Nazir Ahmad Wani, Sharmila Tirumale*
Bangalore University, Department of Microbiology and Biotechnology, Jnanabharathi Campus, Bengaluru-560056, India
Received date: 17/11/2017; Accepted date: 20/11/2017; Published date: 05/01/2018
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Objective: The present study investigates the effect of soil isolated fungus C. cupreum on alpha glucosidase, alpha amylase and HepG2 cancer cells.
Methods: The antidiabetic effect of C. cupreum extracts was determined by α-glucosidase and α-amylase inhibition assays and anticancer activity was done by MTT assay.
Results: The inhibitory effect on α-glucosidase enzyme by C. cupreum extracts was observed in ethyl acetate extract with the value of 57.88 ± 0.05, followed by n-butanol extract 21.27 ± 0.04, methanol extract 15.19 ± 0.05 and chloroform extract 12.34 ± 0.01 at 100 μg/ml. Whereas, the inhibitory effect on α-amylase enzyme by n-butanol extract with a value of 53.70 ± 0.10, followed by ethyl acetate extract 31.81 ± 0.05, methanol extract 20.51 ± 0.05 and chloroform extract 16.00 ± 0.05 at 100 μg/ ml concentration. In anticancer activity, n-butanol extract of C. cupreum showed significant cytotoxic effect against HepG2 cancer cells with IC50 value at 300 μg/ml.
Conclusion: Our study concludes that C. cupreum extracts possess potential phytochemicals that will be useful in antidiabetic and anticancer treatment after their purification and characterization
α-glucosidase, α-amylase, MTT, Chaetomium cupreum, postprandial, hyperglycemia, diabetes, cancer activity
Diabetes is a complex metabolic disorder characterized by hyperglycemia caused by lack of insulin hormone affecting numerous people worldwide. In 2011 it was estimated that 366 million people are suffering with diabetes and by 2030 it is predicted to be 552 million reports by International Diabetes Federation 2030 . In India 40.9 million people had diabetes, which is predicted to grow to 60.9 million by 2025 . In 2014, 387 million people were suffering with diabetes and are predicted to be 592 million by 2035 [3-5]. It was estimated that in the year 2013 in Africa 19.8 million people had diabetes and is predicted to be 41.4 million by 2035 . In 2014 in Eritrea, the diabetic rate in adults was found to 4.89% . In the United States, 8% of the adult population is suffering with diabetes and cause two-fold increase in age dependent mortality . A diabetic study in Saudi Arabia reported that 4004 out of 16917 (23.7%) were suffering with diabetes between the ages groups of 30-70 years and was found more in urban areas of 25.5% compared to rural Saudis of 19.5% . One possible approach for the prevention of diabetes is by controlling postprandial hyperglycaemia by the inhibition of α-glucosidase and α-amylase enzyme present in the gastrointestinal tract . The inhibition of these two enzymes reduces carbohydrate hydrolysis, glucose absorption and consequently reduces the postprandial plasma glucose rise .
The function of enzymes α-amylase is the digestion of starch whereas α-glucosidase helps in absorption of glucose and thus both enzymes are involved in the control of postprandial hyperglycaemia [12-15]. α-amylase secreted by pancreas and salivary glands catalyses the hydrolysis of α-D-(1-4)-glucosidic linkages of starch to produce maltose and glucose, whereas another intestinal enzyme α-glucosidase (maltase, isomaltase and sucrose) releases glucose from maltose and sucrose [16,17]. Thus, the inhibition α-amylase and α-glucosidase is one of the method to control glucose level [18,19]. There are many synthetic inhibitors such as acarbose, voglibose and miglitol are used for type II diabetes . However, these inhibitors have several side effects, such as liver disorders, flatulence, diarrhea and other intestinal disturbances.
According to the global cancer statistic, hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide  and it is estimate that out of 782,000 people diagnosed for liver cancer, among them 746,000 people died . The liver cancer incidence in Western countries is 4 to 15 per 100,000, whereas Asia and Africa it is 120 per 100,000 . Liver carcinogenesis is caused by various factors such as Hepatitis-B virus, Hepatitis-C infection, nonalcoholic fatty liver disease (NAFLD), aflatoxin- B1, diabetes, alcohol and iron accumulation .
Filamentous fungi have been used for the production of different natural products, such as enzymes, antibiotics, colorants, additives and many others which are used in cosmetic, food and pharmaceutical applications . The natural products derived from plant and animal source exhibit some disadvantages such as low solubility and instability . On the other hand, due to the environmental and toxicity issues of synthetic pigments/compounds industries are looking for microorganisms for the alternative source of natural compounds [27-31]. The fungal members of Fusarium, Monascus, Aspergillus, Penicillium and Chaetomium species are reported to produce high pigment production both intracellular and extracellular pigments with different colors ranging from red, yellow and orange [29-35]. Most of these fungal strains are non-pathogenic and non-mycotoxigenic to humans. Fungi produce different types of enzymes and secondary metabolites with various activities to survive and compete with the environment.
Due to the increasing drug toxicity and drug resistance it is important to search an alternative for new drugs from natural sources, such as fungi. Natural compounds are the one of the research alternative with potent inhibitory properties and minimal side effects. Chaetomium species produce a class of secondary metabolites called as azaphilones. Azaphilones are fungal pigments with pyrone-quinone structures and a highly oxygenated bicyclical core with a chiral quaternary centre [36,37]. Azaphilones possesses antimicrobial, anticancer, antiviral, antioxidant, antifungal and anti-inflammatory activities by the production of vinylogous χ-pyridones [38,39]. The aim of the study was to investigate the different extracts of C. cupreum for their antidiabetic and anticancer activities.
Isolation and Identification of fungus
Chaetomium cupreum was isolated from a litter soil sample collected from the GKVK campus, Bangalore, Karnataka, India. The isolation was carried out by the serial dilution method on potato dextrose agar (PDA) medium. The isolated fungus was identified as C. cupreum -SS02 based on morphological and microscopic characteristics . The morphological identity was confirmed by NFCCI, Agharkar Research Institute, Pune, India. For species identification based on the molecular phylogenetics of the internal transcribed spacer (ITS) region using universal primers; ITS-1 (TCCGTAGGTGAACCTGCGG) was the forward primer and ITS-4 TCCTCCGCTTATTGATATGC) was the reverse primer was performed . Search for homologous sequences in Gen Bank was performed using BLAST (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/blast) . BLAST search performed for the sequence of ITS analysis, showed 99% homology with other strains of C. cupreum available in Gen bank. The sequence was deposited in NCBI Genbank with accession Number KF668034. The Culture was deposited in the National Fungal Culture Collection of India (NFCCI), with accession Number NFCCI 3117. The isolated fungus was maintained on potato dextrose agar (PDA) plates and potato dextrose agar slants at 4°C.
Inoculum preparation and fermentation
For inoculum preparation, the fungus was grown at 25°C on a PDA plate for 7 days, then 5 mm mycelial discs were bored out from the periphery of the colony and transferred to 250 ml Erlenmeyer flasks containing 100 ml of potato dextrose broth (PDB) and incubated at 26 ± 2°C on a rotary shaker at 120 rpm for 20 days to achieve the highest pigment production.
Extraction of extracellular pigments
The extraction of pigments was carried out according to the method described previously . After 20 days of incubation biomass was removed by filtration through Whatman No. 1 filter paper and the broth containing the extracellular pigment/ metabolites was obtained. The cultural broth obtained was used for extraction of pigments/compounds by liquid-liquid method in 500 ml of separating funnel using four different organic solvents from non-polar to polar (chloroform, ethyl acetate, n-butanol and methanol) in the ratio of 1:1. The 50 ml of filtered broth and 50 ml of solvent was taken in separating funnel and shaken well for 20 minutes and allowed to stand until the aqueous layer and organic layers separated. The organic layer was collected, filtered through Whatman No. 1 filter paper and transferred to 250 ml of beaker. This process was repeated three times with the same broth and same solvent until no more pigment diffused into the solvent. The whole broth was extracted by using similar procedure with chloroform, ethyl acetate, n-butanol and methanol. Then organic layer was evaporated using vacuum rotary evaporator at 45°C. The crude dried extract was obtained and stored at 4°C for future use.
α-Glucosidase inhibition assay
The effect of C. cupreum extract on the inhibition of α-glucosidase activity was determined according to the method as described by Kim . Fungal extracts were prepared as a stock concentration by dissolving 5 mg/ml of DMSO. The reaction mixture consists of 250 μl of 3.0 mM 4-nitrophenyl α-D-glucopyranoside (PNPG) as a substrate dissolved in 20 mM phosphate buffer (pH 6.9), 100 μl (100 μg/ml in DMSO) from each C. cupreum extract (chloroform, ethyl acetate, n-butanol and methanol extract) and 100 μl of α-glucosidase solution (1.0U/ml) in 0.01 M phosphate buffer (pH 7) and the reaction mixture was incubated at 25 °C for 20 minutes. After incubation, the reaction was stopped by adding 2 ml of 0.1M sodium carbonate (Na2CO3). The α-glucosidase activity was determined by measuring the yellow colored paranitrophenol release from paranitrophenol glucopyranoside (PNPG) at 420 nm. The control sample included all the reagents with enzyme, replacing the sample with DMSO. The inhibitory activity of α-glucosidase is calculated by the following formula:
% Inhibition = ((A0 -A1 ) /A0 ) *100 ……1
where, A0 is the absorbance of the control sample and A1 is the absorbance of the test extract.
α-Amylase inhibition assay
The effect of C. cupreum extracts on the inhibition of α-amylase activity was determined according to the method as described previously . The reaction mixture consists of 250 μl (100 μg/ml DMSO) of C. cupreum extract (chloroform, ethyl acetate, n-butanol and methanol extract), 250 μl of 0.02 M sodium phosphate buffer (pH 6.9) and 100 μl of α-amylase solution (0.5mg/ml). This solution was incubated at 250C for 10 minutes. After incubation 250 μl of 1% starch solution in 0.02M sodium phosphate buffer (pH 6.9) was added and was further incubated at 25 °C for 10 minutes. The reaction was terminated by adding 500 μl of 3,5-dinitrosalicylic acid (DNS) reagent. The reaction tubes were then incubated in boiling water for 5minutes and cooled to room temperature. The α-amylase activity was determined by measuring the absorbance at 540 nm using spectrophotometer. Control sample included all the reagents with enzyme, replacing the samples with DMSO. The α-amylase inhibition by C. cupreum extracts was calculated by the given formula:
% Inhibition = ((A0 -A1 ) /A0 ) *100 ……2
where, A0 is the absorbance of the control and A1 is the absorbance of the test extract.
Cell culture and maintenance
The cancer cell lines HepG2 (Hepatocellular carcinoma) was obtained from National cell culture science (NCCS), Pune, India. The cells were cultured in a cell culture flask using Dulbecco’s Modified Eagles Medium (DMEM) Nutrient Mixture F-12 HAM supplemented with 10% fetal bovine serum (FBS), 15mM HEPES, NaHCO3, pyridoxine and L-glutamine. The cells were maintained in a 5% CO2 incubator at 37ºC.
Cell viability assay
The cytotoxicity of C. cupreum extracts on HepG2 liver cancer cells was assessed using a MTT (3-(4,5-dimetylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay as described previously . HepG2 liver cancer cells were plated (1 × 104 cells/well) by adding 200 μl cell suspension to each well of a 96-well microtiter plates (NUN96ft-Nunclon-96 Flat transparent). The plate was incubated at 37°C in humidified atmosphere with 5% CO2 for 24 hrs to assure attachment and 80% to 100% confluence. After 24 hrs, media was aspirated off and replaced with fresh media (200 μl) containing C. cupreum extract of different concentrations (25 μg/ml, 50 μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml, 250 μg/ml and 300 μg/ml). The plate was incubated at 37°C with 5% CO2 for 24 hours. After incubation, the media was aspirated off and replaced with fresh media containing 20 μl MTT reagent (2 mg/ml PBS) for a total volume of 200 μl was added to each well and incubated at 37°C, with 5% CO2, for 2 to 3 hours. After incubation, MTT containing medium was removed gently and 200 μl of DMSO per well to dissolve the formazan crystals was added. The control sample included all the reagents with HepG2 cells replacing the sample with DMSO. The absorbance was measured at 585 nm using a Model 680 Microplate Reader (Shimadza UV-1800). Percentage of cell viability was calculated according to the following equation .
Percentage of cell viability = (OD of treated cells/OD of control cells) *100 ...….3
The concentration of drug that inhibits 50% of cells (IC50 values) for these samples were calculated.
All the measurements were taken in triplicate and expressed as mean value ± standard deviation. The data were analyzed for statistical significance using one-way analysis of variance (ANOVA) followed by Bonferroni Multiple Comparisons Test. with Graph Pad Prism 6 software (Graph Pad Software, Inc., USA). The probability values of p<0.05 were considered as statistically significant.
Identification of C. cupreum
The fungus was identified on the basis of morphological, microscopic and molecular phylogenetics involving an ITS-5.8S region of the rDNA and named as C. cupreum SS02. In morphology C. cupreum exhibited typical white cottony mycelial colonies on the PDA.
Effect of C. cupreum extract on α-Glucosidase Inhibition
The C. cupreum extracts were studied for their α-glucosidase inhibition property. The percentage inhibition displayed by each C. cupreum extracts are presented in Figure 1. Among the four different extracts of C. cupreum studied, ethyl acetate extract showed inhibition percentage with a value of 57.88 ± 0.05, followed by n-butanol extract 21.27 ± 0.04, methanol extract 15.19 ± 0.05 and chloroform extract 12.34 ± 0.01 at 100 μg/ml. The α-glucosidase inhibitory activity was displayed by ethyl acetate extract with IC50 value at 86.38 μg/ml concentration. The highest inhibition percentage of α-glucosidase enzyme was shown by ethyl acetate extract, whereas methanol extract shows lowest α-glucosidase inhibition percentage. The C. cupreum extracts shows a significant difference (p<0.0001) followed by Bonferroni’s multiple comparison test.
Effect of C. cupreum extract on α-amylase inhibition
The percentage inhibition of α-amylase enzyme displayed by each C. cupreum extracts are presented in Figure 2. Among the different extracts of C. cupreum studied, the inhibition percentage on α-amylase enzyme was shown by n-butanol extract with the value of 53.70 ± 0.10, followed by ethyl acetate extract 31.81 ± 0.05, methanol extract 20.51 ± 0.05 and chloroform extract 16.00 ± 0.05 at 100 μg/ml. The highest α-amylase inhibitory activity was shown by n-butanol extract of C. cupreum with IC50 value at 93.10 μg/ml concentration. The highest inhibition percentage of α-amylase was shown by n-butanol extract whereas chloroform extract shows the lowest α-amylase inhibition percentage. In the results, it was observed that C. cupreum extracts shows a significant difference (p<0.0001) followed by Bonferroni’s multiple comparison test.
Effect of C. cupreum extract on HepG2 cells
The in-vitro effect of C. cupreum extracts on HepG2 is presented in Figure 3. A significant decrease was observed in percentage cell viability against HepG2 cancer cells at different concentrations after 24 hours of treatment. The maximum decrease in percentage cell viability by C. cupreum extracts was observed in n-butanol extract with the value of 51.68 ± 0.01, followed by ethyl acetate extract 43.45 ± 0.01, methanol extract 30.94 ± 0.01 and chloroform extract 24.04 ± 0.07 at 300 μg/ ml concentration after 24 hours. The IC50 values of n-butanol extract of C. cupreum was found at 300 μg/ml after 24 hours of treatment. The results from our study revealed that the compounds from n-butanol extract of C. cupreum could be a possible target against hepatocellular carcinoma treatment. There was a significant difference (p<0.0001) followed by Bonferroni posttests test.
In diabetes the therapeutic approach involves the inhibition of α-amylase and α-glucosidase this will result in delaying the carbohydrate digestion and reduction in glucose absorption thereby lowering postprandial glucose level hyperglycemia . Therefore, the inhibition of carbohydrate digesting enzymes could be one of the most effective approaches to control diabetes . At present, the inhibitors of α-glucosidase and α-amylase used are acarbose, miglitol and voglibose . In carbohydrate digestion they inhibit the conversion of maltose to glucose and thus decrease the rate of glucose into the blood circulation . Our study showed that all the C. cupreum extracts have exhibited inhibition of α-glucosidase and α-amylase activity. However, the maximum α-glucosidase inhibition was shown by ethyl acetate extract of C. cupreum whereas, the maximum α-amylase inhibition was shown by n-butanol extract of C. cupreum. In the literature there was no information available about the α-glucosidase and α-amylase inhibitory activity of the fungus C. cupreum. Hence, our study evaluated the in vitro α-glucosidase and α-amylase inhibitory activity of soil isolated fungus C. cupreum. The inhibition of α-glucosidase and α-amylase by C. cupreum extracts would result in the slow hydrolysis of carbohydrates thereby reduces the rate of glucose uptake from small intestine and thus control of hyperglycaemia condition could be achieved through the inhibition of carbohydrate digesting enzymes [52,53].
In our report, we have demonstrated for the first time that C. cupreum extract acts as an anticancer agent by induction of dose dependent apoptosis in HepG2. The results of this study suggest further in depth in vitro and in vivo studies. The positive outcomes of future studies could be useful for developing C. cupreum compounds as a novel compound for hepatocellular carcinoma treatment. Thus, these biological activities of C. cupreum extracts may be due to the phytochemicals present in it. The positive outcomes of future studies could be useful for developing C. cupreum compounds as a novel compound for antidiabetic and hepatocellular carcinoma treatment.
These findings suggest that the C. cupreum extracts are effective inhibitors for α‑glucosidase and α‑amylase enzymes, which may helpful to reduce the postprandial glucose levels. Also, C. cupreum has displayed significant anticancer activity against HepG2 cancer cells. The results of this study suggest further in depth in-vitro and in-vivo studies on C. cupreum extracts and purification and characterization of their compounds.
The authors declare no conflict of interest.
The authors are grateful to the UGC (University Grants Commission), New Delhi, Govt. of India, for UGC-MRP Grants for financial support and also to the Head, Department of Microbiology and Biotechnology (MB & BT), Bangalore University, Bengaluru (BUB), Karnataka, India, for the use of laboratory facilities.