Antioxidant, anti-inflammatory, antimicrobial and anticancer activities of the pods of green broad bean (Vicia faba L.), in vitro

DOI: https://doi.org/10.21203/rs.3.rs-2591126/v1

Abstract

This study aimed to benefit from the pods’ byproduct of the broad bean (the outer shell). The chemical composition of the green pods was estimated. The methanolic extract of the broad bean pods (BBPs) activity was investigated in vitro as an antioxidant, anti-inflammatory, antimicrobial and anticancer against prostate cancer (Pc3) and liver cancer (HepG2) cells. The findings revealed that BBPs have high contents of carbohydrates, fiber, protein, potassium, calcium, and magnesium. The total phenols and flavonoids of BBPs extract were 286 mg GAE / g and 105 mg QE/g, respectively. The antioxidant activity of the BBPs methanolic extract was measured by 1, 1- diphenyl-2-picryl hydrazyl (DPPH) assay. The highest value of DPPH scavenging activity (80.5%) was achieved by the extract concentrations of 1000 ug/ml. The concentrations of 500 and 250 ug/ml of BBPs extract also showed high levels of DPPH scavenging activity (73.7 and 65.7%), respectively. The methanolic extract of BBPs has an anti-inflammatory effect as it significantly reduces RBCs hemolysis. The maximum inhibition percentage reached 66.7% with a concentration of 1000 ug/ml. Regarding the antimicrobial activity, it was noticed that BBPs extract prevented the growth of B. Subtilis, Staph. aureus and E. coli in addition to the two fungi, Pseudomonas aeruginosa and Candida albicans. The effect of the methanolic extract of BBPs against human hepatocarcinoma (HepG2) and prostate cancer (PC3) cells showed significant decreases in their cell viability in a concentration-dependent manner. It also caused significant changes in cell shape compared to the control.

1. Introduction

Due to the disadvantages of drugs and their high cost, people usually seek cheap sources through natural plant products as a source of nutrients and phytochemicals to fight diseases. Legumes are well known as an important source of protein, carbohydrates, and fibre and are low in fat [1, 2]. They are also inserted into human food for their nutritional benefits, especially their content of phenolic compounds and oligosaccharides [3], enzyme inhibitors, phytosterols and saponins [4]. In addition, it was investigated that legumes intake lower the risk of cancer [5], cardiovascular diseases [6], hypertension and diabetes [7].

Most people consume fresh vegetables known as the broad bean (Vicia faba L.). It belongs to the Fabaceae (Leguminosae) family and the Vicia gene cluster [8]. The fruit is also known as fava beans, broad beans, Windsor beans, horse beans, tick beans, and other names. The name of V. faba in Hindi, the language of India, is "kalamatar and bakala" [9]. It has four subspecies: paucijuga, equaine, major, and minor [10].

It is commonly used in many regions of the world, including Egypt, India, the Netherlands, Spain, Sudan, Saudi Arabia, and China. The seed coat has various hues: white, buff (or beige), purple, green, and red. However, buff-colored beans are the most accepted color for human consumption.

One of the micro components receiving more attention for its health-improving qualities is phenolic compounds, particularly for its antioxidant activity. Numerous phenolic chemicals, including procyanidins, catechins, flavanols, isoflavones, phenolic acids, and tannins—all of which act as natural antioxidants—have been identified in faba beans [11, 12, 13, 14]. According to reports, phenolic chemicals produced from plant materials have the power to impede the digestion of lipids and carbohydrates, hence inhibiting their absorption. These may aid in the reduction of postprandial hyperglycemia in diabetic patients and aid in the weight loss of obese individuals [15].

The Vicia faba peels have antioxidant and anticancer activities due to their content of flavonoids and phenolic acids [16]. The acetone extract of the seed coat revealed antioxidant, antibacterial, anti-inflammatory, and anticancer properties [17]. In a recent study, Mejri [18] reported that the methanolic extract of broad bean pods decreased the high levels of ALT, AST, creatinine, and uric acid in the serum of diabetic rats. Additionally, the pod's methanol extract was found to reduce oxidative stress by activating antioxidant enzymes like catalase, glutathione peroxidase, and superoxide dismutase [18, 19]. It was mentioned that the faba bean exhibited good effects in reducing blood sugar and total cholesterol [20, 21] and preventing heart diseases [22], eye diseases [23], cancer diseases [24, 16] and dysfunction of kidney and liver [25, 23].

Egypt is one of the most consumers of broad bean seeds, known as fool, considered the main dish in Egyptian breakfast, either in its stewed form (Fool Medames) or frying form (Falafel). The pods of the broad bean are usually separated as wastes, although in the early ripening stage, many Egyptian villagers cook the whole green broad bean pod with its outer wall, called a pod.

However, we aimed in our work to study the effect of the methanolic extract of the pods isolated from the broad bean as an antioxidant, antimicrobial, anti-inflammatory and anticancer in vitro.

2. Materials And Methods

2.1. Materials:

2.1.1. Broad bean (Vicia faba L): The premature broad bean pods were purchased freshly in their green form from the local market in Mansoura city.

2.1.2. Chemicals: All chemicals were purchased from Al-Gomhoria Company for medicines and medical supplies in Mansoura City, El- Dakahlia Governorate, Egypt.

Permission to conduct the experiment was obtained from the Scientific Research Ethics Committee of the Faculty of Specific Education, Mansoura University.

2.2. Methods:

2.2.1. Preparation of the pods' powder of broad bean:

Broad beans were cleaned and thoroughly washed in water. Afterwards, the seeds were separated, and the green pods (the fruit wal1l) were oven-dried at 40°C until constant weight, ground to a fine powder and stored at − 20°C.

2.2.2. Preparation of the methanolic extract of broad bean pods:

250 g of pod powder was soaked in 1L methanol, mixed well, then left overnight, and filtered through filter paper. The filtrate was kept in a dark bottle. Another portion of methanol was added to the residue, shaken well, left overnight, and then filtered, and the filtrate was added to the previous filtrate. Finally, the residue was resoaked in methanol overnight and filtered. The three filtrates were collected to make the methanolic extract solution. The solvent was removed by means of evaporation using a rotary evaporator. The obtained extract was collected and dried in a desiccator to a constant weight, then kept in the dark bottles until use.

2.2.3. Chemical analysis:

Ash, fat, fibre, protein, and moisture percentages contents were carried out according to the methods of AOAC [26]. Carbohydrates were calculated by difference. Using optimal experimental conditions, inductively coupled plasma-atomic emission spectrometry (ICP-AES; Horiba Jobin-Yvon Ultima 2 CE) was used to determine the mineral composition [27].

2.2.4. Phytochemical screening of the pods’ extract:

The pods’ extract was subjected to phytochemical tests for qualitative identification of glycosides, phenolics, tannins, alkaloids, flavonoids and saponins by the methods described by Trease and Evans [28] and Harborne [29].

2.2.5. Determination of total phenolics and total flavonoids content:

The colorimetric Folin-Ciocalteu method of Singleton and Rossi [30] was used to determine the total phenolic content (TPC). The results were expressed as mg gallic acid equivalents per g of extract using gallic acid as the standard (mg GAE / g). Using the Dehpour et al. [31] method, the total flavonoid content (TFC) was calculated and represented as mg quercetin equivalents per g extract (mg QE/g).

2.2.6. Evaluation of the antioxidant activity of pods' extract:

2.2.6.1. DPPH radical scavenging method:

Using 1, 1-diphenyl-2-picryl hydrazyl, the methanolic extract of broad bean pods was tested for its capacity to scavenge free radicals (DPPH). In a nutshell, a DPPH solution in methanol at 0.1 mM concentration was made. Three millilitres of the extract in methanol were mixed with one millilitre of this solution at various concentrations (3.9, 7.8, 15.62, 31.25, 62.5, 125, 250, 500, 1000 g/ml). The mixture was briskly shaken before being left to stand at room temperature for 30 minutes. A UV-visible spectrophotometer was then used to detect absorbance at 517 nm [32]. The Log dosage inhibition curve was used to determine the sample's IC50 value, or the sample concentration needed to block 50% of the DPPH free radical. The higher free radical activity was shown by the reaction mixture's decreased absorbance [33]. The percentage of the DPPH scavenging effect was calculated by using the following equation:

DPPH scavenging effect (%) or Percent inhibition = A0 – A1/A0 × 100,

where A0 was the absorbance of the control reaction and A1 was the absorbance of the extracted samples.

2.2.6.2. Reducing power method:

The antioxidant capacity of the sample extract was evaluated using the reducing power, according to Debnath et al. [34] as follows; one mL of the tested sample solution was combined with 2.5 mL of sodium phosphate buffer (0.2mM, 6.6pH) and 2.5 mL of 1 percent K3[Fe (CN)6], then incubated for 20 minutes at 50 degrees Celsius. To halt the reaction, aliquots of 10% CCl3 COOH (2.5mL) were added. 2.5 mL of reaction mixture were added to 2.5 ml distilled water, along with 1 ml of freshly made 0.1% FeCl3 solution, and the reaction was completed after 10 minutes at room temperature. At 700 nm, the absorbance was measured. Higher absorbance indicates that the reducing power is higher.

2.2.7. In vitro Anti-inflammatory assay:

2.2.7.1. Preparation of erythrocyte suspension: Healthy volunteers provided about 3 ml of whole blood, which was collected into heparinized tubes and centrifuged at 3000 rpm for 10 min. The red blood pellets were dissolved in a volume of normal saline equal to the supernatant. Red blood pellets that had been dissolved were measured in volume and reconstituted in an isotonic buffer solution (10 mM sodium phosphate buffer, pH 7.4) as a 40% v/v suspension. Red blood cells that had been reconstituted (resuspended supernatant) were utilized in this way.

2.2.7.2. Hypotonicity-prompted hemolysis: In hypotonic solution (distilled water), the extracts from the pods were dissolved at concentrations of 100, 200, 400, 600, 800, and 1000 ug/ml in centrifuge tubes. Isotonic solutions (5 ml) were prepared in centrifuge tubes with 100–1000 ug/ml extract concentrations. In addition, the vehicle control tube contained 5 ml of distilled water. Each tube received 0.1 ml of erythrocyte suspension, which was lightly mixed. The tubes were centrifuged for three minutes at 1300 g after incubating for one hour at 37°C. The supernatant's haemoglobin content was determined by measuring the absorbance (OD) at 540 nm.

The inhibition percentage of hemolysis by the pods’ extract was calculated as follows:

% Inhibition of hemolysis = 1 – ((OD2 – OD1)/ (OD3 – OD1)) * 100,

where OD1 = absorbance of the extracted sample in the isotonic solution

OD2 = absorbance of the extracted sample in the hypotonic solution

OD3 = absorbance of control sample in the hypotonic solution.

2.2.8. Antimicrobial activity of broad bean pods methanolic extract

Agar well diffusion method

Agar well diffusion was performed to assess the antibacterial activity of the broad bean pod extract. The entire surface of the agar was covered with a volume of microbial inoculum. The extract solution from the pods was then diluted to the necessary concentration, and a well with a diameter of 6 to 8 mm was aseptically drilled using a sterile drill. The agar plates were then incubated in the correct environment for each microbe. The widths of the acquired inhibition zone surrounding the wells (in mm) were measured after incubation durations of 16 to 24 hours (Mucoraceae), 24 hours (A. fumigatus, A. flavus, and A. niger), or 48 hours (other species). The studied microbial strain cannot develop in the agar media due to the extract's potent antimicrobial ingredient, which diffuses into the medium [35].

2.2.9. Effect of the methanolic extract of broad bean pods against Pc3 and HepG2 cells:

Cell viability and proliferation assay (MTT)

The MTT test was used to assess the cytotoxic activity of the methanolic extract of broad bean pods against HepG2 and PC3 cells. A 96-well culture plate was used to conduct the MTT experiment following a previously described technique [36].

A full monolayer sheet formed after 24 hours of incubation at 37°C with 1x105 cells/ml (100 ul/well) in the 96-well tissue culture plate. After the confluent sheet of cells had formed, the growth medium was decanted from 96-well microtiter plates, and the cell monolayer was twice washed with wash media. Dilutions of the extract twofold were created in RPMI medium with 2% serum (maintenance medium). Three wells served as controls and received only maintenance medium after receiving 0.1 ml of each dilution for testing in various wells. The plate was tested after being incubated at 37° C. Cells were evaluated for any indications of toxicity, such as shrinkage, granulation, or a partial or total loss of the monolayer. After adding 20 µl of MTT solution (5 mg/ml) to each well, each well's media was mixed with the MTT using a shaking table at 150 rpm for 5 minutes. The MTT was then allowed to be metabolized during a subsequent 1–5-hour incubation period at 37°C and 5% CO2, after which the medium was discarded (dry plate on paper towels to remove residue if necessary). The metabolic byproduct of MTT, formazan, was then resuspended in 200 ul DMSO and agitated at 150 rpm for five minutes to combine the formazan with the solvent thoroughly. The optical density at 560 nm and subtract background at 620 nm were read. The number of cells and optical density were directly connected.

2.3. Statistical analysis:

The collected data were presented as means with standard deviations. All tests were completed using the computer program of statistical analysis program (SPSS, version 24), according to McCormick and Salcedo [37].

3. Results And Discussion

3.1. Proximate chemical analysis of broad bean (Vicia faba L.) pods

The data in Table (1) showed the chemical composition of the broad bean pods in their green state after oven drying at 50o C. The chemical analysis revealed that the dried pods contain moisture (9.27%), protein (8.38%), fat (0.38%), ash (7.22%), fibers (14.59%) and carbohydrates (60.16%). Our findings indicated that the dried green pods have a high content of carbohydrates and dietary fiber, which differs from the results of Mateos-Aparicio et al. [38], who reported that the broad bean pods contain high contents of protein (13.6%), and dietary fiber (40.1%), fat (1.3%), and ash (6.3%) on a dry weight basis. As demonstrated by Mejri et al. [18], broad bean pods had a high moisture content (79.26% on a wet weight basis), while their content of proteins, carbohydrates, lipids, and dietary fiber was 13.81, 18.93, 0.92 and 57.46% (on a dry weight basis), respectively. Also, Vernaleo et al. [39] stated that fava beans are a rich source of dietary fiber and high in phytonutrients such as isoflavone and plant sterols. The differences in the chemical composition could be attributed to the geographical location, handling and processing or the variety of the broad bean.

Table (1): Proximate chemical composition of broad bean pods (g/100g DW):

components

Moisture

Protein

Fat

Ash

Fibers

Carbohydrates
 

9.27 ± 0.08

8.38 ± 0.06

0.38 ± 0.03

7.22 ± 0.10

14.59 ± 0.03

60.16 ± 0.16
Each value is the mean ± SD

3.2. Mineral contents of broad bean pods (mg/100g DW)

Results in Table (2) revealed that the dried broad bean pods contain 937.2, 32.09, 340.4, 2.189, 3483, 4.442 and 340.1 mg/100g of calcium (Ca), iron (Fe), magnesium (Mg), manganese (Mn), potassium (K), copper (Cu) and phosphorus (P), respectively. It is evident that the broad bean pods are a good source of K, Ca, Mg and Fe. Results are in the same line with Vernaleo et al. [39], who stated that fava beans are a good source of phosphorus, iron, copper, manganese, calcium, magnesium, and potassium. Mateos et al. [38] found that the broad bean pods (byproducts) contain the most important minerals; potassium, calcium, and iron.

Table (2): mineral contents of broad bean pods (mg/100g DW):

ة Mineral

Concentration (mg/100g)

Ca

937.2

Fe

32.09

Mg

340.4

Mn

2.189

K

3483

Cu

4.442

P

340.1

3.3. Phytochemical screening of broad bean pods methanolic extract

As shown in Table (3) the phytochemical screening revealed that the methanolic extract of broad bean pods contain phenolic compounds, flavonoids, glycosides, tannins, alkaloids and saponins.

All phytochemicals were found in the ethanolic extract of Vicia faba L., with the exception of anthracenosides, sterols, and triterpenes (Fabaceae). The aqueous extract included fewer phytochemicals like tannins, alkaloids, glycosides, sterol, triterpenes, and saponins than the ethanolic extract. Additionally, reducing sugars were found in the ethanolic extract exclusively [40]. Faba beans contain polyphenols in a variety of plant components, including the leaves, roots, and seeds [41]. The contents of cotyledons from faba beans are higher than those of their hulls or complete seeds. According to recent studies, faba beans and their derivatives may be an appropriate meal for the treatment of hypertension, diabetes, and cardiovascular disease [42].

Table (3): Phytochemical screening of broad bean pods methanolic extract:

Glycosides

Phenols

Tannins

Alkaloids

Flavonoids

Saponins

+++

+++

+

+

++

+

3.4. Total phenols and total flavonoids of the broad bean pods methanolic extract

The total phenols and flavonoids of the methanolic extract of broad bean pods are represented in Table (4). Data showed that the phenol content was 286 mg GAE/g, and the total flavonoid content was 105mg QE/g. Our results are higher than that of Mejri et al. [18], who reported that the total phenolic compounds (TPC) in the methanol extract of broad bean pods were 115.21 mg GAE per g extract, while the total flavonoids compounds (TFC) were 47.34 mg QE per g extract. Valente et al. [43] found that the total free phenols in the dried pods ranged from 10.87 to 26.34 mg/100g in different varieties of faba bean pods, while the total esterified phenolics ranged from 8.76 to 26.72 mg/100g dry weight. According to Chan et al. [44], the methanolic extract of broad bean pods included a high concentration of total phenolics and flavonoids, including numerous polar aglycones and flavonoid glycosides. The literature needs to characterize the phenolic content sufficiently. According to one study on thirteen genotypes of broad bean pods grown in the same area and subjected to the same conditions, TPC ranged from 56.97 to 149.21 mg EGA per g. The genotypic effect of TFC in the same study had values ranging from 10.23 to 45.92 mg RE per g [45].

Table (4): Total phenols and flavonoids of broad bean pods methanolic extract

Total phenols

(286 mg GAE / g)

Total flavonoids

105 mg QE/g

3.4. Antioxidant activity of broad bean pods methanolic extract

3.4.1. DPPH assay.

The antioxidant activity of the broad bean pods' extract was measured by 1,1-diphenyl-2-picryl hydrazyl (DPPH) assay. The results in Table (6) and Fig. (1) revealed that the DPPH scavenging percentage increased by increasing the pods' extract concentration. The highest value of DPPH scavenging activity which reached 80.5%, was achieved by the extract concentrations of 1000 ug/ml. The concentrations of 500 and 250 ug/ml of the pod extract also showed high levels of DPPH scavenging activity, reaching 73.7 and 65.7%, respectively. The IC50 is a parameter for measuring the concentration of the antioxidant substance needed to decrease the initial DPPH concentration by 50%. Therefore, the low levels of IC50 indicate high antioxidant activity. The obtained results showed that the IC50 concentration of 87.35 ug/ml is small, which means that the broad bean pods have high antioxidant activity. Our results agree with Mateos-Aparicio et al. [19], who reported that the polyphenols extracted from broad bean pods exhibited high reducing power and free-radical scavenging activity. The antioxidant activity of the broad bean pods is attributable to their high content of phenolic compounds [46]. It is well known that most plants contain natural products which play essential roles in fighting diseases including cancer. The broad bean pods are rich in fiber, phenolic acids, and flavonoids, which can prevent the oxidation of cell membranes and protect the cells from free radicals and toxic substances. It has also been found that the tannins in faba beans could provide hydroxyl radical scavenging activity [47]. It was hypothesized that the extract of the pods prevents the reaction of hydroxyl radicals with the hydrogen atoms of the sugar moiety of DNA and hence protects DNA from damage [48].

Table (6): Antioxidant activity of broad bean pods methanolic extract by using DPPH assay.

Extract Conc. (µg/ml)

OD

DPPH scavenging %

1000

0.295

80.5

500

0.397

73.7

250

0.518

65.7

125

0.687

54.5

62.50

0.879

41.8

31.25

0.978

35.3

15.625

1.158

23.4

7.8125

1.224

19.0

3.90

1.305

13.6

1.95

1.356

10.3

IC50 (87.35 µg/ml)

3.4.2. Antioxidant activity of broad bean pods methanolic extract using reducing power

Table (5) shows that the reducing power of broad bean pods' methanolic extract increased by increasing the concentration. It varied from 33.9 to 79.1% by the extract concentrations of 100, 200, and 400 mg/ml. The IC50 values reached 177.32 mg/ml.

Any substance with a reducing power will combine with potassium ferricyanide (Fe3+) to generate potassium ferrocyanide (Fe2+), which will then react with ferric chloride to form ferric- ferrous complex, or pearl's Prussian blue, which is absorbed at 700 nm [49]. Reductive action and antioxidant activity are connected [50]. As mentioned before, the BBP methanol extract demonstrated potent antioxidant activity (60.72%). It was also shown that the lowest IC50 for the DPPH and ABTS assays is accompanied by the compound's highest free radical scavenging activity [18].

Table (5): Antioxidant activity of broad bean pods methanolic extract using reducing power

Concentration

mg/ml

100

200

400

Inhibition %

33.9

61.04

79.1

IC50 (177.32mg/ml)

  

3.5. Anti-inflammatory activity of broad bean pods methanolic extract

HRBC (human red blood cells) method was used for the in vitro study the anti-inflammatory effect of the broad bean pod extract. The erythrocyte membrane and the lysosomal membrane are comparable; therefore, by stabilizing the erythrocyte membrane, the extract from broad bean pods may stabilize the lysosomal membrane.

The data in Table (7) showed that all the extract concentrations exhibited a significant reduction in RBCs hemolysis, where the maximum inhibition percentage reached 66.7% with a concentration of 1000 ug/ml. However, the lowest concentration (400 ug/ml) of pod extract showed an inhibition percentage of about 50.8%. It is clear from these results that the pods' extract of broad beans has anti-inflammatory activity in the studied models.

The hypotonic solution causes hemolysis of RBCs because of fluid accumulation in the cells, which leads to rupturing of their membranes. The injury occurred in the RBCs makes them more susceptible to lipid oxidation via the free radicals causing the moving of some components, such as protein and fluids, to the tissues, which are similar to that produced during inflammation [51].

The extract of broad bean pods prevents the oxidation of RBCs membranes lipids and preserves them. In addition, it stabilizes the RBCs membrane by preventing the production of lytic enzymes and active inflammatory mediators.

Our results revealed that the pods of broad bean extract contain flavonoids, alkaloids, and saponin, to which the anti-inflammatory is attributed. Many studies showed that the plants' flavonoids have antioxidant and anti-inflammatory effects [52, 53, 54]

Their anti-inflammatory effect may be due to their ability to inhibit the enzymes which contribute to the production of inflammatory mediators and the enzymes of arachidonic acid metabolism [55, 56]

Table (7): Anti-inflammatory activity of broad bean pods methanolic extract

Conc. (ug/ml)

Hypotonic Ab. Mean

Sample with Isotonic solution Ab.

Hemolysis Inhibition %

Control

0.759

  

0

1000

0.291

0.057

66.7

800

0.341

0.051

59.0

600

0.367

0.045

54.9

400

0.394

0.041

50.8

200

0.429

0.039

45.8

100

0.503

0.036

35.4

3.6. Antimicrobial activity of broad bean pods methanolic extract:

The antimicrobial activity of the methanolic extract isolated from broad bean pods was assessed in vitro by the agar well diffusion method against four pathogenic bacteria strains and two kinds of fungi. The bacteria strains included two- gram-positive (Bacillus subtilis and Staphylococcus aureus) and two gram-negative (Escherichia coli and Pseudomonas aeruginosa), while the two fungi were Candida albicans and Aspergillus fumigatus.

Table (8) results revealed that the pods' extract prevented the growth of B. Subtilis, Staph. aureus and E. Coli in addition to the two fungi, Pseudomonas aeruginosa and Candida albicans. Their corresponding inhibition zones were 16, 17, 15, 28 and 23 mm, respectively. On the other hand, the Asp. fumigatus was resistant to the pod extract, which showed no activity in this respect. It was also noticed that the antimicrobial effect of the pod extract was more effective than the gentamycin (reference control) against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. These results agreed to a less extent with those of Peyvast and Khorsandi [57], who stated that the ethanolic extract of the seed hull of broad bean exhibited antimicrobial activity against E. Coli, B. subtilis and staph. aureus.

Table (8): Antimicrobial activity of broad bean pods methanolic extract against some microorganisms

Pathogenic microorganism

Diameter in mm

Sample

Control

Bacillus Subtilis (ATCC 6633)

16

25

Staph.aureus (ATCC 6538)

17

15

Escherichia coli (ATCC 8739)

15

17

Pseudomonas aeruginosa (ATCC 90274)

28

22

Candida albicans (ATCC 10221)

23

21

Aspergillus Fumigatus

NA

15

*Antimicrobial activity was determined by using agar diffusion, Disc diameter: 6.0 mm (100 µl was tested).

* NA: No activity

* All samples were dissolved in normal saline (0.9% NaCl).

* Saline didn’t have antimicrobial activity against all tested pathogenic strains

* Inhibition zones were represented as mm.

* Control: Gentamycin

3.7. Anticancer activity of broad bean pods methanolic extract:

Liver cancer is the world's fourth most common cause of death [58]. It is the most cause of mortality and morbidity-concerning cancer because of the widespread hepatitis C in the last decades. HCV infection is the leading cause of cirrhosis [59] which is one of the risk factors for liver cancer [60]. Because of the side effects of the drugs used in cancer treatment, we searched for natural products as novel anticancer remedies.

In this study, the effect of methanolic extract of broad bean pods on human hepatocellular carcinoma (HepG2) and prostate cancer (PC3) cells was investigated. The results in Table (9) and figures (2&3) showed that the methanolic extract of broad bean pods decreased the cell viability and increased cell toxicity of both HepG2 and PC3 in a concentration-dependent manner. It was noticed that the low extract concentration of 31.25 has no significant effect on the two mentioned cells' viability. On the other hand, all the other concentrations caused significant decreases in the viability of the two kinds of cells, increasing cell toxicity. The viability percentage of the doses 125, 250, 500 and 1000 ug/ml was 49. 54%, 35.52%, 18.46% and 4.07%, respectively in HepG2 cells, while it was 48.63% 21.32%, 11.59% and 3.43% in PC3 Cells, respectively. The methanolic extract of the pods under study exhibited high toxicity to the HepG2 and PC3 cells where the toxicity percentage reached more than 96% with a concentration of 1000 ug/ml. The IC50 values of the pod extract were observed at concentrations of 126.97 µg/ml for HepG2 cells and 125.12 µg/ml for PC3 cells, which are good results as an anticancer agent.

Morphological analysis:

As seen in Figs. 2 and 3, the methanolic extract of broad bean pods caused remarkable alterations in the shape of the cells compared to the control. Furthermore, the changes in the cell’s morphology increased with the increase of extract concentration, where a large amount of dead and detached cells indicate the toxic effect of the pods extract on the proliferation of the tumor cells after 24h incubation of the HpeG2 and PC3 Cells with the methanolic extract of the broad bean pods. It was observed that the low concentration of 31.25 ug/ml did not cause any significant alterations. In contrast, the higher concentrations caused substantial changes in the morphology of the tumor cells, which increased by increasing the extract concentration.

Many studies focused on benefiting from the plant byproducts that burden the environment. For example, some fruits and vegetable wastes can be used as feed for cattle and sheep; others can be used in fertilization [61]. Recent studies tried to benefit from the bioactive components found in plant and vegetable wastes as nutraceuticals used for human health as antioxidants and anticancer [62]. Other studies reported that the legumes' polyphenols and micronutrients have important biological values [63, 64]. The role of polyphenols in protecting the human body from chronic diseases such as cardiovascular diseases, diabetes, asthma, cancer, and inflammation was assessed [65]. The anticancer activity of the broad bean pod extract is attributed to its content of phenolic compounds.

P-coumaric and ferulic acids which are among the phenolic acids found in the pods of the broad beans are known for their anticancer activity against different types of cell lines [66].

In a recent study, Ceramella et al. [67] showed that three extracts of broad bean pods (acetone, methanol and ethanol 70%) have excellent antioxidant activity via studying DPPH and ABTS assays. They also found that the three extracts exhibited an enjoyable anticancer activity against melanoma SK- Mel-28 cells.

Table (9): Effect of broad bean pods methanolic extract against liver and prostate cancer in vitro

Pods extrac

Conc. (µg/ml)

Liver (HepG2)

Prostate (PC3)

Viability

Toxicity

Viability

Toxicity

Control

100.0 a

0.00f

100.00a

0.00f

1000

4.07f ± 0.12

96.02a ± 0.26

3.43f ± 0.78

96.44a ± 0.59

500

18.46e ± 2.79

81.54b ± 2.79

11.59e ± 0.98

88.41b ± 0.98

250

35.52d ± 7.21

64.48c ± 7.21

21.32d ± 2.83

78.68c ± 2.83

125

49.54c ± 2.81

50.40d ± 2.91

48.63c ± 3.58

51.38d ± 3.58

62.5

89.99b ± 1.49

10.01e ± 1.49

89.52b ± 2.62

10.48e ± 2.62

31.25

99.83a ± 11.90

0.17f ± 11.90

99.17a ± 2.52

0.83f ± 2.52

IC50 dil.

126.97 µg/ml

125.12 µg/ml

Many studies of plant extracts revealed that polyphenols could play a crucial role in preventing and progressing chronic illnesses related to inflammation, such as cardiovascular diseases, obesity, neurodegeneration, cancers, and diabetes, among other conditions [68, 69]. Polyphenols can suppress toll-like receptor (TLR) and pro-inflammatory genes’ expression. The antioxidant activity of polyphenols is attributed to their ability to inhibit enzymes that contribute to the production of eicosanoids and their anti-inflammation properties. For example, they inhibit certain enzymes producing reactive oxygen species ROS such as xanthine oxidase and NADPH oxidase (NOX). At the same time, they increase other endogenous antioxidant enzymes like superoxide dismutase (SOD), catalase, and glutathione (GSH) peroxidase (Px). On the other hand, they inhibit phospholipase A2 (PLA2), cyclooxygenase (COX) and lipoxygenase (LOX), causing reductions in the production of prostaglandins (PGs) and leukotrienes (LTs) and inflammation antagonism. These effects on the immune system led to health benefits for different chronic inflammatory diseases [69].

4. Conclusion

It can be concluded that the outer shell (fruit wall) of the broad bean, which is mentioned here as the pod, contains bioactive compounds such as phenolic compounds, flavonoids, tannins, and alkaloids, in addition to dietary fiber. Our results revealed that the methanolic extract of the dried fresh green pods has a potent antioxidant activity towards DPPH radicals and anti-inflammatory activity. Also, the pod extract showed antimicrobial activity against some food-born pathogenic microorganisms and anticancer activity against HepG2 and PC3 cell lines. These activities are attributed to phytochemicals and soluble fibers in the methanolic extract of the pods. Therefore, it is recommended to use fresh, immature broad beans with their outer shells on as an edible vegetable or in cooking. At the same time, the dried ripening pods can be solvent extracted to obtain the bioactive components to be used as additives in food manufacturers.

Declarations

Data availability

The datasets generated or analyzed during the current study are available from the corresponding author upon reasonable request.

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