In vitro Assessment of Cytotoxic Activity of Bioactive Peptides from Momordica dioica and Solanum trilobatum against Human Colon Cancer Cells

Plant peptides have gained attention in the medicinal field due to their high antimicrobial and anti-cancer properties. Various plant sources are being used to extract proteins and peptides to be used as a cure for a variety of diseases. The latest studies show that plant peptides are effective in the treatment of cancer due to their ability to preferentially bind to the receptors or membranes of cancer cells leading to tumour growth inhibition, cytotoxicity, decreased proliferation, and apoptosis. The peptides get internalized into the cells causing cancer cell agglutination and aggregation. In addition to acting as therapeutic agents, plant peptides are also used for the targeting of drugs specific to cancer cells. In this study, bioactive peptides were isolated from the seeds of Momordica dioica and leaves of Solanum trilobatum. They were screened and identified using HPLC and MALDI-TOF techniques. In this study, apoptosis was analyzed by the Hoechst 33342 staining method which detected the presence of condensed pycnotic nuclei in apoptotic COLO320DM and COLO205 colon cancer cells at the maximum concentrations of 150 and 175 μg/mL of plant peptides. Further DCFH-DA staining indicated the intracellular ROS production in the treated COLO320DM and COLO205 colon cancer cells. Thus, the isolated bioactive plant peptides can be formulated towards the development of effective anticancer drugs for the treatment of colon cancer in humans.

Cancer is defined as an uncontrolled division of cells and the cell's ability to invade other tissues, causing vascularization, generation of the tumour mass and metastasis 1,2 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 balance 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 . Peptides represent a unique class of pharmaceutical compounds with distinctive therapeutic and biochemical characteristics 4,5 Peptides have been used as potential drugs that are capable of inhibiting intracellular molecules such as receptor tyrosine kinases. Peptides can be used directly as drugs (e.g., as angiogenesis inhibitors), as agents that target tumours carrying radionuclides, and cytotoxic drugs (targeted radiation therapy and chemotherapy), vaccines, and hormones 6 Solanum trilobatum 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 Momordica dioica is a plant with high medicinal values belonging to the genus Momordica and family Cucurbitaceae. M.dioica possesses anti-hyperglycemic and renal protective activities. The fruits of this plant have diuretic, laxative, hepatoprotective, anti-venomous, antihypertensive, anti-inflammatory, anti-asthmatic, antipyretic, antidiabetic, and antidepressant properties 9,10 The aim of this study is to examine the cytotoxic properties of bioactive peptides from the seeds of Momordica dioica and leaves of Solanum trilobatum.

Plant material & reagents
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 were procured from NCCS, Pune, India, and were maintained at a temperature of 37°C and 5% CO 2 atmosphere. The cell lines were cultured in RPMI-1640 medium consisting of 1% antibiotic (penicillin and streptomycin cocktail) and 5% Fetal Bovine Serum.

Preparation of Peptide extracts
The seeds of Momordica dioica were washed with distilled water, shade dried and powdered. About, 30 g of the seed powder was extracted with 300 mL of Sodium Phosphate buffer and was kept in a rotary shaker at 100 rpm, 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 10kDa to isolate peptides with a molecular weight less than 10KDa 11 The leaves of Solanum trilobatum 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 partitioned 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 The extracted peptides from both the plant sources were lyophilized and stored at -20 0 C for further use.

Identification of Peptides in the plant extracts
The lyophilized peptide samples of selected plant sources were fractionated by High-Performance Liquid Chromatography (HPLC) on a C18 column (4.6*250mm*5µm, 300ÚA) 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 and S. trilobatum 13 The mass spectrum of the extracted peptides of M. dioica and S. trilobatum were obtained by MALDI-TOF analysis.

Hoechst staining
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. The cells were stained with Hoechst 33342 dye for 15 mins at room temperature and were visualized using a fluorescence microscope 14 Acridine Orange/Ethidium Bromide Staining COLO 205 and COLO 320 DM cells were grown at the density of 1x10 6 18 Bioactive compounds isolated from plants possess anti-microbial, anti-inflammatory, antitumour and anti-pyretic properties 19 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 and S. trilobatum against human colon cancer cells.

Cell Viability Analysis of COLO 320DM and COLO 205 cells
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 and S.trilobatum for 48 hours, which indicates the potential anti-cancer activity of these peptides. The COLO 320DM cells exhibited increased cytotoxicity of 81% when treated with the maximum concentration of 150 µg/mL of the peptides extracted from M. dioica after 48 h of incubation [ Fig.7]. The COLO 205 cells treated with the maximum concentration of 175 µg/mL of the peptides extracted from S. trilobatum after 48 h showed increased cell cytotoxicity of 75% [ Fig.8] 20,21 The anti-cancer properties of the novel peptides extracted from M. dioica and S. trilobatum were studied against human colon cancer cell lines,

Examination of cell death and oxidative stress in human colon cancer cells
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 at the concentration of 150 µg/mL as seen in [ Fig.9] 23,24 Characteristic features of apoptosis include nuclear changes such as chromatin condensation and nuclear fragmentation 25 This was studied in COLO 320DM indicating changes in the morphology of the nucleus of the cells treated with peptides isolated from M. dioica, 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 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 at the maximum concentration of 150 µg/mL and COLO 205 cells treated with peptides extracted from S.trilobatum at the maximum concentration of 175 µg/mL [ Fig.10 and Fig.11] 27 Further Acridine Orange / Ethidium     activity of cobra venom polypeptide Cytotoxin-II against MCF-7 cell lines 29,30 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 and S.trilobatum. The novel isolated peptides are proved to be potential drug for the treatment of human colon cancer. The oxidative stress in COLO 205 cells treated with peptide extracts from S. trilobatum at the concentration of 175 µg/mL and the changes in the morphology of the nucleus was studied using the 2',7'-dichlorofluorescein diacetate (DCFDA), an oxidation sensitive dye 31,32 Fluorescent microscope images of the cells treated with the peptides isolated from S. trilobatum 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

disCussion
The peptides obtained from M.dioica and S.trilobatum were identified with a C-18 HPLC column. Similar work was performed by Pritchard et al., 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 al., 2017, in which the enzyme hydrolysates of Salmo salar 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 and S.trilobatum which were concordant with the results of Ahmed et al., 2015, in which the molecular mass of the antioxidant peptides from goat milk were also studied in a similar manner 36 .
In this study the cytotoxic properties of the peptides extracted from M.dioicaand S.trilobatum were evaluated. In , human colorectal cancer cell lines were treated with the crude peptide extracts of Ipomoea batatas. The cell proliferation was observed to decrease by 49% with increase in the peptide dosage at 40 µmol/L 37 . Another study by Kumar et al., 2019, examined the in vitro anticancer properties of collagen peptides isolated from Aluterus monoceros 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 studying the morphological changes that occur in the nucleus. In the present study, the COLO 320DM cells treated with peptide extracts from M.dioica 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 Jin et al., 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 .
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 from M.dioica and the Colo 205 cells treated with peptide extracts from S.trilobatum. 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., 2020 where amaranth seed protein hydrolysates were observed to induce apoptosis in breast cancer cell lines 41 .
2',7' -dichlorofluorescin diacetate (DCFDA) is 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 were observed to exhibit increasing levels of ROS in a dose dependent manner. Similar results were also obtained by Brodská et al., 2011,where the leukaemia cell line(CML-T1) was treated with various anti-cancer drugs 42 .

ConClusion
The peptides extracted from Momordica dioica and Solanum trilobatum 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 apoptosis pathway. ROS generation was detected in COLO 205 cells treated with peptides extracted from Solanum trilobatum by DCFDA staining technique, which indicates the onset of apoptosis of the COLO 205 cells through the production of ROS.
The results obtained from this study suggest that the peptides extracted from Momordica dioica and Solanum trilobatum can be used as potential novel drugs against human colon cancer.

aCknowledgeMents
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.

Conflict of Interests
Authors declare that there is no conflict of interest.