Research J. Pharm. and Tech. 14(7): July 2021
ISSN
0974-3618 (Print)
0974-360X (Online)
www.rjptonline.org
RESEARCH ARTICLE
Phytochemical Screening, Volatile Compound Analysis, and Antioxidant
Activity of Mangifera quadrifida Jack (Isem Kembang) Baby Fruit collected
from Lampung, Indonesia
Candra Irawan1, Hanafi2, Henny Rochaeny1, Lilis Sulistiawaty1, Andita Utami1*
Imalia Dwi Putri2
1
Department of Chemical Analysis, Politeknik AKA Bogor,
Jalan Pangeran Sogiri, Tanah Baru, Bogor 16154, Indonesia.
2
Department of Food Industry Quality Insurance, Politeknik AKA Bogor,
Jalan Pangeran Sogiri, Tanah Baru, Bogor 16154, Indonesia.
*Corresponding Author E-mail: andita.utami@gmail.com
ABSTRACT:
Mangifera quadrifida Jack (Isem Kembang) is a rare fruit native to Lampung, Southern Sumatera, Indonesia.
Phytochemical composition and antioxidant activity in Mangifera quadrifida Jack baby fruits are still unknown.
This study aims to analyze the phytochemical compounds and antioxidant activity contained in Mangifera
quadrifida Jack baby fruit extracts. In this study, the baby fruit of Mangifera quadrifida Jack was extracted using
n-hexane, ethyl acetate, and methanol as the solvent in a separate experiment. Phytochemical tests were carried
out for all extracts, followed by analysis of volatile compounds using GC-MS (Gas Chromatography – Mass
Spectrometry). Then the antioxidant activity test was carried out on all three extracts to assess the inhibitory
activity against DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals. Phytochemical test results of Mangifera
quadrifida Jack extracts revealed the presence of various bioactive compounds. GC-MS analysis providesl
information about volatile compound content for each extract, which involves alkanes, alkenes, esters,
carboxylates, phenolic compounds, aromatics, and aldehydes. Antioxidant activity investigation of the extracts
obtained from n-hexane, ethyl acetate and methanol showed the IC50 values of 430.63ppm, 24.52ppm, and 23.67
ppm, respectively. This shows that the baby fruit of Mangifera quadrifida Jack can be used as an alternative
source of natural antioxidants.
KEYWORDS: Mangifera quadrifida Jack, Phytochemical Screening, Antioxidants, Gas ChromatographyMass Spectrometry, and DPPH.
INTRODUCTION:
Indonesia with thousands of islands has a wealth of
biological resources, especially tropical fruits and their
supporting genetic components1. One of them is
Mangifera quadrifida Jack, a rare fruit originating from
Lampung, Southern Sumatera, Indonesia. The baby fruit
of Mangifera quadrifida Jack by Lampung tribe is
commonly used as a mixture of chili sauce because it
enhances the taste and aroma. The parts of Mangifera
quadrifida Jack can be seen in Figure 1.
Received on 15.07.2020
Accepted on 18.11.2020
Modified on 27.09.2020
© RJPT All right reserved
Research J. Pharm. and Tech. 2021; 14(7):3523-3527.
DOI: 10.52711/0974-360X.2021.00610
Figure 1. Mangifera quadrifida Jack Plant
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Research J. Pharm. and Tech. 14(7): July 2021
From the literature, the phytochemical composition and
antioxidant activity of Mangifera quadrifida Jack baby
fruit has never been reported. Phytochemical tests for
medicinal plants are needed, usually phytochemical tests
are used to refer to secondary metabolite compounds
found in plants that have beneficial effects on health or
have an active role for disease prevention, one of which
is antioxidant potential2,3.
filtered and evaporated.
Antioxidants are substances that inhibit the oxidation
process. Oxidation of a molecule can cause a radical
reaction that can start a chain reaction and eventually
cause cell damage or death. Antioxidants can stop this
chain reaction by extinguishing free radical activity.
Antioxidants play a major role in health maintenance and
disease prevention4,5,6. Many foods can be sources of
natural antioxidants, such as spices, tea, chocolate, seeds,
vegetables, and fruits. Most natural sources of
antioxidants are plants and generally contain phenolic
compounds that are spread throughout the plant. Among
other phenolic or polyphenolic compounds can be in the
form of flavonoids. The ability of flavonoids as
antioxidants has been widely studied, where flavonoids
have the ability to change or reduce free radicals and
also as an anti-free radicals7.
Volatile Compound Analysis with GC-MS:
Volatile compounds analysis of hexane, ethyl acetate and
methanol extracts was conducted by GC-MS in order to
determine their chemical composition. GC-MS is a
useful tool for the analysis of various relatively volatile
compounds and this technique has been widely applied
in several research fields12. Chemical compounds were
identified by comparing the mass spectra of targeted
peaks with the mass spectra in compound library (Wiley
W10N11). The conditions of the analysis can be seen in
table 1.
Phytochemical Test:
Phytochemical tests were carried out on each extract
using standard procedures that have been reported in
previous studies9,10,11. These tests include the tests for
alkaloids, tannins, saponins, flavonoids, phenols and
sterol-triterpenoids.
Table 1. GC-MS Analysis Condition
Type
Column
Carrier Gas
Flow Rate
Injector Temperature
Injection Volume
Injection Technique
Temperature Program (Column)
Initial Temperature
Temperature Rate
Final Temperature
Information
Agilent 7890A GC system
DB-5 Capillary Column
Helium
1 mL/min
250C
2 uL
Split
Antioxidant activity can be determined by the IC50 value,
the lower the IC50 value, the higher antioxidant activity.
The antioxidant activity of various fruit sources was
generally extracted with water, ethanol, methanol, ether,
40C hold for 2 min
ethyl acetate, and butanol8. In this study, phytochemical
10C/ menit
tests and characterization of volatile compounds will be
280C in 30 min
carried out on each hexane, ethyl acetate and methanol
extracts on the baby fruit of Mangifera quadrifida Jack. Antioxidant Activity Test:
Then the antioxidant activity test was performed on the The antioxidant activity test on methanol, hexane and
extracts to see its ability to inhibit free radicals.
ethyl acetate extracts was carried out by DPPH method
based on the Molyneux13. A total of 1mL of methanol
MATERIALS AND METHODS:
and ethyl acetate extract each with a concentration of 10
Plant material:
μg/Ml, 20μg/mL, 30μg/mL, 40μg/mL and 50μg/mL were
The dry fruits of Mangifera quadrifida Jack were added to 2mL DPPH 0.1mM. The mixture is then
collected from local area of Lampung, Indonesia. Then homogenized and incubated at room temperature for 30
they were washed thoroughly in distilled water and the minutes in the dark. This solution was then measured for
surface water was removed by air drying under shade.
absorbance at λmax 516nm. The same treatment was
also carried out for blank solutions (DPPH solutions that
Sample Extraction:
did not contain test material) and hexane extracts with
Extraction was done by maceration using several organic concentrations of 50μg/mL, 100μg/mL, 250μg/mL, 500
solvents. Baby fruit of Mangifera quadrifida Jack was μg/mL, and 1000μg/mL. Absorbance measurement data
peeled and separated between the skin and fruit, then were analyzed by percentage of antioxidant activity
dried at room temperature. Mangifera quadrifida Jack using the following equation:
baby fruit that have been refined then soaked in nhexane solvent for three days, then filtered and
evaporated to dry. The results obtained are crude extracts
from n-hexane. The residue from the first immersion is
completely immersed again in ethyl acetate and
methanol respectively to obtain crude extracts of ethyl A = Absorbance
acetate and methanol, each for three days, which is then
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Research J. Pharm. and Tech. 14(7): July 2021
RESULTS AND DISCUSSION:
Extraction using n-hexane, ethyl acetate and
methanol:
Extraction of baby fruit from Mangifera quadrifida Jack
with maceration technique in various solvents for several
days produces a green solution. After evaporation, the
extract changes color to dark brown, as shown in Figure
2. Extraction using n-hexane, ethyl acetate, and methanol
solvent each produces crude extracts weighing 19.3859
g, 36.5893g and 37.2151g.
Figure 2. Extract of Mangifera quadrifida Jack baby fruit in (a) nhexane, (b) ethyl acetate, and (c) methanol solvent
Phytochemical Test:
Phytochemical testing is widely known as a preliminary
method that successfully shows various phytochemical
contents in plant parts, such as alkaloids, flavonoids,
tannins, phenols, saponins and terpenoids14,15,16. The
results of phytochemical tests on each extract of nhexane, ethyl acetate, and methanol can be seen in Table
2.
Table 2: Phytochemical tests on Extract of Mangifera quadrifida
Jack baby fruit
Component Test
Extract
Ethyl
n-Hexana
Methanol
Acetate
Alkaloids
Meyer
Dragendorf
Wagner
Flavonoids
++
+
+++
Phenol
+++
+
+++
Saponin
Tannin
+++
+
+++
Terpenoids
+
+
Unsaturated Steroids
Steroids Glycoside
+
+
+
Table 2 shows that the flavonoid levels were higher in
the methanol and ethyl acetate extracts than in the nhexane extract. The same results were shown in the
phenol and tannin tests. Saponin was not found in all
extracts. The terpenoid test only gave positive results in
ethyl acetate and n-hexane extracts. Steroid glycosides
was found in low levels in all extracts. Phenolic and
flavonoid compounds are common in plants and are
known to have various biological activities. These
beneficial properties include antimicrobial, antiviral,
antiulcerogenic, cytotoxic, antineoplastic, mutagenic,
antioxidant, antihepatotoxic, antihypertensive, antiplatelet, and anti-inflammatory activity. Many of these
biological functions are associated with free radical
activity and antioxidant activity17.
GC-MS Analysis:
The results of GC-MS analysis of baby fruit extracts of
Mangifera quadrifida Jack are shown in Tables 3, 4 and
5 respectively for n-hexane, ethyl acetate and methanol
extract. A total of 8 compounds were found in ethyl
acetate extract, 13 compounds were found in n-hexane
extract and 9 compounds were found in methanol
extract.
Table 3. GC-MS analysis of the n-hexane extract of Mangifera
quadrifida Jack baby fruit
No
Compounds
Retention %
Composition
Time
Area (%)
(Min)
1
Caryophyllene
7.358
3.56
99
2
Bicyclo(3,1,1)hept- 7.561
0.24
96
2-ene
3
7.781
0.7
95
-Humulene
4
8.415
0.21
98
-Bisabolene
5
Phenol, 2,48.810
0.47
98
Bis(1,1Dimethylethyl)
6
5-Oxatricyclo
9.270
0.66
96
7
Trans-Farnesol
10.970
0.74
93
8
Hexadecanoic acid
13.626
1.00
99
9
Octadecadienoic
17.201
0.69
99
acid
10
1.3-Benzenediol
20.463
2.60
90
11
Cyclotetracosane
20.876
3.03
98
12
9-Hexacosene
22.430
1.73
96
13
Stigmasta-5-en-323.422
3.07
99
ol
Table 3 shows that n-hexane extract contains phenolic
compounds and alcohol (Phenol, 2,4-Bis (1,1Dimethylethyl),
Trans-Farnesol,
1.3-Benzenediol,
Stigmasta-5-en-3-ol), hydrocarbons long chains (Bicyclo
(3,1,1) hept-2-ene, Hexadecanoic acid, Octadecadienoic
acid, 5-Oxatricyclo, and 9-Hexacosene), and terpenoids
with the highest percentage (Caryophyllene, -Humulene,
and -Bisabolene). The presence of phenolic compounds
in n-hexane extract is in accordance with the results of
phytochemical tests.
Caryophyllene as a major terpenoid is a major
component of essential oils from medicinal plants,
especially from the aromatic species of the Lamiaceae
family; has been considered a natural antioxidant with
high potency, which can be used as an additive in food
supplements to prevent oxidative stress which
contributes to the emergence of degenerative diseases18.
Caryophyllene and its oxides are the most important
biological compounds that affect various metabolisms.
Caryophyllene has been reported to show significant
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Research J. Pharm. and Tech. 14(7): July 2021
anti-cancer properties. Many other effects such as antibacterial, anti-fungal, immunomodulatory and antiinflammatory have also been reported to show an
association with caryophyllene19.
The literature does not present studies of the biological
activity of long chain alkanes, but the mixture of these
compounds with other substances with different polarity
can act in synergistic effects as antioxidants20.
Table 4: GC-MS analysis of the ethyl acetate extract of Mangifera quadrifida Jack baby fruit
S. No
Compounds
Retention Time (Min)
% Area
1
1,2-Benzenediol
6.200
3.04
2
Phenol, 2,4-bis (1,1 dimethylethyl)
8.397
0.63
3
1,2,3-Benzenetriol
13.975
6.14
4
1-Cyclobutylcyclobutene
15.197
2.14
5
Hexadecanoic acid
18.726
3.55
6
Thiosulfuric acid
19.259
0.64
7
9,12-Octadecadienoic acid
20.913
2.23
8
23.394
4.11
-Sitosterol
Phytosterols contained in vegetable oils are known to
have the function of hypocholesterolemia. Previous
studies have suggested the antioxidant activity of
phytosterol and its components (beta-sitosterol,
stigmasterol,
and
campesterol)
against
lipid
peroxidation21. In table 4. shows a phytosterol derivative
in the form of γ-sitosterol compound, which is an
important plant sterol and has been reported for the first
time in Girardinia. The γ-sitosterol can be used in the
treatment of diabetes mellitus. The docking study of γsitosterol ligand with four different target proteins shows
that this compound is a good molecule that matches the
various targets associated with Diabetes mellitus, so γ-
sitosterol can be developed into an antidiabetic protein
drug22.
1.2 Benzenediol compound or known as catechol is a
phenolic compound, showing a fairly large inhibitory
activity of free radical DPPH. Phenolic compounds are
good electron donors, which can accelerate the
conversion of H2O2 to H2O. Phenolic compounds are
known as strong chain-breaking antioxidants. Phenols
are important plant constituents because of their ability
to suppress free radicals, due to their hydroxyl groups
which can contribute directly to antioxidant activity23.
Table 5: GC-MS analysis of the methanol extract of Mangifera quadrifida Jack baby fruit
S. No
Compounds
Retention Time (Min)
1
Caryophyllene
7.276
2
Phenol, 2,6-Bis(1,1-dimethylethyl)
8.810
3
1,2,3-Benzenetriol
10.685
4
Hexadecanoic acid
14.242
5
9,12-Octadecadienoic acid
16.254
6
9,17-Octadecadienal
19.526
7
Vitamin E
22.273
8
Stigmast-5-en-3-ol
23.303
9
2,5-Furandione
24.801
Table 5 shows that the methanol extract of Mangifera
quadrifida Jack baby fruit contains many polar
compounds such as phenol and alcohol compounds. In
addition, it was found the content of the Vitamin E
compound in the methanol extract. Most fruits and
vegetables contain vitamin E in low to moderate levels.
Vitamin E is known as an excellent antioxidant. The
antioxidant activity of vitamin E (α-tocopherol) during
unsaturated lipid peroxidation has been reviewed based
on its reaction product. Extinguishing reactions of free
radical activity of α-tocopherol occur through αtocopheroxyl radicals as intermediaries. Non-radical
products can be formed by combining free radicals with
α-tocopheroxyl radicals. Tocopheroxyl radicals from γ
and δ-tocopherol prefer to react with each other to form
dimeric products which are still effective as
antioxidants24.
Composition (%)
90
92
95
92
95
92
96
91
% Area
0.29
0.13
0.64
4.80
18.65
1.90
1.14
9.12
0.16
Composition (%)
90
93
95
97
99
97
95
99
97
Antioxidant Activity:
Antioxidant activity tests of Mangifera quadrifida Jack
baby fruit extract was carried out to determine the initial
potential of the compound as an antioxidant. Table 6.
shows the results of antioxidant activity tests based on
the reduction of DPPH radical activity by active
compounds.
Table 6: Antioxidant activity test
S.
Sample
No
1
n-Hexana extract
2
Ethyl acetate extract
3
Methanol extract
IC50 Value
(ppm)
430.63
24.52
23.67
Antioxidant
Level
Weak
Strong
Strong
The IC50 values are obtained from the calculation of
linear regression equations. The "y" coefficient in this
equation is IC50, while the "x" coefficient obtained is the
amount of concentration needed to reduce 50% of DPPH
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Research J. Pharm. and Tech. 14(7): July 2021
radical activity. The results of this study indicate an
increase in percent inhibition by increasing sample
concentration. The antioxidant activity test gave IC50
values of 430.63ppm, 24.52ppm and 23.67ppm for nhexane, ethyl acetate and methanol extracts,
respectively. The test results indicate that ethyl acetate
and methanol extracts have antioxidant activity that is
quite strong compared to n-hexane extracts. According
to Molyneux13, a compound is categorized as having
antioxidant activity if the IC50 value obtained ranges
from 200-1000ppm, where the n-hexane extract of
Mangifera quadrifida Jack baby fruit is classified as a
compound with weak activity but still has potential as an
antioxidant.
Associated with the presence of secondary metabolites
found in the extracts of Mangifera quadrifida Jack baby
fruit, polar groups in phenolic compounds play an
important role as electron donors25,26,27. In addition, the
presence of terpenoids and also vitamin E is predicted to
have a role in inhibiting DPPH free radicals.
6.
7.
8.
9.
10.
11.
12.
13.
14.
CONCLUSION:
Phytochemical test results of Mangifera quadrifida Jack
baby fruit extracts revealed the presence of various
bioactive compounds: flavonoids, tannins, phenols, and
steroid glycosides found in all extracts, saponins in nhexane extracts, and terpenoids in ethyl acetate and nhexane extracts. GC-MS analysis provides information
in the form of the main constituent structure of each
extract, which involves alkanes, alkenes, carboxylates,
phenolic compounds, and aromatics. The antioxidant
activity test on n-hexane, ethyl acetate and methanol
extract obtained IC50 values of 430.63ppm, 24.52ppm
and 23.67ppm, respectively. The considerable
antioxidant activity is thought to be a contribution of the
phenol group, terpenoids and also vitamin E which is
contained in the extracts of Mangifera quadrifida Jack
baby fruit.
ACKNOWLEDGEMENT:
Authors are thankful to Politeknik AKA Bogor for
providing financial support for laboratory and
instrumental facilities.
15.
16.
17.
18.
19.
20.
21.
22.
23.
CONFLICT OF INTEREST:
The authors declare that they have no conflict of interest.
25.
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