CHAPTER ethanol. Around 30 g of grounded

CHAPTER 3

METHODOLOGY

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3.1 Collection of samples

Honey,
propolis and beebread of Trigona sp.
will be collected at Kiulu, Sabah.  

3.1.1 Honey

Honey samples
are harvested directly from the nests using sterile syringes, filtered and
stored in dark glass bottles. A volume of 16 mL of honey are transferred into a
centrifuge tube and 4 mL of distilled water are added to dissolve the honey. After
dissolve the honey in 70 % ethanol, the samples are filtered using Whatman
paper No. 1 and stored in dark glass bottles. The concentration of the honey
after dilution is 80 % (v/v). Subsequent dilution will be done to obtain the
concentration of honey in 5, 10, 20 and 40 % (v/v). The method is adapted from
Muli et al. (2008)

Concentration of honey, % (v/v)

Volume of honey, mL

Volume of distilled water, mL

Total volume, mL

5

1

19

20

10

2

18

20

20

4

16

20

40

8

12

20

80

16

4

20

 

3.1.2 Propolis

Propolis
samples are cut into small pieces and ground to powder before extraction. The
propolis are then extracted with 70 % ethanol. Around 30 g of grounded crude
propolis are dissolved in 100 mL of 70 % ethanol. The solution is put in shaker
(130 rpm) and left at room temperature for 7 days. The solution is filtered
through Whatman paper No.1 and dried under pressure using rotary evaporator. The
extracts were kept in -20 °C until further analysis. This method is adapted
from Muli et al. (2008).

3.1.3 Beebread

Bee bread
samples are cut into small pieces and ground to powder before extraction. The
bee bread are then extracted with 70 % ethanol. Around 30 g of grounded crude
bee bread are dissolved in 100 mL of 70 % ethanol. The solution is put in
shaker (130 rpm) and left at room temperature for 7 days. The solution is
filtered through Whatman paper No.1 and dried under pressure using rotary evaporator.
The extracts were kept in -20 °C until further analysis. This method is adapted
from Muli et al. (2008).

3.2 Microorganisms

The bacteria
that will be used to assay antimicrobial activity of honey, ethanol extract
propolis and ethanol extract beebread are Escherichia
coli (G-, ATTC 9027), Salmonella typhimurium
subsp Enterica (G-, ATTC 14028), Staphylococcus aureus subsp. Aureus (G+, ATTC 25923) and Bacillus cereus (G+, ATTC 25923).

3.3 Agar preparation

A volume of
250 mL of distilled water is added to graduated cylinder. Amount of bacto
typtone, bacto yeast extract and sodium chloride needed is 5.0 g, 2.5 g and 5.0
g respectively. The chemicals are weighed and added into the distilled water.
One chemical must be dissolved before the next chemical is added. The pH of
solution is adjusted to pH 7.0. The solution will be top up with distilled
water up to 500 mL and transferred back to conical flask. Then, 7.5 g of agar
are added into the solution. The solution with agar is stirred until agar
powder dissolved. The flask is covered with aluminium foil and autoclave for 20
minutes. The medium is then leave to cool down (50 °C) and pour into 40 plates.
The agar is left to solidify and sealed. 

3.4 Antimicrobial susceptibility test

3.4.1 Disc diffusion

Bacteria are
cultured on agar plate before transfer into nutrient broth. A loopful of
culture is picked from the nutrient agar culture and inoculated into nutrient
broth medium (LB broth) and incubated for 24 hours at 37 °C. The density of
bacterial cells is measured with McFarland’s standard solution. The size is
adjusted to 0.5 McFarland turbidity standard, approximate 108 colony
forming units (CF/mL). McFarland turbidity standard is prepared by mixing 99.5
mL of sulphuric acid and 0.5 mL of barium chloride.

          The cell suspension is introduced into
the nutrient agar and spread thinly on the plates using glass spreader. Discs
of 6mm diameter are impregnated with 25 µL of honey 5, 10, 20, 40 and 80 %
(v/v), ethanol extract propolis and ethanol extract beebread 1, 5, 10, 20 and
30 % (v/v) for every concentrations. The discs are placed on inoculated agar
plates and incubated for 24 hours at 37 °C. Streptomycin is used as control. The
diameter of the inhibition zones around the discs is measured (in millimeters)
after 24 hours and 48 hours respectively. There are 3 replicates for each
samples. Negative control is distilled water (Muli et al., 2008).

3.4.2 Determination of minimum
inhibitory concentration (MIC)

3.4.2(a) Broth microdilution method

Sterile 96
well round bottomed polystyrene microtitre plates is used. A 100 µL of 5 log
CFU/mL bacteria are added to 100 µL of samples in different concentration, in
each well with 3 replicates per dilution (Osés et al., 2016). Positive control is streptomycin with bacteria and
negative control is distilled water. Plates are incubated for 24 hours at 37
°C. The MIC value is taken.

3.5 Antioxidant assay

Radical
scavenging activity of samples was measured using 2,2- diphenyl-1-picrylhydrazyl
(DPPH). The extract (0.4 mL) is mixed with 3.6 mL of DPPH solution (0.025 g
DPPH in 100 mL ethanol). After 10 minutes of resting in dark place, absorbance
of the sample extract is determined using the spectrophotometer) at 515 nm.
Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid) (10–100 mg/L;
r2 =0.9881) is used as the standard and the results are expressed in
mg/g Trolox equivalents (Godo?iková et al.,
2017).

3.6 Quantitative phytochemical
screening

3.6.1 Total phenolic content

Modified
spectrophotometric Folin-Ciocalteu method is used to measure the total phenolic
content of samples. A volume of 1 mL samples is mixed with 1 mL Folin and
Ciocalteu’s phenol reagent. After 3 minutes, 1 mL of sodium carbonate solution
(10 %) is added to the mixture and top up to 10 mL with distilled water. The
reaction is kept in the dark for 90 minutes. After that, the reading of
absorbance is taken at 725 nm using UV/VIS spectrophotometer. Gallic acid is
used to calculate standard curve (20, 40, 60, 80 and 100 µg/mL, r2 =
0.996). This method is adapted from Moniruzzaman et al. (2014).

3.6.2 Total flavonoid content

The total flavonoid content (TFC) of honey samples is
determined using the method from Chua et
al. (2013). A volume of 5?mL of sample solution (0.1?g/mL) are mixed with
5?mL of 2% aluminium chloride (AlCl3). After
incubation for 10 minutes at 25 °C, a flavonoid-aluminium complex is formed. The
formation of the complex is measured at 415?nm by using an UV-Visible
spectrophotometer.  A calibration curve is created using a standard
solution of catechin (20, 40, 60, 80, and 100??g/mL; r2 = 0.998). 

3.7 HPLC
analysis

Detection and
quantification of various phenolic compounds present in the extracts is done
using reverse-phase HPLC technique. A 15 cm hypersil C18 reverse phase column
with 5 ? particle packing is used for the separation of phenolic compound. The
mobile phase consists of 20 % methanol, 1 % acetic acid and 79 % water is
passed through the column at the rate of 1 ml/min. Five phenolic standards (Gallic
acid, Catechol, p-Coumaric acid, Ferulic acid and Caffeic acid) are used for
their detection and quantification in the plant extracts. Each phenolic
standard stock is prepared (1 mg/ml) in HPLC grade methanol and stored at 0 °C
till further use. All five phenolic standards are mixed freshly for analysis to
obtain 100 ppm strength. The phenolic mix is injected as a single injection to
obtain standard chromatogram. The analysis is run for 50 minutes. The extract
are first diluted to 100 mg/mL for quantification of phenolic compounds in
individual samples. All the extracts are then filtered through cellulose syringe
filters (Sigma-Aldrich) with 0.45 ?m pore size. The volume for each sample and
standard to be injected is 20 ?l. A linear gradient elution scheme is used in
this method and detection is done at 280 nm. The output is given in the units
of ppm and converted to ?g/g (Ranade et
al., 2016).

 

3.8 Statistical analysis

The
experiment is done with three replicates, and all the experimental data is
expressed in mean ± standard deviation. Analysis of Variance (ANOVA) is used to
analyze the result with the probability p= 0.05 as the critical value for all
test. Tukey’s post hoc test is used for separation of statistically significant
means (Muli et al., 2008).