Bioactive compounds produced by fungal endophytes have potential benefits, such as low cost, rapid growth, facile genetic manipulation, and industrial scale-up with fewer negative effects. Millingtonia hortensis is a valuable medicinal plant found throughout South Asia. To the best of our knowledge, no reports are available for the screening of endophytic fungal taxol in this medicinal plant. Hence, seven previously reported endophytic fungi from Millingtonia hortensis L. were used in the present study. Of these, five were found to produce taxol. The agar well diffusion method was used to assess the antibacterial and antifungal activities of partially pure fungal taxol against human and fungal pathogens. Fungal taxol effectively inhibited all tested pathogens. Based on the significant antibacterial activity, the sorting of bacterial cells against the compounds (MID6 and MID9) was analyzed by flow cytometry. MID9 showed a moderate level of cell death compared to the control. The MTT assay was used to examine the cytotoxic effects of fungal taxol against the human liver cancer cell line HepG2. A significant decrease (0.05–0.5 μM) was observed in the cell viability and IC50 dosage was fixed as 0.25 μM after 24 h of treatment. Morphological changes were also observed. From these results, we conclude that this is the first description of the taxol-yielding potential of Cochliobolus hawaiiensis. In addition, fungal taxols exhibit potential antibacterial, antifungal, and anticancer effects. In the future, Cochliobolus hawaiiensis could be a new source of taxol, a revolutionary therapeutic drug.
Microorganisms are natural compounds with a wide range of potential for developing active medications, and traditional medicine is used to treat 80% of the world’s population [1,2]. Since their popularity as complementary, predomination in human health care from prehistoric times and their considerable role in curing illness has increased in recent decades . More than 60–70% of antimicrobials and anticancer medicines presently used in clinical trials are derived from natural materials or have been produced from them . In recent years, researchers have focused on the discovery of new natural bioactive compounds derived from medicinal plants . Paclitaxel (taxol) is the most well-known taxane, and it is employed in the treatment of a wide range of human cancers, including ovarian, breast, lung, prostate, and head and neck cancers . The drug can be obtained from yew (Taxus sp.) but leads to the destruction of trees, resulting in deforestation. Alternative paclitaxel production methods, such as chemical synthesis and Taxus sp. tissue and cell cultures, are more expensive and offer lower yields . Hence, several recent investigations have shown considerable production of plant secondary metabolites by fungal endophytes, raising the prospect of exploiting them as bioactive metabolite sources .
Plant-associated microorganisms, particularly endophytic fungi, remain relatively untapped in terms of natural product discovery . Approximately one million fungal endophytes can produce new compounds, but these are relatively underexplored . Secondary metabolites from plants have a wide range of biological functions, such as antibacterial agents (hypericin),  acetylcholinesterase inhibitors (huperzine A),  and antitumor agents (taxol),  which are also produced by prolific endophytes. Approximately 7,300 medicinal plant species are available in India . The most important constituents present in them are alkaloids, tannins, flavonoids, terpenoids, phenolic compounds, and numerous microbes. Millingtonia hortensis is an important medicinal plant distributed across Southern Asia, particularly India, Burma, Thailand, and South China. It is used to treat many diseases, including cancer, skin disorders, gastrointestinal disorders, respiratory tract disorders, hepatic disorders, epilepsy, ulcers, constipation allergies, and fever. Some of the other known medicinal properties of Millingtonia hortensis have been reported earlier [15,16,17]. However, to the best of our knowledge, no reports are available on the screening of taxol-producing endophytes from Millingtonia hortensis. As a result, finding alternate sources of taxols, such as fungal endophytes, has spurred attention .
Therefore, further research is required in this area. According to our research, no attempt has been made in Asia to identify endophytes capable of producing taxols from Millingtonia hortensis. Hence, the goal of this study was to use multiple spectral and analytical approaches such as thin layer chromatography (TLC), ultraviolet (UV), infra-red spectroscopy (IR), high-performance liquid chromatography (HPLC), NMR, and liquid chromatography-mass spectroscopic (LC-MS) to screen, extract, and characterize taxol from seven distinct endophytic fungi isolated from Millingtonia hortensis. The antibacterial activity of fungal taxol against four plant pathogenic fungi and three human pathogenic bacteria was evaluated using the agar well diffusion method. In addition, the death rate of human pathogenic bacteria was determined using flow cytometry. The anticancer properties of fungal taxol were also tested in vitro against the human liver cancer cell line, HepG-2.
2 Materials and methods
2.1 Fungal taxol extraction
The isolation and identification of endophytic fungi from Millingtonia hortensis L. were previously reported by authors. Among the fungi, seven different endophytic fungi, i.e., Alternaria solani (MID/12), Diaporthe pseudomangiferae (MID/5), Paraconiothyrium brasiliense (MID/15), Phoma macrostoma (MID/6), Cochliobolus hawaiiensis (MID/9), Pseudofusicoccum ardesiacum (MID/2), and Nigrospora sphaerica (MID/10) isolated from Millingtonia hortensis L. were used in the present study. The endophytic fungi were grown in 1,000 mL conical flasks containing 250 mL of the M1D medium supplemented with soy tone. Agar plugs (5 mm diameter) containing sterile mycelia were used as the inoculums. The composition of the MID medium is as follows:
|Ammonium tartrate||5 g|
|Yeast extract||0.5 g|
|Distilled water||1,000 mL|
The isolates were grown for 21 days at 25°C (±2°C) at 12 h light/12 h dark cycle under immobile conditions. After 3 weeks, the culture fluid was passed through four layers of cheesecloth to remove solids and the extracellular fungal compounds in the liquid medium were extracted with the organic solvent dichloromethane (DCM) at a ratio of 1:2 (fungal extract/DCM, v/v). The organic phase was collected, and the solvent was then removed by evaporation under reduced pressure at 40°C using a rotary vacuum evaporator. For subsequent separation and analysis, methanol was used to re-dissolve the solid residue [19,20].
2.1.1 Authentic taxol (standard taxol)
The commercially available authentic taxol (standard taxol) was purchased from Sigma-Aldrich Company (Bangalore) for comparison purposes.
2.2 TLC and UV and IR spectroscopy
Aluminum-pre-coated silica gel plates (Merck, Germany) were used for TLC. Commercially available taxol (Sigma, authentic) and our samples were manually spotted on a TLC plate and subsequently run with chloroform (9.2)/ methanol (0.8, v/v). Taxol was visualized using a spraying substance containing 1% (w/v) vanillin in sulfuric acid, after moderate warming. Then, at the proper R f value, the region of the plate comprising putative taxol (dark gray color) was scraped from the silica plate and then re-eluted with methanol, which is equal to that of authentic taxol. The UV absorption ranges of partially purified fungal taxol and authentic taxol were analyzed using a BeckmanDU-40 spectrophotometer. The IR spectra of the compounds were obtained using a Shimadzu FT IR 8000 series spectrometer in the range of 4,000–500 cm−1 [21,22].
A Shimadzu 9A model HPLC instrument was used to analyze the fungal samples and commercial authentic samples. Twenty microliters of each sample were loaded and detected at 232 nm. The mobile phase of the column consisted of methanol, acetonitrile, and water (25:35:40, v/v) at 1.0 mL/min. The mobile phase was filtered using a 0.2 mm PVDF filter before entering the column [21,22].
Subsequently, an LC-MS study was conducted to validate the presence of taxol. The residue was dissolved in a 9:1 (v/v) mixture of methanol/water. A loop-injection approach was used to inject each sample [21,22].
2.5 1H NMR
On a Bruker (Avance) 300 MHz NMR spectrometer with tetramethylsilane (TMS) as an internal standard, 1H NMR spectra were obtained to verify the presence of taxol. CDCl3 was used as a solvent to dissolve the fungal sample and authentic taxol. Bruker’s standard software was used throughout [21,22].
2.6 Minimum inhibitory concentration (MIC) of fungal taxol against bacterial pathogens
The broth microdilution susceptibility test was used to determine the MIC, and the results were interpreted in accordance with the guidelines established by the National Council for Clinical Laboratory Standards (NCCLS, 2009) . Gram-positive bacteria, such as Staphylococcus aureus MTCC 3160, and Gram-negative bacteria, such as Salmonella typhi MTCC 733 and Escherichia coli MTCC 443, were obtained from MTCC (Chandigarh, India) and used for this study. Fungal taxol was subsequently diluted in the Mueller Hinton broth by a factor of 2 on a 96-well microtiter plate to get a range of concentrations (10–100 µg/mL). After adding 100 µL of the liquid culture medium to each well, a bacterial inoculum of 10 µL was used to achieve a final concentration of 105 CFU/mL in each well. Following 24 h of incubation at 37°C, the microtiter plate was loaded with Resazurin (30 L of 0.015%) and incubated for 4 h at 37°C. The MIC was determined as the lowest concentration of the extract that inhibited microbial growth.
2.7 MIC of fungal taxol against plant fungal pathogens
The agar well diffusion method was used to assess the antifungal activity of partially pure fungal taxol against fungal pathogens . The plant fungal pathogens Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina, and Fusarium oxysporum were obtained from the Department of Microbial Technology, School of Biological Science, MKU (Madurai, Tamil Nadu, India). Carbendazim was used as a positive control, and fungal taxol was used at concentrations of 10–80 µg/mL in 10% dimethyl sulfoxide (DMSO). The potato dextrose agar (PDA) was prepared, and 10–80 µg/mL fungal taxol was loaded in different wells made on the PDA at the corner of the PDA plate with a proper label. A mycelial disc of 9 mm of different plant fungal pathogens was inoculated at the center of the plate and the test and control plates were incubated at 25°C for 2 days. After 2 days, the plates were observed and the results were recorded.
2.8 Flow cytometry
Based on the significant antibacterial activity, partially purified fungal taxol MID6 and MID9 were selected for the flow cytometry study through which we could explore the efficiency of fungal metabolites in terms of the rate of death against human pathogens. Bacterial cultures were grown overnight and treated with compounds MID6 and MID9 at a concentration of 50 µg/mL and then incubated for 6 h. After incubation, the culture was centrifuged at 10,000 rpm for 10 min. The pellet was suspended in 1× phosphate buffer, and the supernatant was discarded. The suspension was stained with 1 µL of propidium iodide, and the results were visualized using a BD FACSAriaTMIII flow cytometer .
2.9 Cell line
The human liver cancer cell line HepG2 was procured from the National Center for Cell Science, Pune, India. The cells were then cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (10%), penicillin, or streptomycin (1%) and incubated at 37◦C in a humidified incubator with 5% CO2.
2.9.1 Cell viability test
The MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay was used to determine cell viability . In 96-well plates, HepG2 cells (5 × 103 cells/mL) were plated in DMEM with 10% FBS and then incubated under 5% CO2 at 37°C for 24 h. The medium was withdrawn, cleaned with PBS, replaced with fresh serum-free medium, and incubated for 1 h. After starvation, the cells were treated with varying amounts of fungal taxol (0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, and 0.5 μM) and incubated for 24 and 48 h, respectively. After incubation, each well was filled with 10 μL of 5 mg/mL MTT solution and incubated for 4 h. The supernatant was aspirated after incubation, and 100 μL of DMSO was added to the crystals for solubilization. The absorbance of each well was measured at 570 nm wavelength using a microplate reader. The following formula was used to calculate the percentage of cells that were viable:
2.9.2 Cell morphology observations
Light microscopy was used to examine morphological changes in fungal taxol-treated HepG2 cells. In a controlled environment, cancer cells (5 × 104 cells/mL) were plated in 100 mm dishes and incubated for 24 h. The spent medium was then withdrawn, followed by the addition of the fresh medium containing either DMSO alone or 0.25 µM fungal taxol and incubated for 24 or 48 h, respectively. The cells were observed under a 20× inverted light microscope (Nikon, Sclipse TS100) after incubation.
3.1 TLC and UV
Seven isolates were inoculated into the M1D medium and extracted with DCM for further investigation. TLC analysis revealed the presence of fungal taxol in the extracts when compared to authentic taxol, which possesses an R f value of 0.4 when visualized under UV light (Figure 1). Five of the seven fungal isolates tested positive for taxol production. The area of the plate containing the putative taxol (at the required R f value) was carefully removed from the silica and re-eluted with methanol and they were used in further studies. The presence of fungal taxol was also detected by UV and IR analyses in comparison of re-eluted samples with authentic taxol. The UV spectra (λ max) of both fungal and authentic taxol showed similar absorption and characteristic peaks at 220 and 273 nm (Figure 2).
3.2 IR spectroscopy
The fungal and authentic taxols were further analyzed and matched with IR. The IR spectra of the fungal taxol showed a broad peak in the region 3400.62–3416.05 cm−1 and was identified as the hydroxyl group (−OH); the amide (–NH) group showed a value of 2926.11–2955.04 cm−1. The aromatic (C═C) group stretch was observed in the regions 1454.38, 1452.45, and 2858.60 cm−1, whereas C and H bonds were observed in the regions 1026.16, 1076.78, and 3408.33 cm−1. The aliphatic CH stretch was observed in the range of 2833.52–2852.81 cm−1. When compared to authentic taxol, the IR spectrum of the fungus taxol was similar (Figure 3). Different functional groups were observed in the IR analysis of both fungal and authentic taxols, which effectively demonstrates the identical nature of the taxol. Based on these findings, we primarily confirmed that the five fungal isolates produced taxol, even though Cochliobolus hawaiiensis has never been documented to produce taxol before, and were therefore chosen for further investigation and confirmation of taxol production.
3.3 HPLC and LC-MS
In the HPLC examination, the fungal taxol showed a peak with a retention time similar to that of authentic taxol (Figure 4). The amount of taxol generated by the fungus was quantified and was estimated to be 282 µg/L. The mass spectrum of the fungal taxol was identical to that of authentic paclitaxel. Authentic taxol produced an (M + H) + peak at 854 and an (M + Na) + peak at 876, as expected. Similar peak ranges were reported for fungal taxol (Figure 5). From the results, we found that taxol was present in the purified sample.
3.4 1H NMR
The 1H NMR spectra revealed strong signals ranging from 1.0 to 8.4 ppm. In the region of 1.0–4.6 ppm, the three proton signals induced by the methyl and acetate groups were observed, including other aliphatic protons. Protons in the skeleton and side chain of taxanes were found in the range of 4.6–6.00 ppm, and between 6.5 and 8.5 ppm, aromatic proton indications developed. 1H-NMR (300 MHz, CHCL3) δ: 8.929 (d, 2H, J = 8.7 Hz), 7.971 (d, 2H, J = 7.2 Hz), 7.926 (2H, d, J = 6.6 Hz), 7.701–7.250 (1H, m), 7.635–7.659 (m, 2H), 7.395 (d, 2H, J = 3.9 Hz), 7.219 (m, 2H), 6.29 (s, 2H), 6.199 (d, 1H, J = 7.8 Hz), 5.89 (1H, s), 5.39–5.431 (m, 3H), 4.904–4.955 (2H, m), 4.718 (2H, s), 4.578 (m, 2H), 4.02 (m, 3H), 2.499–2.511 (m, 5H), 2.228 (s, 2H), 2.101 (d, 2H, J = 8.7), 1.790 (s, 3H), 1.505 (s, 3H), 1.236 (s, 2H), 1.016 (s, 3H) (Figure 6), which are similar to that of authentic taxol.
3.5 Antimicrobial activity against human and plant pathogenic microorganisms
Compared to the positive and negative controls, all five fungal taxols had distinct MIC values against three human pathogenic bacteria. Furthermore, the activity of the compounds was shown to be dose-dependent, with high concentrations having a substantial effect. The MID6 and MID9 extracts showed potential antibiotic effects against E. coli (50 and 40 µg/mL), S. aureus (30 and 20 µg/mL), and S. typhi (15 and 20 µg/mL). Compared with the other extracts, the commercial antibiotic tetracycline had the highest activity, ranging from 15 to 25 µg/mL (Table 1). The MIC of five fungal taxol compounds exhibited different levels of inhibition (ranging between 10 and80 µg/mL) against four tested plant pathogenic fungi compared to carbendazim and DMSO, which were used as negative controls. Furthermore, the antifungal activity of the compounds was dose-dependent and effective at high doses. In comparison to the fungal taxol and commercial fungicide carbendazim, compound MID15 exhibited substantial action against the tested pathogens such as R. solani, S. rolfsii, M. phaseolina, and F. oxysporum in the range of 10–30 µg/mL. Compounds MID12 and MID15 showed remarkable antifungal activity, ranging from 10 to 40 µg/mL, compared to the controls. Moreover, other fungal taxols showed antifungal activity ranging between 10 and 80 µg/mL. Relative to the reference control, the MIC values for fungal taxol from MID5, MID6, and MID9 were relatively low (Table 2).
|Tested fungal extract||MIC (µg/mL)|
|E. coli (MTTC 443)||S. aureus (MTTC 3160)||S. typhi (MTTC 733)|
|Tested fungal extract||MIC (µg/mL)|
|R. solani||S. rolfsii||M. phaseolina||F. oxysporum|
3.6 Flow cytometry analysis
The sorting of bacterial cells against the fungal taxol (MID6 and MID9) was analyzed by flow cytometry. Fluorescence-activated cell sorting (FACS) was performed for 50,000 events. P1 represents live cells and P2 represents dead cells. Compound MID6 showed a death rate of 1.3% (644) against E. coli when compared to commercial antibiotic tetracycline (1.4%, 723). Compound MID9 showed a moderate death rate of 0.9% (462) in E. coli. In the case of S. aureus, MID6 and MID9 showed death rates of 0.3% (139) and 0.6% (279), respectively, compared to tetracycline 0.1% (74). Compound MID6 showed a significant death rate of 1.4% (700) compared to tetracycline (1.3%, 627) against S. typhi. Compound MID9 showed a moderate death rate of 1.2% (608) compared to the control against S. typhi (Figure 7a–c).
3.7 Anticancer efficacy of fungal taxol against a HepG-2 human liver cancer cell line
The MTT assay was used to determine whether fungal taxol was cytotoxic. The effect of fungal taxol on cell viability in the HepG-2 cell line was evaluated for 24 and 48 h. The study found that the viability of control cells was consistently 90–95%. At concentrations of fungal taxol ranging from 0.05 to 0.5 µM, cell viability was shown to be significantly reduced (Figure 8). At 0.25 μM of fungal taxol treatment, only 50% of cells were viable after 24 h of treatment. The IC50 of the fungal taxol was found to be 0.25 µM for 24 h. At 0.3 μM of fungal taxol treatment, 50% of cells were viable after 48 h of treatment. The IC50 of the fungal taxol was found to be 0.3 µM for 48 h. Furthermore, light microscopic examination revealed changes in the morphology of HepG-2 cells treated with fungal taxol for 24 and 48 h. Control HepG-2 cells formed irregular confluent aggregates with rounded and polygonal shapes (Figure 9a). After 24 h (Figure 9b) and 48 h (Figure 9c), fungal taxol exposure caused the polygonal cells to shrink and become spherical. Increased progressive cell shrinkage was evident upon treatment with fungal taxol when compared to control cells, validating the anticarcinogenic activity of fungal taxol.
Endophytic fungi are becoming well-known potential sources of new secondary metabolites with bioactive properties for biological applications . Endophytes that produce taxol have potential benefits such as low cost, rapid growth at high cell density cultivation, ease of genetic manipulation, and the ability to scale up on an industrial scale with fewer negative effects [28,29,30,31,32]. Apart from the fact that studying endophytic fungi opens up new biotechnological paths, it also necessitates the isolation, culture, and bio-prospecting of these organisms. Hence, the goal of this study was to isolate, screen, and characterize taxol-producing endophytic fungi. The antibacterial and anticancer effects of fungal taxols have also been investigated. Seven endophytic fungi were screened for taxol production. TLC was used to preliminarily demonstrate the presence of fungal taxol in the extract. Among the seven fungi, five were able to produce taxol. It was clear from this that the fungi produced taxol positively, as confirmed by TLC. Similar evidence has been reported earlier in different endophytic fungi for positive taxol production [28,29,30,31,32].
Fungal taxols were identified using spectral and analytical techniques such as UV and IR to discern patterns of taxol assignments. In UV spectrum, trace concentrations of various chemicals other than taxol can cause disturbances in the absorbance values, resulting in weight estimations for taxol to be higher than the real value. When compared to the standard fungal taxol, UV absorbance values for the test fungi (220–270 nm) were associated with the real taxol; IR analyses also revealed appropriate peak development of different functional groups. Based on the results of the UV and IR analyses, it was confirmed that the reported fungi produced taxol in the M1D medium, which was also evident in previous publications [22,31,32,33].
HPLC, high-resolution 1H NMR, and MS are among the techniques used to confirm the anticancer chemical taxol obtained from endophytic fungi, the production of which varies widely and has been reported in the literature [34,35,36]. In the HPLC examination, the taxol isolated from Cochliobolus hawaiiensis produced a peak with a retention period close to that of the authentic taxol. The amount of taxol generated by the fungus was quantified and was estimated to be 282 µg/L. The mass spectrum of the fungal compound was identical to that of authentic paclitaxel. Therefore, it was clear that the endophytic fungus produced the diterpene taxol, which showed positive results in mass spectrum analysis [37,38,39,40,54]. The 1H-NMR spectra (in CDCl3) of the isolated fungus taxol were identical to the standard in every way [22,32].
In the present study, five fungal taxa exhibited different MIC values against three human pathogenic bacteria that were tested compared to positive and negative controls. Furthermore, the activity of the compounds was shown to be dose-dependent, with high concentrations having a significant effect. MID6 and MID9 extracts showed potential antibacterial activity against the tested pathogens. The reason for variations observed during the assessment of antibacterial activity might be the presence of active compounds in minimal quantities. The potency of these compounds may be increased after further purification . Literature reports provide substantial evidence that many associated medicinal plants and endophytes can produce natural compounds [42,43,44,45]. Compared with carbendazim and DMSO, the MIC values of five fungal taxol compounds inhibited four plant pathogenic fungi at varying concentrations (ranging from 10 to 80 µg/mL). Furthermore, the antifungal efficacy of these compounds was dose-dependent and significant at high concentrations. Among the five compounds tested, MID15 showed significant activity against the tested pathogens. Similarly, it has been reported earlier by different authors [46,47,48,49] that the metabolites derived from endophytic fungi have antifungal activity in different pathogenic fungi.
Mixtures of live and dead (80°C for 15 min) bacteria were used to standardize the flow cytometry method. Flow cytometry, a new microbiological technology, was used to assess the efficiency of the five disinfection agents. This method is based on kinetic assessments of the effects of a disinfecting agent on the fraction of live/dead cells measured with propidium iodide . In this study, the sorting of bacterial cells against the fungal taxol (MID6 and MID9) was analyzed by flow cytometry. Fluorescence-activated cell sorting (FACS) was performed for 50,000 events. P1 represents live cells and P2 represents dead cells. Compounds MID6 and MID9 had different death rates against the tested pathogens when compared to commercial antibiotics. MTT assay was used to assess the cytotoxic effects of fungal taxol, which revealed the effect of fungal taxol on cell viability in HepG-2 cells for 24 and 48 h. Taxol prevents the production of proper spindles at the metaphase, which is essential for mitosis and cell proliferation, by interfering with the regular restructuring of the microtubule network. These actions cause cells to enter the G2/M phase of the cell cycle and finally die as apoptotic cells [31,50,51]. Taxol and baccatin III have been reported to have in vitro anticancer activities in multiple cancer cell lines [21,36] and potato disc tumor induction assays . It was also shown earlier that the antiproliferative efficacy of paclitaxel occurs via apoptosis in HCC cell lines .
We infer that this is the first report on the taxol-producing capacity of Cochliobolus hawaiiensis based on the findings. Furthermore, fungal taxol exhibited potential antibacterial, antifungal, and anticancer effects against human cancer cell lines. Using microorganisms as a source of taxol production is an alternative method and helped to reduce the cost rate; large-scale production of the drug is possible through fermentation technology and strain improvement of fungus. Based on our findings, Cochliobolus hawaiiensis could be a new source of taxol, a revolutionary pharmaceutical agent. In the future, the optimization of the fungal taxol will be done on a large-scale production, and in vitro/in vivo studies will be carried out for the evaluation of the anticancer activity of taxol obtained from the fungal source.
The authors gratefully acknowledge the “Centre for Excellence in Flow Cytometry” DST- FIST, School of Biological Sciences and “DST- PURSE and RUSA” Madurai Kamaraj University, Madurai, India for the instrument facility.
Funding information: The authors state that no funding was received.
Author contributions: The authors contributed equally to the work.
Conflict of interest: The authors declare that there is no conflict of interest.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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