University of San Agustin

COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS

General Luna St. Iloilo City, Philippines 5000

Chapter 3

MATERIALS AND METHODS

3.1. Research Design

To obain the objectives of this study, the researchers will be collecting 4 kg

of Munggo beans (Vigna radiata) around Barotac Viejo, Iloilo City. It will then be

brought to the Department of Agriculture Region VI Iloilo City for the plant

authentication. The samples will undergo drying in an oven, will then be pulverized by

an herbal grinder and then will be decocted and filtered to obtain the aqueous

extract. The prepared extract will then be freeze dried to obtain the dried crude

extract. The extract will then be screened for its antibacterial activity using agar well

diffusion assay to confirm its bioactivity. It will then undergo solvent partitioning with

hexane and methanol. After which, the antibacterial activity of the sub-extracts will be

determined and the most active subextract will be used for further investigation.

Minimum inhibitory concentration determination of the most active subextract will be

performed using microbroth dilution assay and then will be screen for the presence of

antibacterial phytochemicals using thin layer chromatographic separation and

identification techniques. Bioautography will then be conducted to identify the band

that exhibits antibacterial activity Prep TLC will then be performed to isolate the

bioactive band for FTIR analysis. For a clearer understanding on the design of this

study, a schematic diagram is presented below (Figure 1).

 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

Collection of plant samples

Mung beans (Vigna radiata)

Plant authentication

Processing of Samples (cleaning and oven drying)

Pulverization

Decoction for 30minutes

Suction

Freeze-drying

Confirmatory test for Antibacterial Activity:Agar Well

Diffusion Method

Solvent partitioning (Hexane and methanol)

Identification of the Antibacterial

Subextract: Agar well diffusion method

Antibacterial Profiling of the most active subextract

Thin Layer Chromatography

Minimum Inhibitory Concentration separation and Identification of
antibacterial phytochemicals
determination

Bioautography

Prep TLC

Figure 1. The Schematic Diagram

FTIR analysis
 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

3.2. Sampling Collection and Drying

The researchers will randomly hand-pick the beans of Munggo around

Barotac Viejo, Iloilo City which will then be authenticated at the Department

of Agriculture- Region 6. The plant sample will then be cleaned and dried

using an oven at 44.5 oC for 4 hours. (Azwanida NN.,2015)

3.2.1. Plant Extraction (Decoction method)

The dried Mung bean sample will be be pulverized by an herbal grinder

and 5 grams of dried powder will be decocted with100ml of water at 100 ˚C

for about 30 min until about 50% of the water is lost. After decoction, the

extract is then removed from heat and strained using a filter (Arunkumar

Nagalingam, 2017).

3.3 Freeze-drying

The aqueous extract will be subjected to freezing at -40 ˚C for 24 hours (Kunal et

al., 2015), and will then undergo freeze-drying. The drying is done by clearing the air

with vacuum pump to lower down the pressure to about 10 – 4 atm with the

temperature of -45 to -20 ˚C (Kunal et al.,2015). The temperature of the aqueous

extract is raised to about 38 ˚C (Smith and Brian, 1994) by direct conduction through

the bottom trays radiation from the heat lamp. When the chamber is already emptied

by air, pressure will be below the threshold at which water can simultaneously exist in

a solid, liquid, and gaseous phase (vapour); commonly known as triple point of water.

Once the pressure already below this point, the heat causes the ice crystals trapped

in the frozen aqueous extract to change directly into water vapour. Then vapour is

drawn off and condensed, living the freeze – dried aqueous extract behind. (World

Journal of Pharmaceutical Research, 2015)

 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

3.4 Data Gathering Procedure

3.4.1 Confirmatory Antibacterial Screening (Agar Well Diffusion Method)

In preparing the agar, streak plate method will be used and Mueller

Hinton Agar will be used as the medium since it supports the growth of most

non-fastidious pathogens. The agar will be cooled in a temperature of 40-50

˚C and poured in a petri-dish/plate. Allow to set on a level surface, to a depth

of approximately 4 mm. A 150 mm plate requires approximately 25 mL of

medium. When the agar has solidified, the plates will be dried for 10-30

minutes at 35 ˚C by placing them in the upright position in the incubator with

the lids tilted . (Acharya, 2013).

Twenty-four (24) hours cultures of the test organisms namely

Staphylococcus aureus and Escherichia coli will be prepared. A loop full of

the culture will be streaked uniformly over the surface of sterile Muller Hinton

Agar (MHA) for S. aureus and E. coli. With pipette method, 2mg, 5mg, 10mg

of freeze dried samples of each various fractions will be prepared in pure

Dimethyl sulfoxide (DMSO). Tetracycline 10mg/ml will be used as the

positive control for the assay.. About 30µl of the crude extracts from freeze-

drying will be used used to fill holes bored by 65mm cork borer in the

inoculated agar. The plates where made triplicate and incubated 37oC for 24

hours and the evaluation for antibacterial activity of the extract will confirmed

by the zone of inhibition. (Oseni Lateef, 2013).

 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

3.4.2 Solvent Partitioning

The aqueous extract will undergo solvent partitioning with hexane ,

and methanol. The dried extract will be suspended in methanol and

equal volume of hexane will be added to separate the compounds.

Using a separatory funnel, the two immiscible liquids will be thorough

shaken with frequent releasing of pressure inside the funnel. The

mixture will then be allowed to stand for several minutes until a distinct

separation line between the solvents is observed. The methanol fraction

will be drained off onto a clean and properly labeled Erlenmeyer flask

and the hexane fraction will be poured onto a separate flask. The

exhaustive solvent partitioning will be done. until the color of the

hexane fraction becomes light. The fractions will then be concentrated

to about 5 mL volume in a rotary evaporator and will be further dried

using nitrogen gas to obtain dried subextracts. The dried sub- extracts

will be stored at 4°C in air tight containers. (H. Usman, M.Sc., 2000)

3.4.3 Determination of the Most Active Antibacterial Subextract: Agar

Well Diffusion Method

The same procedure wth 3.4.1 will be done to identify the subextract

that will exhibit antibacterial activity. The dried subextracts will be

reconstituted with pure DMSO to obtain a concentration of 100 mg/mL stock

solution and a volume of the stock solution will be transferred onto their

corresponding wells to evaluate their antibacterial activity. The same positive

control will also be used in this assay. (M. Aldawsari,2014)

 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

3.4.4. MIC Determination of the Most Active Subextract

Exponential phase grown cultures in agar plates of the test bacterial

strains will be prepared by inoculating 2-3 colonies onto a MHA plate

and incubate it for 18-24 hrs at 37 0C. After which, a loop full of colonies

will be inoculated onto a 5 mL MHB medium and the bacterial density

will be determined using a microplate reader at 620 nm. The bacterial

density of the suspension will then be adjusted to 0.5 OD620nm (1 x 106

cfu). The dried extract will be prepared by reconstituting it in DMSO to

obtain a concentration of 100 mg/mL. Then 10 µL (per well) of the stock

solution will be delivered on the wells (first column) of a 96-well plate.

After which, 5 µL of DMSO will be delivered from column 2-9. A volume

of 5 µL of the extract will then be transferred from column 1 to 2 and will

be mixed thoroughly by sucking in and out carefully the mixture by

means of a micropipettor. Then 5 µL of the mixture in column 2 will then

be transferred and mixed onto the third column and the 2-fold serial

dilution procedure will continue until column 9. Then 5 µL of the mixture

in column 9 will be discarded. In the wells of column 10 and 11, 5 µL of

DMSO (negative control) and Tetracycline 10mg/ml (positive control) will

be pipette respectively. After which, 195 µL of the bacterial suspension

(OD620nm 0.5) will be added and the mixture will be incubated at 37 0C for

18-24 hr. After incubation, absorbance of the reaction mixture in each

well will be determine using a microplate reader and % growth inhibition

of each of the concentration and controls will be calculated using the

following formula:
 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

Absnegative - Absextract 23

% growth inhibition = -------------------------------------------------- x 100

Absnegative

High absorbance values suggest growth of the test organisms and

low absorbance values suggests antibacterial activity of the treatments.

(R. Wever 1980)

3.5. Identification of Antibacterial Phytochemicals using Thin Layer

Chromatographic Separation and Identification Techniques

TLC plates will be prepared by cutting the plates into 1 in (width) by

3 in (height) dimensions. Then lines will be marked by a pencil 1 cm from the bottom

and top of the plate. Bottom line will serve as the marker for the origin spot and top

line will serve as the solvent front height. Then a stock solution of the most active

subextract will be prepared by reconstituting the dried subextract in methanol to

obtain a concentration of 2.5 mg/mL. Then 10 µL of the sample will be spotted onto

the TLC plate at the origin line and the chromatogram of the sample will be

developed using Butanol: Acetic acid: water (60: 15: 25 v/ v) as mobile phase. The

separated compounds will then viewed under UV light at 254 nm and 365 nm. The

distance traveled by each band will then be measured and retention factor (Rf)

values of each band will then be calculated using the follo2wing formula:
 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

To determine presence of antibacterial phytochemicals namely

flavonoids, phenolic compounds and amine-containing compounds, the following

spray reagents will be used onto the prepared TLC chromatogram of the most active

subextract :

3.6. Bioautography

Two TLC chromatogram of the most active subextract will be

prepared using the same mobile phase. One plate will serve as

reference plate and the other chromatogram will be overlayed with a

soft agar seeded with the test bacterial strain to about 1 mm thickness

and then will be incubated for 18-24 hours at 37 degree Celsius. After
 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

incubation period, the TLC plate will be flooded with resazurin dye

(0.15mg/mL) and will be allowed to stand for 1-2 hours inside a

biosafety cabinet. A visible blue color indicates antibacterial activity

while pink color indicates no antibacterial activity (Choma et. al., 2010).

3.6.1 Prep TLC procedure

From the TLC, the Researchers will scrape the desired band which travelled

the farthest in the TLC plate. Then collect the scraped material and place it in a

centrifuge tube then suspend it in 500 microliter pure methanol. After which, the

Researchers will centrifuge the sample at 8000 rpm for 5 minutes, then collect

the supernatant liquid. Purge with nitrogen gas to concentrate to about 10

microliter.

3.7. Fourier-transform Infrared Spectroscopy

The prepared sample from prep TLC procedure will then be

subjected to FTIR analysis to identify the functional groups present.

Radiation emitted from the source will split into two with a big splitter in

the interferometer. The fix and moving mirrors will then be reflect of

each of the beam back to the beam splitter, where the two beams

recombine into one and fall to the detector. Then, the two beams will

combine constructively or destructively varying as the optical path

difference, when the moving mirror is moved combined beam will be

transmitted through the sample and will be detected as an interferogram

and contains all infrared information of the sample. The infrared

spectrum will be obtained from the interferogram of fourier. Alcohol,

 University of San Agustin
COLLEGE OF HEALTH AND ALLIED MEDICAL PROFESSIONS
General Luna St. Iloilo City, Philippines 5000

aldehydes, alkyne, amines and ester are the functional groups present.

Alcohols have the highest number of waves and Esters has lowest

number of waves. (Visvesshwari M. 2017)