{"id":60162,"date":"2024-09-30T10:16:53","date_gmt":"2024-09-30T10:16:53","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=60162"},"modified":"2024-10-09T18:50:07","modified_gmt":"2024-10-09T18:50:07","slug":"antiproliferative-and-apoptotic-effects-of-solenostemma-argel-leaf-extracts-on-colon-cancer-cell-line-hct-116","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/antiproliferative-and-apoptotic-effects-of-solenostemma-argel-leaf-extracts-on-colon-cancer-cell-line-hct-116\/","title":{"rendered":"Antiproliferative and Apoptotic Effects of Solenostemma argel Leaf Extracts on Colon Cancer Cell Line HCT-116"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Colorectal cancer (CRC) ranks as the third most prevalent cancer. In 2023, approximately 1.63 million new cases of colorectal cancer were diagnosed globally, resulting in 694,000 deaths. Morbidity and mortality rates from this disease continue to rise rapidly in less developed countries, while highly developed countries tend to be seen stable or decrease, albeit remaining among the highest globally. The global burden is anticipated to surge by 60%, reaching over 2.2 million new cases and 1.1 million deaths by 2030 1, 2<\/sup>. In Sudan, colon cancer is the fifth most common cancer with an incidence rate of 7.1 per 100,000 of 6548 registered cancer cases during the 2009-2010 periods. Its incidence rate is increased per year, according to data collected at the Radiation and Isotope Centre in Khartoum (RICK) from 2009-2013(3). Generally, the incidence of CRC is increased due to increasing age, family history, smoking, excessive alcohol consumption, obesity, and dietary factors 3<\/sup>. <\/p>\n\n\n\n

Conventional therapies of CRC are surgery, chemotherapy, radiotherapy and targeted therapy. However, their dangerous adverse effects and the developing resistance by tumor cells as well as the increasing mortality rate associated with colorectal cancer highlight the need to search for more effective therapies4,5<\/sup>. Plant-based products have provided a valuable resource for finding and developing of unique anticancer drugs that act on several signaling pathways in tumor cells and have limited or no adverse effects. These include the alkaloids Vinca, Vinblastine and Vincristine 6,7<\/sup>. <\/p>\n\n\n\n

In Sudan, the utilization of medicinal and aromatic plants and their derivatives is deeply ingrained in daily life, playing a vital role across various aspects of society and culture8<\/sup>. Solenostemma argel <\/em>(locallyknown in Sudan as \u201cHargel\u201d) belongs to the family Asclepiadaceae. It is widely distributed in Sudan which is its richest source and in Egypt9<\/sup>. It stands as a crucial Sudanese medicinal plant, widely employed in traditional medicine to address various aliments including diabetes, jaundice, measles, syphilis, gastrointestinal disorders, urinary tract infections, and some diseases of liver and kidney10,11<\/sup>. <\/p>\n\n\n\n

Recently, there are a lot of evidences about a wide spectrum of pharmacological effects of S. argel<\/em> and its active compounds with low toxicity include analgesic, anti-ulcerogenic, hypoglycemic, antioxidant and antispasmodic effects12,14<\/sup>. Additionally, several previous studies reported that S. argel <\/em>extracts have anticancer activity against different types of cancer, e.g., Ehrlich ascites carcinoma (EACC), acute myeloid leukemia (AML), acute lymphocyte leukemia (ALL), human hepatocellular cancer (HepG2) and Kaposi\u2019s sarcoma cell 15-18<\/sup>. Therefore, based on the previous studies, it can be expected that S. argel <\/em>also has antitumor activity on other types of human cancer. Therefore, the objective of this study is to assess the in vitro<\/em> anticancer potential of the methanolic extract from S. argel<\/em> leaves against the colon cancer cell line (HCT-116).<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Plant material <\/strong><\/p>\n\n\n\n

The dried Solenostemma argel<\/em> leaves were procured and meticulously identified, and authenticated by the herbarium of the Medicinal and Aromatic Plants Research Institute (MAPRI) in Sudan.<\/p>\n\n\n\n

Chemicals and Reagents <\/strong><\/p>\n\n\n\n

Folin\u2013Ciocalteau reagents, gallic acid, quercetin, aluminum chloride hexahydrate and potassium acetate were obtained from Sigma Chemical Co., St. Louis, MO, USA. Human colorectal carcinoma cells (HCT-116) were acquired from ATCC, USA. The cell culture materials, including penicillin G sodium, Fetal bovine serum (FBS), streptomycin sulphate, L-glutamine, amphotericin B, trypsin\/ EDTA and dimethyl sulphoxide (DMSO) were obtained from Cambrex BioScience (Copenhagen, Denmark). Acridine orange\/ ethidium bromide (AO\/EB) was acquired from Biovision (Mountain View, CA, USA).<\/p>\n\n\n\n

Cell Culture<\/strong><\/p>\n\n\n\n

Human colon cancer cells (HCT-116) were used to assess the antiproliferative and apoptotic effect of the plant extract. The cells were cultured in McCoy’s 5a modified medium which was enhanced by L glutamine (2 mM), penicillin G sodium (100 units\/ml), fetal bovine serum (10%), 250 ng\/ml amphotericin B and streptomycin sulphate (100 units\/ml). The cells were stored in an atmosphere of 5% CO2<\/sub> and 37\u00b0C. For subculture, cell monolayers were harvested after trypsin\/ EDTA treatment. Dimethyl sulphoxide (DMSO) was used to dissolve the extract and diluted to the intended concentration for the test. All experiments were repeated three times unless otherwise stated.<\/p>\n\n\n\n

Extraction of Solenostemma argel<\/em> (Hargel) leaves<\/strong><\/p>\n\n\n\n

The dried Hargel leaves were cleared from other plant pieces and milled manually. After that, 100 grams of these leaves were extracted using 80% methanol via a Soxhlet apparatus. The solvent was then removed through evaporation under pressure using a Rotary evaporator. The resulting extract was stored at a temperature of 4\u1d52<\/sup>C and maintained in a dark place. The obtained extract showed a yield of 20%19<\/sup>.<\/p>\n\n\n\n

Preliminary Phytochemical analysis <\/strong><\/p>\n\n\n\n

The preliminary phytochemical tests for the methanolic extract of S. argel<\/em> were conducted following the procedures outlined by Trease and Evans20<\/sup>. Briefly, a preliminary screening was carried by the application of various testing methods of Draggendorff\u2019s, Liebermann-Bur chard test, Foam formation test, Lead acetate test, Keller-Killani test, Borntrager\u2019s test and Braymer\u2019s test for determining the presence of alkaloids, terpenoid, saponins, flavonoids, cardiac glycoside, anthraquinone and tannins, respectively.<\/p>\n\n\n\n

Determination of the total content of phenols and flavonoids <\/strong><\/p>\n\n\n\n

Folin Ciocalteu reagent was used for analysis of total phenolics content21<\/sup>. Briefly, 0.5 ml of the extract was mixed with 0.5 ml of Folin-Ciocalteu reagent. The solution was kept at 25o<\/sup>C for 5-8 min before adding 2 ml of sodium carbonate solution 7.5 % and adjusting the volume to 8 ml with water. After 2 h, the absorbance was measured at 725 nm. Gallic acid was used as standard for the calibration curve. Total phenolic content was expressed as mg gallic acid equivalents per gram of sample (mg\/g). All samples were analyzed in triplicate.<\/p>\n\n\n\n

The total flavonoid content was measured by a colorimetric assay 22<\/sup>. In brief, 50 \u03bcL of crude extract (1 mg\/mL ethanol) were made up to 1 mL with methanol, mixed with 4 mL of distilled water. Then, 0.3 ml of 5% NaNO2<\/sub> was added. After 5 min, 0.3 ml of 10% aluminium chloride was added, and the mixture was allowed to stand for 6 minute. Then, 2 ml of 1 M sodium hydroxide was added to the mixture, and the final volume of the mixture was brought to 10 mL with distilled water. The absorbance was determined at 420 nm versus a blank. Quercetin was used as standard for the calibration curve. The total flavonoid content of the extract was calculated from a calibration curve, and the result was expressed as mg quercetin equivalents per gram of sample (mg\/g). All samples were analyzed in triplicate.<\/p>\n\n\n\n

Antiproliferative activity assay by Sulfo-Rhodamine-B (SRB) <\/strong><\/p>\n\n\n\n

The SRB assay23<\/sup> was performed to evaluate the antiproliferative efficacy of the leaf extract on HCT-116 cell lines. Cells were seeded in a 96-multiwell plate at a density of 104<\/sup> cells per well, 24 hours prior to treatment, allowing for attachment to the plate. Various concentrations of the extract (0, 25, 50, 100, and 200 \u00b5g\/ml) were added to the cells, with triplicate monolayer wells established for each concentration. Following treatment, the cells were cultured for 48 hours under standard conditions of 37\u00b0C and 5% CO2<\/sub>. Subsequently, the cells were fixed and stained using Sulfo Rhodamine B stain. Excess stain was removed with acetic acid, and the remaining stain was dissolved with Tris EDTA buffer. Absorbance measurements were taken via<\/em> ELISA. Survival curves for each tumor cell line were generated by plotting the relationship between surviving fraction and drug concentration.<\/p>\n\n\n\n

Detection of Apoptosis by Acridine Orange\/ Ethidium Bromide (AO\/EB) Staining<\/strong><\/p>\n\n\n\n

To investigate the onset of apoptosis in HCT-116 cells, a series of treatments were administered using Solenostemma argel<\/em> leaf extract at varying concentrations: 85.3 \u00b5g\/ml (IC50<\/sub>), as well as lower and higher concentrations of 50 and 200 \u00b5g\/ml, respectively. Following a 48-hour incubation period, cells were meticulously washed with cold PBS and subsequently stained with a mixture containing 20 \u03bcg\/ml ethidium bromide and 20 \u03bcg\/ml acridine orange, immediately preceding microscopic examination. A small aliquot (10 \u03bcL) of the gently agitated cell suspension was then placed onto microscope slides, covered with glass slips, and observed under a fluorescence microscope. For each data point, 300 cells were counted in randomly selected fields and quantified in duplicate24<\/sup>.<\/p>\n\n\n\n

Statistical Analysis<\/strong><\/p>\n\n\n\n

Statistical analysis was conducted on all experiments, which were performed in triplicate. The results were then averaged to obtain a representative value. The cell viability percentages were plotted using Microsoft Office Excel 2010 to create graphical representations of the data.<\/p>\n\n\n\n

Results<\/strong><\/p>\n\n\n\n

Phytochemical Analysis <\/strong><\/p>\n\n\n\n

The phytochemical analysis of the methanolic extract of S. argel<\/em>\n(see Table 1) revealed the presence of various groups of secondary metabolites,\nincluding saponins, flavonoids, alkaloids, and tannins, all of which possess medicinal\nsignificance.<\/p>\n\n\n\n

Table 1: Phytochemical screening of the methanol extract of S. argel leaves<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Class of compound<\/strong><\/p>\n<\/td>\n

\n

Result<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Terpenoids<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

Tannins<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

Saponins<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

Flavonoids<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

Alkaloids<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

Anthraquinone<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n

\n

Cardiac glycosides<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

(+<\/strong>): present, (–<\/strong>): absent.<\/p>\n\n\n

Total phenolic and flavonoid contents in the methanolic extract of S. argel <\/strong><\/p>\n\n\n\n

Data on total phenolic and flavonoid contents in the methanolic\nextract of S. argel<\/em> are presented in Table 2. The results showed that\nthe total phenolic was (24.53 \u00b1 0.35 mg), while the total flavonoid was (8.73 \u00b1\n0.025 mg).<\/p>\n\n\n\n

Table 2: Total phenolic and flavonoids contents of S. argel methanolic extract<\/strong><\/p>\n\n\n\n\n\n\n\n
\n

Test<\/strong><\/p>\n<\/td>\n

\n

Amount  <\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Total Phenolics<\/p>\n<\/td>\n

\n

24.53 \u00b1 0.35 mg GAE \/g of dry extract<\/p>\n<\/td>\n<\/tr>\n

\n

Total Flavonoids<\/p>\n<\/td>\n

\n

8.73 \u00b1 0.025 mg QE \/g of dry extract<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The data are presented as the mean \u00b1 standard deviation (SD) of three replicates; GAE: Gallic acid equivalent; QE: Quercetin equivalent.<\/p>\n\n\n\n

Antiproliferative effect of Hargel extract<\/strong><\/p>\n\n\n\n

The antiproliferative activity of S. argel<\/em> leaf extract was\nassessed through the SRB assay, and the half-maximal inhibitory concentration\n(IC50<\/sub>) values were determined from the dose-response curves (refer\nto Figure 1). HCT-116 cells were subjected to varying concentrations of S.\nargel <\/em>methanolic extract (25, 50, 100, and 200 \u03bcg\/ml). Analysis of the SRB\nassay results revealed a concentration-dependent reduction in the proliferation\nof HCT-116 cells upon treatment with the methanolic extract of S. argel<\/em>,\nwith an IC50<\/sub> value of 85.3 \u00b1 0.15 \u03bcg\/ml. As depicted in Fig. 1,\ntreatment with the leaf extract at a concentration of 100 \u03bcg\/ml resulted in a 37%\ninhibition of cancer cell proliferation after 48 hours, while at double the\nconcentration (200 \u03bcg\/ml), the inhibitory effect diminished, although the leaf\nextract continued to induce cell growth arrest.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: <\/strong>Anti<\/strong>proliferative activity of Solenostemma argel<\/em> leaf methanolic extract<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Hargel extract induced Apoptosis<\/strong><\/p>\n\n\n\n

The Acridine orange\/ ethidium bromide (AO\/EB) assays were conducted\nto discern between live and dead cells, revealing that S. argel leaf extract\ninduced cell death in a dose-dependent manner across the studied cell lines.<\/p>\n\n\n\n

Human colon cells (HCT-116) were cultured with leaf extract of Solenostemma\nargel<\/em> at concentrations of 50, 85.3, and 200 \u03bcg\/ml. To detect apoptosis,\ncells were fluorescently stained with acridine orange\/ethidium bromide (AO\/EB)\nand observed under a fluorescence microscope. As depicted in Fig. 2, no\nsignificant apoptosis was observed in the control group (Fig. 2A). However, in\nthe experimental group (Fig. 2B), early-stage apoptotic cells characterized by\ncrescent-shaped or granular yellow-green AO nuclear staining were evident, with\nstaining localized asymmetrically within the cells. Interestingly, increasing\nthe concentration and duration of treatment resulted in a higher number of\nearly-stage apoptotic cells. Furthermore, late-stage apoptotic cells were also\ndetected, exhibiting concentrated and asymmetrically localized orange nuclear EB\nstaining (Fig. 2C). Necrotic cells exhibited increased volume and displayed\nirregular orange-red fluorescence at their periphery, with cells appearing\ndisintegrated (Fig. 2D).<\/p>\n\n\n\n\n\n
\"\"<\/td>\nFigure 2: Morphological changes in HCT-116 cells resulting from the apoptotic effects of the methanolic extract of S. argel <\/em>leaves were detected using AO\/EB staining. <\/strong>

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Discussion <\/strong><\/p>\n\n\n\n

Chemical components of plants or crude extracts are known to be biologically active components. They are directly responsible for various activities such as antioxidant, antimicrobial and anticancer effects 25,26<\/sup>. Therefore, the phytochemical screening is essential to understand their role on treatment of colorectal cancer and subsequently may lead to drug discovery and development. In our study, Solenostemma argel <\/em>methanolic extract was analyzed using the method described by Trease and Evans 20<\/sup>. The obtained results exhibited the existence of terpenoids, flavonoids, tannins, alkaloids, saponins, and cardiac glycosides compounds (Table 1), which is in agreement with previous studies 12,13<\/sup>. Indeed, flavonoids are recognized for their diverse biological activities, which include exhibiting anticancer properties 27-29<\/sup> Numerous studies have reported on the anticancer activities of flavonoids against various types of cancers 30-32<\/sup>. Given that the methanolic extract of S. argel<\/em> leaves contains these secondary metabolite compounds, it is plausible to infer that it possesses a wide range of pharmacological benefits, such as antioxidant, anti-inflammatory, and anticancer activities. This suggests the potential of S. argel<\/em> extract as a valuable resource in the development of therapeutic interventions for cancer and other related conditions.<\/p>\n\n\n\n

The phenolic and flavonoid compounds are renowned for their diverse biological activities, including antioxidant, anti-inflammatory, and anticancer properties33,34<\/sup>; hence it is important to determine the total phenolic and flavonoid contents of S. argel <\/em>leaves extract. In our study, the total phenolic content was measured by the Folin-Ciocalteu reagent method and described as GAE (Gallic Acid Equivalent), whereas the total flavonoid content was estimated using the aluminum chloride colorimetric method and described as QE (Quercetin Equivalent). The total phenolic content in methanolic extract of S. argel<\/em> leaves was found to be 24.53 \u00b1 0.35 mg\/g GAE, while the total content of flavonoid was 8.73 \u00b1 0.025 mg\/g QE (Table 2). This finding is almost near to results obtained by Muddathir et al.<\/em> (2017) 35<\/sup>, who stated that the amount of total phenolic content measured in methanolic extract of S. argel<\/em> (grown in Sudan) was 32.90 \u00b1 3.42 mg\/g GAE. Inconsistently, a previous study conducted in Egypt reported different quantities of total phenolic and total flavonoid in the methanolic extract of S. argel<\/em> leaves, measuring at 6.32 mg\/g GAE and 2.40 mg\/g QE, respectively36<\/sup>. Discrepancies in these quantities may be attributed to variations in environmental factors such as temperature, UV radiation, day length, rainfall, altitude, and climate. These factors significantly influence plant development and subsequently affect the biosynthesis of secondary metabolites with biological activity 37<\/sup>. Therefore, differences in the environmental conditions between regions could contribute to variations in the composition of phenolic and flavonoid compounds in S. argel<\/em> leaves. Furthermore, the total phenolic and flavonoid content can be significantly influenced by the extraction method and the choice of solvent 38,39<\/sup>. Indeed, several studies have indicated a positive correlation between the total content of phenols and flavonoids and the biological effects of the extracts 40-42<\/sup>. Therefore, it is reasonable to speculate that phenolic and flavonoid compounds play a crucial role in the observed anticancer activity of S. argel<\/em> leaves in vitro. <\/p>\n\n\n\n

The present study also evaluated the antiproliferative activity of the methanolic extract of Solenostemma argel <\/em>leaves on colorectal cancer cells (HCT-116) using the Sulfo-Rhodamine B (SRB) assay. The obtained results showed that the extract exhibited antiproliferative effects dependent on doses with IC50<\/sub> of 85.3 \u00b1 0.15\u03bcg\/ml. This finding is consistent with the results reported by Alaa Eldin (2008) 43<\/sup>, who observed a significant reduction in colorectal cancer cell proliferation with increasing doses of the methanolic extract of S. argel<\/em>. Furthermore, Nassr-Allah et al<\/em>. (2009) reported that both aqueous and ethanolic extracts of S. argel<\/em> reduced tumor growth induced by Ehrlich ascites carcinoma cells and prolonged animal survival by 29 days (15). In addition, other plant extracts such as ginger leaf extract, Chinese herbal extract and turmeric root extract have exhibited anticancer properties on HCT-116 cells 44-46<\/sup> in agreement with these findings. <\/p>\n\n\n\n

Although crude extracts can have greater effects than a single ingredient, combinations of ingredients in plant extracts can be very important for the final biological activity; its different fractions contain compounds with different chemical compositions, and therefore, can exhibit different types of activities 47<\/sup>. The previous study conducted by Plaza et al<\/em>. (2005) found that seven novel 15-keto pregnane glycosides, isolated from Solenostemma argel<\/em>, exhibited the ability to decrease the proliferation of Kaposi\u2019s sarcoma cells induced by vascular endothelial growth factor (VEGF) in a dose-dependent manner supporting our results 18<\/sup>. Previous phytochemical screening revealed that leaf extracts of Solenostemma argel<\/em> contain various bioactive compounds like kaempferol and quercetin 48,49<\/sup>. Several previous studies have reported that Kaempferol has antiproliferation activities in various human cancer cell lines, including those derived from colon cancer 50-52<\/sup>. Furthermore, the combination of quercetin and kaempferol exhibited a greater cytotoxic efficacy on HCT-116 cells than did either quercetin or kaempferol alone53<\/sup>. <\/p>\n\n\n\n

Apoptosis is a natural and intricate process of cellular self-destruction, occurring to eliminate damaged or atypical cells 24<\/sup>. However, Cancer cells have the ability to resist apoptosis, allowing them to continue rapidly growing without control. As a result, it would be ideal to have a compound that can effectively modulate apoptosis as a potential treatment for cancer 54<\/sup>. Many plant-based chemicals have been discovered to possess the capacity to trigger programmed cell death in a wide range of cancerous cells derived from humans 5-57<\/sup>. In the present study, the apoptotic effects of Solenostemma argel<\/em> leaf methanolic extract on colorectal cancer cells (HCT-116) were assessed using the acridine orange\/ethidium bromide (AO\/EB) staining method. As shown in Fig. 2, treatment of HCT-116 cells with the Hargel extract at concentrations corresponding to the IC50 <\/sub>value (85.3\u00b5g\/ml), as well as lower and higher concentrations (50 and 200 \u00b5g\/ml), resulted in the staining of early and late apoptotic cells with condensed nuclei, appearing bright green in color at concentrations of extract \u2264 85.3 \u00b5g\/ml (Figure 2b & c). Necrotic cells, on the other hand, appeared red in color at the higher concentration of extract (200 \u00b5g\/ml) (Figure 2d). The results of the current study suggest that the methanolic extract of S. argel<\/em> induces apoptosis in HCT-116 cells in a concentration-dependent manner. The apoptotic effect observed with the methanolic extract of S. argel<\/em> leaves in our study is consistent with previous findings reported by Hanafi and Mansour (2010) (16), they demonstrated that the aqueous extract of S. argel<\/em> leaves induced widespread zones of apoptotic cells in Ehrlich carcinoma tissue. Moreover, several previous studies investigating pure compounds isolated from S. argel<\/em> have also been conducted to evaluate their apoptotic effects, with results confirming their ability to induce apoptosis in various cancer cells (58, 59), findings supported this result. Since apoptosis is considered a new target in cancer drug discovery, this result confirms the potential of S. argel<\/em> as an agent with chemotherapeutic and cytostatic effects against colorectal cancer cells.<\/p>\n\n\n\n

Conclusion <\/strong><\/p>\n\n\n\n

In conclusion, S. argel<\/em> leaf methanolic extract exhibited antitumor effect on colorectal cancer cells (HCT-116) and this effect was shown to be dose dependent. The extract also induced significant apoptosis on HCT-116 cells. Therefore, the leaf extract of Solenostemma argel<\/em> has a promising potential as preventive chemotherapy agent against colorectal cancer cells.<\/p>\n\n\n\n

Acknowledgment<\/strong><\/p>\n\n\n\n

The authors would like to express their gratitude to all staff members of the Laboratory of Cancer Research at the National Cancer Institute from Egypt for providing the infrastructural facilities for conducting the experiments. <\/p>\n\n\n\n

Conflict of Interests<\/strong> <\/p>\n\n\n\n

The authors declare no conflict of interest. <\/p>\n\n\n\n

Funding Source<\/strong> <\/p>\n\n\n\n

There is no funding sources<\/p>\n\n\n\n

Authors\u2019\nContribution<\/strong><\/p>\n\n\n\n

AHM, AHA, and KS performed the experimental work, analyzed the\nfindings, and wrote the manuscript. MS supervised the research. MR revised the\nmanuscript and edited the final version. <\/p>\n\n\n\n

Data availability<\/strong><\/p>\n\n\n\n

Data will be available upon request.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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