Republic of the Philippines

Department of Education
REGION III – CENTRAL LUZON
SCHOOLS DIVISION OF BULACAN
SAN MIGUEL NATIONAL HIGH SCHOOL

IMMUNOMODULATORY, ANTICANCER, AND ANTIMICROBIAL EFFECTS OF

MUNG BEAN PODS GROWN: AN IN VITRO STUDY

A Research Paper Presented to the Faculty of Junior High School Department San Miguel
National High School San Miguel, Bulacan

NICA ROSE V. ISIDRO

MA. CAZANDRA AYEZA R. LALO
Researchers

FARRAH M. RAMOS
Research

April 2023

San Miguel National High School

Scuala St., San Juan, San Miguel, Bulacan
Telephone Nos: (044) 327-1123 / (044) 327-1104
 1

INTRODUCTION

Mungbean (Vigna radiata L.) Wilczek) is an important grain legume grown in the

tropical and subtropical regions of the world. It is one of the important sources of protein for

both man and domestic animals. Another important feature of mungbean is its ability to fix

atmospheric nitrogen in symbiosis with nodule forming rhizobium bacteria. Nutrient stress

soil was defined soil contains nutrient below the critical level (FRG, 2005).¹

Mungbean productivity is constrained by biotic and abiotic factors. Mung beans are high in

nutrients and antioxidants, which may provide health benefits. In fact, they may protect

against heat stroke, aid digestive health, promote weight loss and lower “bad” LDL

cholesterol, blood pressure and blood sugar levels.²

Legume, also called pod, fruit of plants in the pea family (Fabaceae). Most legumes are

dehiscent fruits that release their seeds by splitting open along two seams, though some, such

as peanuts (Arachis hypogaea) and mung bean (vigna radiata), do not naturally open.³ The

fruits come in a variety of sizes and shapes; many, however, are long and narrow and bear

their seeds in a single line. The largest legumes are borne by the monkey ladder (Entada

gigas) and can reach up to 2 metres (6.6 feet) in length. At maturity, legume fruits are usually

dry and papery or hard and woody; the legumes of certain food crops, such as snow peas

(variety of Pisum sativum), edamame (Glycine max), and green beans (Phaseolus vulgaris),

are harvested while still green and fleshy.⁴

Peterson, C.; Denlinger, N.; Yang,
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 2

1. Ziska, Lewis H., Robert Palowsky, and Danielle R Reed. “A Quantitative and Qualitative Assessment of Mung Bean (Vigna Mungo (L.) Wilczek) Seed in
Response to Elevated Atmospheric Carbon Dioxide: Potential Changes in Fatty Acid Composition.” Journal of the Science of Food and Agriculture 87, no. 5
(April 15, 2007): 920–23. https://doi.org/10.1002/jsfa.2818.

2. Talib WH, Mahmod AI, Awajan D, Hamed RA, Al-Yasari IH. Immunomodulatory, Anticancer, and Antimicrobial Effects of Rice Bran Grown in Iraq: An
In Vitro and In Vivo Study. Pharmaceuticals (Basel). 2022 Dec 1;15(12):1502. doi: 10.3390/ph15121502. PMID: 36558953; PMCID: PMC9782048.

3. Wikipedia contributors. “Mung Bean.” Wikipedia, March 11, 2023. https://en.m.wikipedia.org/wiki/Mung_bean.

4. Singh, B., Singh, N., Thakur, S. et al. Ultrasound assisted extraction of polyphenols and their distribution in whole mung bean, hull and cotyledon. J Food
Sci Technol 54, 921–932 (2017). https://doi.org/10.1007/s13197-016-2356-z

Consequently, Mungbean requires hot and dry climate. Cloudy weather, continuous and

heavy rains adversely affect the flowering and podding in mungbean, causing low yields.

Mungbean can be grown on well-drained loamy sand to sandy loam soils. The crop is

sensitive to alkaline, saline or waterlogged soil. Being a short duration crop, mungbean is

cultivated in all three seasons (kharif, rabi and zaid) in different parts of country as a pure

crop as well as an associate crop in various cropping systems.⁵ However, Mung bean has a

well-developed root system. The lateral roots are many and slender, with root nodules

grown.Stems are much branched, sometimes twining at the tips. Young stems are purple or

green, and mature stems are grayish yellow or brown. Mung bean is one of the a few legumes

that is composed of unique macro and micronutrients as well as numerous phytochemicals,

including phenolic acids, flavonoids, sterols, terpenes, alkaloids, and organic acids.⁶ The

majority of these products are water-soluble or biodegradable phyto-films, mainly composed

of biopolymers (proteins, polysaccharides, and lipids) and hydrocolloids (carrageenan)

gums). Furthermore, utilization of plant extracts combined with the phyto-film has shown

good effects, as they could increase the stability of the stored product and could provide a

barrier against spoilage and pathogenic microorganisms. They could also control the

biochemical degradation in food induced by enzymes.⁷

Peterson, C.; Denlinger, N.; Yang,
Y. Recent Advances and
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 3

5. Antioxidant and Antimicrobial Properties of Mung Bean Phyto-Film Combined with

Longkong Pericarp Extract and Sonication - ProQuest,” n.d.
https://search.proquest.com/openview/93705cfeadeac179057c8dc3c8413d7b/1?pq-
origsite=gscholar&cbl=2032363.

6. Das, A., Parida, S.K. Advances in biotechnological applications in three important food
legumes. Plant Biotechnol Rep 8, 83–99 (2014). https://doi.org/10.1007/s11816-013-0299-7

7. Diao, Jingjing, Chi Zhiping, Zengwang Guo, and Liping Zhang. “Mung Bean Protein
Hydrolysate Modulates the Immune Response Through NF‐κB Pathway in
Lipopolysaccharide‐Stimulated RAW 264.7 Macrophages.” Journal of Food Science 84, no.
9 (September 1, 2019): 2652–57. https://doi.org/10.1111/1750-3841.14691.

A cytotoxic compound can cause cell damage or death, either through necrosis or apoptosis.

Some substances are more cytotoxic than others and researchers aim to measure a chemical's

cytotoxicity levels to ensure that it is not harmful and/or fatal to patients.⁸ Treating cells with

the cytotoxic compound can result in a variety of cell fates.⁹ Cytotoxicity studies are a useful

initial step in determining the potential toxicity of a test substance, including plant extracts or

biologically active compounds isolated from plants.¹⁰ The purpose of this research is to

evaluate the cytotoxicity and the Preliminary screening of mung bean pods grown.¹¹ A

preliminary screening is conducted to determine if a proposed project might be cause for

public concern or might have a significant adverse impact on the environment. The

preliminary screening will determine whether the project should proceed without

environmental assessment or if it will be referred to environmental assessment. ¹²

10. Brahmer, Julie R., Karen L. Reckamp, Paul Baas, Lucio Crinò, Wilfried Eberhardt, Elena
Poddubskaya, Scott J. Antonia, et al. “Nivolumab versus Docetaxel in Advanced Squamous-
Cell Non–Small-Cell Lung Cancer.” The New England Journal of Medicine 373, no. 2 (July
8, 2015): 123–35. https://doi.org/10.1056/nejmoa1504627.

Peterson, C.; Denlinger, N.; Yang,

Y. Recent Advances and
Challenges in Cancer
Immunotherapy. Cancers 2022,
14, 3972. [Google Scholar]
[CrossRef] [PubMed]
 4

11. Chacon, E, Daniel Acosta, and John J. Lemasters. “Primary Cultures of Cardiac
Myocytes as In Vitro Models for Pharmacological and Toxicological Assessments.” Elsevier
EBooks, January 1, 1996, 209–23. https://doi.org/10.1016/b978-012163390-5.50010-7.

12. Hobley L, Ostrowski A, Rao FV, Bromley KM, Porter M, Prescott AR, MacPhee CE, van
Aalten DM, Stanley-Wall NR. BslA is a self-assembling bacterial hydrophobin that coats the
Bacillus subtilis biofilm. Proc Natl Acad Sci U S A. 2013 Aug 13;110(33):13600-5. doi:

Statement of the Problem

1. What does the effect of Mung Bean in cytotoxic?

2. What does the effect of Mung Bean in Phytochemical screening?

3. What does the effect of Mung Bean in Total phenolic content (TPC)?

4. What does the effect of Mung Bean in Antioxidant assay?

5. What does the effect of Mung Bean in

Antibacterial assay?

Hypothesis

The Mung Bean pods has no significant effect.

Significance of the Study

Peterson, C.; Denlinger, N.; Yang,

Y. Recent Advances and
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 5

Cancer patients. This study is will help cancer patients to fight cancer by multiple

mechanisms including apoptosis induction, angiogenesis inhibition, and immune system

activation and preventing cancer and stimulating the immune system.

Community. It will benefit the community to reduce cases of cancer and These will

prevent bacteria may infect humans and animals.

Mung Bean cultivators. This study will benefit the Mung Bean cultivators because

they can sell the Mung Bean pods instead of throwing them away.

Scopes and Delimitations

This project is limited to use of Mung Bean pods as a Cytotoxic. The researchers will
focus on evaluating the potential of Mung Bean as a Cytotoxic by identifying its
Phytochemical screening,

Total phenolic content (TPC),Antioxidant assay,and Antibacterial assay.

Collection of Mung Bean will take place at Salangan, San Miguel Bulacan.
Meanwhile the Phytochemical screening,

Total phenolic content (TPC), Antioxidant assay, and Antibacterial assay will take place at
the Science Laboratory in Saint Mary university located at Ponce, Bayombong,Nueva
Vizcaya..
Peterson, C.; Denlinger, N.; Yang,
Y. Recent Advances and
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[CrossRef] [PubMed]
 6

Definition of terms

Preliminary screenings: the gathering of initial information to be used in determining a

person's need for assessment or for referral.

Mung bean pods: The mung bean (Vigna radiata L.) is one of the most important edible
legume crops, grown on more than 6 million ha worldwide (about 8.5% of the global pulse
area) and consumed by most households in Asia.

Phytochemical screening: is another name for this process.These extracts are made from
plant samples rich in secondary metabolites such as the leaves and stems, roots, and bark of
the plants studied. The secondary metabolites, such as alkaloids, terpenes, and flavonoids, are
then examined in the plant extracts.

The TPC assay, also known as the Folin-Ciocalteu (FC) method: is well established and
uses the FC reagent to oxidise phenolic compounds. The reaction results in a blue-coloured
reduced FC reagent, which is measured at 760 nm [9] with the intensity of the blue colour
correlating with the sample's phenolics content.

Antimicrobial assays are important tools: to test and screen the inhibitory effects of myriad
compounds against microorganisms before establishing their inhibitory spectra (broad vs.
narrow).

Brine shrimp lethality bioassay: a simple high throughput cytotoxicity test of bioactive
chemicals. It is based on the killing ability of test compounds on a simple zoological
organism-brine shrimp (Artemia salina).

Peterson, C.; Denlinger, N.; Yang,

Y. Recent Advances and
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 7

METHODOLOGY

General Process:

Phytochemical
Mung Beans TPC Assay
Screening
Pods Extract

Anti Bacterial Anti Oxidant

BsLA Assay
Assay Assay

Waste
Disposal

Figure 1. Flow Chart of Activities

Presented in Figure 1 is the complete procedure of the study. The researchers will

start by gathering materials needed for the study followed by the Mung Bean Pods Extract.

Peterson, C.; Denlinger, N.; Yang,

Y. Recent Advances and
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 8

Then, phytochemical screening and to achieve successful assay, will be complete the TPC

assay. After that, the anti oxidant assay to be experiment. And then, Anti bacteria assay

detects and quantifies the cellular events associated with programmed cell death. And the last

assay is BsLA that have different concentrations. After the Experimentation the data will be

gathered, and results will be interpreted.

Mung Bean pods extract

Based on previous studies, they are protein-rich and contain many essential amino

acids we need to maintain our health. We can easily digest mung bean protein and isolates,

too.

Mung Bean pods was provided by local mills in (location). Mung Bean compressed

place beaker, add 80% ethanol or water, solid-liquid ratio 1:10,50 ℃ are stirred and extracted

120 minutes, after filtration, sucking filtration, revolve the inspissation contracting, get the

Mung Bean extracting solution 1.

Materials:

1Ziska, Lewis H., Robert Palowsky, and Danielle R Reed. “A Quantitative and Qualitative Assessment of Mung Bean (Vigna Mungo (L.) Wilczek) Seed in
Response to Elevated Atmospheric Carbon Dioxide: Potential Changes in Fatty Acid Composition.” Journal of the Science of Food and Agriculture 87, no. 5
(April 15, 2007): 920–23. https://doi.org/10.1002/jsfa.2818.

2. Talib WH, Mahmod AI, Awajan D, Hamed RA, Al-Yasari IH. Immunomodulatory, Anticancer, and Antimicrobial Effects of Rice Bran Grown in Iraq: An
In Vitro and In Vivo Study. Pharmaceuticals (Basel). 2022 Dec 1;15(12):1502. doi: 10.3390/ph15121502. PMID: 36558953; PMCID: PMC9782048.

3. Wikipedia contributors. “Mung Bean.” Wikipedia, March 11, 2023. https://en.m.wikipedia.org/wiki/Mung_bean.

4. Singh, B., Singh, N., Thakur, S. et al. Ultrasound assisted extraction of polyphenols and their distribution in whole mung bean, hull and cotyledon. J Food
Sci Technol 54, 921–932 (2017). https://doi.org/10.1007/s13197-016-2356-z

Peterson, C.; Denlinger, N.; Yang,

Y. Recent Advances and
Challenges in Cancer
Immunotherapy. Cancers 2022,
14, 3972. [Google Scholar]
[CrossRef] [PubMed]
 9

Mung Bean pods

Water

Beaker

Quantitative analysis of Mung Bean pods extract

Samples have been prepared by dissolving them in dimethyl sulfoxide and completed

the volume to 50 mL with acetonitrile. Samples were centrifuged at 4000 rpm for a minute,

then 1mL has been taken to the autosampler. The injection volume was 3 μL. The test was

carried out using the Bruker Daltonik, impact II ESI-Q-TOF system equipped with the

Bruker Daltonik Elute UPLC system. The apparatus was activated using the Ion Source

Apollo II ion funnel electrospray source, nebulizer gas, 2 bar, dry gas flow, 8 L/min, dry

temperature, 200•C, mass accuracy, less than the 1ppm; mass resolution, 50,000. FRS 2.

Chromatographic separation was performed using Burker solo 2-C-18 UHPLC column (100

mm × 2.1 mm × 2 μm) at a flow rate of 0.51 mL/min and a column temperature of 40 ◦C. All

standards were used for identification of ms/z and the retention time ²⁸.

Materials:

Dimethyl Sulfoxide

Acetonitrile

Autosampler

2
Coşkun, Özlem. “Separation Tecniques: CHROMATOGRAPHY.” İstanbul Kuzey Klinikleri, November 11,
2016. https://doi.org/10.14744/nci.2016.32757.n.
Peterson, C.; Denlinger, N.; Yang,
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 10

Bruker Daltonik

Ion Source Apollo

Ion Funnel Electrospray

Nebulizer gas

2bar

Dry Gas flow

Cell culturing condition

To achieve a successful cell culture, many factors were followed such as using

completed medium and incubating cells in 5% CO2 at 37 ◦C. Based on the type of the cells,

the type of culturing medium varied. For MCF-7 and T47D, complete RPMI 1640 medium

was used, while complete MEM medium was used for culturing EMT6/P. High-glucose

DMEM medium was utilized for MDA-MB-231 and fibroblast cell lines. A complete culture

medium was prepared through adding the following supplements with the required

percentage of each type of tissue culture medium. The supplements are: 1% L-glutamine,

10% fetal bovine serum, 1% penicillin-streptomycin, 0.1% non-essential amino acids and

0.1% gentamicin solution 3.

Materials:

Complete RPMI 1640

3
“Cell Culture Conditions,” n.d. https://www.qiagen.com/us/knowledge-
and-support/knowledge-hub/benchhttps://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-
guide/animal-cell-culture/cell-culture-conditions/cell-culture-conditions guide/animal-cell-culture/cell-culture-
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Y. Recent Advances and
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 11

Complete MEM

High-glucose DMEM

Glutamine

Fetal bovine serum

Penicillin-streptomycin

Non-essential amino acids

Gentamicin

MTT cell viability Assay

The different cell lines were prepared and cell viability was checked using trypan blue

stain. Then, the cells were seeded in 96-well plate at a density of 15,000 cells per well in

complete medium. Following 24 h of incubation, the medium in each well was discarded and

the attached cells were treated in triplicate with mung bean extracts. At the beginning, the

extracts were dissolved in DMSO, then a serial dilution method was used to achieve graduate

concentrations from 5 to 0.03 mg/mL. After 48 h incubation, the old medium was removed

and replaced with 100 μL fresh medium in each well. Then, 20 μL of MTT [3-

(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution was added, and

incubated for 3 h at 37 ◦C and 5% CO2. The developed formazan particles were dissolved

using DMSO

(100 μL/well), and incubated for 1 h. The absorbance was measured using a microplate

reader at 550 nm. The percentage of survival was also estimated and compared to the control

Peterson, C.; Denlinger, N.; Yang,

Y. Recent Advances and
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 12

results 30.

Materials:

Trypan blue stain

Dimethyl sulfoxide

Dimethyl thiazol

Diphenyl tetrazolium bromide

Incubated

Microplate reader

Apoptosis Assay

³⁰ Chacon, E, Daniel Acosta, and John J. Lemasters. “Primary Cultures of Cardiac Myocytes as In Vitro Models
for Pharmacological and Toxicological Assessments.” Elsevier EBooks, January 1, 1996, 209–23.
https://doi.org/10.1016/b978-012163390-5.50010-7..
An apoptosis assay detects and quantifies the cellular events associated with

programmed cell death, including caspase activation, cell surface exposure of

phosphatidylserine (PS) and DNA fragmentation 4. For this assay, a T47D cell line was

chosen depending on the results of the MTT assay and the IC50 values. The cells were

cultured in 25 cm2 tissue culture flasks at a density of 15,000 cells/mL of complete RPMI

medium. Following 24 h of incubation, the old medium was removed and the attached cells

were treated with mung bean pods extract with the following concentrations: the ethanol

extracts of mung beans with the concentration of (0.4 μg/ml), had the best antioxidant effect,

4
Hassan M, Watari H, AbuAlmaaty A, Ohba Y, Sakuragi N. Apoptosis and molecular targeting therapy in
cancer. Biomed Res Int. 2014;2014:150845. Doi: 10.1155/2014/150845. Epub 2014 Jun 12. Retraction in:
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 13

n-hexane extract (0.3 mg/mL), methanol extract (1.2 mg/mL) 5. The treated cells were

incubated for 48 h at 37 ◦C. After incubation, the medium was discarded and the attached

cells were collected and centrifuged. The formed cell pellet was lysed using lysis buffer

followed with different steps according to the instructions of the caspase-3 assay kit 6.

Materials:

RPMI medium

Ethanol

Hexane

Methanol

Lysis Buffer

VEGF Assay

Based on the study, Vascular endothelial growth factor (VEGF) is a 27KDa signaling

protein produced by cells that stimulates vasculogenesis and angiogenesis 34. T47D cells were

seeded at a concentration of 15,000 cells/mL in 25 cm2 tissue culture flasks. After 24 h of

incubation, the culture medium was discarded and replaced with fresh medium, obtaining

mung bean pods extracts with the following concentration: ethanol extract (0.4 mg/mL),

nhexane extract (0.3 mg/mL), and methanol extract (1.2 mg/mL). Following 48 h of

incubation, the supernatant was transferred into sterile tubes and VEGF level was measured
5
“Apoptosis Assays | Apoptosis Detection | Apoptosis Markers,” n.d.
https://www.promega.com/products/cellhealth-assays/apoptosis-assays/.
6
Oancea, Marcela, Suparna Mazumder, Meredith E. Crosby, and Alexandru Almasan. “Apoptosis Assays.”
Humana Press EBooks, November 4, 2006, 279–90. https://doi.org/10.1385/1-59745-213-0:279.
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 14

using a VEGFA ELISA kit. The standard curve was estimated using a human VEGFA

standard at different concentrations. The result will be expressed in each extract 35.

Materials:

Culture Flasks

Culture medium

Sterile tubes

VEGF ELISA kit

Waste Disposal

After conducting this study, the chemicals, reagents, and solvents used will be

labelled and disposed into their organic and inorganic containers. Solid wastes will be

collected in a waste container and will be disposed properly 36.

34
³⁴ Takahashi, Hidenori, Yoko Nomura, Junko Nishida, Yujiro Fujino, Yasuo Yanagi, and Hidetoshi
Kawashima. “Vascular Endothelial Growth Factor (VEGF) Concentration Is Underestimated by EnzymeLinked
Immunosorbent Assay in the Presence of Anti-VEGF Drugs.” Investigative Ophthalmology & Visual Science
57, no. 2 (February 1, 2016): 462. https://doi.org/10.1167/iovs.15-18245.
35
Stacker, Steven A., Michael M. Halford, Sally Roufail, Carol Caesar, and Marc G. Achen. “A Simple
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