INTERNATIONAL UNIVERSITY

School of Biotechnology
Biochemistry Department

NUTRACEUTICALS LABORATORY
REPORT 2: DETERMINATION OF TOTAL PHENOLIC
CONTENT
INSTRUCTOR: MSC. LE TRAN HONG NGOC
TEACHING ASSISTANT: HA THI NGOC VY
DATE OF SUBMIT: 19/12/2019

GROUP MEMBER
1. Nguyễn Tấn Phú BTBCIU16057
2. Trương Thị Ngọc Hằng BTBCIU16041
3. Lê Thu Trang BTBCIU16019
4. Nguyễn Hữu Anh Minh BTBCIU16081
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School of Biotechnology
Biochemistry Department
I. ABSTRACT
Phenolic compounds were known as a secondary metabolite which contained in plants,
antioxidant properties, and essential functions of the human body. The determination of
phenolic content in strawberry and pomelo pulp was the main aim of the experiment. Gallic
acid solution with different concentration was considered as standard reagent, Follin-Ciocalteau
reagent was detectable with a spectrophotometer, and sodium carbonate. Pipette technique
was mainly used in the experiment to transfer solution sample accurately. The standard curve
of samples in the absorbance at 765 nm was given by= 0.0037x +0.5266 with R 2=0.98993. The
total phenolic compounds content in strawberry was 7181 μg GAE/g, and the Pomelo was 738
μg GAE/g. This methodology used the Folin-Ciocalteu reaction complies with the requirements
for analytical use and for ensuring the reliability of the results .

II. INTRODUCTION
Phenolic compounds are chemically defined as compounds containing hydroxylated
aromatic rings, the hydroxy group being attached directly to the phenyl, substituted phenyl, or
other aryl group. They are found mainly in plant tissues, including fruits and vegetables.
Phenolic compounds can be divided into different subgroups, such as phenolic acids, flavonoids,
tannins, coumarins, lignans, Quinones, Stevens, and curcuminoids. Since phenolic compounds
are secondary metabolites, which have a vital function in the interaction between a plant and
its environment. In plants, phenolic compounds combined with mono‐ and polysaccharides,
linked to one or more phenolic group, or can occur as derivatives. Phenolic compound's ability
to reduce lipid oxidation, chronic disease. In defense, these molecules related to their
antimicrobial, antinutritional or unpalatable properties. The antioxidant activity of phenolic
compounds is attributed to the capacity of scavenging free radicals, donating hydrogen atoms,
electrons, or chelate metal cations. There some factors can be influenced directly on phenolic
content such as environmental factors, processing, and storage. The degree of ripeness could
make the concentration of phenolic acid decrease, it could be converted into anthocyanin. The
quality of fruits could be decreased due to the oxidation reaction.
The absorption and bioavailability of phenolics in humans is an absorptive process of these
molecules across the intestine into the circulatory system, after food ingestion. After these
absorptive processes the phenolics go through four possible pathways. First, Phenolics are
excreted through feces, Second, Phenolic molecules are absorbed by intestine/ colon mucosa,
and pass through the portal vein and reach the liver, Third, Phenolics are further conjugated in
the liver with methyl, glucuronide or sulfate groups and released into the blood stream for
tissue absorption. Finally, the phenolic content is excreted in urine.
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School of Biotechnology
Biochemistry Department

Figure 1: Predicted routes of absorption of dietary phenolics

The total phenolic content of the samples was determined by the Folin–Ciocalteu's
reagent method. An aliquot of a dried extract ethanolic solution was added to 375 μl sodium
carbonate, and 375 μl of the Folin–Ciocalteu reagent also added to react with phenolic
compounds and non-phenolic reducing substances (including ascorbic acid) to form
chromogens that can be detected spectrophotometrically under 765 nm. The Galic acid was
used as standard, which is a representative and prominent phenolic acid. After experimenting,
the data were collected and used it for conducting standard curve to determine the total
phenolic content of Strawberry and Pomelo sample.
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Biochemistry Department
III. Material and Method
 Material
Chemicals and Reagents: Methanol 80% (v/v) were prepared from the stock solution and
was used as diluent. Gallic acid was used for constructing a standard curve. Folin-Ciocalteau
reagent and 7.5% Na2CO3 were used as color developing. 2g of Pomelo and Strawberry were
prepared to determine the phenolic content in these fruits.

Equipments: Centrifuging machine, plate reader and water bath used were located in the
laboratory belonging to the facilities of the International University, VNU. The 96-wells plate,
micropipettes and the corresponding tips, Eppendorf tubes 1.5 ml, beakers with volume of 250
ml and 500 ml (for the containing of methanol and for wastes) were available in the laboratory.

 Method
1. Prepare standard solutions for constructing the standard curve:

Firstly, Gallic acid solution was prepared with different concentrations from 50µg/ml to
250µg/ml of stock solution of 2000 µg/ml, and diluted with 80% Methanol. Secondly, using
micropipette, 150µL of different concentration gallic acid was pipetted to 375µL Folin-
Ciocalteau reagent and 375 μL 7.5% Na2CO3 in different eppendorfs and was incubated at
room temperature. It was noted that 7.5% Na2CO3 must be added before adding Folin-
Ciocalteau reagent. Lastly, after incubating for 30 minutes, 200µL of 5 mixtures in each
eppendorf was added to 96-well plate, then were measured the absorbance at 765 nm by plate
reader.
Table 1: Different Gallic acid concentration in detail.

Gallic acid Volume of stock Volume of 80% Total volume (mL)

concentration solution (mL) Methanol (mL)
(µg/mL)
50 5 195 200
100 10 190 200
150 15 185 200
200 20 180 200
250 25 175 200

2. Prepare samples:

Firstly, 2g of Pomelo and 2g of Strawberry were well grounded by mortar pestle, then were
mixed with 10mL of 80% Methanol in centrifuge tubes and were placed in a water bath at 60 oC.
Secondly, two other centrifuge tubes that contained the amount of water which was equal to
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two Pomelo and Strawberry tubes in weight. After 25 minutes, the mixtures were centrifuged
by centrifuge machine for 10 minutes at 4000g then were collected the supernatant.

Secondly, the supernatant of Pomelo was diluted with 80% Methanol at 1:2 dilution factor and
the supernatant Strawberry were diluted at 1:10 dilution factor. Lastly, 200µL of 2 mixtures
were added to 96-well plate in triplicate, then were measured the absorbance at 765 nm by
plate reader. Micropipette was required to obtain the correct amounts of those solutions.

IV. RESULT
Absorbance of blank at 765nm = 0.719
Table 2: Absorbance of standards at 765nm

Gallic Acid concentration Absorbance Absorbance – blank

(μg/ml)
50 1.441 0.722
100 1.613 0.894
150 1.760 1.041
200 2.025 1.306
250 2.158 1.439

Figure 2: The standard curve with the absorbance against concentration of glucose
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Table 3: Absorbance of samples at 765nm after minus blank

Sample 1 2 3 Average
Strawberry 1.045 1.049 1.080 1.058
Pomelo 0.834 0.777 0.788 0.800

*Calculate

Σ (xi −x avg)2
 Standard deviation: 
SD strawberry = 0.019
√ N −1
SD Pomelo = 0.030

 Equation: y=0.0037x + 0.5266

 Coefficient of determination: R2 = 0.99
Thus, the concentration of sample: x = (y – 0.0.5266) / 0.0037
x: concentration of phenolic content (μg /ml)
y: absorbance at 765nm

Sample Concentration (μg /ml)

Strawberry 143.6
Pomelo 73.8
Table 4: Concentration of sample
*Calculate the total phenolic content in strawberry
Since the sample included 2g of raw strawberry grinded and 10ml methanol, then diluted with
diluted factor 1:10, the total phenolic content in strawberry must be calculated followed the
below formula.

TPC strawberry =
Conc ( mlμg ) x 10 ml x 10 (dilute factor ) = 143.6 μg /ml2 gx 10 ml x 10 = 7181 μg GAE/g
2g
Similarly, the total phenolic content in pomelo must be calculated:

TPC pomelo =
Conc ( mlμg ) x 10 ml x 2( dilute factor) = 73.8 μg /ml2 gx 10 ml x 2 = 738 μg GAE/g
2g
Table 5: The total phenolic content in strawberry and pomelo

Sample TPC (μg GAE/g)

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Biochemistry Department
Strawberry 7181
Pomelo 738

V. DISCUSSION
Phenolic compounds are not only one of the most common metabolites in plants, but they
also possess a variety of sub-groups with different structures and functions that can benefit
human in many areas. Among those sub-groups, polyphenol - that presents in diverse species of
the Plantae kingdom-has recognizable features with the advantageous health benefits for
human as a powerful antioxidant. Therefore, it is considered as an important parameter to
appropriately measure the phenolic contents in the fruits, functional food or dietary
supplement. With the information from the phenolic acid content, many more conclusions
about the quality and health benefits of the subject can be established.
In this experiment, in order to determine the polyphenol’s content in some types of
common fruits, the Folin-Ciocalteu (F-C) method was utilized to analyze the total phenolic
content in plant extracts. In the most basic definition, F-C reaction is an antioxidant assay that
based on the use of F-C reagents and the principle of transferring electrons between oxidant
and reductant to quantify the concentration of either in the two[1]. Within the course of this
experiment, the reaction conducted was used to measure the reductive capacity of an
antioxidant, here is, polyphenol-containing samples. Although the process is not new and
technologically favorable, the procedure of measuring concentrations of polyphenol by the F‐C
method in fruit extracts proved to be simple, inexpensive and can quickly determine the
phenolic concentration in the desired samples.
Despite being uncomplicated, the procedure of F-C method may be encountered by some
problems, mostly related to the reagent’s sensitivity to external factors and the handling of the
experimental steps, like lab skills for example. In particular, F-C reagent is very sensitive to
sunlight and a degrade in this substance can lead to fallacy in the final result. Hence, all
containers that used to keep F-C reagent are covered completely with aluminum foil in
prevention of exposure to sunlight. Besides, the repeated opening of the container’s cap as
each group alternatively used the reagent may have caused the unwanted exposure to external
factors and as a result, the quality of F-C reagent was degraded before the reaction and
measuring step. Another noticeable point of the F-C method that has been observed was Gallic
acid concentration. This acid was used as a standard to plot the standard curve and
absorbance-concentration function for determining the samples’ concentrations of unknown
phenolic content. From the stock of 2000 µg/ml, the Gallic acid needed to be diluted with
methanol into different concentration that can fit well to the standard curve. The reason
methanol was used as dilution solvent is that the original samples (fruits) were extracted by
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methanol solvent already, and using any other agent for diluting with cause problem as the
solvent is not uniform. For this experiment the decided dilution of Gallic acid were: 5, 10, 15,
20, 25 µl Gallic acid/ 200 µl total. Five evenly distributed points of the standard curve were used
to make it visually easier to observe the concentration of unknown samples, and the total
volume of this standard was optimized based on its availability for 4 groups with chances of
error that lead to overuse. As a result of trace amount of reagent volumes, the requirement for
mixing them together can not be met by hand shaking, thus pipette mixing and vortex were
used to guarantee the reactions can occur with evenly mixed substances. Triplication for the 2
samples were also conducted to minimize errors.
From the result of absorbance and concentration plot, the calculated R 2 = 0.9899 indicated
for a well-fit regression prediction of the data on the trend line. That by means, shown the
trivial error which has been made in this process. The standard curve established from gallic
acid was concluded to be suitable for determining concentrations of unknown phenolic
contents in the samples. Results, therefore, suggested for a good procedure by using gallic acid
and F-C method to plot the function of absorbance-concentration and further to determine
phenolic content of desired samples. Generally, the utilization of F-C method for determining
polyphenol content quantitatively in fruits extract can be achieved by basic lab equipment and
reagents within little amount of time and high accuracy.
The total phenolic contents (TPC) in fruit extracts varied by many factors [2]. For instant,
although belonging to the same Citrus maxima family, the Vietnamese pomelo possesses
different TPC than Taiwanese or Malaysian pomelo. Theoretically, varied types of nutritious
fertilizers, soil compounds content, weather conditions can affect TPC before harvesting. And
for after harvesting activities, the processing and storage stage are also possible sources of TPC
differences from many origins. With that being said, the normal reference range for TPC in
pomelo pulp and peel may not be fully correlated with results from this experiment.
By the same procedure based on the F-C method, previous studies suggested the range of
TPC from 6.2 to 7.39 mg GAE/g in dry weight (DW) of Malaysian pomelo’s pulp and 4.50 to 9.99
mg GAE/g DW of Taiwanese pomelo pulp [3]. However, the TPC values of the pomelo from this
experiment located at ~0.74 mg GAE/g DW. It can clearly be seen, that there was a significant
gap between the reference value and the experimental result from this experiment. The same
results-reference value comparison can be applied to the strawberry extract sample. In some
studies, the TPC of strawberry extract can be as low as 3 mg GAE/g DW or as high as 14 mg
GAE/g DW [4,5,6]. Obtained concentration of ~7.2 mg GAE/g DW, the experiment’s result
legitimately fell within the reference ranges and as the reference values are widely varied, this
result was supposed to be excellent.
Regardless the geographical differences of the fruit sources, there were plenty of factors
that can affect the exact TPC in the fruit extract in this experiment. Differences in time exposing
to the external environment can lead to the various levels of oxidation of the polyphenols; or
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Biochemistry Department
the over-simplified extracting process with mortal, pestle and centrifuging may have not totally
extracted the polyphenol in samples, which led to fall offs in the real concentration of the
phenolic content in the fruit versus the measured one. In addition, lab skills, which directly
affected the exact amount of substances in the experiment have been identified as error’s
sources for the final calculated concentrations- for example, pipetting techniques. And
furthermore, to a lesser extend, other chemical-related problems like the decreased quality of
Folin reagent, Na2CO3 and gallic acid can also be traced as the source for the differences
between expected value of TPC from the reference by other studies versus the result from this
experiment. In two sample, while the strawberry shown normal result, pomelo’s pulp was
indicated to be much lower than the range. Because using the same protocol and chemical, the
significant difference of the results compared to references from other studies by pomelo
pulp’s sample could have not derived from the techniques or chemical sources, rather it may
have been a result of oxidative levels of dry fruit samples. Besides differences in geographical
features, the long exposing time in combination with moderate and but not perfect preserving
techniques could have been the problem to lowering in the concentration of phenolic
compound in pomelo sample.

VI. CONCLUSION
In summary, Folin-Ciocalteu method was an advantageous assay for quantitatively
investigate the phenolic contents of fruit extracts. The results have demonstrated a relatively
short experimental procedure with most of the time-consuming steps came from waiting for
incubating or centrifuging, but the effectiveness given from this assay is remarkable. The idea of
determining concentration from absorbance-concentration standard curve was utilized as it is
simple and effective. Within the used samples- strawberry and pomelo pulp- the phenolic
content was measured to be within the range and lower than the range, of other studies in that
order, which was a result from only the geographical and not technological factors. And beside
that, the overall result can aid to the effectiveness of the Folin-Ciocalteu method in determining
total phenolic contents in fruit extract with high accuracy and reproducibility. In the future, this
method will still have its place among other advanced techniques with high technology due to
its remarkable properties and simplicity; and with some modification, increased applications for
this method can be achieved.

VII. REFERENCES:
[1] Lamuela-Raventós, R. M. (2017). Folin-Ciocalteu method for the measurement of total phenolic
content and antioxidant capacity. Measurement of Antioxidant Activity & Capacity, 107–
115. doi:10.1002/9781119135388.ch6 

[2] Experiment 2: Determination of total phenolic content, Instructor MSc. Le Tran Hong Ngoc,
Nutraceuticals, Biochemistry department, International University, VNU

[3] Comparison of antioxidant properties of pomelo [Citrus Grandis (L) Osbeck] varieties, Toh, J.J., 1
Khoo, H.E. and 2*Azrina, A. International Food Research Journal 20(4): 1661-1668 (2013)
 INTERNATIONAL UNIVERSITY
School of Biotechnology
Biochemistry Department
[4] Kadivec, M., Može Bornšek, Š., Polak, T., Demšar, L., Hribar, J., & Požrl, T. (2013). Phenolic Content of
Strawberry Spreads during Processing and Storage. Journal of Agricultural and Food Chemistry, 61(38),
9220–9229. doi:10.1021/jf4035767 

[5] Antioxidant Capacity and Phenolic Content of Selected Strawberry Genotypes (2005) Djamila Rekika,
Shahrokh Khanizadeh,1 Martine Deschênes, Audrey Levasseur, and Marie Thérèse CharlesAgriculture
and Agri-Food Canada, Horticultural Research and Development Centre, 430 Boulevard Gouin, St-Jean-
sur-Richelieu, QC, Canada, J3B 3E6,40(6):1777–1781.

[6] Antioxidant activity and phenolic content of strawberry genotypes from Fragaria x ananassa. (2009)
A.M. Panico,F. Garufi,S. Nitto,R. Di Mauro,R.C. Longhitano,G. Magrì,A. Catalfo,M.E. Serrentino &G. De
Guidi Pages 203-208. Panico, A. M., Garufi, F., Nitto, S., Di Mauro, R., Longhitano, R. C., Magrì, G., … De
Guidi, G. (2009). Antioxidant activity and phenolic content of strawberry genotypes
fromFragariaxananassa. Pharmaceutical Biology, 47(3), 203–208.10.1080/13880200802462337 

[7] Laboratory manual of Nutraceutical by MSC. Le Tran Hong Ngoc, Experimental 2, Determination of
Phenolic content