Abstract
During our previous investigation, bioactive compounds present in the extract of Streptomyces sp. strain 196 were characterized using LC–MS/MS and 1H NMR studies. These compounds were K-252-C aglycone indolocarbazole alkaloid, decoyinine, and cycloheximide; the study of these natural drugs against lung carcinoma is still limited. Focus of the current investigation was to study the anticancer effect of strain 196 extract on lung cancer cells (A549). During in vitro studies, anti-proliferative effect of extract was studied using MTT assay in A549 cells. Effect of extract on cell survival was further evaluated using colony assay. Cell death was qualitatively assessed using apoptosis assay. The aftereffect of extract treatment on metastatic potential of cancerous cells was studied using wound closure assay. Effect of extract on the morphology and cytoskeletal arrangement of A549 cells was studied using phalloidin staining. The extract demonstrated concentration and time-dependent cytotoxicity with IC50 value at 0.5 mg/ml (6 h) and 0.15 mg/ml (24 h). The proliferation and metastatic potential of cells, as characterized by MTT and migration assay, decreased over time in a concentration-dependent manner. Discrete changes in cellular morphology were noted as a result of the induced cytotoxicity. Apoptosis assay demonstrated 98.7% cell death at highest concentration of extract (1 mg/ml). During in silico studies, molecular docking revealed that strain 196 compounds are efficiently binding to mutant EGFR form (T790M/L858R) with release of binding energy (∆G) between − 5 and − 6.9 kcal/Mol. In conclusion, strain 196 extract could be a source of therapeutic drugs to treat lung carcinoma.
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Introduction
Lung cancer is the principal cause of mortality among malignant diseases (Rath et al. 2018). The most common subtype is non-small cell lung cancer (NSCLC) which is found in ~ 85% cases (Herbst et al. 2018), whereas small cell lung cancer (SCLC) accounts for rest of the cases (Kalemkerian et al. 2013). For their treatment radiotherapy and chemotherapy are the most widely used approaches. However, radiotherapy and chemotherapy cause adverse effects such as gastrointestinal toxicity, nephrotoxicity, oral mucositis, hepatotoxicity, cardiotoxicity, hematopoietic system injury and neurotoxicity. These noxious effects lead to a reduction in quality of life in cancer patients and ultimately results in discontinuation of therapy (Shapiro 2016; Turcotte et al. 2017; Zhang et al. 2018). Therefore, there is a need for improvement in available therapeutic options to avoid their detrimental effects.
Natural compounds are rapidly obtaining attention as a potential source of new anticancer drugs (Chin et al. 2006; Robles-Fernanzdez et al. 2013; Newman and Cragg 2014). Microorganisms are rich sources of various bioactive compounds, out of which some are authenticated to be used as therapeutic agents (Berdy 2005; Demain and Sanchez 2009). Among microbes, Streptomyces genus of actinobacteria has attracted scientific world due to its unmatched ability for production of bioactive compounds. Initially, Waksman and Henrici proposed the genus Streptomyces in 1943; at this time discovery of actinomycin from Actinomyces antibioticus (currently Streptomyces antibioticus) took place. After the identification of actinomycin D as an anticancer agent, myriad anticancer drugs such as bleomycin, anthracyclines, mitosanes, etc., have been extracted from Streptomyces sp. (Berdy 2005; Demain and Sanchez 2009). In recent years, the focus of research is shifting towards investigating the biosynthetic potential of Streptomyces sp. from underexplored habitats. Soil-derived microbes could be a potential source of compounds having pharmaceutical value.
In the present study, Streptomyces sp. strain 196 collected from agricultural soil of Yamuna River New Delhi, India, was extensively studied for its anticancer applications against human lung cancer cell line A549 by performing various in vitro assays and in silico studies. K-252-C aglycone indolocarbazole alkaloid, decoyinine and cycloheximide bioactive compounds were extracted in the form of crude extract using cold extraction method. Anticancer potential of strain 196 extract against human lung cancer cell line A549 was experimentally proven by conducting detailed in vitro study including cell proliferation assay, anchorage-dependent colony assay, apoptosis assay, migration-cell wound closure assay, and morphological changes: immunofluorescence. EGFR (epidermal growth factor receptor) is a transmembrane protein found on the surface of cells to which epidermal growth factors bind. This receptor plays role in the proliferation and development of cells, the aberrant expression of this receptor leads to the conversion of normal cells into the cancerous cells (Lynch et al. 2004; Hynes et al. 2005; Lu et al. 2018). Under in silico studies, molecular docking of strain 196 compounds and positive control osimertinib (Tagrisso) was performed against mutant EGFR form (PDB ID: 3W2O) having both drug resistant mutation (T790M) as well as lung cancer activating mutation (L858R). The tool used for this study was AutoDock 4.2. Furthermore, validation of docking results was performed by calculating root-mean-square deviation (RMSD) using PyMOL. In a nutshell, the results of in vitro studies revealed that extract from strain 196 possesses potential to act against human lung cancer. During in silico studies, when we compared the binding pockets of compounds from strain 196 with ligand (TAK-285) co-crystallized with mutant EGFR and positive control ligand osimertinib; it was found that binding pockets of all three compounds from strain 196 are similar to ligand co-crystallized with mutant EGFR and osimertinib. The findings of the current investigation are anticipated to offer a strong base for further development of new drugs to use in therapy having no or very fewer side effects.
Materials and methods
Actinomycete strain
Actinomycete strain under study (Streptomyces sp. strain 196) was isolated from agricultural soil of Yamuna River, New Delhi, India, during our earlier studies (Kapur et al. 2018a). This strain was also screened against various pathogens to assess its bioactive potential; it showed very high activity against Bacillus cereus MTCC 430 (Kapur et al. 2018a). 16S rRNA gene phylogenetic studies revealed that strain 196 belongs to the genus Streptomyces (Kapur et al. 2018b). During our recent report (Kumar et al. 2019), we have elucidated the structure of bioactive compounds present in the crude extract of strain 196.
The present investigation is focused on the anticancer potential analysis of K-252-C aglycone indolocarbazole alkaloid, decoyinine, and cycloheximide containing strain 196 extract against human lung cancer cell line A549.
Extraction of bioactive compounds
Streptomyces sp. strain 196 was streaked on yeast extract–malt extract (YM) medium plates and incubated at 28 ℃ for 15 days for production of bioactive compounds (secondary metabolites). The extraction of compounds was performed by employing the cold extraction method (Kumar et al. 2019; Schimana et al. 2000; Khanna et al. 2015). In this method, well-grown culture plates were chopped into small pieces. These pieces were transferred into flasks; ethyl acetate was added into each flask and kept for 4–5 h incubation at 200 rpm, 28 ℃ temperature in cooling incubator shaker (New Brunswick Scientific Excella E24, Germany). The resultant was filtered using Whatman no. 1 filter paper. Evaporation of solvent was performed by using a rotary evaporator (Buchi R-200, Switzerland). The resulted extract was weighted in two different tubes, in one tube it was dissolved in the culture medium of A549 cell line while in another tube it was dissolved in the culture medium of WI-38 cell line. In each tube, the final concentration was 2 mg/ml. Furthermore, different volumes of these main stocks were added into the media for having a series of different concentrations of extract during anticancer activity analysis against respective cell lines (A549 and WI-38).
Anticancer activity analysis
Culture of cell lines and reagents
Human lung cancer (A549) and human normal lung (WI-38) cell lines were procured from NCCS, Pune, India. A549 cells were sub-cultured in RPMI-1640 media, supplemented with 10% fetal bovine serum (v/v), and 1% antibiotic–antimycotic solution; whereas, WI-38 cells were grown on Eagle (minimum essential medium) with 2 mM L-glutamine and 1.5 g/L Na bicarbonate, 0.1 mM non-essential amino acid and 1.0 mM Na pyruvate 90%; fetal bovine serum (FBS) 10%. Cultured cells were grown at 37 °C and 5% CO2 in a humified atmosphere.
Culture media RPMI-1640, Dulbecco’s modified Eagle medium (DMEM), phosphate buffered saline (1 × PBS), 0.25% trypsin and 0.02% EDTA solution, FBS (fetal bovine serum), antibiotic–antimycotic mix solution, acridine orange (AO), ethidium bromide (EtBr), DMSO and MTT were procured from Himedia, India. Triton X-100 was purchased from Sigma-Aldrich, India. All molecular biology grade chemicals were used in the study. Alexa Fluor 594 phalloidin (A12381) was purchased from Thermo Fisher.
Cell proliferation assay
The effect of bioactive compounds containing extract of Streptomyces sp. strain 196 on the proliferation of human lung cancer cells (A549) and normal human lung cells (WI-38) was evaluated using MTT assay. Briefly, cells at a density of 5000 cells per well were seeded in 96 well plates and allowed to adhere overnight at 37 °C. Next day, culture media were replaced with media containing a series of different concentrations of extract (n = 3 per group) and incubated for another 6 and 24 h at 37 °C. Post incubation, MTT solution (20 µl of 5 mg/ml in PBS) was added, incubated for 3 h and DMSO (200 µl) was added to dissolve the formazan crystals. The absorbance (OD) was measured using a multi-plate reader (Thermo Fisher, USA) at 570 nm and reference at 650 nm. Cell viability (%) was calculated using the following equation (Zhang et al. 2017; Kumar et al. 2018; Kumar et al. 2019a, b)
where control is cells without extract and blank is only media.
Anchorage-dependent colony assay
A549 cancer cells were seeded in 60-mm culture dish at a density of 500 cells per dish. Subsequent to cell attachment, culture media containing different concentrations of extract (0.001 to 1 mg/ml) were added for 24 h. In the post-incubation period, extract containing media were aspirated and fresh media were added to each dish for an additional 7 days. After the respective time point, cells were washed with 1 × PBS (phosphate buffered saline), fixed in 4% paraformaldehyde for 20 min and stained using crystal violet solution (0.4% w/v). The excess solution was washed and colony formation was computed based on visual examination (Washio et al. 2018).
Apoptosis assay
In this assay, acridine orange–ethidium bromide (AO–EtBr) staining was used for qualitative assessment of apoptosis in the treated cancer cells (Kasibhatla et al. 2006; Kumar and Tiku 2018). AO stains both live as well as dead cells and gives green fluorescence, whereas EtBr stains dead cells and gives red fluorescence. Live cells display uniform green fluorescence, while apoptotic and dead cells display orange and red color, respectively. Subsequently, to 24 h of incubation with extract, cells were harvested and single-cell suspension (8 µl) was mixed with AO–EtBr stain (1 mg/ml; 1:1 v/v) and incubated for 2 min. The cells were visualized using a fluorescence microscope (Make- Nikon ECLIPSE Ti-S, Japan) at 10 × magnification.
Migration-cell wound closure assay
A549 cells at a density of 5 × 105 cells were seeded in a 6-well plate and allowed to grow till 80% confluency. Once confluent, a scratch was made in each well with the help of 200 µl micropipette tip and media containing various concentrations of extract (0.001 to 1 mg/ml) was added. The changes in the migration-cell wound closure were noted at 0 h, 24 h and 48 h using a bright-field microscope (Make- Nikon ECLIPSE Ti-S) at 10 × magnification and analyzed using ImageJ software (Washio et al. 2018; Garcia et al. 2016).
Morphological changes: immunofluorescence
For the study the effect of the extract on the morphology and cytoskeletal arrangement of A549 cells, phalloidin staining was performed (Washio et al. 2018; Nersesian et al. 2018). Briefly, cells incubated with different concentrations of extract for 24 h were fixed in 4% paraformaldehyde, using 0.05% Triton X-100 for permeabilization, and blocked with 5% BSA. Alexa Fluor 594 Phalloidin (A12381, 1:100 dilution) was added for 40 min followed by nuclear staining with DAPI. Mounted slides were examined under a fluorescent microscope (Make- Nikon ECLIPSE Ti-S, Tokyo, Japan) at 60 × magnification.
Statistical analysis
Each experimental group consisted of 3 replicates. Statistical analysis was performed using one-way ANOVA and two-way ANOVA, followed by Tukey post hoc (*p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant). Data are represented as mean ± standard error of the mean.
Molecular docking
Nearly 70–80% cases of non-small cell lung cancer (NSCLC) are due to activating mutation in EGFR tyrosine kinase domain (Lynch et al. 2004). Molecular docking studies were performed to check whether three compounds from strain 196 can bind to mutant EGFR.
Molecular docking of compounds K-252-C aglycone indolocarbazole alkaloid, decoyinine, and cycloheximide present in strain 196 extract was performed against mutant (PDB ID: 3W2O) form of epidermal growth factor receptor (EGFR) tyrosine kinase domain by taking osimertinib (Tagrisso) as positive control. After docking, comparison of the binding pockets of compounds from strain 196 with ligand (TAK-285) co-crystallized with mutant EGFR and positive control ligand osimertinib was done. AutoDock 4.2 with several supporting tools such as MGL tools, Cygwin, Discovery Studio Visualizer, Binary files, and Java was used to perform the docking. The steps for the usage of AutoDock 4.2 were followed as per the protocol given by Rizvi et al. (2013). For validation of docking results root-mean-square deviation (RMSD) was calculated using PyMOL.
Results
Actinomycete strain
Streptomyces sp. strain 196 was grown on YM medium at 28 ℃, after 5 days it showed white colored aerial spores. This grown culture was used for further studies reported in this paper.
Extraction of bioactive compounds
The cold extraction method was employed for isolation of bioactive compounds using ethyl acetate as solvent. Solvent evaporation in rotary evaporator resulted in bioactive compounds containing semi-solid residue (extract). Furthermore, this extract was dissolved in the culture media of cell lines (A549 and WI-38) and used in the anticancer activity analysis.
Anticancer activity analysis
Cell proliferation assay
In vitro anti-proliferative effect of the extract on A549 human lung cancer cells demonstrated a decrease in cell viability in a dose and time-dependent manner (Fig. 1a). Reduction in the viability of cancer cells was initiated at 0.1 mg/ml after 24 h of treatment; while after 6 h, the extract was effective on cells at the only higher dose (Fig. 1a). Extract inhibited proliferation of cells up to 50% at 0.15 mg/ml (24 h) and 0.5 mg/ml (6 h). This suggests that the anti-proliferative effect of the extract was effective after 6 h of treatment. Normal human lung cells (WI-38) treated with the same doses of the extract showed no significant reduction in cell viability as compared to extract-treated cancer cells (Fig. 1b). This suggests that the anti-proliferative effect of the drug specifically restricted to cancer cells only.
Anchorage-dependent colony assay
The effect of the extract on cell survival was further evaluated using colony assay, where a group of at least 50 cells was referred to as a colony. As clearly shown in Fig. 2, 0.001 mg/ml and 0.01 mg/ml extract concentrations showed similar colonies in terms of number and size as compared to the control group. Whereas, higher concentrations of extract from 0.1–1 mg/ml; significantly diminished the number of colonies in a dose-dependent manner.
Apoptosis assay
Cell death was qualitatively assessed using AO–EtBr staining on treated cells. As can be seen in Fig. 3, control (untreated cells) displayed green fluorescence suggesting that all cells were live. In contrast, cells demonstrated relatively higher death as the concentration of extract increased (Fig. 3a). At higher concentration (1 mg/ml), 98.7% cell death was observed (Fig. 3b).
Migration-cell wound closure assay
The aftereffect of extract treatment on the metastatic potential of cancerous cells was studied using wound closure assay. At 0.01–1 mg/ml, the extract demonstrated a clear dose-dependent reduction in the motility of cancer cells. The rate of wound healing of control cells (untreated) was 57% and 92% after 24 h and 48 h, respectively (Fig. 4a, b). In contrast, only 15% and 18% of cells migrated after 24 h and 48 h, respectively, at 0.1 mg/ml. A significant reduction in cell motility was observed at concentrations below the anti-proliferative inhibitory concentration (IC50) values. These results suggest that the extract inhibited the metastatic potential of the cancerous cells A549 in a dose and time-dependent manner.
Morphological changes: immunofluorescence
The morphology of cancer cells studied using phalloidin staining demonstrated dose-dependent changes in morphology. At lower doses (0.001 mg/ml and 0.01 mg/ml), cell morphology was similar to control group (Fig. 5), while higher doses of extract triggered phenotypical changes in terms of rounded morphology, reduction in cell size, cytoskeleton aggregation at the periphery and cytoskeleton disruption after 24 h (Fig. 5).
Molecular docking
The compounds from strain 196 were used during molecular docking against mutant EGFR (T790M/L858R). The results revealed that K-252-C aglycone indolocarbazole alkaloid release binding energy (∆G) of − 6.9 kcal/Mol with mutant EGFR (Fig. 6a and Table 1). In case of decoyinine released ∆G with mutant EGFR was -5.17 kcal/Mol (Fig. 6b and Table 1); whereas, in cycloheximide the released ∆G with mutant EGFR was -5.63 kcal/Mol (Fig. 6c and Table 1). The positive control osimertinib released ∆G -6.28 with mutant EGFR (Fig. 7 and Table 2). The docking results suggested that the compounds present in the extract from strain 196 are efficiently binding to the mutated site of EGFR receptor (T790M/L858R). When we manually compared the binding pockets of compounds from strain 196 with ligand (TAK-285) co-crystallized with mutant EGFR and positive control ligand osimertinib; it was found that binding pockets of all three compounds from strain 196 are similar to ligand co-crystallized with mutant EGFR and osimertinib. All the ligands (i.e., experimental and controls) were observed to bind the pocket which contains mutated site ‘Met790’ in the receptor. Validation of docking results using PyMOL revealed that the complexes K-252-C aglycone indolocarbazole alkaloid + mutant EGFR, decoyinine + mutant EGFR, cycloheximide + mutant EGFR, and osimertinib (positive control) + mutant EGFR when superimposed on mutant EGFR co-crystallized with ligand TAK-285, each showed RMSD value 0 (Fig. 8). Thus, validation results suggested that compounds from strain 196 are efficiently binding to the mutated site of EGFR receptor (T790M/L858R).
Discussion
In recent years, natural products from actinomycetes have received unprecedented attention because of their wide range of biological activities such as antitumor, antimicrobials, antihypertensive, immune-suppressant, etc. Actinomycetes belonging to Streptomyces genus were found to possess exceptional bioactive natural products synthesis abilities. Thus, the bacteria from this genus are worth to be investigated.
In this study, K-252-C aglycone indolocarbazole alkaloid, decoyinine, and cycloheximide containing extract from Streptomyces sp. strain 196 was studied for its anticancer potential against lung cancer cell line A549. Cytotoxicity studies have played a pivotal role in the discovery of new anticancer drugs over the past three decades. In the present study, the cytotoxic activity of strain 196 extract revealed that strain 196 extract suppressed proliferation of cancer cells in a concentration and time-dependent way. It was observed that after 24 h of the incubation period, strain 196 extract-treated cells at a concentration of 0.15 mg/ml showed only 28% cell viability. Moreover, the cytotoxic study evaluated on normal human lung cells (WI-38) revealed no significant reduction in cell viability compared to cancer cells (A549). Therefore, strain 196 extract could potentially be used as anticancer therapeutics for human.
Cytotoxic mechanism of natural bioactive compounds, interfere with basic cellular functions such as cell cycle, apoptosis, inflammation, angiogenesis, invasion and metastasis. Many anticancer drugs from marine organisms cause cytotoxicity and cell death through the induction of apoptosis. In the present study, the long-term cytotoxic effect of the extract on the proliferation of a single cell into a viable colony was investigated and results suggested that extract significantly reduced clonogenic capability of A549 cells in a concentration-dependent manner. Strain 196 extract-treated cells induced apoptosis further confirmed the concentration-dependent cytotoxic effect of the extract on A549 cells. As the concentration of extract increased, cell death also increased.
Besides cell death, the metastatic potential of cells was decreased in a concentration-dependent manner. It is noteworthy that beyond a particular concentration (0.1 mg/ml), the extract showed a similar cytotoxic effect on cells. A significant reduction in cancer cell motility was observed at a concentration below the anti-proliferative inhibitory concentration (IC50) values. This cytotoxic behavior of strain 196 extract was reflected in the phenotypical changes of cells in terms of nuclear morphology, reduction in cell size, cytoskeleton aggregation, loss of normal nuclear architecture and cytoskeleton disruption indicating deterioration of cells, which was not observed in untreated cells.
Moreover, overexpression of EGFR is directly related to lung cancer development, the mutation (L858R) in EGFR tyrosine kinase domain has been detected as the driver for the development of non-small cell lung cancer (NSCLC) (Hynes et al. 2005). In recent years, therapy with EGFR tyrosine kinase inhibitors (EGFR-TKI) has played a vital role in the improvement of survival of NSCLC patients with mutations in EGFR (Lu et al. 2018). Gefitnib and erlotinib are the first generation inhibitors applied clinically and provided a great response in the treatment of NSCLC patients with mutations in EGFR. After 9–14 months, in these types of cases, an acquired resistance observed against gefitinib and erlotinib (Lu et al. 2018). The secondary mutation T790M (threonine790 to methionine790) in EGFR is responsible for 55–70% acquired resistance in NSCLC patients with EGFR mutations. Some second-generation inhibitors as afatinib and dacomitinib have been developed to treat resistance caused by EGFR (T790M) mutation (Lu et al. 2018).
In the present investigation, molecular docking studies were performed to check whether compounds from strain 196 can bind to mutant EGFR. Results suggested that the compounds K-252-C aglycone indolocarbazole alkaloid, decoyinine and cycloheximide can efficiently bind to the EGFR having T790M/L858R mutations. When we compared the binding pockets of compounds from strain 196 with ligand co-crystallized with mutant EGFR and positive control ligand osimertinib; it was found that binding pockets of all three compounds from strain 196 are similar to ligand co-crystallized with mutant EGFR and osimertinib. All the ligands (i.e., experimental and controls) were observed to bind the pocket which contains mutated site ‘Met790’ in the receptor. Validation of molecular docking data suggested that the binding of these compounds on mutated EGFR is efficient as the RMSD value is 0 in each case. So, it can be suggested that there was no forced docking. Docking results were compared with positive control osimertinib which is a third-generation inhibitor of mutant EGFR. It was suggested that K-252-C aglycone indolocarbazole alkaloid binding mutant EGFR more efficiently than the osimertinib; whereas, decoyinine and cycloheximide were found to have less efficient binding in contrast to osimertinib.
Altogether, these findings suggested that strain 196 extract may serve as a potential therapeutic agent against lung cancer. Overall our study warrants further investigation of the extract for subsequent in vivo and clinical trials studies to establish the strain 196 extract as a potentially safe agent for the treatment of lung cancer.
Conclusion
K-252-C aglycone indolocarbazole alkaloid, decoyinine, and cycloheximide containing strain 196 extract reduced A549 cell viability in a dose and time-dependent manner. The extract influenced the proliferation, colony formation, and migration of A549 cells in the respective assays. So, the results of in vitro anticancer effect analysis of strain 196 extract revealed that it has high cytotoxicity against human lung cancer cells (A549); whereas normal human lung cells (WI-38) treated with the same doses of the extract showed no significant reduction in cell viability. In silico studies (molecular docking) suggested that compounds present in extract from strain 196 bind efficiently to mutant EGFR (T790M/L858R).
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Acknowledgements
We thank the National Centre for Cell Science (NCCS), Pune, for providing cell lines. Acharya Narendra Dev College (ANDC), University of Delhi is warmly acknowledged for providing infrastructural facilities. We are also thankful to Dr. Swati Midha (DST-Inspire faculty, Special Centre for Nanoscience, JNU), Km. Anjaly (JNU), Ms. Neha Verma (JNU), Mr. Ravi Kumar (JNU) and Mr. Shani Kumar (University of Delhi) for their valuable suggestions.
Funding
This study was funded by Council of Scientific & Industrial Research (CSIR), Government of India (grant numbers 08/529(0007)/2017-EMR-I and 08/529(0005)/2017-EMR-I).
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PK, AC, MK, BKK and MKK conceived and designed the experiments. PK and AC performed all experiments. PK analyzed the data reported in this study. PK, AC, MK, BKK, AK, RS, and MKK contributed in writing. All authors read and approved the manuscript.
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Kumar, P., Chauhan, A., Kumar, M. et al. In vitro and in silico anticancer potential analysis of Streptomyces sp. extract against human lung cancer cell line, A549. 3 Biotech 11, 254 (2021). https://doi.org/10.1007/s13205-021-02812-w
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Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-021-02812-w