Production of Childinan SF-2 as Bioactive Compound from Daldinia childiae: A Survey on Antioxidant, Antibacterial and Antitumor Properties

Document Type : Research Paper


1 Department of Microbiology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran

2 Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Science, Tehran, Iran

3 Department of Biotechnology, Engineering School of Lorena, University of São Paulo, Lorena, São Paulo, Brazil


Background: Fungal extracts have received increased attention due to their great medicinal applications including antitumor, immune-modulating, antioxidant, radical scavenging, antiviral, antibacterial, antifungal and detoxificating properties.
Objectives: This study is the first report on a novel bioactive compound, namely Childinan SF-2 which was isolated from soil ascomycete fungus. The significant antibacterial, antioxidant and cytotoxic properties of the extract may lead to development of novel, safe and useful substances.
Materials and Methods: The isolate was identified on the basis of molecular approach. Spore suspension was inoculated in the culture medium and the bioactive compound was isolated from the viscous fermented broth via ethanol precipitation of the extracellular compound. The basic chemical composition of the extract including protein, carbohydrate, sulfate radical and uronic acid content were measured. FTIR (Fourier-transform infrared spectroscopy) and GC–MS (Gas chromatography–mass spectrometry) analysis were used for further structural characterization. The extract was utilized for treatment of AGS and MDA cell lines to assess the cell cycle and apoptosis. The antioxidant activity was examined using DPPH, hydroxyl radicals scavenging, β-carotene bleaching inhibition and ferric reducing power assay methods. The extract was tested for evaluation of antibacterial activity using different Gram-positive and Gram-negative bacterial strains
Results: The fungal isolate was identified as the new strain Daldinia childiae SF-2. Initial biochemical characterization of the extract showed that the fungal biopolymer was composed of total sugars, protein, uronic acids and sulfated groups with values of 91.6%, 2.15%, 2.25% and 1.05% (w/w), respectively. FTIR and GC–MS analysis revealed that Childinan SF-2 might be mainly constructed from D-glucose, D-mannitol and D-galactofuranose. The in vitro experiments revealed that Childinan SF-2 enhanced the percentage of necrosis and apoptosis of cancer cells and blocked the cell cycle progression as shown by flowcytometry. Childinan SF-2 represented a considerable antioxidant and antibacterial activity.
Conclusions: These results indicated that Childinan SF-2 can serve as a potential source in medicinal applications.


Main Subjects

1. Background

Different biopolymers, especially exopolysaccharides have been found in nature. The majority of these biocompounds have potential biological functions ( 1 ). The isolation of bioactive biopolymers has received substantial attention due to the fact that most of them are eco-friendly and nontoxic with minimum side effects ( 2 , 3 ). In recent decades, a large group of bioactive biopolymers have been isolated from a variety of endophytic fungal species with showing some activities including anti-oxidant, anti-tumor, anti-viral, anti-bacterial and immune modulating properties ( 4 - 7 ). Chitin, Schizophyllan and β-glucan are the known fungal biopolymers with the unique applications such as anti-cancer and anti-tumor properties ( 8 - 10 ). Daldinia is an ascomycetal genus belonging to the family Xylariaceae ( 11 ). The researches on the genus Daldinia have attracted a lot of interest since it is known as a potential source of polyketides with a strong biological activity ( 12 ). Meanwhile, the fungus D. childiae identified by Rogers et al. ( 13 ) has rarely been investigated as a biopolymer producer. Much less work has been carried out regarding the chemical constituents of bioactive substances extracted from the D. childiae with therapeutic effects ( 13 ).

2. Objectives

The current study aimed to isolate and identify a fungal strain, namely Daldinia childiae SF-2 which could produce an extracellular polymer (Childinan SF-2) with considerable bioactive properties. The cytotoxicity and apoptosis effect against different cancer cell lines along with anti-oxidant and anti-bacterial efficiency was evaluated. Furthermore, the biochemical structure of the fungal biopolymer was evaluated. The current work is the first investigation on extracellular biopolymer extracted from D. childiae SF-2 in terms of antioxidant, antibacterial and antitumor effects.

3. Materials and Methods

3.1. Fungal Identification

The fungus was isolated from the soils of forest area in the North of Iran, [Golestan province (E 50°50´59.6˝ N 36°44´13.3˝)]. The isolation of the samples was carried out using Potato Dextrose Agar (PDA) at 28 °C during 7 days. Samples were preserved at 4 °C and subcultured monthly. The genomic DNA extraction was carried out from the fresh mycelia using cetyltrimethyl ammonium bromide (CTAB) buffer and glass beads ( 14 ). Polymerase chain reaction (PCR) using the ITS1 and ITS4 primers was utilized to amplify this region in the 5.8S rDNA of the genomic DNA. The PCR product was sequenced (Bioneer Corporation, South Korea), assembled and deposited in GenBank. The phylogenetic tree was originated using the neighbor-joining algorithm of MEGA software version 7.0.21 ( 15 ). Confidence levels of the clades were evaluated from bootstrap analysis according to 1000 replications.

3.2. Fungal Biopolymer Preparation

Fungal extract was produced by fermentation according to our previous reported methods ( 16 ). The ethanolic precipitate of the extract was called as Childinan SF-2 and used for further experiments.

3.3. Partial Characterization of the Biopolymer

3.3.1. Biochemical Characterization

Biochemical characteristics of the biopolymer were evaluated in terms of protein content according to the Bradford method, reducing sugars content using DNS (3, 5–dinitro salicylic acid) method and total carbohydrate content according to the phenol-sulfuric methods ( 16 ). Furthermore, the amount of uronic acid and sulfate radicals were evaluated according to methods defined by Blumenkrantz and Hansen ( 17 ) and Dodgson and Price ( 18 ), respectively.

3.3.2. Fourier Transform Infrared (FTIR) Spectroscopy

In order to find out the functional groups within biopolymer structure of the fungal extract obtained, Fourier transform infrared (FTIR) was performed by a Bruker Optics spectrometer (OPUS 3.1) in the frequency range of 4000–400 cm−1 ( 2 ).

3.3.3. Monosaccharide Analysis

The components of the crude extract were analyzed for monosaccharide composition using a GC-MS by the reported method of Jamshidian et al. ( 19 ) with some modifications. The extract was used for alditol acetates preparation. Briefly, the crude extract (10 mg in 2 M trifluoroacetic acid) was hydrolyzed at 121 °C for 3 h, reduced by NaBH4 at room temperature and acetylated with acetic anhydride and pyridine. The obtained alditol acetates were analyzed by gas chromatography coupled with mass spectrometer (Agilent Technologies) which was attached with HP-5 capillary column (0.25 mm × 30 m × 0.25 µm). Peak assignments were made based on retention times and mass spectra by the library and confirmed with the standard reference sugars.

3.4. Cells and Cultures

MDA-MB-231 cell line (human breast cancer) and AGS gastric carcinoma cell line (human stomach cancer) were a gift from Dr. R. Ramezani (Women Research Center, Alzahra University, Iran). Cell lines were cultured in DMEM supplemented with 10% (v/v) FBS at 37 ºC in 5% CO2. Cells were digested by 1 mL trypsin and treated with different concentration of extracts.

3.5. Flow cytometry Analysis

3.5.1. Cell Apoptosis Assessment Using Annexin-V/ Propidium Iodide

The MDA and AGS cells were cultured in the presence or absence of the fungal biopolymer SF-2 (5 mg.mL-1). The cells apoptosis was evaluated using commercially available FITC Annexin V Apoptosis Detection Kit (BD Bioscience; Franklin Lake, NJ) according to the manufacturer’s instructions and subsequently analyzed by a FACS/Calibur flow cytometer (Becton Dickin-son, NJ, USA).

3.5.2. Cell Cycle Arrest Analysis

AGS and MDA cells (5×105 cells/ well in 6-well plates) were exposed to biopolymer SF-2 (5 mg.mL-1) using DNA staining solution (Cell Cycle Staining Kit (MultiScience Biotech Co., Ltd). After treatment, the cells were collected with trypsin and re-suspend in PBS and added to pre-cooled ethanol (75%, v/v) for 4 h. Subsequently, cells were incubated in dark for 30 min before being analyzed by flowcytometry. The cell population was defined by the control (cells were not exposed to biopolymer).

3.6. Determination of Antioxidant Capacity

3.6.1. DPPH Radical Scavenging Assay

The DPPH assay, as previously reported by Shimada et al. ( 20 ) was applied to assess the radical scavenging ability of the biopolymer from strain SF-2. The absorbance was measured at 517nm and the scavenging ability was calculated according to the equation used by Fooladi et al. ( 16 ).

3.6.2. Hydroxyl Radical Scavenging Assay

The hydroxyl radical scavenging activity of the fungal biopolymer was measured according to the method described by Zhang et al. ( 21 ). The biopolymer was dissolved in the distilled water to form the final concentrations of 2-10 mg.mL-1. The hydroxyl radicals were detected by monitoring absorbance at 510 nm. Ascorbic acid was used as the positive control. The hydroxyl radical scavenging activity was calculated according to the mathematical equation used by Fooladi et al. ( 22 ).

3.6.3. βeta-Carotene Bleaching Assay

In order to assess lipid peroxidation capacity, βeta-carotene bleaching assay was carried out according to the method of Mayouf et al. ( 23 ) with some modifications. Vitamin C and ethanol were used as positive and negative controls, respectively. The absorbance of the solution was measured at 460 nm and the percentage of the βeta-carotene bleaching inhibition was calculated in accordance with the mathematical equation used by Fooladi et al. ( 22 ).

3.6.4. Ferric Reducing Antioxidant Power (FRAP) Assay

The antioxidant capacity of the biopolymer from the fungal isolate was evaluated to reduce Iron (III). The FRAP assay was performed according to the method of Sobeh et al. ( 24 ). Ascorbic acid was used as a positive control and FRAP values were expressed as optical density at 700nm.

3.7. Antibacterial Activity Assay

Antimicrobial activity of the biopolymer was assayed by the minimum inhibitory concentration (MIC) method. Four Gram-negative (Salmonella typhimurium ATCC 14023, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 43071 and Escherichia coli ATCC 25922) and five Gram-positive (Enterococcus faecalis ATCC 33186, Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923, Streptococcus agalactiae ATCC 12386 and Listeria monocytogenes ATCC 7644) were utilized for antibacterial activity evaluation. Each well of 96-well micro-titer plates contained Mueller–Hinton Broth (100 μL), bacterial suspension (100 μL, approximately 106 CFU.mL-1) and fungal biopolymer (100 μL, 1% w/v) with concentration from 0.4 mg.mL-1 up to 6.6 mg.mL-1 ( 25 ). After incubation at 37 °C for 24 h, MIC values were measured by visual detection of the turbidity and absorbance measurement at 620 nm. Antimicrobial activity tests were carried out according to the Clinical Laboratory Standard Institute (CSLI) guideline 2012 ( 26 ).

3.8. Statistical Analysis

All data were performed in triplicate and presented as means ± SD. The data were subjected to an analysis of variance (ANOVA) and SPSS (Version 17.0, USA). Statistical significance was determined at P < 0.05.

4. Results

4.1. Fungal Identification

The fungal strain was identified according to the molecular characteristics by sequence analysis of ITS1 and ITS4 regions in the 5.8S rDNA of the genomic DNA. A BLAST exploration of the database and the ITS sequences length (480 bp) indicated a close genetic relation with other isolates of Daldinia spp. ( 27 ). According to the pair wise sequence alignments, the strain revealed a high sequence similarity value (99.8%) to Daldinia species. Phylogenetic analysis obtained base on the ITS dataset showed that the fungal isolate SF-2 was closely related to Daldinia childiae using Neighbor-Joining method (Fig. 1).

Figure 1. Phylogenetic evolutionary relationship of Daldinia Childiae strain SF-2 based on ITS 1-5.8S-ITS2 regions (MEGA Version 7). The isolated strain showed as MN216319. Daldinia Childiae strain SF-2.

4.2. Production of Childinan SF-2

The results showed that maximum biopolymer (1.12 ± 0.11 g.L-1) and biomass (10.12 ± 1.52 g.L-1) productions were obtained during 122 h and 168 h of the fermentation, respectively. The separated and purified biopolymer, namely, Childinan SF-2, was obtained from the fungal isolate that identified as Daldinia childiae SF-2.

4.3. Chemical Characteristics

The biochemical composition of the Childinan SF-2 demonstrated a total protein content of 2.15% (w/w) and a high content of total sugars 91.6% (w/w). Moreover, the results obtained from the DNS evaluation showed that 22.5% (w/w) of the total sugars of the fungal extract contained reducing sugars. In addition, the extract showed a high amount of uronic acids (2.25%) and sulfated groups (1.05%).

The IR spectrum of Childinan SF-2 (Fig. 2A) revealed the intense peaks at 3424, 2929, 1730, 1647 and 1050 cm-1. The broad IR band at 3424 cm-1 was assigned to hydroxyl groups in carbohydrates and the band at 2929 cm-1 was considered as C-H groups. The band in region of 1647 cm-1 was attributed to the stretching vibration of C=O. The bands at 1200-1000 cm-1 were considered the stretch vibration of C-O-C and C-O-H in polysaccharides ( 2 ). Moreover, the presence of absorption peak at around 1730 cm-1 may revealed the presence of uronic acids ( 16 ).

Figure 2.A) The Infrared spectra of the Childinan SF-2 , B) GC/MS spectrum of the derivative compound analyzed for Childinan SF-2, C) The mass spectra of the peaks were characterized by comparison of the spectra of the standard sugars treated by the same reaction.

GC-MS analysis revealed that D-glucose, D-mannitol and D-galactofuranose were the monosaccharides figured out in the molecular structure of the Childinan SF-2 with the mass-to charge-ratio of m/z 429, 441 and 456 based on GC–MS library (Wiley 2007) (Fig. 2B) and comparison of the peaks obtained from the fungal biopolymer with the standard sugars treated with the same reactions (Fig. 2C).

4.4. Effect on Cell Cycle

In cell cycle distribution, the accumulation of the cells in each phase in both treated cells and control sample were depicted in Figure 3. In the case of Childinan SF-2 treatment, the accumulation of cells in G2 was 19.48% (Fig. 3A) and 11.23% (Fig. 3B) for MDA and AGS, respectively, compared to 21.71% of the control groups (Fig. 3C). Moreover, a significant (P<0.05) enhancement in the population of cells was observed in the sub-G1 group for both MDA and AGS after treatment with the biopolymer. Simultaneously, in S-phase, the population of cells was reduced to 30.21% for MDA and 19.65% for AGS cells, compared to 37.89% for the control, inferring that cells were arrested at S and G2 phases of the cell cycle. As shown in Figure 3D, the data represented as mean ± SD of three independent assay which indicated significantly different.

Figure 3. The blocking effect of Childinan SF-2 (5 mg.mL-1) on the cell cycle of A) MDA, B) AGS, and C) control group C). D) Bar graph characterizes the percentage of different cell cycle phases. The data are represented as mean ± SD of three independent assays (One-way ANOVA), indicating significant differences (p < 0.05).

4.5. Effect on Cell Apoptosis

For apoptosis assay, when cells were treated by the biopolymer, the percentage of the cells was reduced to 49.5% and 17.8% for MDA and AGS cells, respectively. In contrast, in the control group, the most of the cells remained healthy (94.7%) in the Q4 area, which indicated that the treated AGS cells tended to undergo apoptosis (P<0.05) more than MDA (Fig. 4). The biopolymer has induced the late apoptosis in both cell lines similarly with the value of 34.0% in Q2 area compared to the 0.41% in the control cells. Consequently, the percentage of the MDA cells significantly (P<0.05) increased (13.5%) in Q3 area, indicating that Childinan SF-2 can enhance early apoptosis of MDA cell line compared to that obtained by control (4.7%) and AGS cells (0.89%) (Fig. 4A). Interestingly, as shown in Figure 4B, Childinan SF-2 could induce a considerable rate of necrosis in the AGS cells in Q1 area with the percentage of 46.1% compared to 2.4% in MDA (Fig. 4C) and 0.22% in the control group (Fig. 4D).

Figure 4. Effect of Childinan SF-2 on the apoptosis of A) MDA, B)AGS, and C) Control group. D) Bar graph characterizing the percentage of apoptotic cells. The data are represented as mean ± SD of three independent assays (One-way ANOVA), indicating significant differences (p < 0.05) .

4.6. Antioxidant Capacity

The antioxidant capacity experiments for Childinan SF-2 showed that DPPH radical scavenging activity increased dramatically in a dose-dependent manner, as the maximum scavenging ability of 77.05% was observed at the highest tested concentration (10 mg.mL-1) of the biopolymer (Fig. 5A). The results showed the remarkable antioxidant potential of the biopolymer compared to that of ascorbic acid (95.25%) used as a positive control. As shown in Figure 5B, the potential antioxidant activity of the Childinan SF-2 for hydroxyl radical scavenging was examined at different concentrations (2-10 mg.mL-1). At the highest concentration, the inhibition rate of the biopolymer on hydroxyl radical was recorded 56.45% compared to the Vitamin C (98.05%) as positive control. Childinan SF-2 had a considerable protection activity for β-carotene bleaching reduction about 59.5% at the highest tested concentration (5 mg.mL-1).

Figure 5. Scavenging capacity of Childinan SF-2 from Daldinia childiae using different assays; A) DPPH, B) Hydroxyl radical scavenging capability, C) bleaching assay, and D) Ferric reducing power. Ascorbic acid (Vitamin C) was used as a positive control. White column, grey column, white doted, grey doted and light horizontal columns showed extract concentrations of 2 mg.mL-1, 4 mg.mL-1, 6 mg.mL-1, 8 mg.mL-1 and 10 mg.mL-1, respectively in DPPH and Hydroxyl radical methods. White, grey, white doted and grey doted columns revealed antioxidant protection ability of the samples versus time in β-carotene method and extract concentrations of 0.01 mg.mL-1, 0.1 mg.mL-1, 1 mg.mL-1 and 2 mg.mL-1, respectively in ferric reducing assay.

As shown in Figure 5C, instantly after starting the experiment, ascorbic acid inhibited the oxidative deterioration of lipids and fatty acids and remained approximately stable, whereas the activity for Childinan SF-2 was observed after 30 min and gradually increased. The effect of Childinan SF-2 was studied corresponding to the ferric reducing power, as depicted in Figure 5D. The Childinan SF-2 showed concentration-dependent antioxidant activity, as the ferric reducing power (wavelength at 700 nm) was elevated by an increment concentration from 0.01 to 2 mg.mL-1. Although the reducing power of the Childinan SF-2 was gently increased, its activity was significantly less than that of the positive control (ascorbic acid).

4.7. Antimicrobial Activity

The results from antibacterial activity, revealed that the biopolymer had antimicrobial activity against both Gram-negative and Gram-positive strains. In the case of Gram-positive bacteria, a higher antibacterial activity was observed against Staphylococcus aureus and Listeria monocytogenes with the MIC value of 6.6 mg.mL-1 and 3.3 mg.mL-1, respectively.

5. Discussion

Daldinia, a genus of fungi, belongs to the family of Xylariaceae with a large number of species easily grown in varying environmental conditions and substrates ( 11 ). In total, 47 taxa in Daldinia were recognized based on morphological and chemotaxonomic evidence. Their biogeography, chorology, ecology, as well as molecular phylogeny based on 5.8S/ITS rDNA and the importance of their secondary metabolites, all provided a basis for more comprehensive identification of these species ( 28 ). The fungus was determined as Daldinia childiae using morphological and molecular methods. The rDNA gene sequence data of the new fungal isolate were deposited in GenBank under accession numbers of MN 216319 as Daldinia chiliae SF-2.

It is widely recognized that the chemical structure and monosaccharide composition are the most important factors defining the bioactivity of polymeric carbohydrate compounds. Herein, Childinan SF-2 was successfully extracted from D. childiae, biochemically characterized and its initial compositional analysis revealed the presence of total sugars, protein, uronic acids and sulfated groups. It is supposed that radical scavenging ability of the Childinan SF-2 may be due to the presence of sulfated groups together with uronic acids ( 29 ).

From the author’s knowledge, there is no evidence for the vast studies on characterization of the extract from D. childiae. According to GC-MS analysis, the m/z values of the alditol acetate derivatives from the fungal biopolymer Childinan SF-2 and the electron ionization (EI) mass spectra were compared to that obtained from the standard sugars based on GC–MS library (Wiley 2007). Although the similarity in the m/z values of the components may lead to the similarity of the fragmentation patterns of the isomers of alditol acetate derivatives, the characterization of each single sugar alone with the similarity of the mass spectra is not adequate to fulfill the structural characterization. Identification of the components was confirmed by comparison of the chromatographic characteristics such as retention time of the individual compounds with standard sugars ( 30 ). Therefore, presence of D-glucose, D-mannitol and D-galactofuranose was figured out in the molecular structure of the Childinan SF-2 according to the library. Each of these components is very common in fungal extracts ( 31 ) but the distribution of the monosaccharaides is different and variable, suggesting that Childinan SF-2 extracted from the fungal isolate was a novel biopolymer that isolated from D. childiae and identified for the first time for their monosaccharide composition. The linkage and comprehensive structural information will be studied through methylation analysis in the future.

It is noteworthy that the increased proliferation and decreased cell death (apoptosis) are two major processes that contribute to the progression of tumor cell growth. The results obtained from treated cells with the highest concentration of Childinan SF-2 (5 mg.mL-1), showed an increment in population of cells in G1 phase compared to that of the control group, which indicated that the fungal extracts arrested the cells in this phase. It is interesting to know that when the accumulation of cells increased in G1, it could not show the efficiency of the compounds for their anticancer activity. Hence, a remarkable bioactive compound should arrest cells at G2 phase, which showed that it was mediated by the mitotic division ( 32 ). The results proposed the induction of apoptosis of cells by Childinan SF-2. Furthermore, the biopolymer Childinan SF-2 prevented the cell division by blocking tumor cell cycle at G0/G1 stage. The results of the study appropriately were compatible with those obtained by Liu et al. ( 33 ) who observed the cytotoxic efficiency of the polysaccharide extracted from Russula griseocarnosa in HeLa and SiHa cells. They observed that a significant increment in both early and late apoptosis at cell lines in a dose-dependent manner. The study conducted by Li et al. ( 34 ) reported the antitumor activity of edible fungal polysaccharide Lentinan on MCF-7 cells. They reported new findings about the mechanisms of Lentinan-antitumor effect for development in functional foods and cancer therapy.

Fungal extracts have been shown to play a significant role as free radical scavengers for the prevention of oxidative damage in living organisms ( 20 , 35 , 36 ). The results of the study correlated well with those obtained by Ren and colleagues ( 37 ) who observed DPPH and hydroxyl radical scavenging activity of 75.4% and 68.5%, respectively for Pleurotus abalonus (PAP) extract. In the study conducted by Reis et al. ( 38 ) the DPPH activity of 17% was measured for methanolic extract from Pleurotus ostreatus that was much lower than Childinan SF-2 (77.5%). In contrast, Penicillium flavigenum CML2965 extract showed a strong DPPH activity of 98.2%. For hydroxyl radical scavenging capacity, Childinan SF-2 showed the scavenging power of 56.45% that was lower than those reported for Daldinia pyrenaica SF-1 ( 22 ) and Neopestalotiopsis SKE15 ( 16 ) extracts with 85.2% and 86.6% activity, respectively. In β-carotene bleaching assay, used as third method, the antioxidant capacity is evaluated by measuring the inhibition of the production of volatile organic compounds and the formation of C=C hydroperoxides arising from linoleic acid oxidation, which results in the discoloration of β-carotene ( 39 ). Bioactive components with different antioxidant effect can prevent or reduce the bleaching of β-carotene.

Regarding ferric reducing power that can help to improve oxidative stress, biopolymer Childinan SF-2 showed a significant activity (OD value of 1.35 nm) that was comparable with those obtained from methanolic extract of Pleurotus ostreatus (OD value of 1.96 nm) and Lentinula edodes (OD value of 1.98 nm) ( 37 ). It was noted that P. flavigenum CML2965 and L. edodes extracts exhibited significant antioxidant activities with 72.2% and 51% of β-carotene protection, respectively. These records were much higher than that of Childinan SF-2 (20.8%) in comparison to the Ascorbic acid (96.05%) and ethanol (11.25%) as positive and negative controls, respectively. Free radical scavenging capacity mostly evaluated by hydroxyl radical and DPPH assays, whereas the β-carotene-linoleic acid system assessment represents the protective features of the antioxidant activities and yet such evaluation is more specific for lipophilic compounds ( 38 ). Therefore, the results collectively showed versatility in antioxidant activity of Childinan SF-2 and dependence of its activity to the chemical composition of reactive compounds.

Resistance of bacterial strains to one or more antimicrobial agents usually increases through mutations and natural selection. Hence, exploring the alternative novel, effective and natural antimicrobial compounds has received much attention ( 39 , 40 ). Although the biopolymer Childinan SF-2 did not entirely inhibit the growth of Gram-negative bacteria including Klebsiella pneumonia and Escherichia coli, it revealed a mild inhibitory effect on the growth of these bacteria with 36% and 45% absorbance reduction, respectively. Consequently, it causes the leakage of vital intracellular constituents and the impairment of the bacterial enzyme systems ( 41 , 42 ). The antimicrobial activity of the biopolymer obtained from fungus Cladosporium cladosporioides was evaluated against various bacterial strains by Yehia et al. ( 43 ). The results showed C. cladosporioides biopolymer had the best antimicrobial activity causing a zone of inhibition ranging from 20.7 to 25.7 mm and a MIC value ranging from 3.90 to 15.62 μg.mL-1 against various tested bacterial phytopathogens ( 43 ). In the other study, the ethyl-acetate extract of the endophytic fungus Penicillium sp. (Stdif 9), exhibited the high inhibition against five Gram-positive bacteria including Bacillus megaterium, Micrococcus luteus, Bacillus cereus, Bacillus subtilis and Micrococcus lysodeikticus, as well as two Gram-negative bacteria of Proteusbacillus vulgaris and Salmonella typhi ( 44 ). Growth inhibition of Staphylococcus aureus is of high importance since methicillin has a widespread resistance to S. aureus and it is assumed that it potentially causes a serious public health threat worldwide ( 26 ).

Low viscosity of the biopolymer solution and high degree of reducing sugars, indicate that Childinan is an oligosaccharide. Probable pharmaceutical applications of the Childinan can be enhanced by fulfilment of structural characterization. The native structure gives a molecular model for new applications and chemical changes and production of semisynthetic designed products may result in more applicable products. Furthermore, Childinan may confer ecological advantages to the fungus. As it is produced along with growth and increasing of the biomass, it can be supposed that secretion of superficial exopolysaccharides protects the mycelia against invading organisms due to its antimicrobial activity and other mentioned bioactive properties. Furthermore, exopolysaccharide production may provide physical advantages such as better water adsorption from the environment and facilitated penetration as the result of its lubricating effect. This may control external microbial populations in the close peripheral region around hyphae. Although, we are not sure whether the same slime production and advantages really happened under natural condition; but it can be much more advanced process under natural condition and external slime may works much more complicated.

6. Conclusion

The novel bioactive compound, Childinan SF-2, was extracted from the fungal isolate Daldinia childiae. The in vitro study indicated that Childinan SF-2 could more effectively elevate the percentage of the apoptosis and necrosis of the cancer cells and block the cell cycle phase. Moreover, the extract showed considerable antioxidant activity via different evaluation methods. The partial characterization of the extract showed that purified Childinan SF-2 had a high content of total and reducing sugars. Further characterization of the chemical structure of Childinan SF-2 and fractionation to suprapure molecules are required to help to unravel the possible mechanism of action and designing of effective probably new pharmaceuticals against cancer.


This study was financially supported by the grant from Vice-chancellor research, Alzahra University, Tehran-Iran (No. 97/3/3630) and the Iran’s National Elites Foundation (No. 96/1/16/551).

Conflict of interests

The author(s) have declared that there is not any conflict of interest.


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