8th day of treatment in the Immunimodulation study.
B. Cyclophosphamide Treated- Liver cells we destroyed
C: New cells were generated in the destroyed portion of the liver.

Histopathologic Examination

Histopathologic studies showed that bile duct adenomas occurred in all cyclophosphamide-treated mice but not in the control group. In the group in which combined P florida and cyclophosphamide was administered, bile duct adenomas were found in only 2 of 6 mice tested (Figure 2).

Antitumor Activity

At the lower dose, aqueous extract of P florida (250 mg/kg) did not reduce tumor burden in mice exposed to EAC cells. However, higher doses of extract (500 and 1000 mg/kg) significantly inhibited tumor growth. The tumor-reducing effect of extract at the higher dose (1000 mg/kg) was approached that of the standard anticancer drug cisplatin (4 mg/kg) (Table 4).

Discussion

Free radicals are molecules with incomplete electron shells, which makes them more chemically reactive than molecule with complete electron shells. Exposure to various environmental factors, including smoking, irradiation, and sunlight, can lead to free radical formation.²² In humans, the most common form of free radical is oxygen. When an oxygen molecule becomes electrically charged, it tries to steal electrons from other molecules, causing damage to DNA and other molecules.²³ Over time, such damage may become irreversible and lead to disease, including cancer. Antioxidants can neutralize the electrical charge and prevent free radicals from taking electrons from other molecules.^24,25

Immunomodulatory agents of natural origin may enhance the immune responsiveness of an organism against a pathogen by activating the immune system.²⁶ Immunity has been shown to be suppressed in cancer. Chemotherapy and radiation therapy, often used in cancer treatment, can contribute to further deterioration in immune function.²⁷ Immunomodulatory effects of the compound β-d-glucan, a heterogeneous group of glucose polymers present in medicinal mushrooms, may represent a coadjuvant for the treatment of malignant tumors.²⁸ It is believed that immunomodulatory agents and antioxidants combined may have a role in the prevention of cancer.

The present study was undertaken to assess the potential action of an aqueous extract of P florida against a cyclophosphamide-induced challenge. Animals administered cyclophosphamide have demonstrated suppressed humoral immunity via reductions in antibody production through interference with helper T-cell activity.^29,30 In the present study, administration of P florida increased total WBC counts in an immunosuppressed group of mice and increased the weights of the spleen. The results indicate that (at least under these conditions) P florida stimulates production of immune cells.³¹ Positive modulation of antioxidant enzymes such as catalase, SOD, and GPX suggests that aqueous extract of P florida also possesses in vivo antioxidant activity. Change in both liver and kidney weight was observed with P florida intervention as compared to saline control, however histopathological study does not shown any clinical sings.

Although the link between antioxidant effect and anticancer activity was explored in our study, edible mushrooms and medicinal plants having strong antioxidant properties also often possess anticancer activity.^32,33 Our findings about antioxidant effects encouraged us to investigate the anticancer potential of our extracts as well. This connection was reiterated by significant inhibition of tumor growth (approaching the effect of the standard anticancer drug cisplatin) that was observed with aqueous extract of P florida (1000 mg/kg). Pleurotus florida is rich in bioflavones and flavonoids,³⁴ which might be responsible for the antioxidant, immunostimulatory, and antitumor activity we observed. Further studies are in progress to isolate and characterize these compounds for their potential anticancer effect.

 

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Table 1: Effect of Aqueous Extract of Pleurotus florida on White Blood Cell (WBC) Counts in 6 Balb/c Mice

Treatment	Maximal WBC Cell Count, mean (SD), cells/µL	Increase in percentage
Control	3.90 (0.06)	…
P florida 100 mg/kg	5.10 (0.07)b	13.3
P florida   250mg/kg	6.00 (0.32)b	24.4
P florida  500mg/kg	9.40 (0.01)b	48.9
Cyclophosphamide, 10 mg/kg	2.80 (0.02)	…
Combined Pleurotus florida, 500 mg/kg, and cyclophosphamide, 10 mg/kg	4.55 (0.08)c	8.9

 

         ^bP < .01 compared with controls.

         ^cP < .001 compared with the cyclophosphamide-treated group.

 

Table 2: Effect of Aqueous Extract of Pleurotus florida on Organ Weights in 6 Balb/c Mice

Treatment	Relative Organ Weight, mean (SD), g/100 g of body weight
	Liver	Kidney	Spleen
Saline Control	0.52 (0.01)	0.24 (0.06)	0.03 (0.01)
P florida 100 mg/kg	0.73 (0.02)a	0.24 (0.05)a	0.04 (0.01)a
P florida 250 mg/kg	0.79 (0.05)a	0.33 (0.06) a	0.05 (0.02)a
P florida 500 mg/kg	0.92 (0.08)	0.34 (0.08)	0.09 (0.02)e
Cyclophosphamide, 10 mg/kg	0.42 (0.02)a	0.19 (0.08) a	0.02 (0.01 a
Combined P florida, 500 mg/kg, and cyclophosphamide, 10 mg/kg	0.76 (0.04)b	0.26 (0.07) b	0.03(0.01)b

a-     Non significant as compared with control at P < 0.05 and P < 0.01

b-    Non significant as compared with cyclophosphamide treated at P < 0.05 and P < 0.01

Table 3: Effect of Aqueous Extract of Pleurotus florida on Antioxidant Enzymes in 6 Balb/c Mice

                 ^aP < 0.01,  ^bP < 0. 05 compared with controls.

                 ^cP < 0. 001 compared with the cyclophosphamide-treated group.

Table 4: Effect of Aqueous Extract of Pleurotus florida on Solid Tumors in 6 Balb/c Mice

Treatment	Final Tumor Volume, mean (SD), cm3	Decrease in percentage
Saline Control	1.48 (0.08)	…
Cisplatin, 4 mg/kg	0.05 (0.02)	95.8
P florida 250 mg/kg	0.77 (0.31)	47.2 a
P florida 500 mg/kg	0.51 (0.28)	70.0a
P florida 1000mg/kg	0.19 (0.05)	84.9a

^a P < .001 vs cisplatin.