TOTAL FLAVONOID CONTENT DETERMINATION

(OFFUNA MARVELOUS).

Materials
Volumetric flask, Beakers, Stirrer, Analytical weighing balance, Pippette,
Spectrophotometer

Chemicals
Methanol, Sodium nitrite (NANO2), Aluminium chloride (AlCl3) Sodium hydroxide
(NaOH), Rutin, Distilled water

Methods

To quantify the total flavonoid content, a modified method of the aluminum chloride
colorimetric method was employed. (Pa’eel et al., 2024). One milliliter of the plant
extract was weighed and transferred to a 10 mL volumetric flask. The remaining
volume was filled with methanol. 0.5 mL of 5% sodium nitrite (NaNO₂) was added to
1 mL of the plant extract and left for 5 minutes. 0.5 mL of 10% aluminum chloride
(AlCl₃) was added. After another 6 minutes, 2 mL of 1 M sodium hydroxide (NaOH)
was then added. Distilled water was added to bring the final volume up to 10 mL. The
solution was incubated at room temperature for 15 minutes. A blank sample was
prepared for comparison. The absorbance of the solutions was then measured at 415
nm using a spectrophotometer, with rutin as the standard.

REFERENCE

Pa’ee1, F., Hamsani, N.N., Wei, Y.J., and Sulaiman, H.A. (2024). Phytochemical
screening and antioxidant activity of selected underutilized plants in Nadir Plot
Taman Botani, Sri Medan, Johor. BIO Web of conferences. 94: 2002
 FERRIC REDUCING ANTIOXIDANT POWER (FRAP)

(OBLA HILARY)

Chemicals

Iron(II) sulfate heptahydrate (FeSO₄·7H₂O), Phosphate buffer, Potassium

hexacyanoferate (K₃[Fe(CN)₆]), Trichloroacetic acid (TCA), Iron(III) chloride
(FeCl₃)

Materials

Cuvette, Spectrophotometer, Water bath or heating block, Analytical balance,

Volumetric flasks, pipettes, Test tubes , Centrifuge

METHODS

The FRAP (Ferric Reducing Antioxidant Power) anti radical activity of the extract was
estimated using a modified method of Rohmah (2022). Different concentrations of the
extract was prepared (20,40,60,80 and 100μg/ml). A 0.5ml of 0.2M phosphate buffer
(pH = 6.6) was added together with 10% pottassiom hexacyanoferate solution and the
entire mixture incubated at 500C for 20 minutes. 0.5ml of 10% TCA solution was then
added and centrifuged. To the supernatant, 0.5ml of distilled water and 0.5ml of 0.1%
Fecl3 were added and incubated at 250C for 5-10 minutes. The absorbance was then
measured at 700nm and the Fe 2+ concentration determined from the Fe 2+ standard
curve.

REFERENCE

Rohmah, J. (2022). Antioxidant activities using DPPH, FIC, FRAP, and ABTS
methods from ethanolic extract of Lempuyang Gajah Rhizome. Jurnal Kimia Riset.
7(2):152-166
NITRIC OXIDE ASSAY (Greiss reagent) (JONATHAN ROSEMARY)

Materials
 Pipetting device.
 Spectrophotometric multiwell plate reader
 Clear flat-bottom 96-well plates.
 Greiss reagent I and II.
 Microcentrifuge.

Reagent Preparation

Nitrite Assay Buffer, Griess Reagent I, and Griess Reagent II: Warm to room
temperature prior to use. Nitrite Standard: Reconstitute with 100 µL of Nitrite Assay
Buffer to generate a 100 mg Nitrite Standard solution. Standard shoukd be kept on ice.
When stored at 2–8 °C standard can stand up to 4 months.

Standard Curve Preparation

1 mg of Nitrite Standard Solution is prepared by adding 5 µL/mg Nitrite Standard to

495 µL of Nitrite Assay Buffer. Nitrite Standard is prepared in 20, 40, 60, and 100µL/
mg.

METHODOLOGY

Griess Reagents and Nitrite Assay Buffer are separately added to each well containing
Standard and test samples in this order 10µL of greiss reagent I, 10µL of greiss reagen
t II and 80µL of Nitrite assay buffer. Griess Reagents are not to be premixed prior to
the experiment. Mix well and incubate at room temperature for 10 minutes. For each
well, 10 µL of Griess Reagent I and 90 µL of Nitrite Assay Buffer are added and well
mixed. Measure absorbance at 540 nm (A540) in end-point mode at room temperature.
The signal is stable for one hour after Reaction Mix was added.

Result
0 Standard reading substracted from all other readings. Nitrite Standard Curve plot. If
the sample background control reading is significant, background control reading is s
ubtracted from its paired sample reading. ΔA540 and Nitrite Standard Curve compared
to get mg of nitrite (B) in the sample well.
Nitrite Concentration (µL/ mg) = (B/V) × D
where: B = the Nitrite amount in the sample from Standard Curve (nmol) V = the
volume of sample added to the reaction well (µL) D= Sample dilution factor.

REFERENCE
Sigma-Aldrich. (2021). Nitric Oxide Assay Kit. Retrieved from ([Link]
[Link])
TITLE: ALPHA-GLUCOSIDASE INHIBITORY ASSAY (ODO PRINCEWILL)

CHEMICALS:
1. potassium phosphate buffer.
2. sodium carbonate.
3. Alpha-D-glucopyranoside (pNPG).

4. α-glucosidase.

EQUIPMENTS:

1. UV-1800 Shimadzu spectrophotometer.

2. Incubator.

MATERIALS:

1. Test tube.
2. Plant extract.
3. Pipette.

METHOD/PROCEDURE:

The methodology as described by Smita et al. (2018) was adopted. A reaction mixture
containing 0.01M phosphate buffer, 0.25ml of 0.5mM pNPG(Alpha-D-
glucopyranoside), 0.1ml of alpha-glucosidase, and 100µl of the extract was incubated
at 37°C for 20 minutes. After halting the reaction with 0.1M sodium carbonate
solution, the absorbance was measured at 400nm depending on the p-nitrophenol
pigment released. The percentage inhibition was determined by;

Inhibition (%) = Absorbance control- absorbance sample

×100
Absorbance control

REFERENCE:
Smita, K., Manjunath, K., and Sarangi, S. (2018). Evaluation of α-glucosidase
inhibitory potential of methanolic leaf extract of ocimum canum. International journal
of pharmacy and pharmaceutical science. 10(1):126-131.
DETERMINATION OF TOTAL PHENOLIC CONTENT (ANEKE HELEN)

EQUIPMENT

UV-Visible spectrophotometer, Water bath, Vortex mixer

MATERIALS:

Plant exact, Pipettes, Microcentrifugation tubes, Glass cuvettes

CHEMICALS and REAGENT

Galic Acid Solution (Standard), Folin Ciocalteu Reagent Sodium carbonate

METHODOLOGY

The Total Phenolic Content (TPC) of the extracts was measured using the Folin–
Ciocalteu colorimetric method, adopted from Phuyal et al. (2020). A stock solution of
gallic acid (1 μg/mL) was prepared by dissolving 10 mg of gallic acid in 10 mL of
methanol, and standard solutions of 20, 40, 60,80 and 100 μg/mL were [Link]
each standard concentration, a reaction mixture was prepared by adding 5 mL of 10%
Folin–Ciocalteu reagent (FCR) and 4 mL of 7% sodium carbonate (Na₂CO₃)
,bringing the total volume to 10 mL. After mixing and incubating at 40°C for 30
minutes,the absorbance was measured at 760 nm using a UV-visible
spectrophotometer, with methanol as the [Link] extract sample (20 40, 60,80 and
100 μg/mL)were analyzed similarly as the standard (in triplicate).A calibration curve
was plotted using average absorbance values versus gallic acid concentrations. The
TPC was calculated and expressed as milligrams of gallic acid equivalents (GAE) per
gram of dry sample (mg/g) using the formula:

V
Total Phenolic Content (TPC)=c
m

Where:

 C= concentration of gallic acid from the calibration curve (mg/mL),

 V = volume of the extract (mL),
 M= mass of the extract (g).

REFERENCE

Phuyal, N., Jha, P. K., Raturi, P. P., & Rajbhandary, S. (2020). Total phenolic,
flavonoid contents, and antioxidant activities of fruit, seed, and bark extracts of
Zanthoxylum armatum DC. The Scientific World Journal. Pg 1-7.
[Link]/10..1155/2020/8780704.
 TITLE: METHODOLOGY FOR FTIR(Fourier Transform Infrared
Spectroscopy) ANALYSIS. (ALFRED WILSON)

MATERIALS/CHEMICALS:

[Link] plant leaves

[Link] blender

3. n-Hexane

4. Mechanical shaker

[Link] paper

6. KBr (potassium bromide)

7. FTIR spectrometer

METHODS/PROCEDURE:

The extract was mixed with a powder called potassium bromide (KBr) and a thin,
transparent discs was formed. These discs were then placed in a machine called a
Fourier Transform Infrared Spectrometer (FTIR), which scanned them to identify the
different chemicals present. The machine looked at the discs over a wide range of
wavelengths (from 600 to 4000) to get the complete extract's composition. The above
methodology was described by Hair, N., and Nair, P.V.(2018).

REFERENCE

Hair, N., and Nair P.V. (2018). FTIR Spectroscopic Analysis of Leaf Extract In
Hexane in Jasminum Azoricum L. Int. Journal of Scientific Research and
Technology.4:170-171.
 METHODOLOGY ON THIOBARBITURIC ACID REACTIVE SPECIES

(TBARS) ASSAY (BY IMADE DIVINE).

Material:

Rat liver homogenate, test tubes, pipettes, plant extract.

Chemicals and Reagents Used:

Tris-HCl buffer, ferrous sulfate (FeSO₄), 8.1% sodium dodecyl sulfate (SDS), acetate
buffer, 0.8% Thiobarbituric acid (TBA), ether and Ethanol.

Equipment:

Centrifuge, homogenizer, surgical knife, water bath, UV-visible spectrophotometer.

Procedure:

The modified method of Ale (2020) was used for the Thiobarbituric Acid Reactive
Species (TBARS) assay. The rat's liver was washed, iced and homogenized in 5mM
Tris-HCl buffer and centrifuged at 4000 rpm for 10 minutes. 100μL of the supernatant
was incubated at 37°C for 1 hour with varying concentrations of plant extract (20–100
μg/mL) in a reaction mixture of 50mM Tris-HCl buffer and 10μM FeSO₄. Lipid
peroxidation was initiated by adding 200μL of 8.1% sodium dodecyl sulfate (SDS),
500μL of acetate buffer, and 500μL of 0.8% Thiobarbituric Acid (TBA). The mixture
was incubated at 100°C for 30 minutes, and the production of TBARS was determined
by measuring absorbance at 532nm.

REFERENCES

Ale, E. (2020). Assessment of antioxidant properties of N-Hexane extract of Morinda

lucida as a link to its pharmacological actions. Pharmacy & Pharmacology
International Journal, 8(3), 174–178. [Link]
DETERMINATION OF ALPHA AMYLASE INHIBITORY (EZEH JUDE)

MATERIALS

Analytical balance, pipettes, test tubes, a water bath, a UV-Visible spectrophotometer,

and a vortex mixer.

CHEMICALS

Phosphate buffer, alpha-amylase enzyme, starch solution, plant extract, 3,5-

dinitrosalicylic acid (DNS), distilled water, and acarbose.

METHOD

This assay was carried out using a modified method from Mahnashi (2022). Different
concentrations of the fraction was prepared (20, 40, 60, 80 and 100 µg/mL). 200µL of
0.02M sodium phosphate buffer (pH 6.9) was added together with 20µL of alpha
amylase enzyme and the entire mixture incubated at 25°C for 10 minutes.
Subsequently, 200 μl of 1% starch solution was added, and the mixture was incubated
again at 25°C for 10 minutes. To terminate the reaction, 400 μl of DNS reagent
(dinitrosalicylic acid) was transferred to the mixture. The resultant solution was kept
in a boiling water bath for five minutes and cooled. After cooling, 15 ml of distilled
water was added to dilute the mixture and the absorbance was noted at 540 nm. A
negative control was conducted by replacing the fraction with buffer, while a positive
control used acarbose instead of the fraction. A blank was also prepared by
substituting the enzyme with buffer. The experiment was repeated in triplicate, and the
percentage inhibition was calculated via the formula shown below.

Inhibition (%) = Abs 540 (control) - Abs 540 (extract) × 100

Abs 540 (control)

A plot was created showing the α-amylase inhibition percentage against the
concentration of the fraction or acarbose, and the IC50 value was determined
graphically.

REFERENCE

Mahnashi, M.H., Alqahtani, Y.S., Alyami, B.A., Alqarni, A.O., Ayaz, M., Ghufran,
M., Ullah, F., Sadiq, A., Ullah, I., Haq, I.U., Khalid, M., and Murthy, H.C.A. (2022).
Phytochemical Analysis, α-Glucosidase and Amylase Inhibitory, and Molecular
Docking Studies on Persicaria hydropiper L. Leaves Essential Oils. Evidence-Based
Complementary and Alternative Medicine. 10:1155
METHODOLOGY FOR LIQUID CHROMATOGRAPHY - MASS
SPECTROMETRY ANALYSIS (EZEH JUDE)

Materials

HPLC system, Analytical column, Cuvettes, Syringes, Volumetric flasks and pipettes.

Chemicals

Water, Methanol, Ammonium formate, Formic acid

METHODS

LC-MS Analysis Protocol (Generic Method) using the LC Waters e2695 Separation
Module with W2998 PDA Coupled to ACQ-QDA MS. The samples were analyzed via
liquid chromatography (LC) coupled with mass spectrometry (MS) following the
method outlined by Vignesh et al. (2018) with some modifications. The plant extract
was reconstituted in methanol and passed through a polytetrafluoroethylene (PTFE)
membrane filter (0.45 μm pore size). A 10.0 μL portion of the filtrate was injected into
the LC system, where it was separated on a Sunfire C18 column (5.0 μm, 4.6 mm x
150 mm). The analysis was performed at a flow rate of 1.0 mL/min with both the
sample and column maintained at 25°C. The mobile phase consisted of 0.1% formic
acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) with
gradient ratio 95:5 A/B ratio was held for 1 minute, then shifted to 5:95 for 13
minutes, maintained until 15 minutes, then returned to 95:5 at 17 minutes, 19 minutes,
and at the end (20 minutes). The PDA detector was set to a wavelength range of 210-
400 nm, with a resolution of 1.2 nm and a sampling rate of 10 points/sec. Mass spectra
were recorded in the range of m/z 100-1250 using an ESI source in both positive and
negative ion modes, with a capillary voltage of 0.8 kV for both modes. The probe
temperature was set at 600°C, with a flow rate of 10 mL/min and nebulizer gas
pressure at 45 psi. The MS operated in automatic mode with a fragmentation voltage
of 125 V. Data was processed using Empower 3, and compounds were identified
based on their elution order, retention time (tR), fragmentation pattern, and base m/z.
(Vignesh et al., 2022).

REFERENCE

Vignesh, A., Selvakumar, S., and Vasanth, K. (2022). Comparative LC-MS analysis of
bioactive compounds, antioxidants and antibacterial activity from leaf and callus
extracts of Saraca asoca. Phytomedicine Plus. 2:100167