Egyptian Pharmaceutical Journal

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 19  |  Issue : 3  |  Page : 274--282

Biological evaluation of ethyl acetate extract of Chaetomium cupreum against Ehrlich ascites carcinoma cells in Swiss albino mice


Nazir A Wani1, Waseem I Khanday2, Sheihk A Ali3, Sharmila Tirumale1,  
1 Department of Microbiology and Biotechnology, Jnanabharathi Campus, Bangalore University, Bengaluru, Karnataka, India
2 PG and Research Centre in Biotechnology, M.G.R. College, Hosur, Tamil Nadu, India
3 Division of Biotechnology, Indian Institute of Integrative Medicine (CSIR), Sanatnagar, Srinagar, Jammu and Kashmir, India

Correspondence Address:
MSc, PhD Sharmila Tirumale
Department of Microbiology and Biotechnology, Jnanabharathi Campus, Bangalore University, Bengaluru 560056, Karnataka
India

Abstract

Background Chaetomium genus is a natural source of different types of secondary metabolites or pigments. These secondary metabolites display a broad spectrum of biological properties including antimicrobial, antioxidant, anti-inflammatory, and anticancer activity. Objective The objective of the study was to evaluate the anticancer activity of ethyl acetate extract of Chaetomium cupreum against Ehrlich ascites carcinoma (EAC) cells in Swiss albino mice. Materials and methods Methods involved are evaluation of acute toxicity study, tumor induction using EAC cells, estimation of various hematobiochemical parameters, and evaluation of antioxidant enzymes and markers of oxidative stress. Results The ethyl acetate extract of C. cupreum-treated EAC-bearing mice at the concentration of 200 mg/kg body weight (bwt) reduced ascitic fluid volume (1.65±0.70 ml) and ascitic fluid weight (1.32±0.69 g) as compared with ascitic fluid volume (4.79±0.52 ml) and ascitic fluid weight (3.93±0.57 g) in EAC control group. Similarly, the cell apoptosis was higher in EAC-bearing mice treated with standard 5-fluorouracil at 50 mg/kg bwt (96.04%) as compared with treatment with ethyl acetate extract at 50 mg/kg bwt (21.92%) followed by 100 mg/kg bwt (36.63%) and increased further at 200 mg/kg bwt (47.48%) in treated groups. In hematological estimation, the EAC-bearing mice treated with ethyl acetate extract at 200 mg/kg bwt showed increased red blood cell count (3.78±0.07×106/μl) and hemoglobin content (6.02±01 g/dl) and decreased white blood cells count (5.45±0.01×103/μl). In biochemical estimation, ethyl acetate extract treatment in EAC-bearing mice at 200 mg/kg bwt decreased aspartate aminotransferase activity (64.10±0.07 U/l), alanine aminotransferase (55.71±0.65 U/l), alkaline phosphatase (107.04±0.02 U/l), cholesterol (124.38±0.04 mg/dl), and triglycerides (155.38±0.04 mg/dl). Similarly, in enzymatic antioxidants and oxidative stress, the ethyl acetate extract-treated EAC-bearing mice at 200 mg/kg bwt increased superoxide dismutase (27.10±0.03 U/mg protein), catalase (20.20±0.02 U/mg protein), and reduced glutathione (24.04±0.03 U/mg protein), whereas decreased glutathione peroxidase (38.04±0.07 U/g hemoglobin) and malondialdehyde content (170.50±0.06 nmol/mg protein) significantly. Conclusion The results of the present finding showed that ethyl acetate extract of C. cupreum possesses significant anticancer potential.



How to cite this article:
Wani NA, Khanday WI, Ali SA, Tirumale S. Biological evaluation of ethyl acetate extract of Chaetomium cupreum against Ehrlich ascites carcinoma cells in Swiss albino mice.Egypt Pharmaceut J 2020;19:274-282


How to cite this URL:
Wani NA, Khanday WI, Ali SA, Tirumale S. Biological evaluation of ethyl acetate extract of Chaetomium cupreum against Ehrlich ascites carcinoma cells in Swiss albino mice. Egypt Pharmaceut J [serial online] 2020 [cited 2020 Nov 26 ];19:274-282
Available from: http://www.epj.eg.net/text.asp?2020/19/3/274/296804


Full Text

 Introduction



In recent years, a significant development has been made in cancer treatment globally. However, still the development of effective cancer prevention remains the greatest challenges in the area of cancer chemotherapy. Cancer causes most of the morbidity and mortality in the world. The incidence of cancer percentage is increasing globally. The main objective of cancer therapy is to induce death of cancer cells without causing damage or little damage to normal cells [1]. Most of the anticancer drugs are carcinogenic, mutagenic, and expensive. One of the most effective methods of treatment of cancer is chemotherapy. Unfortunately, the presently available chemotherapeutic synthetic anticancer molecules used in the treatment of cancer have developed drug resistance and cause various adverse effects in patients [2]. Owing to the limitations of synthetic anticancer drugs, there is an urgent need of molecules derived from natural sources such as microorganisms, plants, marine organisms, and fungi for the development of anticancer drugs for the treatment of human cancer [3]. More recently, it was found that tumor cells develop resistance to a single compound easily than multiple compounds. Therefore, treatment of cancer involving combination of drugs is preferred over a single drug treatment, and combination of specific phytochemicals may be more effective than a single compound in preventing against cancer [4].

The phytochemicals from natural source possess various biological activities such as antimicrobial, antioxidant, and anticancer activity [5],[6]. Approximately 60% of presently used anticancer drugs are derived from natural source including plants, marine organisms, and microorganisms [7],[8]. The recent studies have shown that the naturally occurring phytochemicals are more effective with fewer adverse effects than synthetic drugs [9]. The filamentous fungi are known to produce various phytochemicals such as cyclosporine, lovastatin, and penicillin and also a rich resource of novel compounds of therapeutic importance [10].

Chaetomium species belongs to chaetomiaceae family, which are the largest genus of saprophytic ascomycetes and a natural source of different types of metabolites. Since Kunze was the first to establish this genus in 1817 and till now more than 350 Chaetomium species are known [11]. The Chaetomium cupreum was described by Lawrence Marion Ames in 1949 as part of a military effort to identify the organisms responsible for the biodeterioration [12]. In previous studies, methanol extract of C. cupreum-RY202 exhibited in-vitro anticancer activity against KB cell line having IC50-3.25 μg/ml concentration. Further study revealed that isochromophilonol, ochrephilonol, and azaphilones were isolated from C. cupreum-RY202, and both the compounds exhibit moderate anticancer against KB and NCI-H187 cell line having IC50 of 9.63–32.42 μg/ml [13]. The C. cupreum possess various secondary metabolites with various biological properties [14]. For the present study, Ehrlich ascites carcinoma (EAC) cells were used as the cell line. The Ehrlich tumor is a spontaneous murine mammary adenocarcinoma [15]. The Ehrlich tumor is a rapidly growing carcinoma in all strains of mice. In ascites form in mice, it is used to study the effect of anticancer molecules [16]. To the best of our knowledge, there are no previous reports on the anticancer activity of C. cupreum extracts. The purpose of the study was to investigate the anticancer properties of ethyl acetate extract of C. cupreum against EAC cells in Swiss albino mice.

 Materials and methods



Procurement of fungal culture

The C. cupreum fungus was procured from National Fungal Culture Collection of India (NFCCI), Agharkar Research Institute, Pune, with accession number NFCCI 3117.

Inoculum preparation and extraction

The C. cupreum was inoculated on potato dextrose agar plates and slants and maintained at 4°C for future use. For inoculum preparation, the culture was inoculated into potato dextrose broth and incubated at room temperature for 20 days on a rotary shaker at 120 rpm. The extraction involves liquid-liquid method [17]. After incubation, broth was filtered to remove mycelium. Then solvent and broth were taken in equal volume and shaken well for 15 min in a separating funnel. Then solvent layer was collected, and a crude dried extract powder was obtained.

Animal in-house testing

Male adult Swiss albino mice weighing between 25 and 30 g (n=60) were housed in well-ventilated large spacious polypropylene cages under standard conditions of humidity (50–60%), temperature (30±5°C), and light (12 h light/dark). Animals were provided with dry pellet rat feed along with tap water ad libitum. Animal experiments were done at the animal facility of KM College of Pharmacy, Madurai, Tamil Nadu, India. The present study involving animals was approved by Institutional Animal Ethical Committee (IAEC) and was conducted according to the guidelines of the committee for the purpose of control and supervision of experiments on animals, with Regd. No.: 661/02/C/CPCSEA/29/09/2017.

Toxicity study of ethyl acetate extract

The toxicity test in male Swiss albino mice was performed in accordance with the Organization for Economic Co-operation and Development (OECD) guidelines 425 [18]. The Swiss albino mice (n=6) were deprived of food for 18 h and then administered intraperitoneally 0.25 ml of various doses (250, 500, and 1000 mg/kg body weight (bwt)] of ethyl acetate extract of C. cupreum once only. The toxicity symptoms were recorded for 6, 12, 24 h, and 48 h, after which time, the number of survival animals was recorded. The general behaviors that were observed during acute toxicity study are sedative, hypnosis, convulsion, motor activity, lacrimal secretion, ptosis, change in skin color, and stool consistency.

Experimental design for intraperitoneal dosing of ethyl acetate extract

Group I: animals were treated with normal saline.

Group II: animals were treated with 250 mg/kg bwt of ethyl acetate extract.

Group III: animals were treated with 500 mg/kg bwt of ethyl acetate extract.

Group IV: animals were treated with 1000 mg/kg bwt of ethyl acetate extract.

Induction of tumor using Ehrlich ascites carcinoma cells

The EAC cells were procured from Amala Cancer Research Center, Thrissur, Kerala, India, and were maintained and subcultured in Swiss albino mice after every 10th day through intraperitoneally. The cancer formation was done by Jagetia et al. [19] method. After 8 days, ascitic fluid from peritoneal cavity was withdrawn from EAC-bearing mice. The cancer cells were diluted with normal saline and adjusted in a suspension of 1×106 cells/ml. All animals received 100 μl EAC cells except group I. After 2 h, cancer-developed animals were divided into six groups, with six animals in each group (n=6).

Experimental design

Group I (normal control): animals as normal control received with normal saline (100 μl intraperitoneally) from day 1 to 9.

Group II (EAC control): animals received EAC cells (100 μl intraperitoneally) from day 1 to 9.

Group III (standard control): animals received 5-fluorouracil at 50 mg/kg bwt (100 μl intraperitoneally) from day 1 to 9.

Group IV (EAC+extract): animals received ethyl acetate extract at 50 mg/kg bwt (100 μl intraperitoneally) from day 1 to 9.

Group V (EAC+extract): animals received ethyl acetate extract at 100 mg/kg bwt (100 μl intraperitoneally) from day 1 to 9.

Group VI (EAC+extract): animals received ethyl acetate extract at 200 mg/kg bwt (100 μl intraperitoneally) from day 1 to 9.

Collection of ascitic fluid and blood from animals

After 24 h of last dose and 18 h of fasting (12 days of ethyl acetate extract treatment), the blood was collected in an ethylenediaminetetraacetic acid vial from retro-orbital plexus by sterile capillary for estimation of blood markers. The animals were anesthetized with diethyl ether, and ascitic fluid was collected.

Estimation of ascitic fluid volume, ascitic fluid weight, and cell viability

The ascitic fluid was measured in graduated centrifuge tube and expressed in milliliters, whereas ascitic fluid weight was measured by weighing ascitic fluid and expressed in grams. The cell viability was measured by diluting ascitic fluid with phosphate buffer saline, and 10 μl was mixed with 90 μl of Trypan blue dye and then 10 μl was placed on hemocytometer. Then viable and dead cells were counted, and percentage viability was calculated as follows:

[INLINE:4]

Estimation of hematological parameters in control and experimental animals

Various hematological parameters such as red blood and white blood cell count and hemoglobin (Hb) were estimated.

Estimation of red blood cell count

Estimation red blood cell was carried out by using Neubauer hemocytometer counting chambers by Davidson’s method [20]. A volume of 0.5 ml of ethylenediaminetetraacetic acid-anticoagulated blood in red blood pipette was diluted to 1 : 200 with Hayem’s diluting fluid (mercuric chloride 0.25 g, sodium chloride 0.50 g, sodium sulfate 2.50 g, and distilled water 100 ml, pH 5.9). After incubation at room temperature for 3 min, the sample was placed on counting chamber and the number of red blood cell per cubic millimeter (cumm) was calculated.

[INLINE:5]

where the dilution factor is 200, area counted is 4 mm2 and depth is 0.1 mm.

Estimation of white blood count

Estimation of white blood cells was carried by using Neubauer hemocytometer counting chamber method [20]. A volume of 0.5 ml of ethylenediaminetetraacetic acid-anticoagulated blood was diluted to 1:20 with Jurks’s diluting fluid (glacial acetic acid 2.0 ml, gentian violet 1%, distilled water 97 ml, pH 2.2). The sample was placed on counting chamber, and the number of white blood cells per cumm was calculated as follows:

[INLINE:6]

where the dilution factor is 20, area counted is 4 mm2, and depth is 0.1 mm.

Estimation of hemoglobin

The Hb concentration was measured by the acid-hematin method [21]. The graduated Hb tube was filled with 0.1 N hydrochloric acid by a dropper. Then blood was filled in the Hb pipette up to mark 20 mm3 and transferred to graduated Hb tube containing 0.1 N hydrochloric acid and further diluted by 0.1 N hydrochloric acid drop by drop with constant stirring by glass rod till the color matches with that of standard brown glass rod. The results were recorded on the Hb tube showing concentration of Hb in grams per deciliter.

Estimation of biochemical parameters in control and experimental animals

Aspartate aminotransferase

The aspartate aminotransferase was estimated by Reitman method [22]. A volume of 4.0 ml of solution 1 (each of 1.0 ml of tris buffer 100 mmol/l), L-aspartate (300 mmol), lactate dehydrogenase (1.5 U/ml), and malate dehydrogenase (0.53 U/ml)) was added to 1.0 ml of solution 2 (each of 0.05 ml of α-ketoglutarate; 75 mmol/l), and nicotinamide adenine dinucleotide (0.23 mmol/l)). Then 1 ml of this reaction mixture was added to 100 μl of test sample, and sample tubes were incubated for 1 min at 37°C, and absorbance was measured at 340 nm. The enzyme activity was expressed as micromole of pyruvate formed/mg protein/h.

Alanine aminotransferase

The alanine aminotransferase was estimated by Reitman method [22]. A volume of 3.0 ml of solution 1 (each of 1.0 ml of tris buffer 100 mmol), L-alanine (500 mmol/l), and lactate dehydrogenase (1.5 U/ml) was added to 1.0 ml solution 2 (each of 1.0 ml of α-ketoglutarate; 15 mmol/l), and nicotinamide adenine dinucleotide (0.18 mmol/l). Then 1 ml of this reaction mixture was added to 100 μl of test sample, and reaction tubes were incubated for 1 min at 37°C and absorbance was read at 340 nm. The enzyme activity was expressed as micromole of pyruvate formed/mg protein/h.

Assay of alkaline phosphatase

The alkaline phosphatase was estimated by King’s method [23]. A volume of 100 μl of test sample was mixed well with 0.5 ml enzyme solution, 1.5 ml buffer, and 1.0 ml substrate. After incubation for 15 min at 37°C, 1.0 ml of Folin’s phenol reagent was added and centrifuged for 10 min at 3000 rpm. Then supernatant (1.0 ml) was collected and 1.0 ml substrate, 1.0 ml sodium carbonate (15%), and 0.1 ml magnesium chloride were added followed by 37°C incubation for 10 min, and absorbance was measured at 640 nm. The enzyme activity in serum was expressed as micromoles of phenol liberated/min/mg of protein.

Estimation of total cholesterol

The total cholesterol was estimated by Allain method [24]. A volume of 10 μl of serum sample was added to 1.0 ml of reagent 1 (tris buffer 100 mM, pH 7.7, sodium cholate 10 mM, phenol 6 mM, cholesterol hydrolase 33 U/l, cholesterol oxidase 150 U/l, and horseradish peroxidase 200 U/l). The solution was incubated at 37°C for 10 min, and absorbance was measured at 500 nm. The concentration of cholesterol was measured from standard cholesterol calibration curve. The standard cholesterol consists of 100 mg of cholesterol dissolved in 100-ml ice cold acetic acid, and 10 ml was further diluted with 100-ml acetic acid. The cholesterol content was expressed as mg/dl serum or mg/100 g wet tissue.

Estimation of triglycerides

The triglyceride content was estimated by Werner method [25]. A volume of 10 μl of test sample was incubated at 37°C for 5 min and added to 1.0 ml of reagent 1 (tris-HCl buffer 100 mM, pH 7.8, glycerophosphate oxidase 2000 U/l, lipase 1000 U/l, glycerol kinase 800 U/l, peroxidase 150 U/l, sodium azide 0.1%, magnesium chloride 17 mM, 4-aminoantipyrene 0.4 mM, ATP 0.55 mM, and 3-hydroxyl-2,4,6-tribromobenzoic acid 1.6 mM), and absorbance was measured at 540 nm. The triglyceride content was expressed as mg/dl.

Estimation of enzymatic antioxidants and markers of oxidative stress in control and experimental animals

Estimation of superoxide dismutase

The superoxide dismutase was estimated by Misra’s method [26]. A volume of 1.0-ml plasma in potassium phosphate buffer was mixed with 1.0 ml of sodium carbonate (50 mM), 0.2 ml of hydroxylamine hydrochloride (0.1 mM), and 0.4 ml of nitroblue tetrazolium (75 mM). The reaction mixture was mixed well, and development of blue color was monitored at 560 nm spectrometrically. The superoxide dismutase activity was expressed as U/mg protein.

Estimation of catalase

The catalase was estimated by Aebi method [27]. A volume of 0.1 ml of plasma was mixed well with 1.0 ml of hydrogen peroxide (0.5 mM) and 1.9 ml pf phosphate buffer (10 mM). The absorbance was read at 240 nm, keeping 1-min interval for 3 min. The enzyme activity was expressed as micromoles hydrogen peroxide consumed/min/mg protein.

Estimation of reduced glutathione

The reduced glutathione was estimated by Ellman method [28]. A 1.0-ml test sample was added to 1.0 ml of 20% trichloroacetic acid, which contains 1 mM ethylenediaminetetraacetic acid for precipitating proteins. The solution was incubated for 5 min at room temperature and then centrifuged for 10 min at 2000 rpm. In 200 μl of supernatant, 1.8 ml of Ellman’s reagent (0.1 mM, 5, 50-dithiobis-2-nitrobenzoic acid) was added, and absorbance was measured at 412 nm. The reduced glutathione content was calculated from standard graph from known reduced glutathione. The reduced glutathione was expressed as nanomoles/mg protein or U/mg protein.

Estimation of glutathione peroxidase

The glutathione peroxidase activity was estimated by Hafemann et al. [29]. Volumes of 0.1 ml of 5 mM reduced glutathione, 0.02 ml of plasma, 0.1 ml of 1.25 mM H2O2, 0.1 ml of 25 mM sodium azide, and 1.0 ml of 0.05 mM phosphate buffer were incubated for 10 min for 37°C. The reaction was terminated by adding 2 ml of 1.65% of hydrogen phosphite and centrifuged at 1500 rpm for 10 min. Then 2.0 ml of supernatant was added to 2 ml of 0.4 M disodium hydrogen phosphate and 1 ml of 1 mM Ellman’s regent (DTNB) and incubated at 37°C for 10 min, and absorbance was measured at 412 nm. The results are expressed as U/g Hb.

Estimation of malondialdehyde

The malondialdehyde (MDA) content is estimated by Draper method [30]. A volume of 0.5 ml of test sample was added to 1 ml of trichloroacetic acid. This mixture was centrifuged at 4000 rpm for 10 min. Then 0.5 ml of supernatant was mixed with 1.0 ml of 0.67% thiobarbituric acid reagent followed by incubation for 15 min at 95°C, and absorbance was read at 532 nm. The MDA was expressed as nanomoles of MDA/mg protein.

Statistical analysis

The two-way analysis of variance followed by Tukey’s multiple comparison tests with graph pad prism 6 software (Graphpad Software, Inc., United states) was used.

 Results



Toxicity study of ethyl acetate extract

The intraperitoneal toxicity test was investigated to assess the therapeutic index, which is the ratio of lethal dose and pharmacologically effective dose for the survival and treatment of mice. The behavior of the animals was closely monitored for 6, 12, 24 h, till 48 h. It was found that ethyl acetate extract of C. cupreum did not induce behavioral changes such as hypnosis, convulsion lacrimal secretion, ptosis, change in skin color, stool consistency, diarrhea, locomotor ataxia, weight loss, and mortality in mice during the 48 h of observation. However, a mild sedation was observed ([Table 1]).{Table 1}

Ascitic fluid volume, ascitic fluid weight, cell viability (%), and cell death (%) in control and experimental animals

The anticancer activity of ethyl acetate extract of C. cupreum against EAC cancer-bearing mice was evaluated by measuring the ascitic fluid volume, ascitic fluid weight, and cell viability. From the results, the ascitic fluid volume (4.79±0.52 ml) and ascitic fluid weight (3.93±0.57 g) increased significantly in EAC control group ([Table 2]). The treatment of EAC-bearing mice with ethyl acetate extract of C. cupreum reduced ascitic fluid volume to 2.78±0.44 ml and ascitic fluid weight to 2.81±0.27 g at 50 mg/kg bwt, whereas standard 5-fluorouracil decreased ascitic fluid volume to 0.57±0.22 ml and ascitic fluid weight to 0.44±0.22 g at 50 mg/kg bwt. The cell count (% viable) and cell death (% nonviable) were evaluated by trypan blue assay. The cell viability count was higher in EAC control group (98.02%) than the standard 5-fluorouracil-treated group (3.96%). However, ethyl acetate extract treatment reduced cell viability count to 78.08% at 50 mg/kg bwt, followed by 63.37% at 100 mg/kg bwt, and 52.52% at 200 mg/kg bwt in the treated groups. Similarly, the cell death in ethyl acetate extract treatment was 21.92% at 50 mg/kg bwt, followed by 36.63% at 100 mg/kg bwt and 47.48% at 200 mg/kg bwt in treated groups. However, cell death was 96.04% in standard 5-fluorouracil at 50 mg/kg bwt ([Table 2]).{Table 2}

Hematological parameters in control and experimental animals

All hematological parameters were evaluated in treated and control animals and are presented in [Table 3]. In EAC control group, red blood cells count (2.50±1.2×106/μl) and Hb content (5.10±1.0 g/dl) decreased, whereas white blood count increased (8±1.1×103/μl) compared with normal group. Similarly, ethyl acetate treatment at 50 mg/kg bwt increased red blood cells count to 2.10±0.06×106/μl and Hb content to 3.45±0. 07 g/dl, whereas decreased white blood count to 6.91±0.78×103/μl in EAC-bearing mice. However, the 5-fluorouracil treatment at 50 mg/kg bwt increased red blood cells count to 4.43±0.07×106/μl and Hb content to 9.11±0.03 g/dl, whereas reduced the white blood count to 5.42±1.08×103/μl in EAC-bearing mice significantly ([Table 3]).{Table 3}

Biochemical parameters in control and experimental animals

In the results, treatment with ethyl acetate extract of C. cupreum at 50 mg/kg bwt decreased aspartate aminotransferase activity (81.50±0.06 U/l), alanine aminotransferase (66.91±0.78 U/l), alkaline phosphatase (123.45±0.07 U/l), cholesterol (141.01±0.04 mg/dl), and triglycerides (176.01±0.04 mg/dl) significantly. However, standard 5-fluorouracil at 50 mg/kg bwt decreased aspartate aminotransferase (50.43±0.07 U/l), alkaline phosphatase (40.23±1.08 U/l), alanine aminotransferase (70.11±0.03 U/l), cholesterol (112.09±0.07 mg/dl), and triglycerides (145.09±0.07 mg/dl) significantly (P=0.05) ([Table 4]).{Table 4}

Enzymatic parameters in control and experimental animals

The results showed that all enzymatic antioxidant and oxidative stress markers increased in EAC-bearing mice compared with normal group ([Table 5]). The ethyl acetate extract of C. cupreum treatment at 50 mg/kg bwt increased superoxide dismutase (15.50±0.06 U/mg protein), catalase (9.91±0.78 µmol H2O2 consumed/min/mg protein), and reduced glutathione (11.45±0.07 U/mg protein), whereas decreased glutathione peroxidase (54.45±0.10 U/g Hb) and MDA (230.01±0.04 nmol/mg protein) significantly (P=0.05) in EAC-bearing mice. However, standard 5-fluorouracil treatment at 50 mg/kg bwt increased superoxide dismutase (30.43±0.07 U/mg protein), catalase (25.42±1.08 µmol H2O2 consumed/min/mg protein), and reduced glutathione (27.11±0.03 U/mg protein), whereas decreased glutathione peroxidase (35.11±0.02 U/g Hb) and MDA (160.09±0.07 nmol/mg protein) in EAC mice significantly (P=0.05) ([Table 5]).{Table 5}

 Discussion



Previous reports by the same authors demonstrated that extracts of C. cupreum are rich in various phytochemicals such as flavonoids, carbohydrates, saponins, tannins, glycosides, phytosterol, phenolic terpenoids, azaphilones, and coumarins compounds [17]. Another study reported that extracts of C. cupreum have antioxidant potential because of various phytochemicals present [31]. Based on these observations, anticancer activity of ethyl acetate extract of C. cupreum extract against EAC cells was evaluated. The hematological parameters, serum biochemical parameters, and antioxidant enzymes are considered as important tools for the evaluation of physiological and health status of the organism as well as diagnosis and disease treatment. These parameters indicate the changes in the metabolism of the organism [32]. The parameters such as red blood cells and white blood cells and Hb content are used to analyze blood diagnosis. Owing to the clinical importance of various biochemical parameters such as aspartate aminotransferase, alanine aminotransferase, alkaline phosphate, total cholesterol, and triglycerides, they are used for monitoring and are used as diagnostic tools in patients with diseases in liver, pancreas, kidney, and blood. All these biochemical parameters are used for disease detection all over the world, as well as by International Federation of Clinical Chemistry (IFCC) and the Scandinavian Committee on Enzymes (SCE) [33],[34],[35]. Moreover, during overproduction of reactive oxygen species, the antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase, and reduced glutathione provide the measurement free radicals as well as about the oxidative damage caused by the free radical in the cell [36],[37].

The toxicity report of the present study proved that ethyl acetate extract of C. cupreum did not provoke any pharmacological and behavioral changes. Thus, ethyl acetate extract of C. cupreum appeared safe for preclinical trials. At 50 mg/kg bwt, ethyl acetate extract of C. cupreum decreased cancer volume, cancer weight, and cancer cell viability. At 50 mg/kg bwt, ethyl acetate extract showed 21.92% cell death whereas 5-fluorouracil showed 96.04% cell death. The hemolysis results in red blood cell or Hb count reduction and causes anemia [38]. However, ethyl acetate extract treatment replenishes red blood cells and Hb count toward normal level in a dose-dependent manner. Thus, it indicates that ethyl acetate extract of C. cupreum protects cell surface and thus helps in maintaining normal hemopoietic system. The elevated levels of biochemical parameters such as aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, cholesterol, and triglycerides indicate impaired liver function owing to injection of EAC cells [39]. The treatment of EAC-bearing mice with ethyl acetate extract decreased the level of biochemical parameters in ascitic cancer in a dose-dependent manner, which suggests that phytochemicals present in C. cupreum have the capacity to stabilize the plasma membrane of a cell.

The reduced function of enzymatic antioxidants will result in overproduction of free radicals which is detrimental to cell membrane integrity. The superoxide dismutase and catalase catalyze the dismutation of superoxide anion radical to hydrogen peroxide. Then catalase converts hydrogen peroxide into oxygen and water [40]. Previous studies have presented evidence that reduced glutathione has chemoprotective action, and its deficiency is a risk to cells to oxidative damage [41]. Another study demonstrated that C. cupreum extracts contain high levels of different types of phytochemicals [17]. These phytochemicals donate electrons or atoms to free radicals and thus also repair the enzymatic antioxidants of a cell. Similarly, treatment of EAC-bearing mice with ethyl acetate extract to different doses increases the enzymatic antioxidants to normal level in a dose-dependent manner. Oxidative stress damages lipids and induce lipid peroxidation. MDA is a product produced owing to lipid peroxidation and is used as a marker of cell membrane injury [42]. The MDA is a potentially important contributor to DNA damage and mutations, which it generates endogenously through lipid peroxidation [43]. Thus, MDA can be used as main marker for diagnosis for tumors. The level of lipid peroxides in EAC cell-bearing mice was increased and was decreased with ethyl acetate extract treatment in treated groups. The results from this study indicate that ethyl acetate extract of C. cupreum exhibited antitumor activity against EAC cells and can be a candidate drug for screening in an animal model.

 Conclusion



The evaluation of in vivo anticancer activity of ethyl acetate extract of C. cupreum displayed significant anticancer activity against EAC cells in treated animals. In this study, the work was aimed to explore the toxicity study and the anticancer activity of C. cupreum extract. In this study, analyses of blood parameters, biochemical parameters, enzymatic antioxidants, and marker of oxidative stress were examined at the tolerated concentrations of 50, 100, 200 mg/kg bwt in the control and experimental animals, respectively. The results showed that treatment with ethyl acetate extract of C. cupreum inhibited anticancer activity in a dose-dependent manner but reactive oxygen species in an independent manner against EAC cells. The results showed that C. cupreum extracts contain potent phytochemicals that can be used for therapeutic application after their purification and characterization.

Acknowledgements

The authors are grateful to the Head, Department of Microbiology and Biotechnology (MB & BT), Bangalore University, Bengaluru (BUB), Karnataka, India, for the use of laboratory facilities.

Funding: The authors are grateful to UGC (University Grants Commission), New Delhi, Govt. of India, for UGC-MRP Grants (No-43-474/2014-SR) for financial support.

Author contributions: N.A.W. carried out all the assays, analysis, and interpretation of results, and wrote the initial manuscript. N.A.W. was responsible for the idea of research and interpretation of the results and edited the manuscript.

Conflicts of interest

There are no conflicts of interest.

References

1Gerl R, Vaux DL. Apoptosis in the development and treatment of cancer. Carcinogenesis 2005; 26:263–270.
2Karthikeyan R, Karthigayan S, Balasubashi MS. Antitumor effect of snake venom (Hydrophis spiralis) on Ehrlich ascites carcinoma bearing mice. Int J Cancer Res 2007; 3:167–173.
3Cragg GM, Newman DJ. Plants as a source of anticancer agents. J Ethnopharmacol 2005; 100:72–79.
4Harvey A. Natural products in drug discovery. Drug Discov Today 2008; 13:894–901.
5Beta T, Nam S, Dexter JE, Sapirstein HD. Phenolic content and antioxidant activity of pearled wheat and roller-milled fractions. Cereal Chem 2005; 82:390–393.
6Marinova D, Ribarova F, Atanassova M. Total phenolics and total flavonoids in Bulgarian fruits and vegetables. J Univ Chem Technol Metall 2005; 40:255–260.
7Cragg GM, Kingston D, Newman DJ. Anticancer agents from natural products. London: Brunner-Routledge Psychology Press; 2005. 186–205.
8Newman DJ, Cragg GM, Snader KM. Natural products as a source of new drugs over the period 1981-2002. J Nat Prod 2003; 66:1022–1037.
9Sharpe PC, Richardson DR, Kalinowski DS, Bernhardt PV. Synthetic and natural products as iron chelators. Curr Top Med Chem 2011; 11:591–607.
10Keller NP, Turner G, Bennett JW. Bennett. Fungal secondary metabolism − from biochemistry to genomics. Nat Rev Microbiol 2005; 3:937–947.
11Wang XW. Systematic studies on Chaetomium Kunze and its morphologically simialr taxa from China [PhD thesis]. Beijing, China: Institute of Microbiology, Chinese Academy of Sciences, 2005.
12Ames LM. New cellulose destroying fungi isolated from military material and equipment. Mycologia 1949; 41:637–648.
13Natcha P, Somdej K, Kwanjai K, Soytong K. Chemical constituents from the fungus Chaetomium cupreum RY202. Arch Pharm Res 2015; 38:585–590.
14Han X, Shen T, Lou H. Dietry polyphenols and their biological significance. Int J Mol Sci 2007; 8:950–988.
15Dolai N, Karmakar I, Kumar RBS. Evaluation of antitumor activity and in vivo antioxidant status of Anthocephalus cadamba on Ehrlich ascites carcinoma treated mice. J Ethnopharmacol 2012a; 142:865–870.
16Haldar PK, Kar B, Bala A. Antitumor activity of Sansevieria roxburghiana rhizome against Ehrlich ascites carcinoma in mice. Pharm Biol 2010; 48:1337–1343.
17Nazir AW, Waseem IK, Sharmila T. Phytochemical analysis and evaluation of antibacterial activity of different extracts of soil-isolated fungus Chaetomium cupreum. J Nat Sci Biol Med 2020; 11:72–80.
18 Organization for Economic Cooperation and Development (OECD) guidelines for acute toxicity of chemicals. No. 425 (Adopted: 3 October 2008).
19Jagetia GC, Baliga MS, Aruna R, Rajanikant GK, Jain V. Effect of abana (a herbal preparation) on the radiation-induced mortality in mice. J Ethnopharmacol 2003; 86:159–165.
20Davidson I, Henry JB. Todd-Sanford clinical diagnosis by laboratory methods. 14th ed. Philadelphia, London, Toronto: W.B. Saunders Co. 1969. 139–143
21Sahli AF. Determination of haemoglobin by acid haematin method. In: Dacie JV, Lewis SM editors. Practical haematology. 5th ed. London: Churuchill; 1962.
22Reitman S, Frankel S. A colorimetric method for determination of serum glutamate oxaloacetate and glutamic pyruvate transaminase. Am J Clin Pathol 1957; 28:56–58.
23King J. The hydrolases-acid and alkaline phosphatises. In: practical clinical enzymology. London: Nostrand Company Pvt Ltd; 1965.
24Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20:470–475.
25Werner M, Gabrielson DG, Eastman JU. ltramicro determination of serum triglycerides by bioluminescent assay. Clin Chem 1981; 27:268–271.
26Misra HP, Fridovich I. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972; 247:3170–3175.
27Aebi H. Catalase in vitro. Meth Enzymol 1984; 105:121–126.
28Ellman GL, Fiches FT. Quantitative determination of peptides by sulfhydryl groups. Arch Biochem Biophys 1959; 82:70–72.
29Hafemann DG, Sunde RA, Houestra WG. Effect of dietary selenium on erythrocyte and liver glutathione peroxidase in the rat. J Nutr 1974; 104:580–584.
30Draper HH, Hadley M. Malondialdehyde determination as index of lipid peroxidation. Method Enzymol 1990; 86:421–431.
31Wani NA, Tirumale S. Evaluation of antioxidant properties of different extracts of Chaetomium cupreum SS02. Bull Fac Pharma 2018; 56:191–198.
32Adeyemo OK, Okwilagwe OO, Ajani F. Comparative assessment of sodium EDTA and heparin as anticoagulants for the evaluation of haematological parameters in cultured and feral African cathfish (Clariasgariepinus). Braz J Aquatic Sci Technol 2009; 13:19–24.
33Schumann G, Klauke R. New IFCC reference procedures for the determination of catalytic activity concentrations of five enzymes in serum: preliminary upper reference limits obtained in hospitalized subjects. Clin Chim Acta 2003; 327:69–79.
34Lustig V, Diamandis AP, Goldberg DM. Evaluation of commercially formulated aspartate aminotransferase and alanine aminotransferase activity determinations by the scandinavian committee on enzymes and IFCC methods as modified for use with automated enzyme analysers. Clin Biochem 1988; 21:283–290.
35Gella FJ, Olivella T, Pastor MC, Arenas J, Moreno R, Durban R, Gomez JA. A simple procedure for the routine determination of aspartate aminotransferase and alanine aminotransferase with pyridoxal phosphate. Clin Chim Acta 1985; 153:241–247.
36Storey KB. Oxidative stress: animal adaptation in nature. Braz J Med Res 1996; 29:1715–1733.
37Droge W. Free radicals in the physiological control of cell function. Physiol Rev 2002; 82:47–95.
38Haldar PK, Bhattacharya S, Dewanjee S, Majumder UK. Chemopreventive efficacy of Wedeliacaledulaceae against 20-methylcholanthrene-induced carcinogenesis in mice. Environ Toxicol Pharmacol 2011; 31:10–15.
39Dahiru D, Obidoa O. Evaluation of the antioxidant effects of ziziphusmauritianalam: Leaf extracts against chronic ethanol-induced hepatotoxicity in rat liver. Afr J Trad Complement Altern Med 2008; 5:9–45.
40McCord JM. The evolution of free radicals and oxidative stress. Am J Med 2000; 108:652–659.
41Owen JB, Butterfield DA. Measurement of oxidized/reduced glutathione ratio. Methods Mol Biol 2010; 648:269–277.
42Esterbauer H, Schaur RJ, Zollner H. Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radical Biol Med 1991; 11:81–128.
43Cirak B, Inci S, Palaoglu S, Bertan V. Lipid peroxidation in cerebral tumors. Clin Chim Acta 2003; 327:103–107.