Therapeutic peptides of Mucuna pruriens L.: Anti‐genotoxic molecules against human hepatocellular carcinoma and hepatitis C virus

Abstract To assist the development of new therapeutic strategies for several disorders, biologically active peptides/proteins obtained from plant sources can be considered. Current study expected to determine the biological activities of peptide fractions of Mucuna pruriens against hepatocellular carcinoma cell lines (HepG2/ADM, HepG2, SMMC‐7721, and QGY‐7703), as well as normal cell line to prove their selectivity. Moreover, anti‐genotoxicity and antiviral activity against the hepatitis C virus (HCV) were assessed. The methods of this study were to isolate the peptides of M. pruriens and hydrolysate fractionation via fractionated pepsin‐pancreatin hydrolysates by ultrafiltration/high‐performance ultrafiltration cell, identify anti‐hepatoma activity of peptide fractions human liver cancer and normal cells by (3‐(4,5‐dimethylthiazole‐2‐yl)‐2,5‐biphenyl tetrazolium bromide) (MTT) assay, determine anti‐HCV, and assess anti‐genotoxic effect of peptide fractions against damage that induced via alkylating agent methyl methanesulphonate in human mononuclear cells. The results showed that the fraction 5–10 kDa has been reported to exhibit significant cytotoxic activity against HepG2 and QGY‐7703. It was proven that both of 5–10 and >10 kDa fractions are active against HCV. The cytotoxic concentration (CC50) of 5–10 kDa against the cell line was 703.04 ± 5.21 µg/ml. Anti‐genotoxic activities of the peptide fractions were evaluated as mean values for the analyzed comet images. In this regard, the highest activity of protecting DNA damages was observed by the peptide fraction of 5–10 kDa. This study revealed the potential ability of peptide fractions of M. pruriens for the treatment of liver cancer, HCV, and high activities of protecting DNA damages.

145) (Taghizadeh, Rezaee, Mehmandoust, Badibostan, et al., 2019;Taghizadeh, Rezaee, Mehmandoust, Madarshahi, et al., 2019). The antiviral trait of Beetin 27 (BE27) from Beta vulgaris L. leaves was demonstrated. This defense protein has been attributed to its RNA and DNA polynucleotides. BE27 exhibited superoxide dismutase activity and produced the signal compound hydrogen peroxide (Iglesias et al., 2015). Furthermore, lipopeptide based on viral fusion inhibitors such as cholesterylated peptide HIV-1/2 fusion inhibitors with effective and long-lasting antiviral activity can also be used as novel treatment for clinical development (Zhu et al., 2019). Different herbal compounds with biologically activities are of limited therapeutic use due to their carcinogenic, toxicological, and mutagenic properties. Therefore, the genotoxic analysis seems to be necessary for their potential use as a new therapeutic agent. Bio anti-mutagens can inactive the mutagenic mechanisms as well as the effect on DNA damage repair over decrease in mutation rate (Goswami et al., 2019). Méndez-Espinoza et al. reported that the anti-genotoxic activity against direct mutagens was observed in Cantharellus cibarius (Mendez-Espinoza et al., 2013).
Mucuna pruriens belongs to Fabaceae family is used worldwide in complementary medicine. This legume found in India and China.
It has immuno-modulatory potential with significant improvement in immunoglobulin levels. It is also found that several amino acids as well as proteins including globulins and albumins can be associated with therapeutic properties (Rai et al., 2017;Ulu et al., 2018).
Researches have shown the several biological traits of M. pruriens (Yadav et al., 2015), but there is no report in the literature on the beneficial effects of peptides isolates from M. pruriens. In the current study, we tried to provide the practical tools to promote therapeutic peptides usages with a particular focus on liver cancer and HCV.
Due to the fact that we emphasized on peptide fractions derived from M. pruriens to examine whether they can provide the prediction performance of cytotoxicity and antiviral activity. Current study expected to (a) determine the biological activities of peptide frac-

| Plant material
Mucuna pruriens beans were deposited at the herbarium of Mashhad University of Medical Sciences, Mashhad Iran, and were cultured in the research greenhouse of Ferdowsi University of Mashhad, Iran (latitude 36°16′N, longitude 59°36′E, and 985 m altitude). The beans were washed two times and dried at room temperature (25°C). They were packed in glass containers, kept at −20°C, and protected from light before the analysis.

| Chemicals
The analytical grade reagents were supplied by Merck. Pepsin

| Sample preparation
Mucuna pruriens beans were ground with an electric mill (Toos Shekan, Khorasan, Iran). In order to remove the smallest particles, they filtered through a 2-mm mesh size sieve. A fluidizing air bed was used to separate the hull beans and then the flour obtained was easily milled.
In order to isolate the fiber solids from the protein and the starchcontaining liquid portion, the extract was passed through a 0.177mm mesh size sieve. The remaining solids were washed three times using 300 ml of 3% sodium bisulfite. The digested sample was re-

| Hydrolysis of the protein isolates
Enzymatic hydrolysis was performed in a 1,000-ml reaction vessel equipped with a stirrer. The treatments were as follows: pepsin (Sigma, P7000-100G) and pancreatin (Sigma, P3292-100G). In order to prepare a protein solution, the protein isolates were suspended in distilled water (10% w/v). Then, pepsin-pancreatin system was added to the solution for 90 min (each of them was incubated for 45 min).
The hydrolysis parameters were substrate concentration 4%; enzyme/ substrate ratio 1:10. The pH was adjusted to 7.5 and 2.0, for pancreatin and pepsin, respectively, using 1 mol/L sodium hydroxide. The temperature was held at 37°C for analyses. The enzymatic hydrolysis was stopped by increasing the temperature to 80°C for 20 min. In order to eliminate the insoluble portion, the digested sample was centrifuged at 1,317 g for 20 min (Martínez-Leo et al., 2019).

| Degree of hydrolysis (DH) percent
Degree of hydrolysis (%) was measured via the following Equation

| Hydrolysate fractionation
Hydrolysate fractionation was done via a little modified method that was described by Cho et al. Ultrafiltration/high-performance ultrafiltration cell (Millipore Inc.) was used for pepsin-pancreatin hydrolysates fractionation. Several molecular weights cut off membranes including 1, 3, 5, and 10 kDa were used for preparing the five fractions. The hydrolysate process was started with 10 kDa as the largest cartridge and ended with 1 kDa. Therefore, five peptide fractions were as follows: <1, 1-3, 3-5, 5-10, and >10 kDa. All the fractions were freeze-dried and kept at −20°C until biological analysis (Chalé et al., 2014).

| Cell viability
The cells viability was tested by MTT test. The cells (1 × 10 4 /well) were seeded. Each well contains 100 µl DMEM medium and 10% FBS. After 24 hr, serial dilutions of the peptide fractions and cisplatin (positive control) were added. 100 μl of MTT (5 mg/ml in DMEM) was added for 4 hr. After this time, the medium with MTT was removed. Since, to dissolve the formazan crystals, 150 μl DMSO was added. The absorbance was determined by a spectrophotometric microtiter plate reader at 590 nm (Taghizadeh, Rezaee, Mehmandoust, Badibostan, et al., 2019;Taghizadeh, Rezaee, Mehmandoust, Madarshahi, et al., 2019). The minimum inhibition concentration for inhibiting 50% of the viable cells (IC 50 ) was calculated via the relative viability over the peptide fractions concentration curve (Shakeri et al., 2017).

| Antiviral assay
The HCV was propagated in HEp-2 cells. HEp-2 cells were preserved in the condition described above. The virus titer was performed via 50% Tissue Culture Infectious Dose (TCID 50 ) on HEp-2 cells, and then they were kept at −80°C. The antiviral effect of peptide fractions was evaluated due to cell death in HEp-2 and inhibitory activity on virus-induced cytopathogenicity. The HEp-2 cells were seeded at 10 4 cells/well with 5% CO 2 (at 37°C). The culture media was

| Experimental design
The

| Statistical analysis
These experiments used as a random block design in triplicates. The data were presented as mean ± standard deviation (SD). Variance analysis was carried out by SPSS software version 16.0, followed by posthoc Tukey test was performed to the comparison way. Mean values were calculated significantly different at p ˂ .05.

| Cytotoxic effects of peptide fractions
The IC 50 values of peptide fractions against different cancerous cell lines were shown in Table 1. Among five peptide fractions, 5-10 kDa exhibited significant growth inhibitory effects against human HCC. (2) It should also be noted that the fraction 5-10 kDa has been reported to exhibit significant cytotoxic activity against HepG2 and QGY-

| Antiviral effects of peptide fractions
The antiviral effects of different peptide fractions were evaluated against HCV (Table 2). IC 50 value calculated for 5-10 kDa was significantly lower than those of peptide fractions against cell lines. The SIs of peptide fractions for all HCV were >3. The CC 50 of 5-10 kDa against the cell line was 703.04 ± 5.21 µg/ml (Table 3).

| Genotoxic effects of peptide fractions
The antigenotoxic effects of the peptide fractions showed that a peptide fraction of 5-10 kDa exhibited the highest activity of protecting DNA damages. In comparison the means by Dunnett's test, with consider to the positive control, statistically significant differences can be observed among all peptide fractions with respect to tail length (p ≤ .05) ( Table 4).

| D ISCUSS I ON
Our results showed that the hydrolysis process of M. pruriens protein reduced the protein content of hydrolysates. It may be related to removing the insoluble portion of the hydrolysates. It was found that the fraction >10 kDa showed the highest levels of protein due to its molecular weight cut-off. Differences among protein content of plants may be due to the different fundamental peptide, plant species, and different phase of plant maturity, growing conditions, raw material compositions, as well as the kind of the method used to obtain the protein concentrate (Yam et al., 2015). The values obtained in the present study are promising for obtaining bioactive compounds. Based on our results, we found that these peptide frac-  proteins. This phenomenon is not only of concern to the cell biologist but also has implications with regard to scheduling of anticancer agents against human tumors. However, the available data suggest that certain therapeutic peptides may affect liver cancer and HCV, which creates the potential to alter the pharmacokinetics of co-administered drugs. In addition, the change in exposure to peptide substrate drugs can be limited. Therefore, there is a need to better understand the clinical relevance of such interactions during drug development, to guide the safe and effective use of therapeutic peptide. These observations should be emphasized for future research on M. pruriens to evaluate functional roles in potential therapeutic applications of these proteins.

| CON CLUS ION
However, as yet, the evidence available on bioactive peptides has several limitations, further studies on animals and randomized clinical trials are required to confirm these effects and enable these peptides to be used as preventive or therapeutic treatments.

CO N FLI C T O F I NTE R E S T
We declare no conflicts of interest.

E TH I C A L A PPROVA L
Ethical approval for this study was obtained from Mashhad University of Medical Sciences (IR.MUMS.REC.1395.389).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request. The concentration of the sample required for 50% inhibition. c Selectivity index.

TA B L E 4
The tail length (Mean ± SD) and DNA damage reduction