Effects of yellow and red bell pepper (paprika) extracts on pathogenic microorganisms, cancerous cells and inhibition of survivin

Abstract

The present work examined the biomedical value of red and yellow bell pepper extracts (YME and RME) in terms of antioxidant, antibacterial and anticancer activities by in vitro and virtual studies. The yield of extract was 3.49% for RME and 2.92% for YME. The level of total phenols and total flavonoids significantly varied between the type of extracts, and it was higher in RME than that in YME. The extracts showed promising DPPH and ABTS free radical scavenging rates. The extracts showed an excellent antibacterial activity. The minimal inhibitory concentration (MIC) of RME was 0.20 mg mL⁻¹ for Bacillus cereus, 0.30 mg mL⁻¹ for Escherichia coli, 0.50 mg mL⁻¹ for Staphylococcus aureus and 0.60 mg mL⁻¹ and for Pseudomonas aeruginosa, while the MIC of YME was 0.40 mg mL⁻¹ for B. cereus, 0.40 mg mL⁻¹ for E. coli, 0.50 mg mL⁻¹ for S. aureus, and 0.60 mg mL⁻¹ for P. aeruginosa. TEM results demonstrated the cellular damage induced by RME in B. cereus biofilm. The RME did not show any cytotoxicity in normal NIH3T3 cells, but at 125 μg mL⁻¹ did a strong cytotoxicity in human lung cancer cell line A549 as evident by cytotoxicity assay, ROS and AO/EB staining. The virtual biological examination indicated that β-carotene from RME was a potential compound with higher docking energy against both targeted enzymes and proteins as − 14.30 for LpxC and − 15.59 for survivin. Therefore, it is recommended that RME is a better functional food with novel biomedical properties and it deserves further evaluation for its the novel molecules against multidrug resistant pathogens.

Electronic supplementary material

The online version of this article (10.1007/s13197-020-04663-4) contains supplementary material, which is available to authorized users.

Introduction

Although, the advanced medical research enabled the discovery of novel drugs pertaining the emergence of disease incidences, there are several diseases (Gülçin 2012; Bursal and Gülçin 2011) such as pediatric obesity, fatty liver, diabetes, and arteriosclerosis that are yet to be properly diagnosed or treated. Incidence of the chronic diseases are increasing because of the accumulation of high-level free radicals and fat in the human body by over-consumption of meat and ignoring vegetables. In general, the removal of the excess free radicals from the human body can improve the immune function against dangerous diseases. In fact, the vegetable foods contain the antioxidant molecule and different classes of phytochemicals that remove the free radicals in the body (Bacon et al. 2017; Prasanna et al. 2014; Gülçin 2005; Bae et al. 2016). Among the vegetables the bell pepper (Capsicum annuum L. var. grossum (L.) Sendt) is considered to be a real source for phytochemicals with antioxidant properties, in addition to flavor, colorant and taste of the food. Moreover, the consumption of 0.5 g of peppers in daily life can improve the immunity because it contains phenols, flavonoids, and capsaicinoids (Delgado-Vargas and Paredes-Lopez, 2003; Fowles et al. 2001; Krzyzanowska et al. 2010; Loizzo et al. 2015; Pugliese et al. 2013). These compounds have the promising antioxidants properties that prevent the cardiovascular disease, cancer, and tumor. Reactive oxygen species (ROS) are involved in the pathogenesis of many diseases (Gülçin 2010; Gülçin 2006). Consumption of peppers as dietary supplements can lead to reduction of oxidative damage in biological systems (Lobo et al. 2010). The C. annuum L. var. grossum (L.) Sendt is a cultivar of the species Capsicum annuum L. and it is also known as a bell pepper it is widely consumed in Korea and Japan. Particularly one of phytomolecules named capsaicinoids isolated from the peppers is known to be antioxidant, anti-inflammatary, anticancer, cardioprotective, analgesic and antibacterial in activities, and it contributes to 80–90% of the total pungency of most chili peppers (Gurnani et al. 2016; Imran et al. 2017; Lu et al. 2017; Rollyson et al. 2014; Zewdie and Bosland, 2001).

The bacterial pathogens can be eradicated through DNA damage and cellular membrane damage etc. The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylases (LpxC)) is present in outer cell membrane of the Gram negative bacteria and the enzyme is involved in the increased bacterial resistance towards the antibacterial metabolites and antibiotics (Rubab et al. 2018; Saravanakumar et al. 2019a; Tomaras et al. 2014). Moreover, the inhibition of the anti-proapoptosis protein namely survivin can result in the activation of the apoptosis in cancer cells (Saravanakumar et al. 2019b). The present study investigated the antioxidant, antibacterial and anticancer activity of the metabolites extracted from yellow and red bell pepper, and identified bioactive substances from the extracts using the gas chromatography mass spectrophotometry (GCMS) and high-performance liquid chromatography (HPLC). The work also elucidated the molecular mechanism underlying antibacterial and anticancer activities of the yellow and red bell pepper by computational molecular targeting of LpxC and survivin.

Material and method

Materials

Water-soluble tetrazolium (WST)-1 assay kit (EZ-Cytox, Daeil Lab Service, Republic of Korea), phosphate-buffered saline (PBS), Roswell Park Memorial Institute medium (RPMI-1640), Dulbecco’s modification of Eagle medium (DMEM), Fetal Bovine Serum (FBS), Penicillin, and Streptomycin (PS) were purchased from Gibco, Thermo Fisher Scientific, Seoul, Republic of Korea. All the other chemicals used in antioxidant and chemical compounds analysis were of analytical grade obtained from Sigma Aldrich, Republic of Korea. Bacterial strains such as S. aureus (ATCC13150), B. cereus (KNIH28), S. enterica (ATCC14028), P. aeruginosa (ATCC27853), and E. coli (ATCC27853) were obtained from the Korea Culture Center of Microorganisms, Seoul, and Republic of Korea and preserved in 20% of glycerol stock at − 20 °C. For the experimental purpose the strains were recultured in Mueller–Hinton Broth (MHB) at 37 °C for 24 h. The cell lines such as human lung carcinoma A549 (KCLB-10185) and the Swiss albino mouse embryo tissue (NIH3T3) were obtained from Korean Cell Line Bank (KCLB, Seoul, Republic of Korea). The A549 cell line was cultured in RPMI-1640 and the NIH3T3 cell line was cultured in DMEM, both of the media were incorporated with 10% FBS and 1% PS and incubated in 5% CO₂ humidified incubator at 37 °C for 24 h to achieve 70–80% confluent and then the cells were preserved in Cell Freezing Media (DMSO, BCS) in liquid nitrogen in a cryogenic vial for further experimental use.

Preparation of extracts

Bell pepper was purchased during December 2018 from an agricultural cooperative association in Chuncheon, Republic of Korea. Approximately 700 g of bell pepper cultivar—yellow and red colored were ground separately. The powdered samples were extracted with 500 mL of methanol (HM) at 25 °C for 24 h and this process was repeated twice. All the extracts were pooled together for yellow and red peppers separately and the extracts were filtered two times using the Whatman no.1 filter paper. The extract filtrate was named as yellow bell pepper methanol extracts (YME), red bell pepper methanol extracts (RME) and then concentrated using the rotary evaporator. The concentrated extracts were lyophilized for 72 h in the freeze dryer. For the chromatography assay, the RME was dissolved in the methanol and subjected to metabolites analysis using the nano high resolution LC/MSMS spectrometer (Q Exactive Thermo Scientific, USA), followed by the data were analyzed by proteome software.

Determination of total phenolics

Total phenolic content (TPC) was determined by using the Folin-reagent method (Agbor et al. 2014). The gallic acid standard was prepared at different concentrations at 0, 0.5, 1, 1.5, 2, 2.5, 3 mL in a 25 mL brown volumetric flask and made up to volume to 6 mL using double distilled water, then mixed well with addition of 0.5 mL of folin reagent. Further, 1.5 mL of 20% Na₂CO₃ solution was added, mixed well and incubated at 30 °C for 30 min. On the other hand, for the analysis of samples, 0.5 mL of YME or RME solution was added in 25 mL volumetric flask containing 9.5 mL of distilled water and then mixed well with 0.5 mL of folin reagent. Finally 1.5 mL of 20% Na₂CO₃ solution was added, mixed well and incubated at 30 °C for 30 min. The absorbance of reaction mixtures was measured at 760 nm using UV- spectrophotometer. The TPC in the sample was calculated from the regression equation derived from the standard curve.

Determination of total flavonoids

Total flavonoid content (TFC) in the sample was determined according to the aluminum chloride (AlCl₃) colorimetric assay (Agbor et al. 2014). A standard solution of rutin was prepared by dissolving 0.01 g of rutin in absolute ethanol in 100 mL volumetric flask to prepare the final concentration of 0.1 mg mL⁻¹. Then different concentrations 0, 1, 2, 3, 4, 5, 6 ml of rutin in 50 mL volumetric flask were added with 1.0 mL of AlCl₃ solution and 1.0 mL of KCOOH solution and mixed well. Samples were prepared by dissolving 0.7 g of sample in 60 mL of 60% ethanol, connected with vertical condensation return tube, and reflux for 60 min in a water bath at 70 °C. The filtrate was collected and diluted to 100 mL. For the determination, 4 mL of the sample solution was placed in a 50 mL volumetric flask, and various solutions were added again in the above order. After incubating for 1 h, it was filtered through a filter membrane (0.22 μm), and the filtrate was measured at 510 nm using spectrophotometer. Total flavonoids in the sample was calculated according to the regression equation obtained from the standard curve of rutin.

Antioxidant activity

The 100 μL of different concentrations (0.1–50 mg mL⁻¹) of the samples (YME & RME) were dissolved in 100 μL of 0.01 mM methanolic solution of DPPH in 96 well plate and incubation at 25 °C for 30 min in dark environment, then the absorbance was measured at 515 nm using the UV- spectrophotometer (ELx800, Biotec). Meanwhile, the absorbance of blank sample was measured as the control. DPPH scavenging (%) = 100-(A (sample-A (blank)/A (control) × 100). Where A (sample) is the absorbance of the sample; A (blank) is the absorbance of the sample without DPPH solution; A (control) is the absorbance of DPPH solution in methanol (Deuschle et al. 2015).

The ABTS scavenging was measured adopting the method reported earlier (Re et al. 1999) with some modifications. The ABTS⁺ standard was prepared by dissolving 0.2 mL of 7.4 mM ABTS in 2.6 Mm K₂S₂O₈ in phosphate buffer solution (pH 7.4) followed by incubating for 12 h in a dark environment at room temperature. The 0.8 mL ABTS⁺ solution and 0.2 mL of different concentrations of samples were added to the quartz cuvette, shaken for 10 s, and after incubating for 6 min, the absorbance was measured at 734 nm by UV-spectrophotometer. The sample with different concentrations in the above operation can be replaced with a 95% ethanol solution, and the absorbance value A₀ is measured for blank control. ABTS scavenging (%) was calculated using the formula (1 − A₁/A₀) × 100.

A₀—the absorbance measured by the blank group;

A₁—The absorbance measured by the experimental group.

Antibacterial activity

Antibacterial activity of the YME and RME was determined by disk diffusion method (Gülçin et al. 2004; Gulcin et al. 2008; Gülçin et al. 2010). The pathogens were cultured by inoculating 100 μL (10⁵ CFU mL⁻¹) bacterial suspensions in the 5 ml of sterile liquid muller-Hinton broth (MHB) at 25 °C and incubated for 12 h at 37 °C. Meanwhile muller-Hinton agar (MHA) plates were prepared following the standard protocols. Then, 50 μL (10⁷ CFU mL⁻¹) were spread on MHA and 6 mm sample disk was placed on the plates to access the inhibitory activity. After 24 h incubation at 37 °C, the zone inhibition was measured using the ruler. Bell pepper induced cell wall damage was observed using the FETEM according to the protocols described elsewhere (Saravanakumar et al. 2018a).

Cytotoxicity

The cytotoxicity was determined by WST kit assay. The A459 cells were collected using Trypsin-EDTA by adding an appropriate amount of culture medium (RPMI) and then. 100 μL of cell solution was dispensed in each well of 96-well plate. The plate was incubated in a CO₂ incubator for 24 h (at 37 °C, 5% CO₂). After incubation, 10 μL of sample solution at different concentrations was added to various wells and incubated further for 24 h. Adding 10 μL of EZ-Cytox to each well and kept in an incubator for 1 h. After shaking gently for 1 min, the absorbance was measured at 450 nm using a plate reader. Similar handling was made for NIH3T3 cells. The A549 cells were stained by AO/EB and DCFH-DA and then observed under a fluorescence microscope (Olympus, CKX53 culture microscope, Japan) for the production of reactive oxygen species (ROS) (Saravanakumar et al. 2019a).

Virtual studies

The computer programming based modeling strategies was utilized to study the molecular interactions between the metabolites derived from the bell pepper extracts (YME & RME) against bacterial cell outer membrane enzyme protein (LpxC; PDB ID: 3ULY) (Rubab et al. 2018; Saravanakumar et al. 2019a; Tomaras et al. 2014) and anti-pro-apoptosis protein survivin (PDB ID: 1F3H) (Shakeel et al. 2017), retrieved from the protein data bank (https://www.rcsb.org/). The structure of bell pepper compounds was prepared using the canonical SMILES (https://www.ncbi.nlm.nih.gov/pccompound) and the ACD/ChemSketch. The pre-treatments of ligands, receptor including energy minimization and removal of the water molecules were done according to the methods described earlier (Friesner et al. 2006; Negi et al. 2016; Prasanna et al. 2014; Shakeel et al. 2017). The molecular docking scores (Kcal mol⁻¹) were determined using the computer program Argus Lab 4.0.1 (Mark Thompson and Planaria Software LLC). These interactions and distance were also observed using the BIOVIA Discovery Studio 2016 (Accelrys Software Inc., San Diego, CA, USA).

Results and discussion

Analysis of total phenolics and flavonoids in extracts

Bell peppers are rich sources for polyphenols, flavonoids, carotenoids, capsaicin and dihydrocapsaicin (Tundis et al. 2013). The yield of extract was 3.49% obtained from RME and 2.92% from YME. Total phenolic contents (TPC) and total flavonoids contents (TFC) in the RME and YME are shown in Fig. 1a, b. The TPC and TFC increased with increasing concentration of YME and RME. The level of TPC and TFC was higher in the RME than that in YME. This is in agreement with previous work which has recorded higher levels of TPC, beta-carotene, capsanthin, quercetin, and luteolin in red bell pepper than those in yellow bell pepper (Sun et al. 2007). In addition, the red cultivar is well known to be more matured cultivar than yellow cultivar, leading to increased level of the functional molecules such as capsaicin, phenol and ascorbic acid (i.e., red bell pepper) (Barbero et al. 2014; Ghasemnezhad et al. 2011). The higher level of the phytomolecules possibly triggered the bioactivities particularly antioxidant activity.

Fig. 1.

Antioxidant activity of extracts RME and YME derived from Capsicum annuum L. var. grossum (L.) Sendt. DPPH radical scavenging rate (a), ABTS radical scavenging rate (b), total phenols content (c); total flavonoids content (d)

Antioxidant activity

As shown in Fig. 1c, d, the DPPH and ABTS free radical scavenging rates of two cultivars are directly proportional to the sample concentration. Since the scavenging rate of the two samples was not significantly different at concentrations of 5 mg mL⁻¹ and 50 mg mL⁻¹. The samples of both cultivar (YME &RME) reached the maximum scavenging rate at 5 mg mL⁻¹. The reason for this result is that the bell pepper is rich in polyphenols, flavonoids, carotenoids, capsaicin, and dihydrocapsaicin, which exhibit strong antioxidant effects (Tundis et al. 2013). When the two varieties were compared, from the result of DPPH free radical scavenging rates (Fig. 1a), the IC₅₀ concentration of RME was 0.411 mg mL⁻¹, and YME was 0.742 mg mL⁻¹. From the result of ABTS free radical scavenging rates (Fig. 1b), the IC₅₀ concentration of RME was 0.343 mg mL⁻¹ and that of YEM was 0.618 mg mL⁻¹. The free radical scavenging rate was more in RME than that in YME. This is in accordance with earlier reports that ascorbic acid content is higher in red bell pepper than other colored of bell pepper, which is directly related to its strong antioxidant activity (Zhang and Hamauzu 2002). Although, the bell pepper is rich in the antioxidant molecules, food processing such as cooking and freezing can affect the quality therefore consuming the bell pepper as raw is recommended in the human diet (Loizzo et al. 2013, 2015).

Antibacterial activity

Antibacterial activity of bell pepper extracts (RME and YME) was tested against Gram (+) bacterial pathogens such as S. aureus, B. cereus, and Gram (−) bacterial pathogens P. aeruginosa, E. coli. The inhibitory ability of extracts was measured in terms of zone of inhibition and the extracts showed the zone of the inhibition in a dose dependent manner (Fig. 2a–d). Both of the extracts inhibited the tested pathogens at concentration of 1 mg mL⁻¹ (Fig. 2e). The minimal inhibitory concentration (MIC) of RME was 0.20 mg mL⁻¹ for B. cereus, 0.30 mg mL⁻¹ for E. coli, 0.50 mg mL⁻¹ for S. aureus and 0.60 mg mL⁻¹ for P. aeruginosa. Whereas the MIC of YME was 0.40 mg mL⁻¹ for B. cereus, 0.40 mg mL⁻¹ for E. coli, 0.50 mg mL⁻¹ for S. aureus, and 0.60 mg mL⁻¹ for P. aeruginosa (Table 1). The B. cereus was chosen to study the RME induced cell damage by using TEM. The results indicated apparent cellular damage in B. cereus biofilm compared to untreated cells (Fig. 2a, b). RME exhibited the higher antibacterial effects than YME did due to richness of bioactive phytomolecules. This results are in accordance with an earlier report (Koffi-Nevry et al. 2012). Moreover, the extract of bell pepper is reportedly extending the preservation time of mincedbeef meat coated with the extract through inhibiting different E. coli O157: H7(944, E0019, F4564, and Cider), Staphylococcus aureus and Pseudomonas aeruginosa (Aljaloud et al. 2012). The antibacterial activity has also been reported with silver nanoparticles, synthesized by bell pepper extract (Kachhwaha 2014).

Fig. 2.

Antibacterial activity of different concentrations of red cultivar (RME) and yellow cultivar (YEM) against different pathogenic bacteria, Bacillus cereus (a), Escherichia coli (b), Staphylococcus aureus (c): Pseudomonas aeruginosa (d), RME and YME in the inhibition zone of four different pathogens (e). Observation of RME induced cellular dame in Bacillus cereus by TEM (a, untreated control group; b, treated with RME)

Table 1.

Determination of the minimal inhibitory concentration (mg mL⁻¹) of RME and YEM extracts against selected bacterial pathogens

	Gram-positive bacteria		Gram-negative bacteria
	Bacillus cereus	Staphylococcus aureus	Escherichia coli	Pseudomonas aeruginosa
RME	0.20 ± 0.04	0.50 ± 0.02	0.30 ± 0.02	0.60 ± 0.03
YME	0.40 ± 0.02	0.50 ± 0.03	0.40 ± 0.04	0.60 ± 0.02

Anticancer activity

In this study, the WST kit assay was used to detect the cytotoxicity of RME and YME on A549 and NIH3T3 and the results are shown in the Fig. 3a. Both the extracts did not show any cytotoxicity in NIH3T3 cells, whereas the extracts at 125 μg mL⁻¹ exhibited a strong cytotoxicity in A549 cells (Fig. 3b). It is known that pepper extracts induce apoptosis in human cancer cells because it contains a capsaicin that inactivates NADH oxidase on the cell membrane of cancer cells, thereby inducing apoptosis in cancer cells (Macho et al. 1999; Morré et al. 1995; Sun et al. 1992; Wolvetang et al. 1996). Many functional components of capsaicinoids are present in the bell pepper (Yazawa et al. 1989). Therefore, we believe that bell pepper extract has an anticancer activity to A549 cells. The IC₅₀ concentration of the RME was 185.82 μg mL⁻¹ and YME was 517.61 μg mL⁻¹. The cell viability was found to be lower in RME than that in YME. This indicates that the anti-cancer activity of the red cultivar is more effective than the yellow one because of the presence of more total phenolic substances in red cultivar than that in yellow cultivar (Park et al. 2012). In addition Pectin polysaccharide from bell pepper is reported to effectively inhibit the growth of breast cancer cells (Adami et al. 2018). Anticancer activity of the extracts is closely related to polyphenols (Jeong et al. 2011).

Fig. 3.

Cytotoxicity of the RME and YME in NIH3T3 cells (a), A549 cells (b), measurement of ROS generation in A549 cells treated with RME and YME (c, d). AO/EB staining of A549 cells treated with RME and YME (E). IC₂₅, IC₅₀ and IC₇₅ concentration of the RME was ≤ 125 μg mL⁻¹, 185.82 μg mL⁻¹, 603.75 μg mL⁻¹respectively, whereas YME was 166.75 μg mL⁻¹, 517.61 μg mL⁻¹, ≥ 1000 μg mL⁻¹ respectively

Level of reactive oxygen species (ROS) is more prone to oxidative stress. Therefore, it is proposed that any substance that stimulates oxidative stress in cancer cells, promotes ROS production or inhibits the ROS clearance system, which is higher than the original level of ROS in cancer cells, leading to irreversible oxidative damage and cell death (Trachootham et al. 2009). In our experiment, the level of ROS was determined by dichloro-dihydro-fluorescein diacetate (DCFH-DA) method. The DCFH-DA reagent itself does not have fluorescence and can pass through the cell membrane freely. After DCFH-DA enters the cell, it is hydrolyzed into DCFH by the esterase in the cell, while DCFH cannot pass through the cell membrane. If there is enough ROS in the cell, it is capable of oxidizing non-fluorescent DCFH to DCF, which is capable of producing fluorescence. According to the micrograph of Fig. 3c, d, some fluorescent greens are faintly visible in the control group, which is also in line with the theoretical basis that the ROS level in cancer cells is higher than that of normal cells. In the experimental group, the number of fluorescent green cells was proportional to the concentration of the two extracts. This indicates that the extracts of both cultivators induce apoptosis of cancer cells by stimulating oxidative stress in cancer cells. The two experimental groups were compared with each other. At the same concentration, the number of fluorescent green cells in the RME was always higher than that in the YME. This indicates that the anti-cancer activity of RME is stronger than that of YME.

Figure 3E shows the results of staining A549 cells in the control group and the experimental group with the Acridine orange (AO)/Ethidium bromide (EB) stain. AO is a staining agent that can be inserted into the DNA of a cell, which has a complete cell membrane, thereby rendering the cell fluorescent green under the microscope. EB can only pass through the damaged cell membrane, thereby entering the cell, embedding the DNA, and making the cell appearing fluorescent red under the microscope. Therefore, it can be observed in the figure that in the control group, the cells are all fluorescent green under the microscope, and each cell has a normal structure, so the cells used in the experiment were living cells. In the experimental group, the fluorescent red cells increased with the concentration of the extract, and the cells were condensed and beaded, so these cells were late apoptotic cells. This was observed in the experimental group of red and yellow bell pepper methanol extracts, indicating that both extracts induced apoptosis by destroying cancer cell lines. However, the red fluorescent cells in the RME were significantly more than the YME, and it revealed that the anti-cancer effect of RME was better than that of YME.

Virtual biological studies

The phytomolecules derived from the RME extracts (Figure S1 and Table 2) were determined by nano_high resolution LC/MSMS spectrometer and their molecular interactions with bacterial cell outer membrane enzyme protein (LpxC) (Rubab et al. 2018; Saravanakumar et al. 2019a; Tomaras et al. 2014) and anti-pro-apoptosis protein, survivin (Shakeel et al. 2017). The results indicated all the molecules derived from RME showed a remarkable inhibitory activity in terms of the docking score (Table 2) also several compounds from the bell pepper and their biological activity are depicted in the Table 3. However, among the molecules tested, β-carotene exhibited higher docking energy towards both targeted enzymes/proteins as − 14.30 for LpxC and − 15.59 for survivin (Table 2, Fig. 4). The β-carotene showed strong inhibitory activity of LpxC through its interactions with hydrophobic side chain LEU141, LEU 102, LEU6, LEU237, TRP10, PHE101, TRP145, and ALA144, electrically charged side chain ASP105, ASP240, LYS103, and ARG106, polar neutral side chain THR5, unique amino acids PRO7, PRO142, and PRO143 (Fig. 4a, b). In case of the survivin, β-carotene showed the interactions with electronically charged side chain LYS23, GLU348, LYS307, GLU22, GLU393, LYS328, hydrophobic side chain TYR391, Val346, ILE309, ALA97, VAL16, VAL346, Polar neutral side chain SER339, ASN308, THR341, THR14, unique amino acids GLY21, PRO329, GLY15 (Fig. 4c, d). This evidenced the remarkable inhibitory ability of the β-carotene. Similarly antioxidant activity of the β-carotene from the bell pepper is known from previous works (Sun et al. 2007; Zhang and Hamauzu 2002).

Table 2.

Analysis of the volatile and nonvolatile compounds from the extracts of RME Capsicum annuum L by nano high resolution LC/MSMS spectrometer and their inhibitory effect on the targeted bacterial outer membrane enzyme (LpxC) and anti-proapoptotic protein survivin

No.	Name of the compound	Retention time (min)	Mol. Wt/(g·mol−1)	Docking energy (Kcal/mol)
				3ULY	1F3H
1	β-carotene	37.62	536.888	− 14.30	− 15.59
2	Capsanthin	43.71	584.885	− 14.22	− 10.56
3	Violaxanthin	45.64	600.884	− 13.48	− 10.51
4	Lutein	17.33	568.886	− 14.01	− 9.82
7	Luteolin	16.06	286.239	− 7.45	− 7.39
8	Quercetin	17.66	302.238	− 6.80	− 7.21
9	Ascorbic acid	1.25	176.124	− 5.18	− 4.85
11	Galic acid	49.19	424.452	− 9.54	− 7.81
12	Catechin	17.18	290.271	− 7.64	− 7.14
13	Capsaicin	17.46	305.418	− 9.46	− 14.59
14	Dihydrocapsaicin	34.11	307.434	ND	− 14.53
17	Apigenin	51.47	270.24	− 7.96	ND
26	Alkaloids	1.74	348.402	− 8.15	− 7.05
27	Steroids	28.14	530.614	ND	− 7.46
38	Orientin	13.77	448.38	− 7.22	ND
41	Isoscoparin	33.32	462.407	− 7.23	− 5.01

ND not detected. Bacterial cell outer membrane enzyme protein (LpxC; PDB ID: 3ULY), anti-pro-apoptosis protein survivin (PDB ID: 1F3H)

Table 3.

The compound reported from the bell pepper the extracts of Capsicum annuum L and their known biological functions

No.	Name of the compound	Mol. Wt/(g·mol−1)	Bioactivity	Ref.
1	β-carotene	536.888	Antioxidant	Sun et al. (2007), Zhang and Hamauzu (2002)
2	Capsanthin	584.885	Antioxidant	Sun et al. (2007), Zhang and Hamauzu (2002)
3	Violaxanthin	600.884	Antioxidant	(Zhang and Hamauzu (2002)
4	Lutein	568.886	Antioxidant	Zhang and Hamauzu (2002)
5	Chlorophyll a	893.509	Antioxidant	Zhang and Hamauzu (2002)
6	Chlorophyll b	907.492	Antioxidant	Zhang and Hamauzu (2002)
7	Luteolin	286.239	Antioxidant	Lee et al. (1995), Sun et al. (2007) Zhang and Hamauzu (2002)
8	Quercetin	302.238	Antioxidant	Ghasemnezhad et al. (2011), Lee et al. (1995), Sun et al. (2007)
9	Ascorbic acid	176.124	Antioxidant	Ghasemnezhad et al. (2011), Lee et al. (1995)
10	Flavonoids		Antioxidant & hypoglycaemic & Antibacterial	Koffi-Nevry et al. (2012), Lee et al. (1995), Loizzo et al. (2013, 2015),Tundis et al. (2013)
11	Galic acid	424.452	Antioxidant	Ghasemnezhad et al. (2011)
12	Catechin	290.271	Antioxidant	Ghasemnezhad et al. (2011)
13	Capsaicin	305.418	Antioxidant & hypoglycaemic & Antiproliferative	Loizzo et al. (2015), Park et al. (2012), Tundis et al. (2013)
14	Dihydrocapsaicin	307.434	Antioxidant & hypoglycaemic	Loizzo et al. (2015), Tundis et al. (2013)
15	Phenols		Antioxidant & hypoglycaemic & Antibacterial	Koffi-Nevry et al. (2012), Loizzo et al. (2013,2015), Tundis et al. (2013)
16	Carotenoids		Antioxidant & hypoglycaemic	Loizzo et al. (2013, 2015), Tundis et al. (2013)
17	Apigenin	270.24	Antioxidant & hypoglycaemic	Loizzo et al. (2013)
18	Trans-p-Feruloyl-β-D-glucoside		Antioxidant	Materska (2014)
19	Trans-p-Sinapoyl-β-D-glucoside		Antioxidant	Materska (2014)
20	Quercetin 3-O-α-L-rhamnoside-7-O-β-D-glucoside		Antioxidant	Materska (2014)
21	Luteolin 6-C-β-D-glucoside-8-C-α-L-arabinoside		Antioxidant	Materska (2014)
22	Apigenin 6-C-β-D-glucoside-8-C-α-L-arabinoside		Antioxidant	Materska (2014)
23	Lutoeolin 7-O-[2-(β-D-apiosyl)—β-D-glucoside]		Antioxidant	Materska (2014)
24	Lutoeolin 7-O-[2-(β-D-apiosyl)-4-(β-D-glucosyl)-6-malonyl]- β-D-glucoside		Antioxidant	Materska (2014)
25	Quercetin 3-O-α-L-rhamnoside		Antioxidant	Materska (2014)
26	Alkaloids	348.402	Antibacterial	Koffi-Nevry et al. (2012)
27	Steroids	530.614	Antibacterial	Koffi-Nevry et al. (2012)
28	Pectic polysaccharides		Antineoplastic	Adami et al. (2018)
29	Feruloyl hexoside		Antioxidant & Anticancer	Jeong et al. (2011)
30	Sinapoyl hexoside
31	Luteolin 6,8-di-C-hexoside
32	Quercetin O-rhamnosyl-O-hexoside
33	Luteolin C-pentosyl-C-hexoside
34	Vicenin-2	594.522
35	Apigenin C-pentosyl-C-hexoside
36	Luteolin 8-C-hexoside
37	Luteolin 6-C-hexoside
38	Orientin	448.38
39	Kaempferol pentosyldihexoside
40	Luteolin O-(apiosyl)hexoside
41	Isoscoparin	462.407
42	Quercetin 3-O-hexoside
43	Luteolin O-(apiosylacetyl)glucoside
44	Luteolin malonypentosyldihexoside
45	Quercetin 3-O-rhamnoside

Fig. 4.

Investivation of the moelcuar intraction between the compounds from the RME aganinst tragted enzyme (LpxC) or protein (survivin). The 2D and 3D visualization of interaction between the highly potential molecule β-carotene from the RME against LpxC (a,b), survivin (c,d) respectively

Conclusion

This work reports the phytochemical composition, antioxidant, antibacterial and anticancer properties of the extracts derived from the two types of bell pepper (RME and YME). According to the results, although both types of extracts exhibited antioxidant activity, higher activity was exhibited in RME. The RME and YME significantly inhibited the S. aureus, B. cereus, P. aeruginosa and E. coli as shown by in vitro and computational modeling studies. In terms of anticancer activity, both of the extracts showed a strong inhibitory effect on human lung cancer cell A549, but not cytotoxic to normal cell NIH3T3. Antibacterial and anticancer potential of the extracts derived compounds was revealed by molecular docking study. Therefore, these results evidenced that RME is better functional food supplement to improve healthy human diet.

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Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.