Research Article

In Silico and In Vitro Analysis of Red Kidney Beans (Phaseolus vulgaris) and Black-Eyed Beans (Vigna unguiculata) and Their Phytochemical Analysis

Afshan Kaleem*, Iqra Noor, Roheena Abdullah and Mehwish Iqtedar

Department of Biotechnology, Lahore College for Women University, Lahore.

Received | December 22, 2023; Accepted | April 04, 2024; Published | June 20, 2024

*Correspondence | Afshan Kaleem, Department of Biotechnology, Lahore College for Women University, Lahore; Email: [email protected]

Citation | Kaleem, A., I. Noor, R. Abdullah and M. Iqtedar. 2024. In Silico and In Vitro analysis of red kidney beans (Phaseolus vulgaris) and black-eyed beans (Vigna unguiculata) and their phytochemical analysis. Biologia (Lahore), 70(1): 01-07.

DOI | https://dx.doi.org/10.17582/journal.Biologia/2024/70.1.1.7

Keywords | Phaseolus vulgaris, Vigna unguiculata, Disc diffusion method, FTIR analysis, Caspase-3 protein, apoptosis, Beta-amyloid protein

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Red kidney beans (Phaseolus vulgaris) are herbaceous plants belonging to the family Fabaceae. These are significant food crops on account of both their nutritive value and their financial advantages. Kidney beans are vital component of human nutrition as they are a rich source of protein, containing approximately 20% to 25% protein, and carbohydrates, which make up 50% to 60% of their nutritional content. In addition, they are also excellent sources of vitamins, minerals, and unsaturated fatty acids (Hayat et al., 2014). While Black-eyed bean (Vigna unguiculata) is a plant which belongs to Fabaceae. High nutritious values of black-eyed beans make them popular all over the world (Chandrasekaran et al., 2015).

The main difference between red kidney beans and black-eyed beans lies in their shape, flavor and size but they also differ in toxicity. Red kidney beans are large in size while black eyed beans are smaller in size. Red kidney beans have meaty, slightly sweet and nutty taste while Black-eyed beans have dense creamy taste (Gyrisco et al., 1959).

Dry beans, including types like red kidney and black-eyed beans, consist of a variety of advantageous phytochemicals and antioxidants, including coumestrol, phytate, isoflavones, lecithin, phytosterols etc. Studies show, consumption of these beans decreases the chance of various diseases like cancer, aging, cardiovascular diseases, obesity, colon cancer etc. (Djordjevic et al., 2011).

Beans show anti-microbial potential against many important food-borne pathogens. However, antimicrobial effect is associated with phytochemicals present in beans, vegetables such as polyphenols, lectins, peptides, flavonoids and Tannis. Red kidney beans and black-eyed beans exhibit antimicrobial properties against various types of bacteria for example Staphylococcus aureus, Bacillus subtilis and Salmonella typhi, Klebsiella pneumoniae, Escherichia coli, which are Gram-positive and Gram-negative respectively, as well as some fungi such as Aspergillus fuinigatus, Rhizopus stolonifer and Mucor mucedo (Olajide, 2020). In beans, by using different bioinformatics tools, different modifications are done to overcome the factors which affect their yield like climatic condition, occurrence of insect pest (Bello et al., 2014).

The objective of the current study was to analyze the various biological characteristics of Phaseolus vulgaris and Vigna unguiculata to identify new therapeutic leads for specific disorders (colon cancer and memory improvement in Alzheimer’s disease).

Materials and Methods

Sample collection

Red kidney and black-eyed beans were purchased from the local market of Lahore, Pakistan

Extract preparation

Both bean samples were dried and grounded to fine powder and aqueous extracts were prepared. The sample was dissolved in distilled water. The solution was homogenized on the magnetic stirrer for 25-30 minutes, then filtrated and stored at 4°C (Hang et al., 1980).

Qualitative analysis

Different qualitative test for detection of phytochemicals were performed including alkaloid, flavonoids, carbohydrates, proteins, saponins, steroids, cardiac glycosides, tannins, terpenoids, phenol and quinones (Tripathi et al., 2017; Pascale et al., 2018; Jaya et al., 2019).

Anti-microbial activity

Anti-microbial activity was performed using the disk diffusion method by Roy et al. (2020).

FTIR analysis 

Both aqueous extract samples were run on the Fourier transform infrared (FTIR) to analyze their bioactive compounds (Akinpelu et al., 2017). Peaks were matched with FTIR spectra retrieved from the PuBChem database (https://pubchem.ncbi.nlm.nih.gov/) and ligands, which were determined from the FTIR spectra, were downloaded from the PDBeChem software (https://www.ebi.ac.uk/pdbe-srv/pdbechem/).

Bioinformatics investigation

Protein docking was performed by using the Galaxy web server (https://galaxy.seoklab.org/). For docking, protein of interest was downloaded from PDB (Protein Data Bank). Beta amyloid protein (APLP1 (Uniprot ID: P51693), APP (Uniprot ID: P05067)) and Caspase-3 (PDB ID: 1NME) were downloaded from PDB and their charges were removed using the Pymol software.

Results and Discussion

Antibacterial activity of Vigna unguiculata and Phaseolus vulgaris

Antibacterial activity of Vigna unguiculata and Phaseolus vulgaris was evaluated against Staphylococcus aureus and Escherichia coli. Both extracts showed anti-bacterial activity against Staphylococcus aureus and Escherichia coli. The maximum zone of inhibition was reported in both gram-negative and gram-positive strains by Phaseolus vulgaris (Figure 1).

 

FTIR analysis

FTIR analysis of Vigna unguiculata (Black-eyed beans) and Phaseolus vulgaris (red kidney beans) showed the presence various functional groups like hydroxyl, carbonyl, phenol, alkane, alcohol etc. However, peaks matched from PuBChem software at 3334.92 and 3317.56, 1153.43, 709.80 and 1639.49 indicating rutin, kaempferol, benzoic acid and naringenin bioactive compounds (Figure 2).

 

Bioinformatic analysis of Vigna unguiculata and Phaseolus vulgaris

Different detected bioactive compounds (ligand) were docked with respective proteins. Rutin, Naringenin, Benzoic acid and kaempferol were docked with both APLP1 and APP proteins, which were determined in Vigna unguiculata, (Tables 2-4) (Figures 3-6), while in the case of Phaseolus vulgaris, rutin was docked with Caspase-3 (Table 5) (Figure 6). The binding with various amino acids were found to consist of different hydrogen bonding (HB) and hydrophobic interactions (HI). However, kaempferol did not bind with any protein.

 

Table 1: Qualitative phytochemical analysis of extracts.

Phytochemicals	Phaseolus vulgaris	Vigna unguiculata
	Aqueous extract	Aqueous extract
Alkaloids	+	+
Carbohydrates	+	+
Proteins	+	+
Flavonoids	+	+
Steroid	+	+
Saponins	+	+
Phenol	+	+
Quinones	+	+
Tannins	-	+
Cardiac glycoside	+	+
Terpenoids	+	+
Phenol	+	+
Saponins	+	+

 

Table 2: Binding of different ligands with APLP1 (285-499) protein.

Ligand	APLP1 amino acid	Types of bonds	Length of the bond	Ligand amino acid binding sites
Rutin	Arg 540 Pro 541 Leu 547	1: HB 2: HB HI HI	3.13 3.29	NH2-O8 NH1-O8
Naringenin	Arg 480 Gln 486 Leu 547 His 548 Gln 483 Glu 519 Lle 518	HB HB HI HI HI HI HI	3.02 3.00	NE-O6 NE2-O8
Benzoic acid	Glu 519 Gln 486	HB HI	2.87	OE2-O5

 

Table 3: Binding of different ligands with APLP1 (290-495) protein.

Ligand	APLP1 amino acid	Types of bonds	Length of the bond	Ligand amino acid binding sites
Rutin	Asn 461 Glu 494 Leu 464 Leu 485 Ser 493	HB 1: HB 2: HB HI HI HI	3.20 3.32 2.87	OD1-O36 OE1-O25 OE1-O27
Naringenin	Arg 478	HB	3.26	NH2-O10
Benzoic acid	Arg 478 Ala 474 Asp 468	HB HI HI	3.28	NH1-O5

 

Table 4: Binding of different ligands with APP protein.

Ligand	APP amino acid	Types of bonds	Length of the bond	Ligand amino acid binding sites
Rutin (Model 6)	Arg 42 Asn 44 Arg 20 Asp 46 Thr 47	1: HB 2: HB HB HB 1: HB 2: HB 3: HB HB	3.08 2.99 2.82 2.95 2.73 3.05 3.18 2.89	NH1-O16 NH1-O4 ND2-O14 NH2-O16 OD1-O14 OD1-O16 OD2-O20 OG1-O28
Naringenin (Model 5)	Glu 10 Arg 42	HB HI	3.23	OE1-O8
Benzoic acid (Model 3)	Thr 26 Asp 24 Tyr 22 Val 25 Ser 6	HB HB HI HI HI	3.27 3.04	OG1-O5 OD2-O5

 

Table 5: Binding of ligand with caspase-3 protein.

Ligand	Caspase-3 amino acid	Types of bonds	Length of the bond	Ligand amino acid binding sites
Rutin	Lys 271 Lys 186 Leu 194 Leu 269 Leu 273 Lle 187 His 277 Val 189	HB 1: HB 2: HB 3: HB HI HI HI HI HI HI	3.24 3.11 3.07 2.91	NZ-O36 NZ-O34 NZ-O38 NZ-O40

 

 

Both in developing and developed nations, the tendency of diabetes, obesity, cardiovascular illnesses and cancer are rising. It is anticipated that both the diet and nutrition would be crucial in preventing certain illness problems. Beans and other legumes are an excellent source of protein, minerals, dietary fiber, carbohydrates, and bioactive compounds. They also serve as medicinal plants and are used as common food in developing countries. In current study various experiments were done in order to evaluate biological characteristics of the beans (Phaseolus vulgaris and Vigna unguiculata) and their therapeutic effect on specific diseases (colon cancer and memory improvement in Alzheimer’s disease).

 

 

Qualitative analysis of aqueous extracts of Phaseolus vulgaris and Vigna unguiculata showed that both extracts contain alkaloids, steroids, phenols, carbohydrates, proteins, terpenoids, glycosides, saponins, quinones, tannins, flavonoids. All these results were in agreement with the results with the work reported in the literature (Luka et al., 2013; Tripathi et al., 2017; Jaya et al., 2019). However, tannin was not found in Phaseolus vulgaris, which contradicts the results in literature (Jaya et al., 2019).

 

Aqueous extract of Phaseolus vulgaris and Vigna unguiculata, both showed anti-bacterial activity against Staphylococcus aureus and Escherichia. The maximum zone of inhibition was reported in both Gram-Negative and Gram-Positive strains by aqueous extract of Phaseolus vulgaris. However, in Phaseolus vulgaris seeds, lectin which is active phytohemaglutinin is responsible for the antibacterial activity, while in the case of Vigna unguiculata the bioactive component, globulins, has shown comparable antibacterial efficacy (Chidebelu et al., 2019).

The FTIR of Vigna unguiculata, peaks obtained in the area of 3334.92 refer to O-H stretching, peak at 1639 refers to C=C, were similar with the working of Akinpelu et al. (2017). While in the case of Phaseolus vulgaris, peak obtained in the area of 1653 cm−1 this was similar with the results of Liu et al. (2013).

FTIR analysis of Phaseolus vulgaris confirms the presence of rutin, which was docked with the protein Caspase-3, an apoptotic marker in colon cancer and binds to various amino acids through hydrogen bonding like Lys 271, Lys 186. These results showed good docking with lowest docking energy of –13.461 Kcal/mol. Rutin have anti-cancerous and anti-proliferative properties, and it prevents colon cancer cells from multiplying by triggering apoptosis, which is achieved by cell cycle arrest and activation of the Caspase protein. Furthermore, Caspase-3 expression was enhanced after treatment with rutin (Jayameena et al., 2018).

FTIR analysis of Vigna unguiculata confirms the presence of rutin, kaempferol, benzoic acid and naringenin. These bioactive compounds were docked with beta amyloid protein (APLP1 and APP). These bioactive compounds bind with various amino acids through hydrogen bonding like Arg 540 (Rutin), Arg 480, Gln 486 (Naringenin) and Arg 478 (Benzoic acid) in the case of APLP1 (285-499) as shown in Table 3. Similarly, Asn 461, Glu 494 (Rutin), Arg 478 (Naringenin) and Arg 478 (Benzoic acid) in APLP1 (290-495) (Table 4). All these interactions take place in the E2 domain, the collagen binding region and the compositional bias region which include acidic and basic residues. In the case of APP, different bioactive compounds form bond with amnio acids like Arg 42, Asn 44, Arg 20, Asp 46, Thr 47 (Rutin), Glu 10 (Naringenin) and Thr 26, Asp 24 (Benzoic acid) as shown in Table 5. Rutin and naringenin interaction take place in the GFLD subdomain and E1 domain.

Rutin has potential to reduce oxidative stress, to prevent Aβ aggregation and cytotoxicity and proinflammatory cytokines. Oral administration has shown to significantly reduce interleukin IL-1 and IL-6 levels in the brain and alleviated memory problems in AD transgenic mice (Xu et al., 2014). Moreover, administration of naringenin may lead to an enhanced spatial learning and memory in a rat model of Alzheimer’s disease through controlling the PI3K/AKT/GSK-3 pathway (Nouri et al., 2019).

The studies by Aduema (2016, 2019) also indicate that consumption of these beans improves learning and memory. The determined bioactive compounds of Vigna unguiculata have beneficial effects on Alzheimer’s disease treatment and hence can become a potential drug for memory improvement.

Conclusions and Recommendations

In conclusion, the current study suggests that Phaseolus vulgaris and Vigna unguiculata are excellent source of proteins, carbohydrates and various bioactive compounds. They have good antimicrobial activity which make them potent agent against several microbes. FTIR analysis indicated the presence of several functional groups. Phaseolus vulgaris contain rutin, a glycoside of the flavonoid quercetin and rutinose, that showed good docking results with lowest docking energy of –13.461 Kcal/mol against Caspase-3 protein, which is an apoptotic marker in colon cancer. It might serve as a standard therapy for colon cancer. Vigna unguiculata contains rutin, naringenin and benzoic acid, which showed good docking activity against Beta-amyloid (APLP1 and APP) protein for memory improvement in Alzheimer’s disease. These results suggest that Vigna unguiculata could be beneficial and may act as a therapeutic agent in Alzheimer’s disease and hence could become a potential drug for memory improvement.

Author’s Contribution

Afshan Kaleem: Conceptualisation, formal analysis, administration, writing, review and editing.

Iqra Noor: Conceptualisation, data curation, formal analysis and writing.

Roheena Abdullah: Conceptualisation, administration, review and editing.

Mehwish Iqtedar: Data curation, administration, review and editing.

Conflict of interest

The authors have declared no conflict of interest.

References

Aduema, W., 2016. Neurobehioural effects of chronic consumption of uncooked black eye beans on spatial learning and memory in mice. Curr. Trends Biomed. Eng. Biosci., 1(2): 555556. https://doi.org/10.19080/CTBEB.2016.01.555556

Aduema, W., 2019. Effects of long-term administration of cooked beans (Vigna unguiculata) diet on learning and memory in mice. Int. J. Food Sci., 2(1): 65-75. https://doi.org/10.47604/ijf.168

Akinpelu, L.A., Adegbuyi, T.A., Agboola, S.S., Olaonipekun, J.K., Olawuni, I.J., Adegoke, A.M. and Olayiwola, G., 2017. Antidepressant activity and mechanism of aqueous extract of Vigna unguiculata sp. Dekindtiana (L.) walp dried aerial part in mice. Int. J. Neurosci. Behav. Sci., 5(1): 7-18. https://doi.org/10.13189/ijnbs.2017.050102

Bello, M.H., Moghaddam, S.M., Massoudi, M., McClean, P.E., Cregan, P.B. and Miklas, P.N., 2014. Application of in silico bulked segregant analysis for rapid development of markers linked to Bean common mosaic virus resistance in common bean. BioMed. Central Fenomics, 15(1): 1-13. https://doi.org/10.1186/1471-2164-15-903

Chandrasekaran, S., Rajkishore, V.B. and Ramalingam, R., 2015. Vigna unguiculata-an overall review. Res. J. Pharma. Phytochem., 7(4): 219-222. https://doi.org/10.5958/0975-4385.2015.00033.3

Chidebelu, P., Onovo, I. and Nweze, E.I., 2019. Antimicrobial activities of Cajanus cajan L., Phaseolus vulgaris L. and Vigna unguiculata L. against some bacterial and fungal isolates. J. Basic Pharmacol. Toxicol., 3(1): 29-33.

Djordjevic, T.M., Šiler-Marinkovic, S.S. and Dimitrijevic-Brankovic, S.I., 2011. Antioxidant activity and total phenolic content in some cereals and legumes. Int. J. Food Prop., 14(1):175-184. https://doi.org/10.1080/10942910903160364

Gyrisco, G.G., Muka, A.A. and Briant, A.M., 1959. Studies of flavors and odors of potatoes and red kidney beans grown in rotation with lindane-treated red clover. J. Econ. Entomol., 52(3): 473-475. https://doi.org/10.1093/jee/52.3.473

Hang, Y.O., Steinkraus, K.H. and Hackler, L.R., 1980. Amino acid composition of high protein fractions prepared from mung beans, pea beans, and red kidney beans. J. Food Sci., 45(2): 388-389. https://doi.org/10.1111/j.1365-2621.1980.tb02622.x

Hayat, I., Ahmad, A., Masud, T., Ahmed, A. and Bashir, S., 2014. Nutritional and health perspectives of beans (Phaseolus vulgaris L.): An overview. Crit. Rev. Food Sci. Nutr., 54(5): 580-592. https://doi.org/10.1080/10408398.2011.596639

Jaya, J.S., Das, J.L. and Sujin, D., 2019. Qualitative analysis of phytochemicals in two legume species. Plant Arch., 19(1): 1391-1394.

Jayameena, P., Sivakumari, K., Ashok, K. and Rajesh, S., 2018. Rutin: A potential anticancer drug against human colon cancer (HCT116) cells. Int. J. Biol., Pharma. Allied Sci., 7(9): 1731-1745. https://doi.org/10.31032/IJBPAS/2018/7.9.4532

Liu, C., Zhao, M., Sun, W. and Ren, J., 2013. Effects of high hydrostatic pressure treatments on haemagglutination activity and structural conformations of phytohemagglutinin from red kidney bean (Phaseolus vulgaris). Food Chem., 136(3-4): 1358-1363. https://doi.org/10.1016/j.foodchem.2012.09.082

Luka, C.D., Olatunde, A., Tijjani, H. and Olisa-Enewe, I.A., 2013. Effect of aqueous extract of Phaseolus vulgaris L. (red kidney beans) on alloxan-induced diabetic Wistar rats. Int. J. Sci. Invent. Today, 2(4): 292-301.

Nouri, Z., Fakhri, S., El-Senduny, F.F., Sanadgol, N., Abd-ElGhani, G.E., Farzaei, M.H. and Chen, J.T., 2019. On the neuroprotective effects of naringenin: Pharmacological targets, signaling pathways, molecular mechanisms, and clinical perspective. Biomolecules, 9(11): 690. https://doi.org/10.3390/biom9110690

Olajide, M.A., 2020. Phytochemical screening and medicinal attributes of crude extracts of soybean (Glycine max) and black-eyed bean (Phaseolus vulgaris) leave. J. Adv. Bot. Zool., 7(4): 1-4.

Pascale, R., Bianco, G., Cataldi, T.R., Kopplin, P.S., Bosco, F., Vignola, L. and Milella, L., 2018. Mass spectrometry-based phytochemical screening for hypoglycemic activity of Fagioli di Sarconi beans (Phaseolus vulgaris L.). Food Chem., 242: 497-504. https://doi.org/10.1016/j.foodchem.2017.09.091

Roy, M., Sarker, A., Azad, M.A.K., Shaheb, M.R. and Hoque, M.M., 2020. Evaluation of antioxidant and antimicrobial properties of dark red kidney bean (Phaseolus vulgaris) protein hydrolysates. J. Food Measur. Charact., 14(1): 303-313. https://doi.org/10.1007/s11694-019-00292-4

Tripathi, I.P., Tripathi, R. and Tiwari, A., 2017. Investigation of biologically active phytoconstituents present in selected plants material of Verbenaceae, Lamiaceae and Fabaceae family. Int. J. Multidis. Curr. Res., 5: 31-37.

Xu, P.X., Wang, S.W., Yu, X.L., Su, Y.J., Wang, T., Zhou, W.W. and Liu, R.T., 2014. Rutin improves spatial memory in Alzheimer’s disease transgenic mice by reducing Aβ oligomer level and attenuating oxidative stress and neuroinflammation. Behav. Brain Res., 264: 173-180. https://doi.org/10.1016/j.bbr.2014.02.002