Khả năng kháng oxy hóa và ức chế tyrosinase của cây dứa (ananas comosus) TT TA

MINISTRY OF EDUCATION AND TRAINING
CAN THO UNIVERSITY

SUMMARY OF THESIS THESIS
Specialization: Biotechnology
Industry code: 7420201
NGUYEN THI THU HAU

ANTIOXIDANT AND ENZYME TYROSINASE
INHIBITOR OF PINEAPPLE (Ananas comosus)

YEAR 2022


WORK COMPLETED AT CAN THO UNIVERSITY
Main instructor: Assoc.Prof.Dr. Tran Nhan Dung
Sub-instructor: Assoc. Prof.Dr. Huynh Van Ba
The thesis is defended before the university doctoral thesis evaluation
board
Meeting at: …………….., Can Tho University).
At ..... hour ..... day ..... month ..... year .....
Review 1:
Review 2:
Review 3:
Confirmation of review by the Chairman of the Council

Thesis can be found at the library:
- Learning Resource Center, Can Tho University;
- Vietnam National Library.

i

LIST OF PUBLICATIONS
Domestic magazine
1. Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Tong
Van Hai., 2020. A survey on antioxidant activation from ethanol
extraction from the leaves, stems and peels of pineapple (Ananas
comosus) at Tac Cau, Kien Giang region. Journal of Agriculture and
Rural Development, No. 8 (2020), 74-80.
2. Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Trinh
Chi Bao, 2020. Survey activity antioxidant from extracts ethanol the stems
and fruit pineapple (Ananas comosus) at Tac Cau, Kien Giang region.
Journal of Medicine, No. 8 (1), 45-49.
3. Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Nguyen
Minh Chon, Nguyen Duc Do, Vo Thi Yen Linh, Le Thi Thu Doan,
Nguyen Thi Truc Anh, 2020. Investigation of antioxidant activity and
tyrosinase inhibition of methanol extract from pineapple leaves at Tac
Cau, Kien Giang province. Vietnam Journal of Agricultural Science and
Technology, No. 10 (2020), 109-114.
4. Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Vo Thi
Yen Linh, Le Thi Thu Doan, Thi Ngoc Quyen, Bui Tran Hoang An, 2020.
A survey on antioxidant activation from ethanol extraction from the peels
and fruit of pineapple (Ananas comosus) of green and ripe stages at Tac
Cau, Kien Giang region. Journal of Tra Vinh University, 12 (2020), 1-11.
5. Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Nguyen
Minh Chon, Nguyen Duc Do, Pham Thi Be Tu, Tran Thi Ngoc Ly, Lam
Huynh Duc, Pham Thi Ai Vi, Ly Le Quyen, Le Thi Ngoc Nga, 2021.
Antioxidant and inhibition of tyrosinase form methanol extraction of stems
and fruits pineapple (Ananas comosus) at Tac Cau, Kien Giang province.
Journal of Agriculture and Rural Development, No. 3+4 (2020), 68-74.

6. Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Nguyen
Minh Chon, Nguyen Duc Do, Pham Thi Be Tu, 2021. Investigation of
antioxidant and tyrosinase inhibitory activities from the methanol extract
of pineapple peel (Ananas comosus (L.) Merr.) collected in Tac Cau area,
Kien Giang. Journal of Medicine and Pharmacy, March 17 (2021), 21-28.
Proceedings of the domestic conference
ii


1. Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Ninh
Khac Huyen Tran, Lam Thi Kim Ngan, Vuong Tu Ky, Tran Ngoc Trong,
Le Thi Thu Doan, 2020. A survey on antioxidant activation from ethanol
extraction from the peels and fruit of pineapple (Ananas comosus) of green
and ripe stages at Tac Cau, Kien Giang region. Proceedings of the
National Biotechnology Conference 2020, 194 – 199.
2. Nguyen Thi Thu Hau, Tran Nhan Dung, Huynh Van Ba, Vo Thi
Yen Linh, Le Thi Thu Doan, Thi Ngoc Quyen, Bui Tran Hoang An, 2020.
A survey on antioxidant activation from ethanol extraction from the peels
and fruit of pineapple (Ananas comosus) of green and ripe stages at Tac
Cau, Kien Giang region. Proceedings of the Conference on Application
of Biotechnology in Agriculture and Fisheries Production (2nd time),
2020, 5.

iii


CHƯƠNG 1: CHAPTER 1: INTRODUCTION
1.1 Pose the problem
Pineapple (Ananas comosus (L.) Merr.) of the bromeliaceae family,
native to Paraguay, is the third most valuable fruit in the world after

bananas and citrus (Vrianty et al. al., 2019). In the body of organisms
(including humans), free radicals are always produced, which leads to the
destruction of organelles and internal structures of cells due to mutations
that lead to dangerous and degenerative diseases. cells (Ghasemzadeh,
2012; Vrianty et al., 2019).
Antioxidant compounds are compounds that slow down or prevent the
growth of free radicals that protect cells and the body. The disadvantages
of cell degeneration can be prevented by supplementing with antioxidants
from food and medicine. (Ghasemzadeh, 2012; Ha & Thu, 2009; Vrianty
et al., 2019).
Tyrosinase is the main enzyme in the biosynthetic pathway of
melanin, a complex of pigment granules in the skin, hair, retinal mucosa,
and melanin surrounding the nerve axons. Melasma is the phenomenon
of increasing the content of melanin to protect the skin, helping the skin
against ultraviolet rays (Bonesi et al., 2019; F. Kong et al., 2000; Pires et
al., 2021). Chemically synthesized tyrosinase inhibitors are not really safe
and effective. Therefore, the problem of finding compounds that inhibit
tyrosinase from nature is studied for application in the fields of medicine,
food industry, environmental treatment and cosmetics. (Lai et al., 2018;
Yuan et al., 2020).
In the world and in Vietnam, there have been many studies on
antioxidant capacity from pineapple fruit pulp, leaves, and skin, but there
has not been an overall comparative assessment of antioxidant capacity
of leaves, stem, fruit pulp and pineapple peel. At the same time, there are
few studies on the ability of pineapple extracts to inhibit tyrosinase
activity.
The purpose of the thesis is to study on “Antioxidant capacity and
tyrosinase inhibition of pineapple plant”. (Ananas comosus (L.) Merr.)”
in order to improve the application value of pineapple in other fields such
as pharmaceuticals and cosmetics.

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1.2 Dissertation Objectives
Evaluation of antioxidant capacity and tyrosinase inhibition of
pineapple leaf, stem, fruit pulp and peel extracts (Ananas comosus (L.)
Merr.). Qualitative and quantitative composition of secondary plant
compounds (of which the most important are polyphenols and flavonoids)
of crude extracts and compare the antioxidant capacity and tyrosinase
inhibition of the extracts of the high sample has the ability to effectively
inhibit tyrosinase.
1.3 Research content
Content 1: Preliminary survey to select high extraction solvent and
raw material areas and identification of pineapple species in Tac Cau area.
Content 2: Investigate the antioxidant capacity (DPPH reduction
activity, Fe3+ ion reduction activity to Fe2+ ions, Cu2+ ion reduction
activity to Cu+ ions) and tyrosinase inhibition of Tac Cau pineapple high
methanol.
Content 3: Fractional separation chromatography of pineapple peel
methanol extract and investigation of antioxidant and tyrosinase inhibition
of highly active fractions.
Content 4: Investigate the ability of inhibiting melanin production of
highly active fraction from methanol extract F1 of pineapple peel on
B16F10 cell line.
Content 5: Investigate compounds in pineapple peel F1 methanol
extract that have antioxidant and tyrosinase inhibitory properties through
GC-MS spectrum analysis.
1.4 Subjects and scope of research
1.4.1 Research subjects

The study was carried out on pineapple plants (leaves, stems, flesh and
pods) collected in Hon Dat (Long Xuyen Quadrangle) and Tac Cau
(Western Hau River ecoregion) of Kien Giang province.
1.4.2 Research scope
The study focused on the antioxidant capacity (conducted by 3 methods
of DPPH reduction, Fe3+ ion reduction, Cu2+ ion reduction) and the ability
to inhibit tyrosinase enzyme activity of extracts from leaves, stems, and
flesh pineapple fruit and bark.
The studies were carried out from May 2018 to January 2021 at the
Biotechnology Research and Development Institute, Can Tho University
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and the Center for Experimental Practice Management, Kien Giang
University.
1.5 Meaning of research
1.5.1 Scientific significance
The thesis provides useful scientific information on the antioxidant and
tyrosinase inhibitory ability of pineapple extracts for application in fields
such as medicine, pharmacy and cosmetic chemistry.
1.5.2 Practical significance
Research results show the potential of using by-products in the pineapple
processing industry, preparing extracts from pineapple peels using raw
materials in medicine and pharmacy to help reduce environmental
pollution and increase value of pineapple products in pineapple farming
industry.
1.6 The novelty of the thesis
The thesis has determined that pineapple leaves, stems, fruit pulp and
peel have antioxidant capacity and inhibit tyrosinase activity. The
pineapple peel methanol extract has higher antioxidant capacity (Cu2+

deionization) and inhibits tyrosinase activity than the methanol extract
from pineapple leaves, stems, and flesh.
The content of total polyphenols and total flavonoids was related to
the antioxidant capacity of pineapple extract and to the ability to inhibit
tyrosinase activity of pineapple extract.
The results of inhibiting melanin production on the B16F10 melanoma
cell line of the F1 fraction extracted from pineapple peel methanol extract
open a new direction of application of pineapple by-products in medicine
and pharmacy (prevention and treatment of related diseases). related to
the formation of melanin such as Alzheimer's, Parkinson's, freckles,
melasma and diseases related to melanoma cell lines).

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CHƯƠNG 2:
CHAPTER 2: RESEARCH METHODS
2.1 Research media
2.1.1 Time and place
Time: From May 2018 to January 2021.
Location: Biotechnology Research and Development Institute, Can
Tho University and Center for Experimental Practice Management, Kien
Giang University.
2.1.2 Materials
Leaves, stem, fruit pulp and peel of pineapple (Ananas comosus (L.)
Merr.) were collected in two different ecological regions of Kien Giang
province, Vietnam (Western Song Hau ecoregion - Tac Cau pineapple
and natural habitat). Long Xuyen Quadrangle - Hon Dat pineapple). The
pineapple samples studied were identified based on morphological
characteristics according to the Vietnamese plant classification system

according to Pham Hoang Ho, (1993). Criteria for selecting experimental
samples: leaves at the top, fruit and pineapple skin are aged from 24 to 26
weeks after flowering, pineapple stems are aged from 3 to 5 years old.
Cell B16F10 was provided by the ATCC cell bank (USA).Phương pháp
nghiên cứu
2.1.3 Content 1: Preliminary survey to select raw materials and
extraction solvents.
Objectives: to determine the raw material area and type of extraction
solvent for further studies of the thesis.
Evaluation of antioxidant capacity of pineapple ethanol extract
in Hon Dat and Tac Cau
a. Extraction from parts of pineapple plants collected at Hon Dat and Tac
Cau in ethanol solvent
Experimental layout: determine the moisture content of raw
materials by drying method (Appendix 9.6, Vietnam Pharmacopoeia V)
by infrared moisture drying balance MX50 AND (Japan).
Experiments were arranged and conducted according to Phụng, 2007;
Rodriguez., 2017; Vrianty et al., 2019 with correction (Table 3.1).
Evaluation criteria: Efficiency (%) = (CT mass (g)/Sample weight
(g)) x 100 (1)
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Table 3.1: Experimental arrangement for extracting pineapple leaf,
stem, flesh and peel extracts in Hon Dat and Tac Cau in ethanol
Experiment
L_EHĐ
T_EHĐ
TQ_EHĐ
V_EHĐ

L_ETC
T_ETC
TQ_ETC
V_ETC

Parts of
the Plant
Leaves.
Trunk
Fruit flesh
Peel
Leaves.
Trunk
Fruit flesh
Peel

Ecological
zone

Solvent

Sample/solvent
ratio (w/v)

Hon Đat

Ethanol

1:4

Tac Cau

Ethanol

1:4

b. Investigation of antioxidant capacity of pineapple ethanol extract in
Hon Dat and Tac Cau areas
Experimental arrangement: The experiment was performed
according to the description of (Jovanović et al., 2018; Li et al., 2014)
with correction.
Dilute 0.1 Mm DPPH solution; Phase the extract to the appropriate
concentration range (investigated, mixed with methanol); Dilute the
standard vitamin C solution to the concentration range (12.5 - 150 µg/mL)
(mix with methanol).
Implementation process
Place 1 mL of the extracts at the prepared concentrations in turn into
a test tube, add 1 mL of 0.1 mM DPPH. After 30 min of incubation in the
dark, the OD was measured using a spectrophotometer at 517 nm. The
control sample was carried out in the same way but replaced the extract
with methanol. Build vitamin C standard curve: build vitamin C standard
curve with the same procedure as for extract.
Evaluation criteria: Based on the OD value of vitamin C and extracts
at 517 nm, calculate the percentage of preparation at different
concentrations according to formula (2). The DPPH-reducing activity
was expressed as a percentage of inhibition:
Percentage inhibition (%) = (A0 - A)/A0 x 100 (2)
From the percentage inhibition value at the concentrations of each extract
sample, construct a calibration curve y = ax + b. From this, infer the IC50
value (the concentration of extract or vitamin C at which 50% inhibition).

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The lower the IC50 value, the higher the crude sample will have the
higher antioxidant activity and vice versa.
Experiment 2: Investigation of antioxidant capacity of
pineapple extract in Tac Cau area extracted in ethanol/methanol
solvent
a. Extraction from parts of pineapple plant collected at Tac Cau in
ethanol/methanol
Table 3.2: Experimental arrangement for preparation of crude extracts
of pineapple leaves, stems, flesh and skin in different ecological regions
and solvents
Experiment
L_ETC
T_ETC
TQ_ETC
V_ETC
L_MTC
T_MTC
TQ_MTC
V_MTC

Parts of the
Plant
Leaves.
Trunk
Fruit flesh
Peel
Leaves.

Trunk
Fruit flesh
Peel

Ecological
zone

Solvent

Ethanol

Sample/solvent ratio
(w/v)

1:4

Tac Cau
Methanol

1:4

Experimental layout and monitoring criteria: similar to experiment 1.
Experiment 3: Sequencing genes to identify Tac Cau pineapple
species
Purpose: This study aims to redefine the species of pineapple in Tac
Cau area (endemic to Kien Giang province.
a. DNA Extraction
ollow the CTAB (Cetyl trimethyl ammonnium bromide) procedure,
described by Rogers and Bendich, (1988). Use a standard commercial kit
for sample purification (PCR Clean up). The process follows the author

Tran Nhan Dung et al ., 2012.
b. Perform PCR reaction
In good quality DNA samples, primers that amplify the ITS sequence
region (Baldwin et al., 1995) are used to perform PCR reactions as
follows:
+ ITS1: 5’-TCC GTA GGT GAA CCT GCG G-3’
+ ITS4: 5’-TCC TCC GCT TAT TGA TAT GC-3’
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- PCR products were checked by electrophoresis on 2% agarose gel
at a potential of 50V. Electrophoresis spectra were observed with a gel
imager (Bio-rad, USA).
c. Sequencing
Samples after performing satisfactory PCR reactions were sent to
First BASE Laboratories (Malaysia) laboratory for sequencing using the
ABI PRISM automatic sequencing machine system.
PCR products after agarose gel electrophoresis and gel imaged and
stored at 4oC. Sequencing on the ABI PRISM 3730xl Genetic Analyzer
system with the BigDye® Terminator v3.1 Cycle Sequencing Kit
sequencing kit.
The DNA sequence of the ITS region after correction will be
compared with the data of NCBI using the BLAST tool. Through the
similarity between the query sequence and the NCBI database to identify
the materials used in the study.
2.1.4 Content 2: Evaluation of antioxidant capacity and tyrosinase
inhibition of Tac Cau pineapple high methanol
Experiment 4: Quantification of total polyphenols and
flavonoids in pineapple extract
a. Quantification of total polyphenols

Purpose: to determine the total polyphenol content in the extracts
extracted from the leaves, stems, fruit pulp and skin of pineapples in Tac
Cau area which were extracted in methanol solvent.
Experimental arrangement: The experiment was performed
according to the description of (Li et al., 2014; Petrillo et al., 2016; Zhou
et al., 2014b) with correction.
Conduct experiments: Preparation: Prepare 10% Folin-Ciocalteu
reagent (mix with deionized water). Dilute 2% Na2CO3 solution (mix
with deionized water). The crude extract is prepared in the following
concentrations: (1 mg/mL) (mixed with methanol). Standard gallic acid
is prepared as a solution with a concentration range of 20-120 µg/mL
(mixed with methanol).
Rating Criteria: based on the measured OD value at 765 nm.
Construct the standard curve equation for gallic acid y = ax + b,
calculate the polyphenol content in the sample according to formula (4).
Total polyphenol content: C = (c * V)/m (4)
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b. Quantification of total flavonoids
Purpose: Determination of total flavonoid content in extracts of
leaves, stems, fruit pulp and pineapple peel from Tac Cau area extracted
in methanol solvent.
Experimental arrangement:: The experiment was performed
according to the description of (Ghasemzadeh, 2012; Petrillo et al., 2016)
with correction.
Rating Criteria: Based on the sample of OD đo giá trị ở bước sóng
415 nm. Build the standard method of rutin, flavonoid function of the total
function has in the sample sample (6).
Total flavonoid content: F = (c*V)/m (6)

Experiment 5: Antioxidative evaluation of Tac Cau pineapple high

methanol
a. Investigation of DPPH reducing activity: similar to experiment 1 and
experiment 2.
b. Investigation of reducing activity Fe3+
Purpose: The experiment aimed to determine the antioxidant activity
through the reaction with potassium ferricyanide (Fe3+) to form kali
ferrocyanide (Fe2+ ) of pineapple methanol extract in Tac Cau area.
Experimental arrangement: The experiment was performed
according to the description of Singhal et al., (2014) with correction.
Conduct experiments: 0.2 M phosphate buffer (pH 6.6); 1% solution
of potassium ferricyanide; 10% solution of trichloroacetic acid; 0.1%
ferric chloride solution; phase pineapple extract into a suitable
concentration range (investigated, mixed with deionized water). Dilute
vitamin C into a range of concentrations (2 - 12 µg/mL) (phase in
deionized water)
Rating Criteria: Based on the OD value of vitamin C and the extract
at 700 nm, calculate the percentage of Fe3+ ion inhibition obtained at
different concentrations according to formula (8). Fe3+ deionization
activity is expressed as percentage inhibition according to formula (6).
Percentage inhibition (%) = (A – Ao)/Ao x 100 (6)
c. Investigation of reducing activity Cu2+
Objective: The experiment aimed to determine the antioxidant
activity through the reduction of Cu2+ to Cu+ ions of the methanol extract
of leaves, stems, fruit pulp and pineapple peel in Tac Cau area.
8


Experimental arrangement: The experiment was performed

according to the description of (Celik et al., 2007; Özyürek et al., 2011)
with correction.
Conduct experiments: Sodium acetate buffer solution 0.004 M (pH
4.5); mixed extracts of leaves, stems, flesh and skin of pineapple with
original concentration of 2000 µg/mL; CuSO4.5H2O solution
concentration 50 µg/mL; ascorbic acid solution (150 - 350 µg/mL);
Extract the extract with the appropriate concentration.
Rating Criteria: Based on the OD value of vitamin C and extract at
450 nm, calculate the percentage of Cu2+ ion inhibition obtained at
different concentrations according to formula (7).
Percentage inhibition (%) = (As – Ao)/As x 100 (7)
Experiment 6: Investigation of tyrosinase inhibitory ability of
pineapple methanol extract in Tac Cau area
Objective: evaluated the inhibitory activity of tyrosinase enzyme
activity in vitro of the extracts of leaves, stems, fruit pulp and skin of
pineapple collected in Tac Cau ecoregion which were extracted in
methanol solvent.
Experimental arrangement: The experiment was performed
according to the description of (Chintong et al., 2019; Phúc, 2020)
with correction.
Conduct experiments: Dilute the crude extract in 1% DMSO solution
to a concentration of 10 mg/mL. Continue diluting the extract to a final
concentration of 2 mg/mL in 1/15 M phosphate buffer, pH = 6.8. Continue
the crude high phase into a concentration range according to the
experimental set-up concentrations for each of the different extracts.
Dilute tyrosinase in 1/15 M phosphate buffer, pH = 6.8 to get
concentration of 250 U; Dilute L_Dopa in 1/15M phosphate buffer, pH =
6.8 to achieve a concentration of 3 mg/mL.
Conduct experiments: Add to the eppendorf tube 0.2 mL of extracts
of different concentrations (according to the concentration range

investigated); Add 25 µL tyrosinase (250 U concentration) and incubate
at 37oC for 10 minutes; Add 50 µL L-Dopa (3 mg/mL concentration);
Continue incubation at 37oC for 10 minutes.
Measure the absorbance of light at 475 nm.
9


Rating Criteria: The percentage of enzyme that was inhibited was
calculated using the formula (8).
% Inhibition = ( A − B ) − (C − D ) 100 (8)
A− B
Construct a standard curve showing the correlation between
percentage inhibition and sample concentration. Based on the standard
curve equation, the IC50 value is determined (the concentration level
capable of inhibiting 50% of the enzyme).
2.1.5 Content 3: Evaluation of antioxidant capacity and tyrosinase
inhibition of pineapple extracts
Experiment 7: Effect of solvent on high extraction efficiency of
pineapple peel fractions
Objective: The experiment was carried out to obtain CPĐ.
Experimental arrangement: The experiment was performed
according to the description of Ikram et al., 2020 with correction.
Table 3.5: Ratio of solvents used in pineapple peel silica gel column
chromatography
Experiment
Solvent
Ratio (v/v)
F1
n-hexane
100%

F2
n-hexane:ethyl acetate
3:7
F3
ethyl acetate
100%
F4
ethyl acetate:methanol
3:7
F5
methanol
100%
Rating Criteria: Recovery efficiency CPĐ calculated according to the formula (9).
Performance (%) = (mass CPĐ/ mass CT) x 100 (9)
Experiment 8: Quantification of total polyphenols and total
flavonoids in pineapple peel fractions
Objective: The experiment was carried out for the purpose of
determining the total polyphenol content and total flavonoid content in
F1, F2, F3, F4, F5 after silica gel column chromatography.
Experimental layout and monitoring criteria: similar to experiment 4.

10


Experiment 9: Investigation of antioxidant capacity and
tyrosinase inhibition of pineapple peel extracts
Objective:: identify the high fraction sample with the strongest
tyrosinase inhibitory activity to test the inhibition of melanin production
on the melanoma cell line B16F10 and analyze the GC-MS spectrum.
Experimental layout and monitoring criteria: similar to experiment 5a, 5b và 6

2.1.6 Content 4: Investigation of the ability to inhibit melanin
production on melanoma cell line B16F10 of high fractions F1
Experiment 10: Investigation of inhibition of melanin
production by pineapple peel F1 fraction on B16F10 cell line
Objective: Determination of the inhibitory activity of melanin
production of pineapple peel F1 fraction on the melanocyte cell line
B16F10.
Experimental arrangement: Samples were sent to the Biological
Laboratory, Institute of Biotechnology under the Vietnam Academy of
Science and Technology. Experiments were carried out according to
(Kim et al., 2014; Lee and Cho, 2018; L. Q. Loan et al., 2018) with
correction.
Conduct experiments: B16F10 cells were grown in a 6-well plate at
a concentration of 1x105 cells/mL, and continued to incubate the cells
with the study sample at different concentrations in the presence of αMSH (10 nM) for 48 h. After 2 days of incubation at 37oC and 5% CO2,
cells were harvested and washed with PBS. The cell residue was then
dissolved in 1N NaOH solution containing 10% DMSO and incubated at
80oC for 1 hour. After measuring the OD value at 405/450 nm.
Rating Criteria: The ability to inhibit melanin production of the studied
samples was compared with the negative control without sample incubation
according to the formula (10):
% Inhibits melanin production =100%- ((ODmẫu – OD– αMSH)/(OD+α
MSH – OD-α MSH)) x 100 (10).
2.1.7 Content 5: GC-MS spectrum analysis of pineapple peel F1 fractions
Experiment 11: GC-MS mass spectrometry analysis of pineapple peel
F1 fractions
Objective: The study was carried out to determine in the F1 fraction
of pineapple peel there are compounds and groups of compounds that
11

have antioxidant and tyrosinase inhibitory properties through a series of
previous studies.
Experimental arrangement: The experiment was performed
according to the description of (Ikram et al., 2020) with correction.
Sample phase: The sample phase is carried out at the Institute of
Biotechnology Research and Development, Can Tho University.
Dilute CPD F1 pineapple peel in acetone (France) or methanol
(Germany) to get 1 mL of a solution with a concentration of 100 ppm.
Method of preparation: Weigh 1mg + 1mL to get a concentration of
1000 ppm; Withdraw 100 microliters of stock solution 1000 ppm + 900
microliters of solvent to obtain a concentration of 100 ppm; 10 ppm and
1 ppm phase.
Performing analysis: After being prepared, samples are sent for GCMS analysis at Can Tho Technical Center for Standards, Metrology and
Quality.
The sample analysis temperature program is installed on the Shimadzu
GCMS-QP2010 system.
Processing GC-MS data: Based on ion spectroscopy combined with
data from NIST, WILEY libraries and previous de-spectral information
to identify compounds present in F1 analytical samples.
2.2 Data processing methods
Raw data were calculated, processed and charted using Excel 2016.
Statistical data were processed by Minitab 16 software such as: analysis
of variance ANOVA, coefficient of variation (CV%), average
comparison. Average the difference using Tukey's test.

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CHƯƠNG 3: CHAPTER 3: KẾT QUẢ VÀ THẢO LUẬN

3.1 Preliminary survey results for raw materials
3.1.1 High antioxidant results of pineapple ethanol extract in two
ecological zones Hon Dat and Tac Cau
Results of sampling, processing and extraction of extracts from
leaves, stems, flesh and skin of pineapple in Tac Cau and Hon Dat in
ethanol solvent
Table 4.1: Results of extracting pineapple extracts at Hon Dat and Tac Cau
Parts of
Experiment
the Plant

Solvent

Weight (g)
Eco
zone Extraction

Humidity (%)

Fresh
samples

Leaves.

L_EHĐ

20,03±0,29 81,17±1,33

Trunk

T_EHĐ

32,6±0,77 63,23±2,05
Hon
Đat 112,68±2,72 84,6±1,05

Fruit flesh TQ_EHĐ
Peel
V_EHĐ

51,69±0,43
Ethanol

80,9±1,25

Leaves.

L_ETC

21,08±0,10 81,03±0,42

Trunk

T_ETC

35,01±0,20 63,7±0,92
Tac
Cau 129,95±6,68 84,3±0,62

Fruit flesh TQ_ETC

Peel
V_ETC

54,35±0,80

81,6± ,96

CT
5,1
5,5
7,3
5,6
5,2
5,1
7,6
5,9

Performance
(%)
2 ± 0,03e
3,3 ± 0,08d
11,27±0,27b
5,17±0,04c
2,11±0,01e
3,5±0,02d
12,99±0,67a
5,44±0,08c

Preliminary survey to select the material area (Ecoregion of Long
Xuyen Quadrangle - Hon Dat - and the West River Hau ecoregion - Tac

Cau) and suitable solvents (ethanol/methanol).
The moisture content of fresh materials showed that the moisture
content in pineapple body was about 81.03 - 81.17%, that of pandan
leaves was 63.23 - 63.7%, that of fruit flesh was 84.3 - 84.6% and that of
peel the result is 80.9 – 81.6%. The extract was dried at 45oC until the
moisture content reached from 5.1 to 7.6% (standard of Vietnam
Pharmacopoeia V).
Statistical processing results show that, when using the same type of
sample, there was no difference in high extraction efficiency.
High antioxidant results of pineapple ethanol extract in two
ecological zones Hon Dat and Tac Cau
The results of investigation of antioxidant activity of pineapple extracts in
two ecological regions extracted in ethanol solvent are shown in Table 4.2.
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Table 4.2: Results of antioxidant activity of pineapple ethanol collected
in Hon Dat and Tac Cau ecological areas by DPPH reduction method
Experiment
L_EHĐ
T_EHĐ
TQ_EHĐ
V_EHĐ
L_ETC
T_ETC
TQ_ETC
V_ETC
Ascorbic acid

Ecological zone

De-DPPH (IC50 µg/mL)
104,08 ± 1,89ef
251,92 ± 7,69b
292,93 ± 8,86a
143,63 ± 2,64d
76,74 ± 1,35g
196,52 ± 17,92c
220,41 ± 9,85c
112,85 ± 8,66e
84,07 ± 2,12fg

Hon Đat

Tac Cau

control sample

Thus, the Tac Cau ecoregion showed a higher antioxidant capacity
than the extracts from the Hon Dat ecoregion. The IC50 index of leaf
samples from Tac Cau area was lowest (76.74 ± 1.35) µg/mL, which was
lower than that of ascorbic acid control (84.07 ± 2.12) µg/mL.
When comparing the antioxidant results on each pair of similar parts
of pineapple in two different ecological regions, the leaf sample of Tac Cau
(IC50 = 76.74 µg/mL) showed a higher antioxidant capacity than 1. 36
times that of Hon Dat leaves (IC50 = 104.08 µg/mL). Similarly, the
antioxidant capacity of Tac Cau pineapple peel (IC50 = 112.85 µg/mL) is
1.27 times higher than that of Hon Dat pineapple peel (IC50 = 143.63
µg/mL); sample of Tac Cau fruit (IC50 = 220.41 µg/mL) was 1.33 times
higher than that of Hon Dat fruit (IC50 = 292.93 µg/mL); Tac Cau

pineapple stem sample (IC50 = 196.52 µg/mL) was 1.28 times higher than
that of Hon Dat pineapple stem sample (IC50 = 251.92 µg/mL).
The research results are consistent with the previous study by
Rodriguez., (2017), which studied TPC, TFC and antioxidant capacity of
pineapple plants in different ecological regions in Bzazil. Results in
different ecological regions showed different results in terms of antioxidant
capacity, total polyphenol content and total flavonoid content.
Tac Cau islet has rich alluvial soil due to the accretion of rivers and
typical topography is brackish water, alkaline and salty soil, but the
quality of juicy pineapple is grown on a large area of more than 2,700
hectares provide a large source of raw materials for production and
research.
14


At the same time, the research results are similar to the previous study
by Yuris and Siow, 2014, which studied the antioxidant capacity of fruit
pulp in 3 pineapple varieties Joseplune, Morris and Sarawak in different
ecological regions of the species Ananas comosus. As a result, different
ecological regions, different material varieties and different solvents, the
ability to reduce DPPH is also different. Therefore, the leaves, stem, fruit
pulp and pineapple peel were selected in the Tac Cau ecoregion to
conduct research for further experiments.Kết quả kháng oxy hóa cao chiết
ethanol/methanol dứa vùng Tắc Cậu, Kiên Giang.
Results of sampling, processing and extraction of extracts from
leaves, stems, flesh and skin of pineapple in Tac Cau in
ethanol/methanol solvent
Each type of solvent will have the ability to extract different groups of
compounds. Two types of solvents commonly used in the extraction of
natural compounds are ethanol and methanol because of their low cost

and high efficiency, large solvent recovery and low toxicity. (Phụng,
2007; Trúc et al., 2020; Vrianty et al., 2019). The results of preparing
extracts from leaves, stems, fruit pulp and pineapple peel in Tac Cau area,
Kien Giang province in ethanol and methanol are shown in Table 4.3.
Table 4.3: Results of sampling, processing and extraction of pineapple
extract at Tac Cau in ethanol/methaol
Weight (g)

Part

Experiment Solvent

Extraction

Leaves.
L_ETC
21,03±0,29
Trunk
T_ETC
32,2±0,77
EtOH
Fruit flesh TQ_ETC
119,34±3,74
Peel
V_ETC
52,36±0,73
Leaves.
L_MTC
22,08±0,99
Trunk

T_MTC
34,35±0,20
MeOH
Fruit flesh TQ_MTC
127,95±8,13
Peel
V_MTC
53,68±1,33

Humidity * (%)
Fresh
CT
samples
80,83±0,99 5,0
62,57±2,1
5,4
84,6±1,0
7,1
81,27±1,3
5,4
81,97±0,76
5,1
64,37±0,77
5,3
83,97±1,19 7,2
81,97±1,0 5,7

Performance
(%)

2,1±0,03d
3,2±0,15c
11,93±0,37a
5,24±0,07b
2,21±0,1d
3,44±0,19c
12,81±0,81a
5,37±0,13b

Thus, when using materials from Tac Cau pineapple leaves, stems,
fruit pulp and peel extracted in ethanol and methanol, the statistical
processing results of extraction efficiency did not differ between samples
of the same set. parts but only the differences between the parts.
Extraction efficiency goes from high to low in the following order: pulp
15


→ rind → stem → leaf. Continue to study on the DPPH reduction activity
of the extracts extracted in ethanol and methanol solvents to choose the
most suitable solvent for further studies of the thesis.
Antioxidative results of pineapple ethanol/methanol extracts
collected at Tac Cau
The results of the investigation of the antioxidant activity of ethanol and
methanol extracts of leaves, stems, fruit flesh and pineapple peels in the Tac
Cau ecoregion are shown by the IC value of 50 (inhibition concentration of
50%) through the reduction activity. DPPH (Table 4.4). The lower the IC 50
value, the stronger the antioxidant activity and vice versa.
Table 4.4: Antioxidant activity of pineapple ethanol and methanol
extract in Tac Cau ecoregion, Kien Giang
Experiment

L_MTC
T_MTC
TQ_MTC
V_MTC
L_ETC
T_ETC
TQ_ETC
V_ETC
Ascorbic acid

Solvent

De-DPPH (IC50 µg/mL)
41,13 ± 0,46g
138,59 ± 11,10d
154,47 ± 4,13c
64,79 ± 4,19f
77,98 ± 1,48f
180,92 ± 6,27b
199,28 ± 3,41a
111,38 ± 7,14e
73,01 ± 2,97f

Methanol

Ethanol
control sample

All treatments showed antioxidant capacity through DPPH free radical
scavenging test results. Pineapple methanol extract showed higher

antioxidant activity than pineapple ethanol extract.
IC 50 of Tac Cau leaf methanol extract was lowest ( 41.13 ± 0.46)
µg/mL, which was 1.8 times lower than that of ascorbic acid control
(73.01 ± 2.12). µg/Ml. The lowest antioxidant capacity was high in fruit
pulp ethanol with the highest IC 50 index of 199.28 ± 3.41 µg/mL.
When comparing the antioxidant activity on each of the extracts
extracted in two different solvents, the highest antioxidant capacity was
high in leaf methanol (IC 50 = 41.13 ± 0.46 µg/mL). the second is pod
ethanol (IC 50 = 64.79 ± 4.19 µg/mL) and foliar ethanol (IC 50 = 77.98 ±
1.48 µg/mL) and the third is rind ethanol (IC 50 =) 111.38 ± 7.14 µg/mL)
fourth was high methanol body (IC 50 = 138.59 ± 11.10 µg/mL), fifth was
high methanol fruit pulp (IC 50 = 154.47 ± 4, 13 µg/mL) and the lowest
16


antioxidant capacity was high ethanol stem and pineapple pulp Tac Cau,
Kien Giang (IC 50 = 180.92 ± 6.27 µg/mL and IC 50 = 199.28 ± 3.41).
µg/mL). The results of antioxidant activity of extracts in different solvents
and organelles showed different results.
The study results are also consistent with the study by Jovanovic et al.,
2018, which evaluated the antioxidant capacity of the peel, apical leaves,
stem, and juice of pineapple (Ananas comosus [L.] Merr.) by DPPH free
radical scavenging method. The results showed that pineapple peel and
stem extracts in absolute methanol had the highest antioxidant capacity
(IC 50 = 1.745 ± 0.046 mg/mL), while the lowest oxidizing capacity was
high in the pulp extract in aqueous solution. The medium is water (IC 50
= 88 ± 2.09 mg/mL). In addition, the difference in DPPH free radical
scavenging capacity is also influenced by soil conditions, climatic
conditions as well as plant age and season (L. Q. Loan et al., 2018).
Results of gene sequencing of Tac Cau pineapple plant

Morphological characteristics
The research object is pineapple in Kien Giang, the sample was
identified by Nguyen Van Phuoc, Department of Crop Science, Kien
Giang University based on morphological characteristics according to
Pham Hoang Ho, (1993) and according to the classification system.
species of Vietnamese herb, identified the research object belongs to the
pineapple genus Queen, belongs to the species Ananas Comosus.
Gene sequencing results
The results of comparing the ITS sequences of pineapple samples used
in this study showed the similarity from 99.07 to 100% with the ITS
sequences of Ananas comosus with the genetic codes HM104185.1,
XM_020251500, HM768296.1. The results also showed the similarity of
the ITS sequence of pineapple samples with related plants..
The results also show the similarity of ITS sequences of pineapple
samples with some other related plants. Specifically, this sequence is
similar to the Guzmania hybrid with 98.56% similarity, Elaeis guineensis
with 98.20% similarity, Cocos nucifera with 85.20% similarity.
According to the morphological characteristics and distribution of
species in the genus Ananas published on the NCBI data bank, pineapple
has similar characteristics to the Tac Cau pineapple tree sample under
17


study. When comparing the morphological characteristics of pineapple
samples from the Tac Cau area with those of Ananas comosus (L.) Merr.
described by the author Pham Hoang Ho found a similarity..
The combination of ITS gene sequence and morphological
characteristics described in the book "Trees of Vietnam" by Pham Hoang
Ho, 2003, can confirm that the pineapple sample under study is anas
comosus (L.) Merr.

3.2 Results of antioxidant capacity and tyrosinase inhibition of Tac
Cau pineapple high methanol
3.2.1 Quantitative results of total polyphenols and flavonoids in
pineapple methanol extract
The biological activity of antioxidant and tyrosinase inhibition is
highly dependent on the polyphenol and flavonoid content of the extract.
Therefore, it is necessary to study to determine the content of total
polyphenols and total flavonoids. The quantitative results of total
polyphenols and total flavonoids in the methanol extracts of leaves,
stems, fruit pulp, and pineapple peel are shown in Table 4.5.
Table 4.5: Contents of total polyphenols, total flavonoids of leaves,
stems, fruit pulp, and skin of Tac Cau pineapple
Experimen
L_MTC
T_MTC
TQ_MTC
V_MTC

Total polyphenol content
(mg GAE/g)
242,00 ± 2,69b
187,63 ± 2,79c
50,32 ± 1,56d
291,43 ± 0,87a

Total flavonoid content
(mg QE/g CPĐ)
44,70 ± 1,97a
3,78 ± 0,39c
4,89 ± 0,64c

35,16 ± 2,34b

The results of Table 4.5 showed that all extracts samples had the
presence of total polyphenols and total flavonoids and there were
statistical differences. The polyphenol content in Tac Cau pineapple peel
methanol was the highest (291.43 mg GAE/g) and the lowest was highest
in Tac Cau pineapple pulp methanol (50.32 mg GAE/g) and ranked from
low to high as follows: fruit pulp stem leaf and the highest sample
was the methanol extract of the fruit rind. The flavonoid content was
highest in leaf methanol extract (44.7 mg QE/g) and lowest in pineapple
stem methanol (3.78 mg QE/g).
18


3.2.2 Antioxidant results of Tac Cau pineapple high methanol
The antioxidant activity of the extract was shown by DPPH reduction
activity, Fe 3+ reduction activity, Cu 2+ reduction activity, and IC value
of 50 (50% free radical inhibitory concentration) (Table 4.6).
Table 4.6: Antioxidant activity of pineapple methanol extract
Experimen
L_MTC
T_MTC
TQ_MTC
V_MTC
Acid ascorbic

De-DPPH(*)
31,27 ± 3,91c
131,14 ± 11,37a
143,55 ± 14,59a

62,63 ± 4,66b
84,07 ± 2,12

Value IC50 (µg/mL)
De-Fe3+ (*)
110,25 ± 2,76c
168,69 ± 4,44b
203,25 ± 3,99a
97,72 ± 0,42d
7,95±0,34

De-Cu2+ (*)
270,65 ± 8,31c
752,56 ± 14,37a
373,53 ± 16,30b
220,95 ± 8,21d
260,33 ± 10,58

In general, the crude extracts have antioxidant activity through
reducing DPPH, reducing Fe 3+, reducing Cu 2+. Tac Cau (L_MTC)
pineapple leaf extract has antioxidant activity with DPPH reduction
activity (IC 50 = 31.27 ± 3.91 µg/mL), 2 times stronger than that of
methanol_shell sample (V_MTC) with value (IC 50) = 62.63 ± 4.66
µg/mL) and 4.6 times higher than that of pineapple pulp methanol extract
(IC 50 = 143.55 ± 14.59 µg/mL), and 4.2 times higher than that of
pineapple pulp. pineapple stem methanol sample (IC50 = 131,14 ± 11,37
µg/mL).
In contrast, investigating the antioxidant activity of the extract by
reducing Fe 3+ , reducing Cu 2+ , the sample V_MTC had the strongest
antioxidant activity through the lowest IC 50 value . Methanol_pineapple

peel has antioxidant activity with Fe 2+ and Cu 2+ ion reduction activitie
(IC50 = 97,72 ± 0,42 µg/mL, IC50 = 220,95 ± 8,21 µg/mL).
In the structure of tyrosinase enzyme, the active site has the
participation of two Cu 2+ ions . Therefore, the method of testing the
antioxidant capacity by reducing Cu 2+ ion method shows the potential
of the method in studying the relationship between antioxidant and
inhibiting tyrosinase enzyme activity.
3.2.3 The relationship between TPC and TFC oxidant capacity of Tac
Cau pineapple plant
The results also showed a positive relationship between total
polyphenol content and total flavonoid content with antioxidant capacity
of extracts from leaves, stems, fruit pulp and pineapple peel through
19


DPPH and deionization ability. Cu 2+. Thus, samples of pineapple leaves
and skin with high polyphenol and flavonoid content showed high
antioxidant activity. Similarly, the polyphenol and total flavonoid content
of the stem and fruit pulp were low, so the antioxidant activity was also
low.
3.2.4 Tyrosinase inhibition results of pineapple high methanol Tac
Cau area
The inhibitory activity of tyrosinase enzyme of Tac Cau pineapple
methanol extracts is shown in Table 4.7.
Table 4.7: Tyrosinase inhibition of Tac Cau pineapple methanol extracts
Experiment
L_MTC
T_MTC
TQ_MTC
V_MTC

Acid Kojic

TYR inhibitory (IC50 µg/mL)
150,16 ± 17,34c
777,68 ± 12,00a
414,94 ± 17,76b
94,12 ± 2,79d
23,35 ± 1,62

Survey results of tyrosinase inhibitory activity showed that
methanol_pineapple peel had the strongest activity with the lowest IC 50
value (IC 50 = 94.12 ± 2.79 µg/mL). Tac Cau pineapple peel methanolic
extract (V_MTC) has a tyrosinase inhibitory activity 1.6 times higher than
that of methanol extract with the value (IC 50 = 150.16 ± 17.34 µg/mL),
8.3 times higher than that of the leaf methanol extract (IC 50 = 150.16 ±
17.34 µg/mL). with the high methanol_body sample with the value (IC
50 = 777.68 ± 12 µg/mL) and 4.4 times higher than that of the pineapple
pulp methanol with the value (IC 50 = 414.94 ± 17.76 µg/mL) ). At the
same time, all four samples of pineapple extract had lower tyrosinase
inhibitory activity than the control Kojic acid (IC 50 = 23.35 ± 1.62
µg/mL).
3.3 Antioxidant and tyrosinase inhibitory results of pineapple peel extracts
3.3.1 Fractional separation chromatography of pineapple peel
methanol extract
From 58 grams of pineapple peel methanol extract, 5 main segments
were obtained: F 1, F 2, F 3, F 4, F5 with the results shown (Table 4.8).
Table 4.8: Fractional high modulation results
Extract segment
F1

Weight (g)
1,99
20

Performance (%)
3,43


Extract segment
F2
F3
F4
F5

Weight (g)
3,57
11,76
19,98
12,69

Performance (%)
6,16
20,28
34,45
21,87

3.3.2 Quantitative results of total polyphenols and total flavonoids in
pineapple peel fractions
Quantitative results Table 4.9 shows that the total polyphenol content,
flavonoid highest total in F 1 value was 430.89 ± 6.44 respectively (mg

GAE / g CPD) and 220.05 ± 6.03 ( mg QE/g CPD). The content of total
polyphenols and total flavonoids was lowest in F 4 with values of 96.17
± 4.76 (mg GAE/g CPD) and 42.17 ± 0.91 (mg QE/g CPD) respectively.
Table 4.9: Quantitative results of TPC and TFC of pineapple peel fractions
Experimen
F1
F2
F3
F4
F5

Total polyphenol
content (mg GAE/g)
430,89 ± 6,44a
167,36 ± 1,43c
124,50 ± 2,58d
96,17 ± 4,76e
243,39 ± 3,09b

Total flavonoid content
(mg QE/g CPĐ)
220,05 ± 6,03a
166,22 ± 4,86b
64,64 ± 1,09d
42,17 ± 0,91e
82,73 ± 1,98c

3.3.3 Antioxidant and tyrosinase inhibitory results of pineapple peel extracts

Results of in vitro antioxidant investigation of pineapple peel extracts

Table 4.10: Antioxidant activity of pineapple peel extracts
Experimen
F1
F2
F3
F4
F5
CT

Value IC50 (µg/mL)
De-DPPH(*)
De-Cu2+ (*)
f
14,52 ± 0,44
18,78 ± 2,55f
55,75 ± 0,74e
138,39 ± 1,71d
141,51 ± 7,56c
371,46 ± 3,16c
288,33 ± 13,38a
550,74 ± 17,63a
b
186,42 ± 9,45
394,20 ± 6,12b
d
98,82 ± 1,05
102,50 ± 0,29e

In vitro tyrosinase inhibition results of pineapple peel extracts

Table 4.11: Tyrosinase inhibitory ability of pineapple peel extracts
CPĐ
F1
F2
F3
F4
F5

TYR inhibitory (IC50 µg/mL)
84,98 ± 5,06cd
111,84 ± 4,03c
174,50 ± 2,46b
232,79 ± 29,34a
185,97 ± 0,50b
21