{"id":39358,"date":"2021-06-30T10:24:23","date_gmt":"2021-06-30T10:24:23","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=39358"},"modified":"2021-07-13T08:25:59","modified_gmt":"2021-07-13T08:25:59","slug":"in-vitro-assessment-of-cytotoxic-activity-of-bioactive-peptides-from-momordica-dioica-and-solanum-trilobatumagainst-human-colon-cancer-cells","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol14no2\/in-vitro-assessment-of-cytotoxic-activity-of-bioactive-peptides-from-momordica-dioica-and-solanum-trilobatumagainst-human-colon-cancer-cells\/","title":{"rendered":"In Vitro Assessment of Cytotoxic Activity of Bioactive Peptides from Momordica Dioica and Solanum Trilobatumagainst Human Colon Cancer Cells"},"content":{"rendered":"

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

Cancer is defined as an uncontrolled division of cells and the cell\u2019s ability to invade other tissues, causing vascularization, generation of the tumour mass and metastasis. [1,2]<\/sup> Chemotherapy is one of the significant treatments for cancer, that functions by administering agents that are cytotoxic to the cancer cells. The most important drawback with the conventional chemotherapy methods is the incapability of directly transporting the required quantity of the drug to the tumour cells without any damage to the healthy cells. Lately, the\u00a0balance of the structural rigidity along with the flexibility has made the small peptides as effective drugs for targeting cancer cells with high affinity and specificity[3]<\/sup>.Peptides represent a unique class of pharmaceutical compounds with distinctive therapeutic and biochemical characteristics. [4,5]<\/sup> Peptides have been used as potential drugs that are capable of inhibitingin tracellular molecules such as receptor tyrosine kinases. Peptides can be used directly as drugs (e.g., as angiogenesis inhibitors), as agents that target tumours carrying\u00a0radionuclides, and cytotoxic drugs (targeted radiation therapy and chemotherapy), vaccines, and hormones. [6]<\/sup><\/p>\n

Solanum trilobatum<\/em> is a predominant medicinal plant that is rich in iron, crude fibre, calcium, carbohydrates, minerals, fat, and phosphorus in the leaves. It is considered as a cure in the reduction of blood glucose level, asthma, several kinds of leprosy, and also in the arrest of blood vomiting. It also contains antifungal, antibacterial, antioxidant, and antimitotic properties. [7,8]<\/sup><\/p>\n

Momordica dioica <\/em>is a plant with high medicinal values belonging to the genus Momordica and family Cucurbitaceae<\/em>. M.dioica <\/em>possesses anti-hyperglycemic and renal protective activities. The fruits of this plant have diuretic, laxative, hepatoprotective, anti-venomous,\u00a0antihypertensive, anti-inflammatory, anti-asthmatic, antipyretic, antidiabetic, and antidepressant properties. [9,10]<\/sup> The aim of this study is to examine the cytotoxic properties of bioactive peptides from the seeds of Momordica dioica <\/em>and leaves of Solanum<\/em> trilobatum<\/em>.<\/em><\/p>\n

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

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

Roswell Park Memorial Institute medium1640 (RPMI-1640), 50X Penicillin and Streptomycin Antibiotic, and Fetal bovine serum (FBS) were acquired from Hi-Media. Ethidium Bromide, Acridine Orange, and DCFDA were purchased from Santa Cruz. Hoechst 33342 was acquired from Thermofisher Scientific. COLO 205 and COLO 320 DM cell lines\u00a0were procured from NCCS, Pune, India, and were maintained at a temperature of 37\u00b0C and 5% CO2 <\/sub>atmosphere. The cell lines were cultured in RPMI-1640 medium consisting of 1% antibiotic (penicillin and streptomycin cocktail) and 5% Fetal Bovine Serum.<\/p>\n

Preparation of Peptide extracts<\/strong><\/p>\n

The seeds of Momordica dioica <\/em>were washed with\u00a0distilled water, shade dried and powdered. About, 30\u00a0g of the seed powder was extracted with 300\u00a0mL of Sodium Phosphate buffer\u00a0and was kept in a rotary shaker at 100\u00a0rpm, overnight. The extract was filtered and subjected to 80% Ammonium Sulphate Precipitation method to precipitate the peptides from the buffer extract. Further, the extracted peptides were dialysed using a membrane with a cut-off MW\u00a010kDa to isolate peptides with a molecular weight less than 10KDa. [11]<\/sup>The leaves of Solanum trilobatum <\/em>were shade dried and powdered. 8g of the leaf powder was extracted with dichloromethane and methanol (1:1). The extract was filtered using a muslin cloth and\u00a0partitioned with an equal volume of dichloromethane and water. The organic layer was discarded and the aqueous layer of methanol – water was concentrated using a rotary evaporator. [12]<\/sup>The extracted peptides from both the plant sources were lyophilized and stored at -20 0<\/sup>C for further use.<\/p>\n

Identification of Peptides in the plant extracts<\/strong><\/p>\n

The lyophilized peptide samples of selected plant sources were fractionated by High-Performance Liquid Chromatography (HPLC) on a C18 column (4.6*250mm*5\u00b5m, 300\u02daA)with a linear gradient of 0-60% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 mL\/min. The absorbance was read at 280 nm for the peptides extracted from M. dioica<\/em> and S. trilobatum. <\/em>[13]<\/sup>The mass spectrum of the extracted peptides of M. dioica <\/em>and S. trilobatum<\/em> were obtained by MALDI-TOF analysis.<\/p>\n

Hoechst staining<\/strong><\/p>\n

The apoptosis of cancer cells depicts nuclear condensation which can be visualized by staining with Hoechst 33342 stain. The COLO 320 DM cells (10^5 cells\/mL) were cultured in a 6-well plate and treated with the peptides isolated from M.dioica<\/em>. The cells were stained with Hoechst 33342 dye for 15 mins at room temperature and were visualized using a fluorescence microscope. [14]<\/sup><\/p>\n

Acridine Orange\/Ethidium Bromide Staining<\/strong><\/p>\n

COLO 205 and COLO 320 DM cells were grown at the density of 1×106<\/sup> on the coverslips in 6-well plates and incubated for 48 h. COLO 205 colon cancer cells were treated with 175 \u03bcg\/mL of peptides extracted from S.trilobatum<\/em> for 24 h and 48h. COLO 320DM cells were treated with 150 \u03bcg\/mL of peptides extracted from M.dioica<\/em> for 24 h and 48 h. A fixative 4%\u00a0paraformaldehyde was used to fix the COLO 205 cells with an incubation of 30 min and permeabilized with 0.1% Triton X-100 for 15 min. The cells were stained with 2\u03bcg\/mL of AO\/EB for 10 min and the dead cells were washed with PBS. Cells were mounted and photographed using fluorescence microscopy. [15]<\/sup><\/p>\n

DCFDA staining<\/strong><\/p>\n

Oxidative stress was analysed by evaluation of ROS using DCFDA staining in COLO 205 colon cancer cells for 24 h and 48 h. COLO 205 colon cancer cells were treated with 175\u03bcg\/mL of peptides extracted from S. trilobatum <\/em>for 24 h and 48 h and were photographed in fluorescence microscopy. [16]\u00a0<\/sup><\/p>\n

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

Structural Analysis of Peptide Extracts<\/strong><\/p>\n

The peptides extracted from M. dioica<\/em>[Fig.1] and S. trilobatum<\/em>[Fig.2]were analyzed using a C-18 HPLC column (4.6*250mm*5\u00b5m, 300\u02daA) at the absorbance of 280 nm.HPLC analysis of the peptides extracted from Momordica dioi<\/em>ca<\/em>at 280nmdepicted the presence of a single peak at the retention time of 13.099 min. This is found to be in concordance with the results\u00a0obtained by the HPLC analysis of antioxidant peptides isolated from \u03b1-Lactalbumin and \u03b2-Lactoglobulin in Hern\u00e1ndez-Ledesma et al<\/em>. 2005. [17]<\/sup> The highest peak for the peptide extracts from Solanum trilobatum <\/em>was observed at the retention time of 33.230 min by eluting in the C-18 HPLC column at the absorbance of 280 nm.<\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig1\"<\/a><\/td>\nFigure 1: HPLC <\/strong>analysis<\/strong> of peptide<\/strong>sextracted <\/strong>from <\/strong>the seeds of <\/strong>Momordica dioica.<\/em><\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

 <\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig2\"<\/a><\/td>\nFigure 2: HPLC analysis of peptide<\/strong>s extracted<\/strong> from <\/strong>the <\/strong>leaves of Solanum trilobatum.<\/em><\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The molecular weight of the peptides extracted from M. dioica<\/em>[Fig.3] and S. trilobatum<\/em>were examined using MALDI-TOF analysis [Fig.4]. The crude sample of M.dioica<\/em> had the peptide with the highest molecular weight of 855.085Da and the peptides from S.trilobatum<\/em> has the highest molecular weight of 1446.911Da. The results are homologous to the results obtained in Hashempour et al<\/em>., 2013 by the characterization of cyclic peptides from Viola ignobilis<\/em>. [18]\u00a0<\/sup><\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig3\"<\/a><\/td>\nFigure <\/strong>3: MALDI-TOF Analysis of peptide<\/strong>s extracted <\/strong>from\u00a0<\/strong>the seeds<\/strong> of Momordica dioica.<\/em><\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig4\"<\/a><\/td>\nFigure 4: MALDI-TOF analysis of peptide<\/strong>s extracted <\/strong>from <\/strong>the <\/strong>leaves of Solanum trilobatum.<\/em><\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Bioactive compounds isolated from plants possess anti-microbial, anti-inflammatory, anti-tumour and anti-pyretic properties. [19]<\/sup> The present study focuses on the isolation, characterization and determination of the anti-cancer properties of the peptides extracted from the two selected plant sources, M. dioica <\/em>and S. trilobatum<\/em> against human colon cancer cells.<\/p>\n

Cell Viability Analysis of COLO 320DM and COLO 205 cells<\/strong><\/p>\n

The reduction in the number of viable cancer cells was observed using a phase contrast microscope [Fig.5 and Fig.6]after being treated with the peptides isolated from M.dioica<\/em> and S.trilobatum<\/em> for 48 hours, which indicates the potential anti-cancer activity of these peptides. \u00a0The COLO 320DM cells exhibited increased cytotoxicity of 81% when treated with the maximum concentration of 150 \u00b5g\/mL of the peptides extracted from M. dioica <\/em>after 48 h of incubation [Fig.7]. The COLO 205 cells treated with the maximum concentration of 175 \u00b5g\/mL of the peptides extracted from S. trilobatum <\/em>after 48 h showed increased cell cytotoxicity of 75% [Fig.8]. [20,21]<\/sup><\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig5\"<\/a><\/td>\nFigure 5: Phase contrast micrographs of COLO320 DM<\/strong>cells<\/strong>(10X) \u2013(a) Control (untreated)\u00a0\u00a0(b) Treated with Peptide<\/strong>s<\/strong> extract<\/strong>ed<\/strong> from M. dioica<\/em> (150 \u00b5g\/mL) \u2013 <\/strong>48<\/strong> hr.<\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

 <\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig6\"<\/a><\/td>\nFigure 6: Phase contrast micrographs of COLO205 <\/strong>cells<\/strong>(10X)(a) control (untreated) (b) Treated with peptide<\/strong>s <\/strong>extract<\/strong>ed<\/strong> from S. trilobatum<\/em> (175\u00b5g\/mL) \u2013 <\/strong>48<\/strong> hr.<\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

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 <\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig7\"<\/a><\/td>\nFigure 7: Determination of cell viability in colon cancer cells treated with peptides <\/strong>of M<\/strong>. dioica<\/em><\/strong> in an increasing concentration from 25\u00b5g\/mL to 150 \u00b5g\/mL<\/strong>using MTT assay<\/strong>. <\/strong>The values are expressed as mean\u00b1SEM and were statistically significant (**P<0.01), compared with control.<\/strong> (a) 24 hrs, (b) 48 hrs.<\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

 <\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig8\"<\/a><\/td>\nFigure 8: Determination of cell viability in colon cancer cells treated with peptides of S. trilobatum <\/em>in an increasing concentration from25\u00b5g\/mL to 175 \u00b5g\/mLusing MTT assay. The values are expressed as mean\u00b1SEM and were statistically significant (**P<0.01), compared with control.<\/strong> (a) 24 hrs, (b) 48hrs.<\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The anti-cancer properties of the novel peptides extracted from M. dioica <\/em>and S. trilobatum<\/em> were studied against human colon cancer cell lines, COLO 320DM and COLO 205 cells. Our findings demonstrate that the peptides extracted from M.dioica<\/em> have potent cytotoxic activity against COLO 320DM cells and the peptide extracts from S.trilobatum<\/em> exhibit cytotoxic activity against COLO 205 cells. Similar results were obtained in Li et al<\/em>., 2013 by treating the human colorectal cancer cell lines with the crude peptide extracts from Ipomoea batatas<\/em>. [22]<\/sup><\/p>\n

Examination of cell death and oxidative stress in human colon cancer cells<\/strong><\/p>\n

Nuclear changes are characteristic to apoptosis and is a major factor that distinguishes apoptosis from necrosis. Hoechst 33342 staining revealed nuclear changes such as nuclear fragmentation and chromatin condensation which are associated with apoptosis in COLO 320DM cells treated with peptides extracted from M. dioica <\/em>at the concentration of 150 \u00b5g\/mL as seen in [Fig.9]. [23,24]<\/sup>Characteristic features of apoptosis include nuclear changes\u00a0such as chromatin condensation and nuclear fragmentation. [25]<\/sup> This was studied in COLO 320DM indicating changes in the morphology of the nucleus of the cells treated with peptides isolated from M. dioica<\/em>, by Hoechst 33342 staining method that are characteristic to apoptosis such as fragmented and condensed apoptotic bodies. Similar results were observed in a study of the mechanism of apoptotic induction by gecko peptide mixtures in treated human hepatocellular carcinoma cells. [26]<\/sup><\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig9\"<\/a><\/td>\nFigure 9: Hoechst staining for COLO 320 DM cancer cells:<\/strong>(a) Control (untreated) (b) Treated with Peptides extracted from M. dioica <\/em>(150\u00b5g\/mL) \u2013 48 hr.<\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Further, Acridine Orange \/ Ethidium Bromide staining was performed to study the morphological changes that lead to cell death in COLO 320DM cells treated with peptides extracted from M.dioica<\/em> at the maximum concentration of 150 \u00b5g\/mL and COLO 205 cells treated with peptides extracted from S.trilobatum <\/em>at the maximum concentration of 175\u00a0\u00b5g\/mL [Fig.10 and Fig.11]. [27]<\/sup> Further Acridine Orange \/ Ethidium Bromide fluorescent staining technique was performed on COLO 320DM cells treated with peptides isolated from M. dioica<\/em> and COLO 205 cells treated with peptides from S. trilobatum <\/em>to study the changes that occur in cellular morphology during apoptosis. [28] <\/sup>The untreated control cells appeared green in colour while the cells treated with the peptides extracted from M. dioica <\/em>and S\u00a0trilobatum appeared orange and red in colour. The cells treated with the maximum concentration of 150 \u00b5g\/mL peptides extracted from M.dioica <\/em>and S.trilobatum <\/em>, showed shrunken nuclei and fragmented apoptotic bodies as opposed to the untreated control cells with large and normal nucleus. A comparative study was performed by Umayaparvathi et al<\/em>., 2014, studying the anticancer effect and antioxidant activity of bioactive peptides extracted\u00a0<\/em>from the enzyme hydrolysates of Saccostrea cucullata<\/em> and by Ebrahim etal<\/em>., 2014, evaluating the anti-cancer activity of cobra venom polypeptide Cytotoxin-II against MCF-7 cell lines. [29,30]<\/sup>This study supports the apoptotic property of the extracted novel peptides against human colon cancer cell lines. These findings clearly suggest the anti-tumour properties of the peptide extracts from M.dioica <\/em>and S.trilobatum. <\/em>The novel isolated peptides are proved to be potential drug for the treatment of human colon cancer.<\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig10\"<\/a><\/td>\nFigure 10: AO\/EtBr staining for COLO 320 DM cancer cells:<\/strong>(a) Control (untreated) (b) Treated with Peptides extracted from M. dioica <\/em>(150\u00b5g\/mL) \u2013 48 hr.<\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

 <\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig11\"<\/a><\/td>\nFigure 11: AO\/EtBr staining for COLO 205 cancer cells:<\/strong>(a) Control (untreated) (b) Treated with Peptides extracted from S. trilobatum <\/em>(175\u00b5g\/mL) \u2013 48 hr.<\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

The oxidative stress in COLO 205 cells treated with peptide extracts from S. trilobatum<\/em> at the concentration of 150 \u00b5g\/mL and the changes in the morphology of the nucleus was studied using the 2\u2019,7\u2019-dichlorofluorescein diacetate (DCFDA), an oxidation sensitive dye. [31,32]<\/sup> Fluorescent microscope images of the cells treated with the peptides isolated from S. trilobatum<\/em> showed an increase in ROS levels when compared to the untreated control cells [Fig.12]. An analogous work was carried out by treating the human hepatocarcinoma cells with peptides isolated from maize with the generation of reactive oxygen species. [33]<\/sup><\/p>\n\n\n\n
\"Vol14No2_Vit_Rup_fig12\"<\/a><\/td>\nFigure 12: DCFDA staining for COLO 205 cancer cells:<\/strong>(a) Control (untreated) (b) Treated with Peptides extracted from S. trilobatum <\/em>(175\u00b5g\/mL) \u2013 48 hr.<\/strong><\/p>\n

Click here to view figure\u00a0<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

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

The peptides obtained from M.dioica<\/em>and S.trilobatum\u00a0<\/em>were identified with a C-18 HPLC column. Similar work was performed by Pritchard et al<\/em>., 2010, where the bioactive peptides in Cheddar cheese were extracted and identified using a C-18 RP-HPLC column[34]. Similar work was carried out by Neves et <\/em>al., 2017, in which the enzyme hydrolysates of Salmo salar\u00a0<\/em>were analysed in a RP-HPLC column[35]. MALDI-TOF analysis was performed to obtain the molecular mass of the peptide extracts from M.dioica<\/em>and S.trilobatum\u00a0<\/em>which were concordant with the results of Ahmed et al<\/em>., 2015, in which the molecular mass of the antioxidant peptides from goat milk were also studied in a similar manner[36].<\/p>\n

In this study the cytotoxic properties of the peptides extracted from M.dioica<\/em>and S.trilobatum<\/em> were evaluated. In Li et al<\/em>., 2013, human colorectal cancer cell lines were treated with the crude peptide extracts ofIpomoea batatas<\/em>. The cell proliferation was observed to decrease \u00a0by 49%with increase in the peptide dosage at 40 \u03bcmol\/L[37]. Another study by Kumar et al<\/em>., 2019, examined the in vitro <\/em>anticancer properties of collagen peptides isolated from Aluterusmonoceros<\/em>[38]. Hoechst 33342and Acridine Orange\/ Ethidium Bromide (AO\/EtBr) are fluorescent staining techniques that binds to the DNA of the cells and thus aids in\u00a0studying the morphological changes that occur in the nucleus. In the present study, the COLO 320DM cells treated with peptide extracts from M.dioica<\/em> were stained with Hoechst 33342 stain and modifications in the nuclear morphology such as nuclear fragmentation, characteristic to apoptotic cells were observed. These results are homologous with the studies of Jinet al<\/em>., 2016, in which human hepatocellular carcinoma cells (HepG2) were treated with different concentrations of gecko peptide mixture which depicted chromosomal condensation in HepG2 cells[39].<\/p>\n

AO\/EtBr staining technique was further used to study the cellular changes that are distinctive to apoptotic cell death in Colo 320DM cells treated with peptide extracts fromM.dioica<\/em>and the Colo 205 cells treated with peptide extracts from S.trilobatum<\/em>. This staining technique can be used to visualize the morphological changes that occur in the cell membrane of apoptotic cells. It can also be used to effectively determine the stages of apoptosis in the treated cells[40]. Similar results were obtained with the studies of Taniya et al<\/em>., 2020 where amaranth seed protein hydrolysates were observed to induce apoptosis in breast cancer cell lines[41].<\/p>\n

2′,7′ \u2013dichlorofluorescin diacetate (DCFDA)\u00a0is another fluorescent staining method that helps to identify the levels of reactive oxygen species (ROS) in live cells. High concentration of ROS is often indicative of apoptosis in cells that is caspase dependent and involves mitochondrial disintegration. The Colo 205 cells treated with peptides extracted from S.trilobatum<\/em> were observed to exhibit increasing levels of ROS in a dose dependent manner. Similar results were also obtained by Brodsk\u00e1et al<\/em>., 2011,where the leukaemia cell line(CML-T1) was treated with various anti-cancer drugs[42].<\/p>\n

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

The peptides extracted from Momordica dioica <\/em>and Solanum trilobatum<\/em> were found to inhibit cell proliferation and cause cell death in COLO 320DM cells and COLO 205 cells respectively. Cell death was examined by fluorescent staining techniques such as Hoescht, DAPI and AO\/EtBr. The results indicated chromosomal condensation and nuclear fragmentation in the treated human colon cancer cell lines which is characteristic of\u00a0apoptosispathway. ROS generation was detected in COLO 205 cells treated with peptides extracted from Solanum trilobatum <\/em>by DCFDA staining technique, which indicates the onset of apoptosis of the COLO 205 cells through the production of ROS.<\/p>\n

The results obtained from this study suggest that the peptides extracted from Momordica dioica<\/em> and Solanum trilobatum<\/em> can be used as potential novel drugs against human colon cancer.<\/p>\n

Acknowledgement<\/strong><\/p>\n

We would like to express our deepest gratitude to the Department of Biotechnology, School of Bioengineering, Faculty of Engineering & Technology, SRM Institute of Science and Technology, Kattankulathur 603203, Tamilnadu, India for helping with the laboratory facilities and also thank SRM-DBT for providing the facility to use the fluorescent microscopy.<\/p>\n

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

Authors declare that there is no conflict of interest.<\/p>\n

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

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