Songklanakarin J. Sci. Technol.

45 (2), 228–235, Mar. – Apr. 2023

Original Article

Evaluation of nutritional and chemical compositions and

morphology of horny little devil (Smilax myosotiflora)
from different regions in Malaysia
Rasmaizatul Akma Rosdi1, Deny Susanti Darnis2, Nik Fakurudin Nik Ali1,
Mohd Dasuki Sul’ain1, Wan Norhasikin Wan Marizam1,
Mohamad Noor Mohamad Roze1, and Wan Rosli Wan Ishak3*
1 Biomedicine Program, School of Health Sciences,
Universiti Sains Malaysia (Health Campus), Kubang Kerian, Kelantan, 16100 Malaysia

2 Department of Chemistry, Kulliyyah of Science,

International Islamic University of Malaysia, Kuantan, Pahang, 25200 Malaysia

3 Nutrition
and Dietetics Program, School of Health Sciences,
Universiti Sains Malaysia (Health Campus), Kubang Kerian, Kelantan, 16100 Malaysia

Received: 3 November 2022; Revised: 11 February 2023; Accepted: 24 February 2023

Abstract

Smilax myosotiflora is a wild creeping plant that was scientifically proven to possess antioxidant, aphrodisiac and
synergistic effects. However, the composition of the plant can be varied by locations and this will affect the efficacy of the plant
bioactivities. Therefore, this study aimed to determine the morphology, and the compositions of nutritional and chemical in the
plant from different regions in Malaysia. S. myosotiflora was collected from Kelantan, Perak, and Pahang rainforests. The
morphology and nutritional composition of the plant were determined through SEM-EDX and proximate analysis accordingly.
Total phenolic compounds (TPC) and total flavonoid compounds (TFC) were determined while the GC-MS analyses were run on
the plant for its chemical profile. It was found that the morphologies of S. myosotiflora tubers were comparable among different
areas. Carbon, oxygen and potassium were the main elements with only a low intensity of calcium detected on the surfaces of the
Kelantan tubers. S. myosotiflora were significantly different in nutritional compounds but not in TPC and TFC between the
samples. There were 15 identical compounds detected in the chloroform extract of S. myosotiflora samples, where 2-methyl-7-
phenylindole was the most abundant. Considering the highest TPC, TFC and the most ingredients obtained through the GC-MS,
Perak is the best location to harvest and promote the cultivation of S. myosotiflora plant. However, more studies should be
performed on S. myosotiflora to explore more benefits for the pharmaceutical and agricultural sectors.

Keywords: Smilax myosotiflora, morphology, nutritional profile, chemical profile

1. Introduction of peninsular Malaysia, southern Thailand, Java, Burma, and

throughout tropical regions in Southeast Asia (SEA). It has
Smilax myosotiflora or the horny little devil is a been known in vernacular as ‘ubi jaga’, ‘ubi besi’, ‘akar
herbaceous creeping plant that grows wildly in the forests tanding’, ‘akar restong’ or ‘itah besi’ in Malaysia and
Indonesia, or as ‘Khao-yen bai bang’ and ‘Lek thong daeng’
in Thailand (Rosdi, Sul'ain, Darnis, & Ishak, 2022). The
*Corresponding author species originated from the monocotyledon family of
Email address: wrosli@usm.my Smilacaceae, the second largest family in the Liliales order.
 R. A. Rosdi et al. / Songklanakarin J. Sci. Technol. 45 (2), 228-235, 2023 229
The hooked thorns allow S. myosotiflora to hang onto and
grow over soils and surfaces up to 10 m height. Its leaves are
light green, smooth and broadly elliptic 4-17 cm long, while
the tubers are dark brown with rough surface, very hard, have
an irregular round shape and are covered with the hairy roots.
S. myosotiflora grows best in moist soil with pH 5-6, rich in
humus and nutrients, sheltering under the bigger trees in
lowland and hilly areas with good drainage (Jones & German,
1993).
The S. myosotiflora tubers have been documented as
the most functional part of the plant, and were used by locals
and the aboriginal people in Malaysia and the southern part of
Thailand for various therapeutic effects (Mohammad, Milow,
& Ong, 2012; Rosdi et al., 2022). Over the generations, S.
myosotiflora was pronounced as an aphrodisiac, a lumbago
reliever, an energy booster, restoring vitality and libido, and
has been used to treat rheumatism and syphilis traditionally
(Nurul Ayuni et al., 2018; Ong, & Azliza, 2015 & Ahmed,
Fatimah, Siti Zaiton, & Parveen, 2015; Zaki, Gandaseca, Figure 1. S. myosotiflora harvesting locations in Kelantan, Perak &
Mohd Rashidi, & Ismail, 2019). In numerous scientific Pahang, in Malaysia
studies, it was proven to possess the aphrodisiac, synergistic,
antioxidant and anthelmintic effects, thus having potential to tubers were broken down into smaller pieces using a mortar
treat several critical medicinal problems (Chyang, Mustapa, & and a pestle. The ground tubers were passed through a 200-
Ambia, 2018; Dasuki, Khaizil, Emylia, Noor Izani, & Mohsin, mesh sieve to obtain the fine powder. The powder samples
2012; Mustaffar Bakri, 2013; Rahman, Fatt, & Sulaiman, were kept in tightly sealed containers at 4ºC until use.
2010; Wan, Ahmad, & Sul’ain, 2013). However, the
composition of the plant may vary by area and this could 2.2 Morphological evaluation
impact its efficacy of the functional activities.
Though the distribution of wild S. myosotiflora Scanning electron microscope (SEM) analysis was
plants in Malaysia shares similar climate, studies have shown carried out to investigate the morphology of S. myosotiflora.
that other factors such as local microclimates, temperature, The powder samples were mounted on carbon stubs and
light intensity, soil composition, and plant collection period sputter coated with gold (Leica, Germany) for 15 min. The
influence the morphology and composition of the plant coated S. myosotiflora samples were viewed at 20 kV with
(Alcántara-Ayala et al., 2020; Bouba et al., 2012; Kosanic, field emission SEM (Fei, Switzerland). Energy-dispersive X-
Anderson, Harrison, Turkington, & Bennie, 2018; Zhang et ray (EDX) spectroscopy was done for quantitative analysis of
al., 2018). The distinctive effects on the plant in response to the elemental composition of S. myosotiflora tubers at 20kV
those variations could lead to an inconsistency in the bioactive on an EDX spectrometer (Quanta, US) equipped with an X-
compounds of S. myosotiflora and its pharmacological flash detector.
activities. Therefore, this study was performed to evaluate the
profiles of S. myosotiflora from three locations in Malaysia by 2.3 Nutritional analysis
investigating its morphology as well as nutritional and
chemical compositions. These data are essential for theoretical Proximate analysis was commenced to determine
foundations, knowledge and technical support in medicinal the total ash, calories, carbohydrates, crude fat, moisture, and
plant quality control, which will contribute to the benefit of crude protein of S. myosotiflora in accordance to Ng et al.
pharmaceutical and agricultural sectors. (2020) and the official standard methods of the Association of
Official Analytical Chemists (AOAC, 2005) with slight
2. Materials and Methods modifications. The total ash content was assayed by
incinerating the powder in a muffle furnace at 550°C for three
2.1 Plant materials and sample preparation hours (AOAC 930.05). For the calorie measurement, a bomb-
calorimeter (IKA-WERKE, Germany) system and software
S. myosotiflora were harvested from the forests of Kelantan were used to determine the calorific values of the samples.
(4°50'55.3"N 102°03'11.5"E), Perak (5°29'31.6"N, The carbohydrate contents of S. myosotiflora tubers were
101°26'26.6"E) and Pahang (4°41'14.2"N, 102°06'33.8"E) as estimated by difference of crude protein, crude fat, moisture
shown in Figure 1, with the help of the aboriginal people and and total ash (Method of Analysis of Nutrition Labelling
local villagers. The S. myosotiflora tubers were cleaned under AOAC). Soxhlet extraction with petroleum ether as its solvent
the running tap water to remove any surface pollutants, and was conducted to determine the amount of crude fat in the
the hairs were discarded concurrently. S. myosotiflora tuber samples (AOAC 936.15). S. myosotiflora moisture was
samples were prepared following a previous study (Wan, determined gravimetrically by drying the sample overnight in
Ahmad, & Sul’ain, 2016). Cleaned samples were dried in a a hot air oven at 105°C (AOAC 931.04). For crude protein
ventilated drying oven (Memmert, Germany) at 40-50°C for a quantification, semi-micro Kjeldahl method was applied using
few days. Then a power grinder (Golden Bull, Malaysia) was a Kjeldahl analyzer (FOSS, Denmark) with the nitrogen
applied to obtain powdered samples. Prior to that, the dried conversion factor of 6.25 (AOAC 991.20).
230 R. A. Rosdi et al. / Songklanakarin J. Sci. Technol. 45 (2), 228-235, 2023

2.4 Total phenolic and flavonoid content (TPC and separated volatile compounds was based on the comparison of
TFC) assays their retention time (RT) with those in NIST17 mass spectral
library. The relative amount of each component was
Fine ground S. myosotiflora tuber sample of 10 mg calculated by comparing its average peak area to the total area
was stirred in 10 mL distilled water (dH2O) at room (%).
temperature for three hours before filtering through Whatman
no. 1. Then, the filtrate was used to determine the total 2.6 Statistical analysis
phenolic and flavonoid contents in the S. myosotiflora
quantitatively. The use of water in this study as the extraction The mean values and standard deviations of
solvent was intended to imitate a common way on how the nutritional contents, TPC and TFC were calculated according
plant is prepared and used in folk medicine as reported by to the duplicate or triplicate readings from three independent
Rosdi et al. (2022). experiments. Data were first assessed for normality using the
The TPC of the plant was determined using a 96- Kolmogorov-Smirnov test. The determination of significant
well microplate with Folin–Ciocalteu method from Mangao et differences was done using one-way ANOVA or Kruskal-
al., 2020 with slight modifications. In brief, 20µL of S. Wallis test, followed by Mann-Whitney when not normally
myosotiflora (1mg/mL) or gallic acid and 100µL of 10% distributed. All statistical analyses were performed using
Folin–Ciocalteu reagent was aliquoted into the 96-well plate. GraphPad PRISM Version 6.0 by Dotmatics, California. P-
After 5 min, 80µL of 700mM sodium carbonate (Na2CO3) value < 0.05 was required to call a significant difference
was added to the same wells, followed by two hours of between groups.
incubation in the dark. The absorbances of samples were
measured at 765nm. The S. myosotiflora TPC was expressed 3. Results and Discussion
in mg GAE/g DW (mg gallic acid equivalent per gram of dry
weight used) based on the calibration curve for gallic acid 3.1 S. myosotiflora sample preparation
(12.5-400mg/L with R2=0.99). The gallic acid dilutions of 10-
400 mg/mL were used as calibration standards. The preparation of S. myosotiflora samples in the
Meanwhile, the TFC of S. myosotiflora was study was done according to Wan et al., (2016). Figure 3
quantified using aluminium chloride colorimetry in 96-well showcases the original powder of tuber samples and after the
plate as described by Sembiring, Elya, and Sauriasari (2018) total ash evaluation in nutritional analysis. Figure 3(A)
with slight modifications. The dH2O, 100µL and 10µL sodium exhibits the S. myosotiflora powder samples after finely
nitrate (50mg/mL) were added to the well followed by 25µL ground and passed through 200-mesh sieve. Even though the
S. myosotiflora filtrate (1mg/mL) or quercetin (10–200 mg/L) samples were prepared through a standard method, the
as the standard. After 5 min, 15µL aluminium chloride pulverized tubers of S. myosotiflora rendered perceivable
(100mg/mL) and 50µL of 1M sodium hydroxide were added. color nature where S. myosotiflora from Kelantan appeared to
The absorbance of the sample mixture was determined at 510 be more brownish while Perak’s was more whitish. All the
nm against a blank, and the TFC value was expressed in mg samples had been collected from their actual habitats, with
QE/g DW (mg quercetin equivalent per gram of dry weight Kelantan’s sample obtained from the forest of Tahan range,
sample used), based on the calibration curve for quercetin while Perak’s from Titiwangsa range, and Pahang’s from
(12.5-400mg/L with R2=0.99). TPC and TFC assays were run Pantai Timur range (Figure 1). Therefore, it can be claimed
in triplicates and the readings were done using a multimode that the S. myosotiflora plants grew in wild and visibly away
microplate reader (Thermo Scientific, US). from the industrial contamination or waste. However, the
variations of the tuber granule’s phenotype and physiology are
2.5 GC-MS analysis yet attributable by the origins of the geographical harvesting
place, climate change, and the divergent collection periods,
S. myosotiflora fine powder was macerated in which may alter the biochemical composition of the soil and
chloroform overnight to form the S. myosotiflora chloroform affect the yield of the plant (Kosanic et al., 2018; Wang,
extract (SMCE). The SMCE was dissolved in methanol and Tang, Fu, Huang, & Zhang, 2016). Other external factors, for
dichloromethane (50:50) before injecting into the GC-MS instance the surrounding temperature, sample storage, and
system equipped with a 5975 Mass Selective Detector handling techniques in the laboratory, also contribute to
(Agilent, USA) and a HP-5 MS capillary column (30 m length variability of physicochemical properties of the plant (Kaur,
x 0.25 mm internal diameter with 0.25 μm film thickness). Singh, Ezekiel, & Sodhi, 2009).
Samples were run simultaneously using a modified method Meanwhile, Figure 3(B) displays the residues of the
based on Zubair et al. (2017) where the carrier gas was 99.9 % samples after an overnight incineration when determining the
helium at a constant flow rate 1.5 mL/min. The temperature of total ash content. As can be seen, the incinerated tuber
the injector was initially 300 °C prior to the injection of a 1.0 samples appeared in different colors than theirs before.
μL sample. The temperature program was as follows: initial Residues of S. myosotiflora from Kelantan were white ash,
temperature 150°C held for 1 min, then ramping at a rate of 10 while the whitish Perak had transformed to firebrick color,
°C/min up to 290 °C held for 5 min. The temperature of mass and Pahang’s to cantaloupe color. Total ash represents the
spectra determination (MSD) transfer line was 300°C. The inorganic residues consisted of minerals that remain after the
MSD was operated in electron ionization (EI) mode, with an combustion of carbon and fibers, evaporation of moisture, and
ionization energy of 70eV, and the mass range scanned was so forth, from the samples (Chanda, 2014). Hence the
3–500 m/z. The temperature of ion source was 230 °C while distinctive colors of ashes from S. myosotiflora as seen in the
the MS quadrupole was at 150 °C. The identification of figure may indicate the presence of various incombustible
 R. A. Rosdi et al. / Songklanakarin J. Sci. Technol. 45 (2), 228-235, 2023 231
minerals such as sodium, aluminum, nickel, calcium, a low percentage of Ca in Kelantan sample. Though this
magnesium, silicon or iron in the plants which variably gained Smilax species signified rather little discrepancy between the
from the soils of their origin places. These minerals are samples, many have reported that the environmental factors,
essential nutrients contributing to sustainability of growth and for example climate, temperature and soil composition, have
yield in the plant (Veeresham, 2012). The present study high correlation with morphology and chemical composition
established that variations in the minerals exist in the same in the plant (Abdelsalam et al., 2019; Alcántara-Ayala et al.,
plant species, dependent on the harvesting area. 2020; Backouchi, Aouida, Khemiri, & Jebara, 2015; Yusuf et
al., 2020). Those factors can modify the mineral contents in
3.2 Morphological evaluation the soil, which eventually affects the nutrient intake of the
plants and alters their morphology and biochemical
The SEM-EDX analysis was applied to the tubers of composition (Kosanic et al., 2018). Therefore, even though
S. myosotiflora to compare the morphologies and elemental the variability were less existed in the tubers of the study, the
compositions among the samples. According to the SEM morphology and biochemical contents in other parts, for
images in Figure 2, the granules of S. myosotiflora tubers were instances leaves, flowers or buds, should be further
relatively irregular in shape and size. However, there was no investigated for further clarification.
distinct observation on the micromorphology of the granules
in the samples, except for Pahang’s sample which appeared
slightly more aggregated than Kelantan’s or Perak’s (Figure 2:
Panels A, B and C). Overall, the individual S. myosotiflora
tuber granules were polygonal, spherical to angular in shape at
the size range of 5-50 µm and possessed roughish
irregularities and fragmented surfaces (Figure 2: Panels D, E
and F). Figure 2 also shows the elemental compositions on the
surfaces of S. myosotiflora ground tubers from the EDX
analysis. The components of carbon (C), oxygen (O) and
potassium (K) were primarily present in all samples at
comparable intensities, with only a small amount of calcium
(Ca) solely presence in the S. myosotiflora from Kelantan.
From these findings, the wild growing plant of Figure 3. S. myosotiflora dried tubers in powder form (A) and after
honey little devil from three different locations possessed ashing at 550°C overnight for the total ash determination
similar micromorphology and chemical constituents except for (B)

Figure 2. SEM-DEX analysis of S. myosotiflora tubers at magnifications 250x and 5000x: Kelantan (A and D), Perak (B and E) and Pahang (C
and F), C K - carbon-K, O K - oxygen-K, K K - potassium-K, Ca K - calcium-K
232 R. A. Rosdi et al. / Songklanakarin J. Sci. Technol. 45 (2), 228-235, 2023

3.3 Proximate analysis compounds and their bioactivities. The data manifested that S.
myosotiflora is a ‘high calorie, low fat’ plant, which not only
Proximate analysis of total ash, crude fat, crude can be used for medicinal purposes but also has potential to
fiber, protein, moisture, carbohydrate, and protein was supplement the diet.
performed on the S. myosotiflora finely ground samples.
Figure 4 outlines the nutritional compositions of the tubers 3.4 TPC and TFC assays
sampled from three states in Malaysia: Kelantan, Perak, and
Pahang. According to the graphs, the S. myosotiflora tubers Phenolic and flavonoid compounds are among the
exhibited significant differences in several nutritional most functional bioactive ingredients from plants with
components between the regions except the TDF and calories. significant health benefits and play crucial role in the
The sample of Kelantan was significantly different with Perak antioxidant, anticancer and aphrodisiac activities (Dasuki et
in the percentages of total ash (1.71 ± 0.02 vs 1.31 ± 0.23), al., 2012; Chittasupho, Manthaisong, Okonogi, Tadtong, &
crude fat (0.18 ± 0.04 vs 0.38 ± 0.10), moisture (8.65 ± 1.61 Samee, 2022; Sembiring et al., 2018; Zubair et al., 2017). The
vs 5.39 ± 0.62) and carbohydrates (82.57 ± 1.72 vs 87.23 ± TPC and TFC in the S. myosotiflora samples were determined
1.33). The percentages of total ash (1.71 ± 0.02 vs 1.04 ± using Folin–Ciocalteu and aluminium chloride colorimetry
0.03), crude fiber (15.27 ± 1.38 vs 10.92 ± 0.81), protein (6.58 methods. In Table 1, S. myosotiflora manifested no significant
± 0.78 vs 3.92 ± 0.12), moisture (8.65 ± 1.61 vs 5.26 ± 0.66) differences in TPC and TFC levels despite having been
and carbohydrates (82.57 ± 1.72 vs 89.50 ± 0.65) were harvested from different places and times. Even so, the S.
significantly different between Kelantan and Pahang. The S. myosotiflora from Pahang had the lowest levels of TPC (3.64
myosotiflora samples of Perak and Pahang were significantly ± 0.26 mg GAE/g DW) and TFC (23.43 ± 1.05 mg QE/g DW)
different only in carbohydrate contents of 87.23 ± 1.33 % vs among the cases. Meanwhile, Perak’s sample possessed the
89.50 ± 0.65 %. The calories seemed comparable with Perak’s highest TPC and TFC levels at 5.15 ± 0.57mg (GAE/g DW)
sample showing the highest 389.3 ± 28.16kcal/(100g) and 27.48 ± 0.44 mg (QE/g DW).
followed by Kelantan’s 387.9 ± 147.20kcal/(100g) and Based on the data from the three examined S.
Pahang’s 385.4 ± 81.96kcal/(100g). myosotiflora samples, ironically, geographical variations and
Based on this study, the variations in nutritional other environmental factors were imperceptible to impacts the
composition also exist in plants growing wild, such as in S. phenolic and flavonoid contents in the tubers, as the levels
myosotiflora, even though their growth was only influenced were fairly similar. Previous studies have evidenced that the
by the natural factors. According to previous studies, type of contents of phenolics and flavonoids in a plant were mainly
soil, moisture, and exposure to environmental factors could influenced by geographical, climate, and other environmental
lead to modifications in the physicochemical and factors such as length of day, temperature, light intensity, and
phytochemical properties, which contribute to the nutritional water content in the soil (Danladi et al., 2015; Morreeuw et
composition in a plant species (Chanda, 2014; Ogundola, al., 2021; Zhang et al., 2018). Studies done on the onion (Bibi
Bvenura, & Afolayan, 2018; Veeresham, 2012). Therefore, for et al., 2022), mulberry (Zhang et al., 2018), millet (Kumari,
the purposes of phytopharmaceutical development, clinical Madhujith, & Chandrasekara, 2017), Melastoma
research, or manufacturing, consistency on the harvesting area malabathricum L. (Danladi et al., 2015) and Moringa oleifera
is critical as divergence could impact the effectiveness of Lam. (Iqbal & Bhanger, 2006) have demonstrated significant

Figure 4. Percentages of the elements in proximate analyses of S. myosotiflora sampled from three places: Kelantan (Kel), Perak (Per) and
Pahang (Phg). Abbreviations: * - P-value < 0.05, ** - P-value < 0.005, *** - P-value ≤ 0.005, **** - P-value < 0.0001
 R. A. Rosdi et al. / Songklanakarin J. Sci. Technol. 45 (2), 228-235, 2023 233
Table 1. TPC and TFC in S. Myosotiflora analysis. According to the data on three examined S.
myosotiflora samples, 15 constituents were detected and
Sample TPC (mg GAE/g DW) TFC (mg QE/g DW) tentatively identified in the SMCEs (Table 2). Although the
SMCEs compounds revealed considerably different
SM Kel 5.13 ± 0.13 24.46 ± 0.31 percentages, overall, 2-methyl-7-phenylindole (RT: 19.772)
SM Perak 5.15 ± 0.57 27.48 ± 0.44 was the major compound accounting for 3.63 to 15.17% of the
SM Phg 3.64 ± 0.26 23.43 ± 1.05 total compound contents. In the SMCE Kelantan, it was
noticeable that there were a few compounds detected at
The values are given as mean ± SD. Abbreviations: mg GAE/g DW; greater levels than in Perak or Pahang SMCEs, for instance
mg gallic acid equivalent per gram of dry weight, mg QE/g DW; mg
quercetin equivalent per gram of dry weight sample used, SM Kel; S.
eicosane (RT: 6.257), pentacosane (RT: 8.130), docosane
myosotiflora of Kelantan, SM Perak; S. myosotiflora of Perak, SM (RT: 9.918), tetracosane (RT: 11.587), 2-methylpentacosane
Phg; S. myosotiflora of Pahang. (RT: 13.139) and heneicosane, 3-methyl- (RT: 14.583). Other
than that, 1-octadecane (RT: 8.901), nonadecyl
variations in their TPC and TFC induced by the trifluoroacetate (RT: 9.659), 1-nonadecene (RT: 10.751),
aforementioned factors. Therefore, the findings from this tris(tert-butyldimethyl silyloxy)arsane (RT: 18.986) and 2-
study are in some contrast with those prior studies. methyl-7-phenylindole (RT: 19.772) were found to be
Nevertheless, before any conclusion could be made, it is comparatively abundant in SMCE Perak, while benzene, (1-
recommended to perform more research on these secondary methylundecyl)- (RT: 7.357) and bis(2-ethylhexyl) phthalate
metabolite contents in the S. myosotiflora to confirm the (RT: 12.799) was the highest in SMCE Pahang. Meanwhile,
findings. butyl 9,12-octadecadienoate (RT: 12.186) and octadecane, 3-
Meanwhile, Dasuki et al. (2012) reported that the ethyl-5-(2-ethylbutyl)- (RT: 16.043) only presented in the
TPC of S. myosotiflora in methanol extract was 6.55mg SMCEs from Perak and Kelantan.
GAE/g DW, which was higher than the content in this study. The chemical constituents of S. myosotiflora traced
The use of a different extraction solvent to determine the TPC using a non-polar solvent, chloroform, displayed almost
and TFC also explains the variability of phytochemical levels identical constituents, which mostly were volatile straight
in the plant. An organic solvent with high polarity, for chain compounds: alkanes, alkenes, fatty acid esters, and
example methanol or ethanol, may enhance the solubility and methyl esters. The secondary metabolites of 2-methyl-7-
extraction of complex and high molecular weight compounds, phenylindole from lactone group, which was discovered to be
such as the polyphenols. However, for reasons of simplicity the most prominent compound in the SMCEs, has been
and applicability, water has been widely used in traditional reported to have strong correlation with antimicrobial and
medicine as the extraction agent. Its ability to extract high antiparasitic activities of plants (Bloch, Vijay, Singh, Minna,
contents of other compounds such as carbohydrates, proteins, & Sougata, 2021; Norouzi, Hejazy, Shafaghat, & Shafaghat,
and organic acids could interfere the quantification of 2021; Raj, Vijayakumari, Jebarubi, & Kavitha, 2022).
phenolics and flavonoids comtents in the plant. More research Meanwhile, eicosane, pentacosane, docosane and tetracosane
should be conducted using the organic solvent to explore the are compounds from alkanes, was the biggest group of
biochemical compounds in the plant qualitatively. phytochemicals detected in the SMCEs. The group is a series
of long chain saturated hydrocarbons with single covalent
3.5 Volatile compounds from GC-MS bonds. In the agricultural sector, alkanes are synthesized as
part of the epicuticular leaf wax for terrestrial plants, and as a
The chemical constituents in the SMCEs were plant chemotaxonomy biomarker (Bush & McInerney, 2013).
investigated qualitatively and quantitatively using the GC-MS Therefore, the alkane compounds established in the SMCE

Table 2. List of identified compounds in SMCE from the three regions

Peak area (%)

No. RT Compound name Formula
Kel Per Phg

1 6.257 Eicosane C20H42 1.04 0.18 0.20

2 7.357 Benzene, (1-methylundecyl)- C18H30 - 0.33 1.05
3 8.130 Pentacosane C25H52 1.56 0.65 0.84
4 8.901 1-Octadecene C18H36 - 1.10 0.47
5 9.659 Nonadecyl trifluoroacetate C21H39F3O2 0.48 0.79 -
6 9.918 Docosane C22H46 2.37 0.41 0.68
7 10.751 1-Nonadecene C19H38 0.94 4.17 1.12
8 11.587 Tetracosane C24H50 2.66 0.74 0.95
9 12.186 Butyl 9,12-octadecadienoate C22H40O2 - 1.48 -
10 12.799 Bis(2-ethylhexyl) phthalate C24H38O4 1.24 1.64 2.58
11 13.139 2-Methylpentacosane C26H54 2.02 0.56 0.72
12 14.583 Heneicosane, 3-methyl- C22H46 1.56 0.31 0.29
13 16.043 Octadecane, 3-ethyl-5-(2-ethylbutyl)- C26H54 1.22 - -
14 18.986 Tris(tert-butyldimethyl silyloxy)arsane C18H45AsO3Si3 1.07 3.60 1.77
15 19.772 2-Methyl-7-phenylindole C15H13N 3.63 15.17 8.56

Abbreviations: RT; retention time, Kel; SMCE of Kelantan, Per; SMCE of Perak, Phg; SMCE of Pahang, -; not detected
234 R. A. Rosdi et al. / Songklanakarin J. Sci. Technol. 45 (2), 228-235, 2023
could be developed as a chemotaxonomy profile for S. Morphological variation of leaf traits in the
myosotiflora plant. Further investigations are suggested to Ternstroemia lineata species complex (Ericales:
determine the chemical compositions of mid- and polar Penthaphylacaceae) in response to geographic and
compounds in the S. myosotiflora plant to solidify the climatic variation. PeerJ, 8, 1–27.
findings. Backouchi, I. Z., Aouida, M., Khemiri, N., & Jebara, M.
(2015). Genetic diversity in Tunisian populations of
4. Conclusions faba bean (Vicia faba L.) based on morphological
traits and molecular markers. Genetics and
The present study established the qualitative and Molecular Research, 14, 7587–7596.
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compositions including TPC and TFC levels of S. S., Iqbal, A., . . . Abbasi, A. M. (2022). Variations
myosotiflora samples from Kelantan, Perak and Pahang states in total phenolic, total flavonoid contents, and free
in Malaysia. Some significant variations were found in the radicals’ scavenging potential of onion varieties
primary metabolite compounds (nutritional contents) among planted under diverse environmental conditions.
the samples, for instance in carbohydrates, crude fiber, crude Plants, 11, 1–31.
fat, total ash and moisture. In the secondary metabolites, no Bloch, K., Vijay, Singh, P., Minna, K., & Sougata, G. (2021).
distinct variations were detected either through EDX Natural compounds from Plumbago zeylanica as
spectroscopy, TPC, TFC or GC-MS investigations. On the complementary and alternative medicine.
contrary, even though the micromorphology of the tubers Singapore: Springer.
seemed almost identical, the features of the ground powder of Bouba, A. A., Njintang, N. Y., Foyet, H. S., Scher, J., Montet,
S. myosotiflora from the three locations were utterly different D., & Mbofung, C. M. F. (2012). Proximate
showing that variability exists in this plant. Despite all, the composition, mineral and vitamin content of some
Titiwangsa range in Perak had the most potential for wild plants used as spices in Cameroon. Food and
harvesting S. myosotiflora plant due to the highest TPC and Nutrition Sciences, 3, 423–432.
TFC, and the most compounds detected in the GC-MS Bush, R. T., & McInerney, F. A. (2013). Leaf wax n-alkane
analysis. It is essential to understand the criteria, correlations distributions in and across modern plants:
and deviations of the medical plant contents, for instance in S. Implications for paleoecology and chemotaxonomy.
myosotiflora, to avoid inconsistent quality in pharmaceutical Geochimica et Cosmochimica Acta, 117, 161–179.
development. Since this is the first study to report on the Chanda, S. (2014). Importance of pharmacognostic study of
variability of the promising honey little devil, future studies medicinal plants: An overview. Journal of
are strongly suggested in order to optimize the utilization of Pharmacognosy and Phytochemistry, 2, 69–73.
the plant. Chittasupho, C., Manthaisong, A., Okonogi, S., Tadtong, S.,
& Samee, W. (2022). Effects of quercetin and
Acknowledgements curcumin combination on antibacterial, antioxidant,
in vitro wound healing and migration of human
Authors would like to thank the Universiti Sains dermal fibroblast cells. International Journal of
Malaysia for financial support through the Research Universiti Molecular Sciences, 23, 1–16.
Initiative (RUI) with grant no RUI/1001/8012209. We also Chyang, P. J., Mustapa, M., & Ambia, K. M. (2018).
appreciate the Science Lab Management Unit team of the Synergistic antimicrobial effects of different ratio
School of Health Sciences, USM, for all the help given during combination of Smilax myosotiflora, Persicaria
the experiments. odorata and Syzygium aromaticum with antibiotics.
International Journal of Research in
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