B I O D I V E R S I T A S ISSN: 1412-033X
Volume 24, Number 12, December 2023 E-ISSN: 2085-4722
Pages: 6934-6941 DOI: 10.13057/biodiv/d241255
Identification, screening, and analysis of secondary metabolite content
in methanol extracts of ant nests plant tubers from Aceh, Indonesia
ROSLIZAWATY ROSLIZAW ATY
1,2,
, GHOLIB GHOLIB
3
, NADIA RAHMI
4
, KHAIRAN KHAIRAN
5
,
RINALDI IDROES
5
, SYAFRUDDIN SYAFRUDDI N
2
, MAHDI ABRAR
6,


1
Graduate School of Mathematics and Applied Sciences, Universitas Syiah Kuala. Jl. Teuku Nyak Arief No. 441, Banda Aceh 23111, Aceh, Indonesia.
Tel./fax.: +62-651-7551536,

email: [email protected]
2
Laboratory of Clinic,

Faculty of Veterinary Medicine, Universitas Syiah Kuala. Jl. Teuku Nyak Arief No.441, Banda Aceh 23111, Aceh, Indonesia
3
Laboratory of Physiology,

Faculty of Veterinary Medicine, Universitas Syiah Kuala. Banda Aceh, Indonesia. Jl. Teuku Nyak Arief No.441,
Banda Aceh 23111, Aceh, Indonesia
4
Program of Biology, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala. Jl. Teuku Nyak Arief No. 441, Banda Aceh 23111, Aceh,
Indonesia
5
Department of Pharmacy, Faculty of Mathematics and Natural Sciences, Universitas Syiah Kuala. Jl. Teuku Nyak Arief No. 441, Banda Aceh 23111,
Aceh, Indonesia
6
Laboratory of Microbiology, Faculty of Veterinary Medicine, Universitas Syiah Kuala. Jl. Teuku Nyak Arief No. 441, Banda Aceh 23111, Aceh,
Indonesia. Tel./fax.: +62-651-7551536,

email: [email protected]
Manuscript received: 16 November 2023. Revision accepted: 30 December 2023.
Abstract. Roslizawaty R, Gholib G, Rahmi N, Khairan K, Idroes R, Syafruddin S, Abrar M. 2023. Identification, screening, and analysis
of secondary metabolite content in methanol extracts of ant nests plant tubers from Aceh, Indonesia. Biodiversitas 24: 6934-6941. This
study aims to identify the chemical components obtained from extracting and partitioning methanol extracts from ant nest tubers
(Myrmecodia sp.) originating from Aceh Jaya and Aceh Besar. These extracts were identified using phytochemical screening, Fourier
Transform Infrared (FT-IR), and Gas Chromatography-Mass Spectroscopy (GC-MS) methods. The identification results showed that the
ant nest plant from Aceh Jaya belongs to the species Myrmecodia tuberosa Jack. The ant nest plant from Aceh Besar belongs to the
species Hydnophytum formicarum Jack. Phytochemical screening results showed that the methanol extract from Aceh Jaya's ant nest
tubers contained flavonoids, tannins, saponins, and terpenoids, while Aceh Besar's ant nest tubers contained flavonoids, tannins,
terpenoids. GC-MS analysis revealed that the major compound found in the methanol extract from Aceh Jaya's ant nest tubers was 2H-
oxecin-2-one,3,4,7,8,9,10-hexahydro-4-hydroxy-10-methyl-,[4S-(4R*,5E,10S*)]- constituting 50.63% of the extract. In comparison, the
primary compound in the methanol extract from Aceh Besar was resorcinol at 17.92%. FT-IR analysis indicated that the FT-IR spectra
of the methanol extracts from Aceh Jaya's ant nest tubers contained O-H, C-H, C=O, C=C alkene, and C=C aromatic groups. At the
same time, methanol extracts from Aceh Besar's ant nest tubers also contained O-H, C-H, C=O, C=C alkene, and C=C aromatic groups.
Keywords: Ant nest plant, GC-MS, medicinal plants, methanol extract, Myrmecodia, secondary metabolites
INTRODUCTION
Indonesia is considered the second most biodiverse
country after Brazil, with an estimated 10% or around
25,000 plant species of flowering plants found within its
borders, while there are approximately 28.000 plant species
(Astirin 2000). Most of these Indonesian flowering plant
species are known to have medicinal properties, which are
deep-rooted in Indonesian society (von Rintelen et al.
2017). Plants serve as a source of chemical compounds,
including primary and secondary metabolite compounds.
Secondary metabolites are chemical compounds typically
known for their bioactivity and role in defending against
unfavorable environmental conditions such as temperature,
climate, pests, and plant diseases. They can also treat
various human illnesses

(Ping et al. 2013). One method
used to assess the content of secondary metabolites is
phytochemical screening.
Phytochemical screening studies the active compound
components in a sample, including their chemical structure,
biosynthesis, natural distribution, biological functions,
isolation, and chemical composition comparison among
various plant species. The common compounds screened in
this experiment are alkaloids, flavonoids, tannins, terpenoids/
steroids, and saponins. One plant known for its medicinal
properties and believed to contain secondary metabolites is
the ant nest plant. Ant nest plants are currently deemed
suitable as raw materials for both modern and traditional
medicine, with tubers being part of the plant used for
medicinal purposes

(Dirgantara et al. 2022).
Therefore, the fractions were tested through phytochemical
screening and Gas Chromatography-Mass Spectroscopy
(GC-MS) analysis to determine the compound content in
the ant nest fractions. This was done to compare the results
of qualitative phytochemical screening with the results of
quantitative tests using GC-MS chromatography. In addition,
GC-MS analysis is used to identify compound components
present in plant cells that cannot be done using ordinary
phytochemical screening (Baeshen et al. 2023). Other
characterization analyses can also use Fourier Transform
Infra-Red (FTIR). This characterization analysis is quite
fast, accurate, and relatively sensitive. This type of analysis

ROSLIZAWATY et al. – Secondary metabolite content in methanol extracts of ant nests plant tubers

6935
can characterize samples in the form of liquids, solutions,
pastes, powders, films, fibers, and gases. This analysis also
makes it possible to analyze material on the surface of the
substrate (Barnes et al. 2023). GCMS results report that the
phenolic structure of Myrmecodia sp. includes kaempferol,
luteolin, routine, quercetin, apigenin, rosmarinic acid,
procyanidin B1, gallic acid, catechin, caffeic acid, p-coumaric
acid, and ferulic acid (Dirgantara et al. 2022), while the
FTIR test on M. pendens extracts from the n-butanol fraction
has succeeded in isolating an isolated xanthine oxidase
inhibitor with an IC50 of 79.77% (Simanjuntak et al. 2010).
Ant nest plants are widely found in the Aceh Province
and are commonly utilized as an alternative traditional
treatment by the local population. The phytochemical
screening of Aceh's ant nest tubers (Myrmecodia sp.) has
revealed the presence of secondary metabolites such as
phenolic compounds, saponins, triterpenoids, and steroids

(Frengki et al. 2014). Ant nest tubers have the potential to
act as anti-hyperglycaemic agents, enhance immunologic
responses,

and exhibit antibacterial properties

(Efimenko et
al. 2020). However, research and scientific publications on
Aceh's ant nest plants are currently limited, and reports on
their medicinal properties are scarce. Therefore, this study
aims to conduct phytochemical screening of methanol
extracts from Aceh's ant nest plant, Aceh Besar (representing
the lowland area) and Aceh Jaya (representing the coastal
area). In this research, methanol extracts from Aceh's ant
nest tubers will undergo partitioning based on differences
in polarity, from non-polar to polar. This research is
expected to serve as an initial step in understanding the
active compound contents within ant nest tubers that play
an active role in disease healing.
MATERIALS AND METHODS
The ant nest tubers used in this research had been
identified (determination test) at Herbarium Bogoriense,
Direktorat Pengelolaan Koleksi Ilmiah BRIN Cibinong.
The research on the screening and analysis of secondary
metabolite content in methanol extracts of ant nest tubers
(Myrmecodia sp.) from Aceh was conducted at the Natural
Products Pharmacy Laboratory, Department of Pharmacy,
Faculty of Mathematics and Natural Sciences, Syiah Kuala
University. FT-IR (Fourier Transform-Infrared) and GC-
MS (Gas Chromatography-Mass Spectroscopy) analyses
were conducted at the Instrumentation Laboratory,
Department of Chemistry, Faculty of Mathematics and
Natural Sciences, Syiah Kuala University. The research
was conducted from July to December 2022.
Preparation of ant nest tubers and sampling method
The ant nest plant specimens used in this research were
the tubers of ant nest plants native to Aceh, obtained from
the tropical forests of Aceh Province, specifically from
Aceh Besar (representing the lowland region) and Aceh
Jaya (representing the coastal area). Lowland areas are
defined as those with an altitude of <400 masl (meters
above sea level, while the coastal Areas fall within the
range of 401-800 masl

(Lestari et al. 2021). The sampling
of these two specimens was based on their respective
altitudes. The sampling method involved observing fresh
ant nest plants that met specific criteria, including the
tubers being neither too young nor old, well-formed tuber
shapes, brown coloration, non-wilted and non-wrinkled
skin, and a firm texture. Details of the plant's habitat, tuber
color, odor, taste, and other distinctive features were recorded.
Preparation of simplicia
Intact tubers of ant nest plants were cleaned, washed,
drained, and then sliced thinly. Drying was carried out
naturally, meaning they were dried indirectly under sunlight.
The duration of drying varied but lasted approximately 10
days. Once the ant nest tubers had dried, they were cut into
small pieces and ground into a fine powder using a blender.
The finely blended result was sieved and placed into clean
jars. Simplicia characterization was conducted following
the guidelines of Sriyanti et al. (2022).

Extraction
Approximately 2,000 g of simplicia powder from Aceh
Jaya's ant nest tubers and 1,500 g of simplicia powder from
Aceh Besar's ant nest tubers were placed in maceration
containers and dissolved in 75 ppm of methanol. The
mixture was occasionally stirred in closed containers and
left undisturbed for 5 days, shielded from light, with
occasional stirring. After 5 days, the sample was filtered,
and the filtered residue was subjected to maceration again
with 25 parts of methanol. This mixture was left for 2 days.
The macerate was evaporated using a rotary evaporator at
45°C until a concentrated extract was obtained. Extract
characterization was conducted following the guidelines of
Heinrich et al. (2022).
Phytochemical analysis of methanol extracts from
tubers ant nest plant from Aceh Jaya and Aceh Besar
Phytochemical screening for identifying flavonoids,
terpenoids, and steroids was conducted following the
guidelines

of Sriyanti et al. (2022). The identification of
alkaloids and saponins was performed according to the
guidelines of Heinrich et al. (2022), while the identification
of tannins was carried out by Sriyanti et al. (2022).
FT-IR analysis (Fourier Transform Infrared) and GC-
MS Analysis (Gas Chromatography-Mass Spectrometry)
FT-IR analysis followed Sriyanti et al. guidelines

(Sriyanti et al. 2022). GC-MS analysis was performed
using Gas Chromatography-Mass Spectrometry with the
Thermo Scientific ISQ 7000 Single Quadrupole GC-MS
instruments.
RESULTS AND DISCUSSION
Phytochemical screening
The results of identifying herbarium samples at Herbarium
Bogoriense, Direktorat Pengelolaan Koleksi Ilmiah BRIN
Cibinong, are presented in Table 1. This identification
needs to be done to determine the differences in ant nest
plant species originating from Aceh Jaya and Aceh Besar.

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Table 1. Identification of ant-nest plant herbarium samples

Identification Aceh Jaya Aceh Besar
Regnum/Kingdom Plantae Plantae
Sub Regnum/Sub
Kingdom
Tracheobionta Tracheobionta
Super Divisio/Super
Division
Spermatophyta Spermatophyta
Divisio/Division Magnoliophyta Magnoliophyta
Classis/Class Magnoliopsida Magnoliopsida
Sub Classis/Sub Class Asteridae Asteridae
Ordo/Order Rubiales Rubiales
Familia/Family Rubiaceace Rubiaceace
Genus/Genus Myrmecodia. Hydnophytum
Species/Species Myrmecodia
tuberosa Jack.
Hydnophytum
formicarum Jack.


Table 2. Phytochemical screening results of Aceh ant nest plant
extracts

Secondary metabolite compounds
Ant nest plant origin
Aceh Jaya Aceh Besar
Flavonoid + +
Terpenoid + +
Steroid - +
Tanin + +
Fenolik + +
Saponin + -
Alkaloid
- Dragendroff - -
- Meyer - -
- Wagner - -


Phytochemical screening conducted on the methanol
extract results included tests for alkaloids, flavonoids,
tannins, saponins, and terpenoids/steroids using reagents.
The phytochemical screening results obtained in this study
are presented in Table 2.
Several internal and external factors influence the
presence of secondary metabolites in a plant. Internal
factors, such as genetic and external factors, including
light, temperature, humidity, pH, soil nutrient content, and
altitude, play a significant role. Surface air temperature on
Earth is relative and represents a region's degree of heat
and cold. Different altitudes result in different
temperatures. Altitude is one of the factors that affect the
growth of a plant. The series of metabolic processes in
plants can be disrupted, leading to variations in compounds
produced at different altitudes

(Nataraj et al. 2022).
The phytochemical screening results indicate that altitude
can affect the content of secondary metabolites in ant nest
tubers. The difference observed is the presence of saponins
in the tubers of the Aceh Jaya ant nest plant, representing
coastal areas with an altitude of 401-800 m above sea level,
while the tubers of the Aceh Besar ant nest plant,
representing lowlands with an altitude <400 m above sea
level, do not contain saponins. This difference is attributed
to variations in physical factors such as temperature and
humidity between lowlands and coastal areas. Higher
altitudes are associated with lower temperatures

(Nataraj et
al. 2022). Another difference in phytochemical screening
results is the presence of steroids in the tubers of the Aceh
Besar ant nest plant. In contrast, the Aceh Jaya ant nest
tubers do not contain steroids. However, GC-MS analysis
results show that both extracts contain steroids.
According to Pasaribu et al.

(2018), ant nest plants from
the genera Hydnophytum and Myrmecodia contain various
compounds, making them biologically active. Hydnophytum
formicarum Folium contains terpenoids, alkaloids, tannins,
glycosides, and saponins, while H. formicarum Cortex
contains flavonoids, alkaloids, terpenoids, and glycosides.
Terpenoids, alkaloids, and phenolics are also found in the
ant nest plant Myrmecodia pendens. Phytochemical
screening of ant nest plants M. Beccarii, Myrmecodia sp.,
and Hydnophytum sp. indicates that all three species
contain flavonoids, triterpenoids/steroids, and saponins
(Mardany et al. 2016; Pasaribu et al. 2018). Pasaribu et al.
(2018) found that the ant nest plant Myrmecodia rumphii
Becc. contains various secondary metabolites, including
triterpenoids/ steroids, phenolics, flavonoids, alkaloids, and
tannins, distributed in methanol, ethyl acetate, and n-
hexane solvents. Mardany et al. (2016) reported that the
ethanol extract of Myrmecodia beccarii Hook.f. ant nest
tubers contain flavonoids, saponins, and tannins. In
addition, based on the phytochemical screening results
conducted in this study, it is evident that ant nest tubers are
positively rich in various types of secondary metabolites
with medicinal properties. Ant nest extract has many
medicine activities, namely antioxidant, antibacterial,
cytotoxic, and anticancer (Dirgantara et al. 2022).
GC-MS analysis
The results of GC-MS chromatogram of methanol
extracts from tubers of Aceh Jaya and Aceh Besar ant nest
plants are presented in Figure 1 and Figure 2. Meanwhile,
the analysis results of chemical compounds in methanol
extracts from the ant nest tubers of Aceh Jaya and Aceh
Besar were presented in Tables 3 and 4, respectively.
Based on the GC-MS analysis of methanol extracts
from Aceh Jaya ant nest tubers, 22 compounds were
identified. The most abundant compound group in the
methanol extract from Aceh Jaya ant nest tubers in this
study was coumarins, especially 2H-oxecin-2-
one,3,4,7,8,9,10-hexahydro-4hydroxy- 10methyl-[4S-
(4R*,5E,10S*)] representing 50.63% of the total
compounds. This result is consistent with the FT-IR
analysis, which indicated the presence of coumarin
compounds characterized by O-H and C=C aromatic
groups. Organic coumarin compounds are known for their
anti-microbial activities

(Widelski et al. 2018).
Based on the GC-MS analysis of methanol extracts from
Aceh Besar ant nest tubers, 31 compounds were identified.
The most abundant compound group in the methanol
extract from Aceh Besar ant nest tubers in this study was
simple phenolics, specifically resorcinol, accounting for
17.92% of the total compounds. This result is consistent
with the FT-IR analysis, which indicates the presence of
resorcinol compounds characterized by O-H, C=O, and
C=C aromatic groups. Phenolic compounds represent the
largest group of compounds that act as natural antioxidants
in plants

(Hamsar and Mizaton 2012). Phenolic compounds

ROSLIZAWATY et al. – Secondary metabolite content in methanol extracts of ant nests plant tubers

6937
from plants have the ability to act as antioxidants, anti-
inflammatory agents, antiproliferative agents, antimutagenic
agents, and anti-microbial agents. Those phenolic compounds
also play a role in preventing and treating degenerative
diseases, cognitive disorders, cancer, premature aging, and
immune system disorders

(Lin et al. 2016).
The differences in compounds obtained in the GC-MS
analysis are due to the choice of solvents; polar compounds
will dissolve in polar solvents such as methanol, while non-
polar solvents like N-hexane

(Nwabueze and Okocha
2008). In this study, compounds such as 2(5H)-furanone at
retention time of 10.84, and hexadecenoic acid at retention
time of 20.317 were identified, whereas in this study, the
methanol extract from Aceh Besar ant nest tubers yielded 2
(5H)-furanone at retention time of 6.946 and 2(5H)-
furanone at retention time of 7.017.
Based on the phytochemical screening results, Aceh
Jaya ant nest tubers tested negative for alkaloids using three
reagents: Meyer, Borchardt, and Dragendorf. This result
was confirmed by FT-IR analysis and GC-MS analysis,
indicating that the methanol extract from Aceh Jaya ant
nests does not contain alkaloids. The phytochemical screening
for flavonoid compounds in the methanol extract from Aceh
Jaya ant nest tubers showed positive results. This is in line
with the FT-IR analysis, which indicated the presence of
flavonoids characterized by the C=O group, a distinctive
feature of flavonoids. However, GC-MS analysis of the
methanol extract from Aceh Jaya ant nest tubers did not
identify any compounds belonging to the flavonoid group.




Figure 1. GC-MS analysis results of methanol extract from Aceh Jaya ant nest tubers




Figure 2. GC-MS analysis results of methanol extract from Aceh Besar ant nest tubers

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Table 3. Analysis results of chemical compounds in methanol extracts from Aceh Jaya and Aceh Besar (Indonesia) ant nest using the
GC-MS method

Sample
origin
Peak
name
Retention time
(min)
SI (%) % area Compound
Aceh Jaya 1 17.913 70.4 1.22 1-Acetoxy-p-menth-3-one
2 18.049 68.6 1.30 1-Acetoxy-p-menth-3-one
3 21.035 76.1 3.22 2(3H)-Furanone, 4,5-dihydro-4-(2-methyl 3-methylene-1-buten-4-yl)-
4 21.562 79.2 0.61 [1,1'-Bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester
5 21.851 88.1 4.43 (4S,4aR,7S,7aR)-4,7-Dimethylhexahydrocyclopenta[c]pyran1(3H)-one
6 23.711 72.1 0.73 6-Nonenal, 3,7-dimethyl-
7 26.310 76.9 3.49 Geranyl vinyl ether
8 26.745 80.7 1.09 [1,1'-Bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester
9 27.262 73.2 50.63 2H-Oxecin-2-one, 3,4,7,8,9,10-hexahydro-4-hydroxy-10-methyl-, [4S(4R*,5E,10S*)]-
10 28.143 75.6 0.90 [1,1'-Bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester
11 32.779 89.9 4.96 n-Hexadecanoic acid
12 35.319 87.1 0.65 11-Octadecenoic acid, methyl ester
13 35.983 88.7 3.57 Linoelaidic acid
14 36.092 90.3 6.91 trans-13-Octadecenoic acid
15 36.265 84.1 2.05 17-Octadecynoic acid
16 36.466 78.7 0.61 [1,1'-Bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester
17 42.479 81.4 0.55 Ethanol, 2-(9-octadecenyloxy)-, (Z)
18 44.894 82.5 1.48 9-Octadecenoic acid (Z)-, 2-hydroxy-1-(hydroxymethyl)ethyl ester
19 52.101 84.7 2.57 Campesterol
20 52.522 89.6 5.36 Stigmasterol
21 53.298 85.8 3.09 Sitosterol
22 54.393 75.6 0.59 Ethyl iso-allocholate

Aceh Besar 1 5.878 81.1 0.87 Ethanol, 2-butoxy-
2 5.963 87.7 1.19 Ethanol, 2-butoxy-
3 6.225 77.3 0.36 Cyclopentanone, 2-methyl-
4 6.592 91.8 17.17 Ethanol, 2-butoxy-
5 6.827 89.2 2.14 2(5H)-Furanone
6 6.946 91.7 14.13 2(5H)-Furanone
7 7.017 92.8 13.84 2(5H)-Furanone
8 13.285 74.2 0.36 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy- 6-methyl-
9 15.197 71.5 1.32 Catechol
10 17.605 88.5 17.92 Resorcinol
11 21.547 78.2 0.47 E-7-Tetradecenol
12 21.860 74.5 0.27 2-Butyl-3-methylcyclopent-2-en-1-one
13 25.210 80.1 0.54 Phenol, 2,6-dimethoxy-4-(2-propenyl)-
14 26.291 74.6 1.25 7-Oxabicyclo[4.1.0]heptane, 1-methyl-4-(2- methyloxiranyl)-
15 26.366 72.2 0.66 4-Chloro-3-n-hexyltetrahydropyran
16 26.733 75.2 0.43 R-Limonene
17 28.693 81.7 0.83 Tetradecanoic acid
18 30.730 69.8 0.59 Pentadecanoic acid
19 31.978 83.1 0.95 Pentadecanoic acid, 14-methyl-, methyl ester
20 32.356 74.6 0.35 Benzenepropanoic acid, 3,5-bis(1,1- dimethylethyl)-4-hydroxy-, methyl ester
21 32.818 89.2 10.58 n-Hexadecanoic acid
22 35.172 83.4 0.33 11,14-Eicosadienoic acid, methyl ester
23 35.284 85.5 0.73 11-Octadecenoic acid, methyl ester
24 35.961 87.9 1.84 Linoelaidic acid
25 36.093 92.3 8.11 cis-Vaccenic acid
26 36.447 78.1 0.76 Octadecanoic acid
27 52.051 68.4 0.36 Ethyl iso-allocholate
28 52.466 71.7 0.44 Cholesta-22,24-dien-5-ol, 4,4-dimethyl-
29 53.238 72.3 0.54 ß-Sitosterol
30 54.092 69.4 0.46 1-Heptatriacotanol
31 55.286 63.0 0.24 Ethyl iso-allocholate


FT-IR Analysis
The phytochemical screening for tannin compounds in
the methanol extract from Aceh Jaya ant nest tubers
yielded positive results. This result is consistent with the
FT-IR analysis, which indicates the presence of tannins
characterized by the aromatic C=C and O-H groups (Table
4). However, GC-MS analysis of the methanol extract from
Aceh Jaya ant nest tubers did not identify any compounds
belonging to the tannin group. This may be due to the
presence of volatile compounds that are not well-suited for
GC-MS analysis. For other compounds like flavonoids and
saponins, it may be due to limitations in standard
compounds available in the GC-MS spectrophotometer
library

(Parastar and Weller 2023).

ROSLIZAWATY et al. – Secondary metabolite content in methanol extracts of ant nests plant tubers

6939
Phytochemical screening for terpenoid/steroid compounds
in the methanol extract from Aceh Jaya ant nest tubers
yielded positive results for terpenoids (Table 2). This result
aligns with the FT-IR analysis, which indicates the
presence of terpenoids characterized by O-H, C-H, C=O,
and C=C functional groups, suggesting the presence of
terpenoids, but also contains O-H, alkane C-H, and C=C,
indicating the presence of steroids. The positive terpenoid
results in phytochemical screening and the functional group
analysis in FT-IR align with the GC-MS analysis results,
which show the presence of monoterpenoids such as
geranyl vinyl ether at 3.49%, 1-acetoxy-P-menth-3-one at
1.30%, and 6-nonenal, 3,7-dimethyl- at 0.73%. Additionally,
the functional group analysis in FT-IR indicating the
presence of steroids aligns with the GC-MS analysis
results, which show the presence of steroids such as
stigmasterol at 5.36%, sitosterol at 3.09%, campesterol at
2.57%, and ethyl iso-alcoholate at 0.59%.
In this study, there were differences in the results of the
content of secondary metabolite compounds tested by
phytochemical screening and GC-MS analysis. This can be
attributed to the qualitative nature of phytochemical screening,
while GC-MS analysis is quantitative. The results analyzed
by GC-MS are more accurate than those tested by
phytochemical screening. Furthermore, GC-MS analysis
has been widely used to identify hundreds of compound
components present in plant cells, which cannot be achieved
with conventional phytochemical screening, as it is limited
to identifying compound groups

(Al-Rubaye et al. 2017).
Based on the phytochemical screening of Aceh Besar
ant nest tubers, the results showed negative results for
alkaloid. This is consistent with the FT-IR and GC-MC
analyses, indicating that the methanol extract from Aceh
Besar ant nest tubers does not contain alkaloids.
Phytochemical screening for flavonoid compounds in the
methanol extract from Aceh Besar ant nest tubers yielded
positive results. This aligns with the FT-IR analysis, which
indicates the presence of flavonoids characterized by the
C=O group, a distinctive feature of flavonoids.
The phytochemical screening for tannin compounds in
the methanol extract from Aceh Besar ant nest tubers also
yielded positive results. This result is consistent with the
FT-IR analysis, which indicates the presence of tannins
characterized by the O-H and C=C aromatic groups.
However, phytochemical screening for saponin compounds
in the methanol extract from Aceh Besar ant nest tubers
resulted in a negative outcome consistent with FT-IR and
GC-MS analysis. The absence of certain secondary
metabolite groups, such as tannins, could be attributed to
the presence of volatile compounds that are not well-suited
for GC-MS analysis. Compounds like flavonoids and
saponins may be due to limitations in the standard
compounds available in the GC-MS spectrophotometer
library

(Parastar and Weller 2023).
The phytochemical screening for terpenoid/steroid
compounds in the methanol extract from Aceh Besar ant
nest tubers yielded positive results for terpenoids. This
result aligns with the FT-IR Analysis, which indicates the
presence of terpenoids characterized by O-H, C-H, C=O,
and C=C functional groups, suggesting the presence of
terpenoids. It also contains O-H, alkane C-Hm, and C=C,
indicating the presence of steroids. The positive terpenoid
results in phytochemical screening and the functional group
analysis in FT-IR align with the GC-MS analysis results,
which show the presence of a monoterpenoid, R-limonene,
at 0.43%. The functional group analysis in FT-IR
indicating the presence of steroids aligns with the GC-MS
analysis results, which show the presence of steroids such
as β-sitosterol at 0.54%, cholesta-22,24-dien-5-Ol, 4,4-
dimethyl- at 0.44%, and ethyl iso-alcoholate at 0.36%.
According to Hertiani et al.

(2013), GC-MS analysis of
ant nest isolates (subfractions obtained from ethyl acetate-
methanol) revealed 78.80% purity of isolates occurring at
10.8 minutes. Stable ion fragments at 110 m/z with 100%
abundance were identified as 1,3 benzediol (Similarity
Index: 81). 1,3 benzediol (resorcinol) belongs to the group
of simple phenolics. This correlates with the findings in
this study regarding the methanol extract from Aceh Besar
ant nest tubers, where the most abundant compound is
resorcinol at 17.92%, which is a simple phenolic compound.
The differences in the results of GC-MS analysis for some
compounds may be due to species, growing locations, and
various solvents with different polarities.


Table 4. FT-IR analysis results of methanol extracts from ant nest tubers of Aceh Jaya and Aceh Besar

Sample origin Functional groups Obtained wavenumber (cm
-1
) Literature wavenumber (cm
-1
) References
Aceh Jaya O-H
C-H
C=O
C=C
C=C aromatics rings
3424.76
2931.93
1704.18
1623.17
1519.32
3200-3600
3000-2850
1725-1700
1680-1600
1500-1600
Skoog et al. (2016)
Pavia et al. (1979)
Pavia et al. (1979)
Pavia et al. (1979)
Skoog et al. (2016)

Aceh Besar O-H
O-H
C-H stretching
C-H stretching
O-H
C=O
C=O
C=C
C=C aromatic rings
3385.22
3385.22
2931.93
2867.31
2733.25
1713.83
1713.83
1610.63
1520.94
3400-3200
3400-2400
3000-2850
3000-2850
3400-2400
1725-1705
1725-1700
1680-1600
1500-1600
Pavia et al. (1979)
Pavia et al. (1979)
Pavia et al. (1979)
Pavia et al. (1979)
Pavia et al. (1979)
Pavia et al. (1979)
Pavia et al. (1979)
Pavia et al. (1979)
Skoog et al. (2016)

B I O D I V E R S I T A S 24 (12): 6934-6941, December 2023

6940
The polarity of each solvent type will affect the
selectivity of the compound types that will be extracted.
The solubility of compounds follows the principle that
solvents with the same polarity will dissolve compounds
with similar polarity. Chemical compounds in plant organs
have varying polarities depending on the location of
hydroxyl groups and the chemical element arrangements.
Areas with an altitude of below 400 m asl. are categorized
as lowlands. These soil types are relatively fertile with high
soil fertility indexes (Mujiyo et al. 2022). Environmental
conditions play a crucial role in plant growth and
development. Differences in altitude can result in
significant differences in environmental conditions.
Altitude can affect media, nutrient retention, water
viability, and erosion (Rahmawaty et al. 2023). Lowlands
have higher light intensity and environmental temperatures
than medium and highlands. Sunlight intensity significantly
affects the plants' photosynthesis, which correlates with the
content of both primary and secondary metabolites (Zhou
et al. 2022). Adequate light intensity enhance the synthesis
of organic compounds in ant nest tubers through
photosynthesis

(Farji-Brener and Werenkraut 2017).
In conclusion, the methanol extract from Aceh Jaya ant
nest tubers contains flavonoids, tannins, saponins, and
terpenoids. The ethyl acetate extract contains flavonoids,
tannins, and terpenoids, while the methanol extract from
Aceh Besar ant nest tubers contains flavonoids, tannins,
and terpenoids. The main compound in the methanol
extract from Aceh Jaya ant nest tubers is 2H-oxecin-2-one,
3,4,5,8,9,10-hexahydro-4-hydroxy-10-methyl-, [4S -
(4R*,5E,10S*)]-, constituting 50.63% of the extract.
Meanwhile, the primary compound in the methanol extract
from Aceh Besar ant nest tubers is resorcinol, constituting
17.92%. FT-IR spectra of the methanol extracts from Aceh
Jaya and Aceh Besar ant nest tubers indicate the presence
of O-H, C-H, C=O, C=C alkenes, and C=C aromatics.
ACKNOWLEDG EMENTS
The authors would like to thank the Chancellor of Syiah
Kuala University, Banda Aceh, Indonesia, for his trust in
providing research funding through the Lector Grant
scheme for the fiscal year of 2021 with contract number:
250/UN11.2.1/SPK/PT.01.03/PNBP/2021.
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