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From Weed to Medicinal Plant: Antioxidant Capacities and Phytochemicals of

Various Extracts of Mikania micrantha

Article in International Journal of Agriculture and Biology · February 2018

DOI: 10.17957/IJAB/15.0522

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
17–0544/2018/20–3–561–568
DOI: 10.17957/IJAB/15.0522
http://www.fspublishers.org

Full Length Article

From Weed to Medicinal Plant: Antioxidant Capacities and

Phytochemicals of Various Extracts of Mikania micrantha
Amirah Haziyah Ishak1, Nurul Husna Shafie1*, Norhaizan Mohd Esa1, Hasnah Bahari2 and Amin Ismail1
1
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia
2
Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang,
Selangor, Malaysia
*
For correspondence: [email protected]

Abstract
Mikania micrantha is commonly consumed as traditional medicine in some countries, including Malaysia. Little is known
about the antioxidant properties and phytochemicals of M. micrantha. This study was aimed to investigate the total phenolic
content (TPC), total flavonoid content (TFC) and antioxidant capacities of the leaves and stems of M. micrantha of hot water,
cold water, 70% ethanol, ethyl acetate, and hexane extracts. Folin-Ciocalteu and aluminium chloride colorimetric assays were
used to determine the TPC and TFC, respectively. The antioxidant capacities were determined using rapid, inexpensive and
small-scale microplate of five different antioxidant assays. Gas chromatography-mass spectrometry (GC-MS) was used to
chemically profile and characterize the phytochemicals. In comparison with different solvents, the ethyl acetate stems (EAS)
and leaves (EAL) extracts of M. micrantha had the significantly greatest TPC (141 ± 0.51 mg gallic acid equivalent/g) and
TFC (70.1 ± 0.92 catechin equivalent/g), respectively. Moreover, EAS extract had the significantly greatest antioxidant
capacities using DPPH (EC50 = 324 ± 61.4 μg/mL), ABTS (0.53 ± 0.01 mmol trolox equivalent/g), FRAP (1.28 ± 0.05 mmol
Fe2+/g), phosphomolybdenum antioxidative power (219 ± 7.03 mg ascorbic acid equivalent/g), and β-carotene bleaching (108
± 2.23%) assays. GC-MS analysis of EAS showed the presence of sesquiterpenes (30.46%), phenol (16.38%), and alkane
hydrocarbons (10.45%), which may contribute to its antioxidant capacities. These findings suggest the stems extract of M.
micrantha using ethyl acetate as the potential source of natural antioxidant agents and its utilization to prevent oxidative
damage-related diseases could be further explored. © 2018 Friends Science Publishers

Keywords: Antioxidant; GC-MS; Mikania micrantha; Phenolic; Phytochemicals; Sesquiterpenes

Introduction Malaysia (Tripathi et al., 2012; Day et al., 2016; Ishak et al.,
2016). This plant is known as American rope, Chinese
Herbal medications have gained much attention for their creeper, mile-a-minute, ‘Chhagalbati’ or ‘Japanilata’ (West
use in relieving symptoms of disease (Shayganni et al., Bengal), ‘Selaput tunggul’ (Malaysia) and ‘Sembung
2015) and important in health care especially in developing rambat’ (Indonesia) (Haisya et al., 2013; Nornasuha and
countries. Studies have reported various plants, fruits and Ismail, 2013; Saha et al., 2015). In agriculture, M.
vegetables to contain a high amount of antioxidants that micrantha is a weed plant that can reduces the growth and
can be utilized to prevent oxidative damage-related productivity of several crops such as rubber, oil palm, and
diseases (Bordoloi et al., 2016; Ozkan et al., 2016). cocoa plantation in Malaysia which cost 8‒10 million
Naturally, antioxidants can be found in parts of plants, i.e., dollars per annum to control its growth (Sankaran, 2008).
flowers, leaves, stems, and roots. However, many factors This is due to its fast-growing habit and production of
including genetics, geographical region, climate/season, allelopathic substances (Sankaran, 2008; Nornasuha and
storage, and even the processing condition can affect the Ismail, 2013; Day et al., 2016). However, this plant is used
antioxidant capacities and amount of bioactive traditionally to treat insect bites and stop minor external
phytochemicals in plants (Li et al., 2012; Arena and bleeding or consumed as a juice as an alternative to reduce
Radice, 2016; Rai et al., 2017). glucose, cholesterol, and high blood pressure (Facey et al.,
Mikania micrantha Kunth (Asteraceae or Compositae) 2010; Ishak et al., 2016).
is a perennial creeping vine and widely distributed in South M. micrantha has demonstrated many health benefits,
and North America and can also be found in Africa, Pacific such as antimicrobial (Facey et al., 2010; Chetan et al.,
Islands and Southeast Asia, including Southern China and 2012; Chetia et al., 2014), anti-diabetic (Wan Nurhayati et

To cite this paper: Ishak, A.H., N.H. Shafie, N.M. Esa, H. Bahari and A. Ismail, 2018. From weed to medicinal plant: antioxidant capacities and
phytochemicals of various extracts of Mikania micrantha. Int. J. Agric. Biol., 20: 561‒568
 Ishak et al. / Int. J. Agric. Biol., Vol. 20, No. 3, 2018

al., 2013), anti-dermatophytic (Jyothilakshmi et al., 2015), Determination of Antioxidant Contents and Capacities
anti-stress (Ittiyavirah and Sajid, 2014), anti-inflammatory
(Pérez Amador et al., 2010), anti-proliferative (Ríos et al., Total phenolic content (TPC): Determination of TPC was
2014), and anti-cancer (Dou et al., 2014; Matawali et al., carried out according to Lee et al. (2014). Samples or a
2016) activities. In fact, M. micrantha is rich in standard (20 µL) were mixed with 100 µL of diluted Folin-
phytochemicals such as terpenoids (sesquiterpene lactones), Ciocalteu reagent (1:10, v/v in distilled water) in a 96-well
alkaloids, flavonoids, steroids, reducing sugars, saponins, plate. After 5 min, 80 µL of 7.5% sodium carbonate
phenolics and tannins (Dev et al., 2015; Dong et al., 2017). (Na2CO3) were added to each well. The plate was covered
In spite of its medicinal benefits as aforementioned, and left in the dark for 30 min on a Stovall belly dancer
the information of the potential antioxidant agent in this (Greensboro, NC, USA). The absorbance was measured at
plant is still scarce especially the comparison between 765 nm against a reagent blank. A standard calibration
different parts and the effect of different solvents which can curve using gallic acid (0.98‒1000 µg/mL) was plotted, and
affect the antioxidant capacities and bioactive compounds in the results were expressed as mg gallic acid equivalent
this plant. Antioxidant agents is important to prevent (GAE)/g extract using the following formula: TPC per 1 g
oxidative damage-related diseases i.e., cancer, extract = [(TPC per mL sample x dilution factor x total
cardiovascular diseases and diabetes etc., (Sharma et al., sample volume used)/sample weight].
2014) thus potential health benefits of this plant should be Total flavonoid content (TFC): The TFC was carried out
further investigated. We hypothesized that the effectiveness according to Belguith-Hadriche et al. (2013) with
and efficiency of secondary metabolites are significantly modifications. Samples or a standard (25 µL) were pipetted
affected by the extraction solvents (Kong et al., 2012). into the 96-well plate. Then, 100 µL of distilled water
Therefore, this study was aimed to determine and compare (dH2O) and 7.5 µL of 5% sodium nitrite (NaNO2) were
the antioxidant properties of various solvent polarities, i.e., added and incubated for 5 min. After that, 7.5 µL of 10%
hot water, cold water, 70% ethanol, ethyl acetate, and aluminium chloride hexahydrate (AlCl3.6H2O) was added
hexane extracts of the leaves and stems of M. micrantha and incubated for another 5 min. Then, 50 µL of 1 M
using rapid and small-scale microplate assays of the sodium hydroxide (NaOH) and 60 µL of dH2O were added
antioxidant procedures. The GC-MS analysis was and the absorbance was read using a microplate reader at
performed to chemically profile and characterize 510 nm. A standard calibration curve using catechin (1.95‒
phytochemicals of M. micrantha and hence, to confirm its 250 µg/mL) was plotted, and TFC were expressed as mg
medicinal values. catechin equivalent (CE)/g extract using the formula: TFC
per 1 g extract = [(TFC per mL sample x dilution factor x
Materials and Methods total sample volume used)/sample weight].
DPPH radical scavenging assay: The DPPH radical
Sample Collection and Preparation scavenging capacity was determined using the method
described by Kong et al. (2012). Samples or a standard (50
Mikania micrantha was collected in August 2015 from μL) at various concentrations (0.98–1000 μg/mL) and 195
Penang, Malaysia, and identified by the Forest Research μL of 100 μM DPPH solution were mixed in a 96-well plate
Institute Malaysia (FRIM), Kepong, Selangor, Malaysia and left in the dark at room temperature for 30 min. The
(Voucher no: SBID 051/15). The leaves and stems of M. absorbance of the reaction mixture was read at 515 nm.
micrantha were separated, washed and dried in a ventilated Butylated hydroxytoluene (BHT) and gallic acid were used
drying oven at 35°C for 72 h. as standards.
ABTS radical scavenging assay: ABTS radical scavenging
Extraction of M. micrantha capacity was determined according to the method described
by Othman et al. (2016) with some modifications. The
Powdered leaves and stems of M. micrantha were samples or a standard (20 μL) were mixed with 200 μL of
extracted separately using solvents of different polarities, the ABTS•+ solution in a 96-well plate, and incubated at
i.e., hot water and cold water (highly polar), 70% ethanol 30°C for 6 min. The results were expressed as mmol trolox
(polar), ethyl acetate (semi-polar) and hexane (non-polar). equivalent (TE)/g extract from the trolox calibration curve
For hot water extraction, 25 g of the samples were (0.02‒0.31 mM).
immersed in 250 mL of distilled water (dH2O) and Ferric reducing antioxidant power (FRAP) assay: The
incubated at 70°C for 18 h. Cold water, 70% ethanol, FRAP assay was carried out to determine the iron-reducing
ethyl acetate and hexane extracts were prepared by capacity of each extract according to the method by Benzie
homogenizing 25 g of the samples with 250 mL of the and Strain (1996), with modifications. Firstly, 300 mM
solvent on an orbital shaker at 100 rpm for 72 h at room acetate buffer (pH 3.6) and 40 mM HCl were prepared.
temperature. The organic solvents (70% ethanol, ethyl FRAP reagent was freshly prepared by mixing the acetate
acetate and hexane) were evaporated at 48°C, while the buffer, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) solution in
filtrate for water extracts was freeze-dried. 40 mM HCl, and 20 mM FeCl3.6H2O at a ratio of 10:1:1

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 Antioxidant Properties and Phytochemicals of M. micrantha / Int. J. Agric. Biol., Vol. 20, No. 3, 2018

(v:v:v), and pre-warmed in a water bath at 37°C. Then, 20 Statistical Analysis

μL of samples/standard/blank were mixed with 180 μL
FRAP reagent in a 96-well plate and incubated at 37°C for Results were expressed as mean ± standard error of the
30 min. The absorbance was read at 593 nm. A standard mean (SEM) of at least three independent experiments.
calibration curve was plotted using iron (II) sulphate One-way analysis of variance (ANOVA) and Tuckey’s
heptahydrate (FeSO4.7H2O) (0.1 – 1.0 mM) and the final multiple comparison test were used to compare means
results were expressed as mmol Fe2+/g extract. between solvents. An independent t-test was used to
Phosphomolybdenum antioxidative power (PAP) assay: compare means between leaves and stems. Pearson
The total antioxidant capacity of the samples was determined correlation test was used to determine the relationship
by green phosphomolybdenum complex formation (Prieto et between antioxidant contents and antioxidant activities. The
al., 1999). Reagent solution was prepared by mixing of data were analysed using IBM SPSS Statistics, version 21
sulphuric acid (0.6 M), sodium phosphate (28 mM) and and were considered statistically significant when p < 0.05.
ammonium molybdate (4 mM) in 50 mL distilled water.
Sample (100 μL) and 1 mL reagent solution were mixed in a Results
microcentrifuge tube and incubated at 95°C for 90 min. The
sample mixture was allowed to cool to room temperature Total Phenolic and Flavonoid Content in M. micrantha
prior pipetting 200 μL of the mixture into a 96-well plate. Extracts
The absorbance was measured at 695 nm against the reagent
blank. The antioxidant capacity was expressed as mg The TPC ranged between 38.3±4.67 to 104±2.50 and
ascorbic acid equivalent (AAE)/g extract from the standard 34.8±1.80 to 141±0.51 mg GAE/g, for the leaves and stems
calibration curve of ascorbic acid (0.001‒0.25 mg/mL) using of M. micrantha, respectively (Table 1). The ethanol leaves
the formula: Antioxidant capacity (AC) = [(AC per mL (ETL), hot water leaves (HWL), and ethyl acetate leaves
sample × dilution factor × total sample volume used) / (EAL) extracts had the greatest phenolic contents but not
sample weight]. significantly (p > 0.05) different when compared with each
β-carotene bleaching (BCB) assay: The β-carotene other. However, ethyl acetate stems (EAS) extract showed
bleaching assay was performed using the procedure significantly (p < 0.05) greater TPC (141±0.51 mg GAE/g)
described by Othman et al. (2016) with some modifications. when compared to EAL. Hexane extracts contained the
The β-carotene (BC) working reagent was prepared by lowest TPC and no significant (p > 0.05) different
mixing 1 mL of BC (0.2 mg/mL in chloroform) with 20 μL (38.3±4.67 mg GAE/g and 34.8±1.80 mg GAE/g) between
linoleic acid and 200 μL Tween 40 in a 100 mL round the leaves and stems, respectively.
bottom flask, and the mixture was evaporated using a rotary The TFC ranged between 8.54±0.69to 70.1±0.92 and
evaporator at 30°C for 1 min. Next, 50 mL of ultrapure 3.47±0.25 to 44.6±4.15 mg catechin equivalent/g (CE/g),
water was added to the mixture and shaken vigorously to for the leaves and stems of M. micrantha, respectively
form an emulsion. An aliquot of 200 μL of the preheated (Table 1). The EAL extract had significantly (p < 0.05)
emulsion (50°C) was transferred into a 96-well plate greater TFC compared to other extraction solvents in the
containing 20 μL of the sample/standard/blank. The zero- following trend: ethyl acetate > hexane > 70% ethanol > hot
time absorbance was measured at 470 nm immediately, and water > cold water. A similar trend was observed for TFC of
the mixture was incubated at 50°C for 2 h. Blank samples the stems extracts. Between parts, ethyl acetate and hexane
(in the absence of β-carotene) were prepared for background extracts of the leaves contained significantly (p < 0.05)
subtraction, and BHT was used as the standard. The greater TFC than the stems extracts. Hot water, cold water,
capacity of the extracts to protect against the oxidation of β- and 70% ethanol extracts of the leaves showed greater but
carotene was determined according to Othman et al. (2016). not significant (p > 0.05) TFC than the stems.

Gas Chromatography-Mass Spectrometry (GC-MS) Antioxidant Capacities

Analysis
DPPH radical scavenging capacity: The EC50 of DPPH
GC–MS analysis was used for phytochemicals identification scavenging capacity of M. micrantha extracts ranged
of the ethyl acetate leaves (EAL) and stems (EAS) extracts between 544±19.1 to 699±21.2 µg/mL and 324±61.4 to
of M. micrantha according to Painuli et al. (2016). The 752±77.7 µg/mL, for the leaves and stems, respectively
samples (100 µg/mL) were prepared in methanol (HPLC (Table 2). EAS was the most efficient extract which had a
grade) and filtered through a 0.2 μm nylon membrane filter significantly (p < 0.05) lower EC50 value (324 μg/mL) when
prior to analysis. Compounds were identified based on the compared to the other extracts. A low EC50 value indicates
retention time (RT) for GC, and interpretation of mass the potent radical scavenging capacity of the extracts as a
spectrum was done by comparing spectral fragmentation low concentration was adequate to inhibit the DPPH radicals.
obtained to the database provided by the National Institute For the standards, the EC50 of the gallic acid (2.33±0.14
Standards and Technology (NIST08.LIB). µg/mL) was lower than BHT (62.27±5.46 µg/mL).

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Table 1: Antioxidant contents of the leaves and stems of M. micrantha extracted using hot water, cold water, 70% ethanol,
ethyl acetate and hexane

Extraction solvent
Hot water Cold water 70% ethanol Ethyl acetate Hexane
Total phenolic content (TPC) (mg GAE/g extract)
Leaves 93.7 ± 0.53a* 69.6 ± 0.64b* 104 ± 2.50a* 83.4 ± 8.89ab 38.3 ± 4.67c
Stems 56.5 ± 2.25b 48.8 ± 2.00bc 44.2 ± 2.53c 141 ± 0.51a* 34.8 ± 1.80d
Total flavonoid content (TFC) (mg CE/g extract)
Leaves 11.6± 1.05c 8.54± 0.69c 12.8± 1.81c 70.1± 0.92a* 38.6± 1.70b*
Stems 5.49± 0.46c 3.47± 0.25c 10.0± 0.89c 44.6± 4.15a 25.7± 1.74b
Results are expressed as the means ± SEM (n = 3). Values with different letters are significant at p < 0.05 between the extraction solvents of the same parts
and * indicates significance at p < 0.05 between different parts of the same solvent. GAE, gallic acid equivalent; CE, catechin equivalent. The concentration of
samples used for TPC and TFC were 1 mg/mL

Table 2: Antioxidant capacities of the leaves and stems of M. micrantha extracted using hot water, cold water, 70% ethanol,
ethyl acetate, and hexane

Extraction solvent
Hot water Cold water 70% ethanol Ethyl acetate Hexane
DPPH assay EC50 (µg extract/mL)
Leaves 544 ± 19.1a* ND 699 ± 21.2a 583 ± 14.3a ND
Stems 752 ± 77.7b ND 640 ± 3.00b 324 ± 61.4a* ND
ABTS assay (mmol TE/g extract)
Leaves 0.18 ± 0.01b 0.13 ± 0.00c 0.26± 0.01a* 0.22 ± 0.00a 0.10 ± 0.00c
Stems 0.15 ± 0.02b 0.09 ± 0.01c 0.14 ± 0.01b 0.53± 0.01a* 0.08 ± 0.01c
FRAP assay (mmol Fe2+/g extract)
Leaves 0.34 ± 0.04b 0.25± 0.01bc* 0.36± 0.03ab 0.48 ± 0.04a 0.18 ± 0.03c
Stems 0.21± 0.01bc 0.09 ± 0.02c 0.31 ± 0.02b 1.28± 0.05a* 0.16 ± 0.04c
PAP assay (mg AAE/g extract)
Leaves 39.4 ± 1.25d 36.8 ± 1.19d 65.6 ± 3.40c 109 ± 6.89a 84.4 ± 2.64b
c* c* c a*
Stems 62.2± 1.60 64.2 ± 5.53 54.1 ± 1.09 219 ± 7.03 124± 3.34b*
BCB assay (% inhibition of bleaching)
Leaves 61.2 ± 11.4a 60.1 ± 10.1a 80.6 ± 8.68a 89.6 ± 3.51a 76.6 ± 3.92a
Stems 63.9 ± 10.5b ND 68.4 ± 6.73b 108 ± 2.23a* 75.8 ± 8.12b
Results are expressed as the means ± SEM (n = 3). Values with different letters are significant at p < 0.05 between the extraction solvents of the same parts,
and * indicates significance at p < 0.05 between different parts of the same solvent; ND, not detected; EC50, concentration of the extracts (µg/mL) required to
inhibit 50% of the radicals; the lowest EC50 indicates the greatest antioxidant capacity; TE, trolox equivalent; AAE, ascorbic acid equivalent

ABTS radical scavenging capacity: For the leaves, Phosphomolybdenum antioxidative power (PAP): Total
ethanol leaves (ETL) and ethyl acetate leaves (EAL) antioxidant capacity of M. micrantha extracts ranged
extracts showed significantly (p < 0.05) greater TEAC between 36.8±1.19 to 109±6.89 and 54.1±1.09 to 219±7.03
(trolox equivalent antioxidant capacity) value compared mg ascorbic acid equivalent/g (AAE/g) for the leaves and
to hot water, cold water, and hexane extracts (Table 2). stems, respectively (Table 2). The ethyl acetate and hexane
For the stems, ethyl acetate stems (EAS) extract showed extracts (semi-polar and non-polar, respectively) had
significantly (p < 0.05) greatest TEAC value (0.53±0.01 significantly (p < 0.05) greater total antioxidant capacities
mmol TE/g) compared to the other extracts. Between compared to the more polar extracts (hot water, cold water,
parts, only ETL and EAS showed a significantly (p<0.05) and 70% ethanol). Between parts, stems extracts had a
greater TEAC value than the stems and the leaves, greater antioxidant capacity than the leaves extracts.
respectively. However, the ethanol leaves (ETL) extracts showed a
Ferric reducing antioxidant power (FRAP): The ethyl significantly (p < 0.05) greater antioxidant capacity than the
acetate extracts of both the leaves and stems (0.48±0.04 ethanol stems (ETS) extract (65.6±3.40 and 54.1±1.09,
mmol Fe2+/g, 1.28±0.05 mmol Fe2+/g, respectively) respectively).
exhibited the greatest iron reducing ability, followed by β-carotene bleaching (BCB): All extracts showed the
70% ethanol and hot water of both parts. However, cold inhibition of β-carotene bleaching except the cold water
water and hexane extracts showed the lowest antioxidant stem extract, which had no detectable inhibition (Table 2).
capacity (Table 2). Similar to other assays, the FRAP EAS had a significantly (p < 0.05) greater antioxidant
assay also demonstrated that EAS exhibited the most capacity (108±2.23%) when compared to the leaves and
significant (p < 0.05) and greatest antioxidant capacity other extracts. Interestingly, EAS extract had a greater
(1.28±0.05 mmol Fe2+/g) when compared to the other antioxidant capacity when compared to the standard BHT
solvents. (94.95%) at the same test concentration of 1000 ppm.

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Table 3: Pearson correlation analyses of the antioxidant Table 4: Phytochemicals identified in the leaves and stems
contents and the antioxidant capacities in the extracts of M. of M. micrantha extracted using ethyl acetate
micrantha
Peak Compounds CAS RT MW Area
DPPH ABTS FRAP PAP BCB (min) (%)
TPC r value -0.685** 0.897** 0.829** 0.490** 0.463* Ethyl acetate leaves
TFC r value -0.459 0.410* 0.485** 0.629** 0.576** 1 2,2-Dimethoxybutane 3453-99-4 3.358 118 15.64
**Correlation is significant at p < 0.01; *Correlation is significant at p < 2 Nonane 111-84-2 5.465 128 2.65
0.05. TPC, total phenolic contents; TFC, total flavonoid contents; DDPH, 3 Undecane 1120-21-4 7.172 156 4.06
2,2-Diphenyl-1-picrylhydrazyl; ABTS, 2’-azinobis-3- 4 Cyclohexyl(dimethoxy)methylsilane 17865-32-6 9.748 188 0.90
ethylbenzothiazoline-6-sulphonic acid; FRAP, ferric reducing antioxidant 5 α-Cubebene 17699-14-8 12.604 204 0.65
power; PAP, phosphomolybdenum antioxidative power; BCB, β-carotene 6 α-Longipinene 5989-08-2 12.710 204 0.51
bleaching assay 7 Copaene 3856-25-5 13.028 204 0.65
8 Germacrene D 23986-74-5 13.176 204 4.26
9 Caryophyllene 87-44-5 13.650 204 4.38
Correlation between Antioxidant Contents and 10 Cedrene 11028-42-5 13.747 204 0.81
Antioxidant Capacities 11 α-Caryophyllene; Humulene 6753-98-6 14.119 204 0.88
12 Acoradiene 24048-44-0 14.234 204 0.41
The correlation coefficients (r) between TPC, TFC, and 13 4,6-Dimethyldodecane 61141-72-8 14.350 198 1.14
antioxidant capacities of M. micrantha extracts are shown in 14 3,5-bis(1,1-dimethylethyl)phenol 1138-52-9 14.610 206 14.74
15 β-Himachalene 1461-03-6 14.685 204 2.71
Table 3. TPC revealed a very strong (r >0.8) positive 16 δ-Cadinene 483-76-1 14.849 204 3.48
correlation with the ABTS (r = 0.897) and FRAP (r = 0.829) 17 Hexadecane 544-76-3 14.918 226 1.73
assays, and strong (r = 0.60-0.79) negative correlation with 18 2,6,11-Trimethyldodecane 31295-56-4 15.170 212 0.98
the DPPH assay (r = -0.685). Meanwhile, TFC exhibited a 19 Eicosane 112-95-8 17.385 282 0.50
Ethyl acetate stems
strong positive correlation with the PAP assay (r = 0.629) 1 2,2-Dimethoxybutane 3453-99-4 3.358 118 4.76
and moderate (r = 0.40‒0.59) positive correlation with the 2 Undecane 1120-21-4 5.489 156 1.60
BCB (r = 0.576) assay. 3 Decane 124-18-5 7.201 142 2.34
4 Cyclohexyl(dimethoxy)methylsilane 17865-32-6 9.756 188 0.61
GC-MS Analysis of Ethyl Acetate Extracts of M. 5 α-Cubebene 17699-14-8 12.607 204 1.52
6 α-Longipinene 5989-08-2 12.720 204 0.17
micrantha 7 Thujopsene 470-40-6 12.824 204 0.18
8 Copaene 3856-25-5 13.032 204 1.28
GC-MS analysis was performed to identify the 9 Caryophyllene 87-44-5 13.653 204 8.20
phytochemicals present in the ethyl acetate extracts of the 10 Cedrene 11028-42-5 13.751 204 1.28
leaves (EAL) and stems (EAS) of M. micrantha which had 11 cis-β-Farnesene 28973-97-9 13.933 204 0.79
12 α-Caryophyllene; Humulene 6753-98-6 14.123 204 2.77
the greatest antioxidant contents and capacities. The EAL 13 β-Cubebene 13744-15-5 14.225 204 0.96
extract had a total of 19 identified compounds (Table 4), 14 Di-epi-α-cedrene; α-Funebrene 50894-66-1 14.239 204 1.12
which are composed primarily of alkane hydrocarbons 15 α-Amorphene 483-75-0 14.324 204 0.52
(26.7%), sesquiterpenes and their derivatives (18.74%), and 16 Germacrene D 23986-74-5 14.439 204 3.78
17 α-Zingiberene 495-60-3 14.525 204 0.51
phenol (14.74%). In EAS, there were a total of 24 18 3,5-bis(1,1-dimethylethyl)phenol 1138-52-9 14.613 206 16.38
compounds identified (Table 4), which included 19 β-Himachalene 1461-03-6 14.690 204 1.66
sesquiterpenes and their derivatives (30.46%), phenol 20 trans-α-Bergamotene 13474-59-4 14.750 204 0.61
(16.38%), and alkane hydrocarbons (10.45%). 21 δ-Cadinene 483-76-1 14.854 204 4.35
22 Eicosane 112-95-8 14.919 282 1.28
23 Nonadecane 629-92-5 15.174 370 0.47
Discussion 24 Cedrol 77-53-2 16.221 222 0.76
CAS = chemical abstract service number; RT = retention time; MW =
Extraction is the main step in medicinal plant study for the molecular weight
recovery and isolation of bioactive plant phytochemicals
prior to analysis. The choice of extracting solvent is The high flavonoid content in the ethyl acetate extracts
important as various solvent polarities affect the yield and demonstrated the presence of semi-polar flavonoids and less
type of compound extracted (Chan et al., 2015). Therefore, polar flavonoids, such as isoflavones, flavanones,
M. micrantha samples were extracted using five different methylated flavones, and flavonols (Martson and
solvents to determine the antioxidant content and capacities. Hostettmann, 2005). Moreover, ethyl acetate and hexane
The greatest TPC content in the ethyl acetate, 70% ethanol, promotes the extraction of lipophilic compounds, such as
and hot water extracts of M. micrantha indicate the presence flavonoids and carotenoids (Bae et al., 2012). Another study
of medium polar to highly polar phenolic compounds in the also showed the ability of ethyl acetate to extract more
extracts. Hexane, the non-polar solvent has a reduced ability flavonoid compounds from medicinal plant compared to
to extract phenolic compounds from M. micrantha other organic solvents (Mohd Hadzri et al., 2014).
compared to the polar solvents (Table 1). However, for Natural antioxidants have various functions and show
TFC, ethyl acetate and hexane were the best solvents to synergistic interactions. Therefore, it is important to use
extract flavonoid compounds from M. micrantha (Table 1). different types of solvent extractions and various methods to

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evaluate the antioxidant capacities of plant extracts to avoid except α-Longipinene, β-Himachalene, β-Cubebene, and α-
a misleading interpretation (Lin et al., 2013). From our data, Zingiberene, which were also reported in the previous study
the ethyl acetate stems (EAS) extract stands out with of the essential oil composition of M. micrantha from China
efficient antioxidant capacities (Table 2), which (Zhang et al., 2004).
significantly correlated with the phenolic and flavonoid In addition, there are an adequate supply of raw
contents (Table 3). A very strong positive correlation material of this plant for the development of modern
between TPC with ABTS and FRAP assays demonstrated medicines in pharmaceutical industry (Jyothilakshmi et al.,
that the phenolic compounds act as hydrogen donors and 2015) since it can be found easily in plenty from its natural
reducing agents in neutralizing the free radicals due to their habitat. Results from this study demonstrated ethyl acetate
hydroxyl groups (Saeed et al., 2012). Meanwhile, as the best solvent for extraction of phytochemicals from M.
flavonoids contribute moderately to the total antioxidant micrantha particularly its stems part, which also showed the
capacity and the inhibition of β-carotene bleaching based on greatest antioxidant capacities. Therefore, these findings
the PAP and BCB assays, respectively. Overall, phenolic provide scientific evidences for the potential utilization of
compounds might act as antioxidant agents in scavenging this plant in the prevention and treatment of oxidative
radicals using the DPPH and ABTS assays, whilst the total damage-related diseases and many more. In fact, ethyl
flavonoids act as chain-breaking antioxidants by donating a acetate has been used as an extraction solvent in the
hydrogen atom to lipid radicals using the BCB assay. production of pharmaceutical which has the lowest toxic
GC-MS analysis of the ethyl acetate extracts of M. potential (Cue and Zhang, 2009). This application could
micrantha indicated the presence of three major compounds increase the value of M. micrantha from weed to medicinal
such as sesquiterpenes, phenol, and alkane hydrocarbons in plant.
the extracts (Table 4). The presence of 3,5-bis (1,1-
dimethylethyl) phenol in the EAS extract could be the Conclusion
contributor to the high phenolic and antioxidant capacities in
the extract, since, in most cases, the inhibitory activity of The present study showed that ethyl acetate stems (EAS)
extracts has been attributed to the most dominant and leaves (EAL) of M. micrantha contained the greatest
compounds (Das et al., 2012). In fact, phenols can act as TPC and TFC compared to other extraction solvents,
antioxidants by donating an H-atom to unstable free radicals respectively. EAS extract had the greatest radical
through the hydrogen atom transfer (HAT) mechanism and scavenging activity, hydrogen donating ability, iron
can also act as an iron chelator. We have also shown that reducing ability, total antioxidant capacity, and inhibition of
there was a positive correlation between the total phenolic β-carotene bleaching based on DPPH, ABTS, FRAP,
content as given by the ABTS and FRAP assays (Table 3) phosphomolybdenum, and β-carotene bleaching assays,
that could be attributed by the presence of 3,5-bis (1,1- respectively. The significant relationship between
dimethylethyl) phenol in the ethyl acetate extracts. antioxidant contents and antioxidant capacities of M.
Moreover, 3,5-bis (1,1-dimethylethyl) phenol has been micrantha indicates that total phenolic and flavonoid
reported as having antioxidant and anti-cancer properties contribute to the antioxidant capacities. The GC-MS
(Rizvi et al., 2014). An alkoxy derivatives of alkane, 2,2- analysis of the ethyl acetate extracts revealed that the
dimethoxybutane found in both stems and leaves of ethyl bioactive compounds are 3,5-bis (1,1-dimethylethyl) phenol,
acetate extracts has been reported to possess anti- an alkylphenol, sesquiterpene and their derivatives, and
dermatophytic activity (Das et al., 2012). Other alkanes alkane hydrocarbons. In summary, ethyl acetate is the best
hydrocarbon compounds detected such as nonane, decane, solvent to extract abundant bioactive compounds with
undecane, 4,6-dimethyldodecane, hexadecane, 2,6,11- strong antioxidant activity in M. micrantha. Hence,
trimethyldodecane, eicosane and nonadecane (Table 4) utilization of M. micrantha, particularly its stem as a source
might possess other potentials. Sesquiterpenes, which were of natural antioxidants should be further explored in future.
found abundantly in the extracts could also act
synergistically as the antioxidant compounds. For instance, Acknowledgements
caryophyllene, one of the bicyclic sesquiterpenes has
potential antioxidant, anti-inflammatory, and antimicrobial NHS thanks Universiti Putra Malaysia for Project Grant
activities (Sharma et al., 2016). Another compounds such as funding [GP-IPS/2016/9489400]. AHI thanks UPM and the
cedrol (a sesquiterpene alcohol), cedrene, and thujopsene Ministry of Higher Education (MoHE) Malaysia for
possess inhibitory effects against cytochrome P450 (CYP) Graduate Research Fellowship (GRF) and My Master
enzyme (CYP2B6 and CYP3A4) in human liver scholarships.
microsomes in vitro as well as anti-inflammatory,
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