Int. J. Biosci.

2016

International Journal of Biosciences | IJB |

ISSN: 2220-6655 (Print), 2222-5234 (Online)
http://www.innspub.net
Vol. 8, No. 1, p. 25-32, 2016

RESEARCH PAPER OPEN ACCESS

GC-MS analysis and evaluation of in vitro antioxidant potential

and total phenolics content of wild hops (Flemingia strobilifera
(L.) W. T. Aiton)

Jhoan Rhea L. Pizon1, Olga M. Nuñeza1*, Mylene M. Uy2, W.T.P.S.K Senarath3

1
Department of Biological Sciences, Mindanao State University- Iligan Institute of Technology
(MSU-IIT), Iligan City, Philippines
2
Department of Chemistry, Mindanao State University- Iligan Institute of Technology (MSU-IIT),
Iligan City, Philippines
3
Department of Botany, University of Sri Jayewardenepura, Nugegoda, Sri Lanka

Key words: Fabaceae, Gas chromatography, Leaf extracts, Scavenging activity.

http://dx.doi.org/10.12692/ijb/8.1.25-32 Article published on January 20, 2016

Abstract
Wild hops (Flemingia strobilifera Linn.) is a shrub belonging to Fabaceae family. The leaves of F. strobilifera
are commonly used by the Subanen, the indigenous group in Lapuyan, Zamboanga del Sur, Philippines to treat
inflammation. In this study, the hydromethanolic (80%) and aqueous leaf extracts of F. strobilifera were
evaluated for their antioxidant activity and total phenolics content. The active semi-volatile components of 80%
methanol leaf extract were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS). DPPH radical
scavenging activity was used to determine the potential of this plant as anti-oxidant. Total phenolics content was
determined using Folin-Ciocalteu reagent and calculated as gallic acid equivalence. GC-MS analysis revealed the
presence of eight compounds. Both the 80% methanol and aqueous extracts of F. strobilifera showed significant
scavenging activity with low IC50 values of 0.299 mg/mL and <0.25 mg/mL, respectively. There was positive
correlation between the scavenging activity percentage and the total phenolics content only in the aqueous
extract of F. strobilifera while 80% methanol extract showed negative correlation between inhibition percentage
and total phenolics content which can be attributed to the solvent used and method used in quantification of
phenolics. Nevertheless, the results suggest that these leaf extracts are potent source of antioxidant compounds
and may serve as natural anti-inflammatory agents.
* Corresponding Author: Olga M. Nuñeza  [email protected]

25 Pizon et al.
 Int. J. Biosci. 2016

Introduction and economically essential, plants are also safe from

Free radicals cause oxidative stress and induce DNA harmful side effects caused by synthetic drugs (Azlim
damage, protein carbonylation, and lipid et al., 2010). Moreover, plants that exhibit free radical
peroxidation which result into several health-related scavenging activity can also inhibit inflammatory
problems such as aging, cancer, Parkinson’s disease agents (Miguel, 2010).
(Garg et al., 2013), and inflammation (Halliwell,
1999). Free radical-induced diseases can be alleviated Flemingia strobilifera (L.) W.T. Aiton (wild hops)
by the application of antioxidants. An antioxidant is a which belongs to family Fabaceae is traditionally used
molecule capable of slowing or preventing the by the Subanen tribe in the treatment or suppression
oxidation of other molecules. Furthermore, there is a of inflammation. Biochemical assessments reveal the
widespread agreement that the synthetic antioxidants presence of chalcone, flavonoid glycosides, aurone
such as butylatedhydroxytoluene, glycosides, epoxy chromenes, lipids, phenolic
butylatedhydroxyanisole, gallic acid esters, and compounds, tannins and phytosterols in F.
tertiary butylatedhydroquinone must be replaced strobilifera (Madan et al., 2008; Ghalot et al., 2011;
with natural antioxidants since synthetic antioxidants Ramcharan et al., 2010). There have been several
impose health risk and toxicity and have low studies conducted on the roots, however, leaves are
solubility along with moderate antioxidant activity less studied. Hence, the present study aims to assess
(Peteros and Uy, 2010; Bhaskar et al., 2009; Garg et the antioxidant activity, total phenolics content, and
al., 2013). Thus, there is a need to develop potential the compounds present in the leaf extracts of F.
sources of natural antioxidants. strobilifera which may validate the traditional use of
this plant as anti-inflammatory agent.
Medicinal plants indeed have great importance to
human health and the society in the treatment of a Materials and methods
wide range of diseases. According to Chanda et al. Collection of Plant Material
(2011), the use of traditional medicine is widespread The leaves of F. strobilifera were collected from the
and plants still represent a large source of natural Municipality of Lapuyan, Zamboanga del Sur,
antioxidants that might serve as leads for the Philippines (Fig. 1.).
development of novel drugs. Aside from being useful

Fig. 1. Map of the Municipality of Lapuyan, Zamboanga del Sur, Philippines (https://maps.google.com.ph/,
2015).

26 Pizon et al.
 Int. J. Biosci. 2016

The leaves were washed properly under running tap Determination of Total Phenolics Content
water and distilled water, air dried, powdered, and Total phenolics content of the extracts was
stored in an airtight container. determined using the protocol of Formagio et al.
(2014). An aliquot of 100µL of extract in methanol
Preparation of Extracts (1.0 mg/mL) was mixed with 1.0 mL of distilled water
Maceration and 0.5 mL of Folin-Ciocalteu’s reagent (1:10 v/v).
Fifteen grams of the dried and powdered leaves were After mixing, 1.5 mL of 2% aqueous sodium
soaked in 150 mL 80% methanol in a mechanical bicarbonate was added and the mixture was allowed
shaker at 100 rpm for seven days. The solution was to stand for 30 minutes with intermittent manual
filtered and the filtrates were collected and shaking. Total phenolics content is expressed as gallic
evaporated using a rotary evaporator at 50 °C. The acid equivalence (GAE) in mg gallic acid per gram of
crude extract obtained was then stored in a freezer extract. The methanol solution served as blank. All
(Amarowicz et al., 2003). assays were carried out in triplicate. The total
phenolics content was determined from a standard
Decoction calibration plot of absorbances of gallic acid
A 400 mL volume of distilled water was added to 15.0 measured at different concentrations (4.0 ug/mL, 8.0
g of powdered leaves of F. strobilifera. The mixture ug/mL, 16.0 ug/mL, 32.0 ug/mL, 63.0 ug/mL).
was boiled to obtain 100 mL. The decoction was
filtered and the obtained filtrates were subjected to GC-MS Analysis of leaf extract
evaporation in a rotary evaporator at 50°C. The For the preparation of extracts for the analysis, 1.0
resultant extract was lyophilized to remove excess mg of the crude extract was diluted with a mixture
water then placed in air tight containers and stored in containing 0.5 mL absolute methanol and 0.5 mL
the freezer for further use. dichloromethane to separate chlorophyll. The upper
layer with no chlorophyll was transferred to another
DPPH radical scavenging activity of leaf extracts tube. A 10.0 µL was then taken and was further
Antioxidant activity of the F. strobilifera leaf extracts diluted with 1.0 mL chloroform. The GC-MS analysis
was quantified by measuring the 2, 2-diphenyl-1- was carried out using Agilent Technologies 7890A GC
picrylhydrazyl (DPPH) radical scavenging activity. system coupled to 5975C Mass Selective detector, and
The DPPH (Sigma) scavenging activity of the extracts driven by Agilent Chemstation software and with HP-
was determined using spectrophotometry which was 5MS 30mx 0.25mm x 0.25 µm df capillary column.
adopted from the method of Perera et al. (2013). The carrier gas was ultra-pure helium at a flow rate of
1.0mL/ min. and a liner velocity of 37 cm/s. The
Reaction mixture was prepared using 2.5mL of temperature of the injector was set at 320°C. The
6.5x10-5 M freshly prepared DPPH solution and 0.5 instrument was set to an initial temperature of 70°C
mL of extract dissolved in methanol. Extracts were which was programmed to increase to 280°C at the
tested in four concentrations (0.25 mg/mL, 0.5 rate of 10°C/ min with a hold time of 4 min. at each
mg/mL, 1.0 mg/mL, and 2.0 mg/mL) with three increment injections. An aliquot of 1 µL sample was
replicates at room temperature in a dark condition. injected in a split mode 100:1. The mass spectrometer
Absorbance was measured at 540 nm using UV-Vis was operated in the electron ionization mode at 70eV
spectrophotometer (SHIMADZU UV mini 1240). and electron multiplier voltage at 1859V. Other MS
Methanol was used as control. Ascorbic acid was used operating parameters were: ion source temperature
as the reference standard. The percentage of DPPH 230°C, quadruple temperature °C, solvent delay 3
radical scavenging activity was determined using the min and scan range 22-550 amu (automatic mass
equation: unit). The compounds were identified by direct
% scavenging activity= A0-As/ A0 x 100 comparison of the mass spectrum of the analyte at a

27 Pizon et al.
 Int. J. Biosci. 2016

particular retention time to that of a reference (version 20.0). Difference at p <0.05 was considered
standard found in the National Institute of Standards statistically significant.
and Technology (NIST) library. Total GC running
time was 45 min (Chipiti et al., 2015). Results and discussion
2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical
Statistical analysis scavenging activity of leaf extracts
All experiments were carried out in triplicates. Data The leaf extracts of F. strobilifera exhibited
were expressed in Mean ± standard deviation. significant radical scavenging activity (Table 1).
Statistical analysis was done with SPSS software

Table 1. Average DPPH scavenging activity of hydroalcohol and aqueous leaf extracts of F. strobilifera at
different concentrations.
Extracts % Activity
0.25 mg/mL 0.5 mg/mL 1 mg/mL 2 mg/mL IC50 mg/mL
80% methanol 42.00± 2.36 64.57± 0.53 73.09± 0.50 77.73± 1.25 0.299
aqueous 61.73± 1.54 67.16± 1.14 71.30± 1.67 74.28± 1.41 <0.25
Ascorbic acid* 65.17± 3.95 72.20± 0.39 73.39± 0.30 83.86± 1.32 <0.25
*standard.

The scavenging activity of the leaf extracts was dose- (<0.25 mg/mL) is the same with the standard
dependent wherein the % activity increased as the (ascorbic acid) with IC50 value of 0.25 mg/mL. The
concentration of the leaf extract was increased. Both IC50 of the methanol extract (0.299 mg/mL),
80% methanol and aqueous leaf extracts exhibited however ,is slightly higher than the standard but is
high activity which is more or less comparable to the still considered low. According to Figueroa et al.
standard (ascorbic acid) wherein the aqueous extract (2014), low IC50 value denotes high ability of the
has values ranging from 61.73± 1.54 to 74.28± 1.41 extracts to act as DPPH scavenger. Thus, the results
mg/mL and the methanol extracts with values indicate that the 80% methanol and the aqueous leaf
ranging from 42.00± 2.36 to 77.73± 1.25 mg/mL. extracts of F. strobilifera are potent antioxidants.
Moreover, the low IC50 value of the aqueous extract

Table 2. Total phenolic content of extracts expressed in mean gallic acid equivalence (GAE).
Sample Extracts GAE (mg/g extract)
F. strobilifera 80% methanol 12.49± 0.020
aqueous 102.98± 0.003

Total Phenolics Content Phenols comprise the largest group of secondary

In order to further confirm the antioxidant activity of metabolites that have been reported to have
the extracts of F. strobilifera, their total phenolics antioxidant properties which play an important role
content was evaluated. Table 2 shows the total in lipid peroxidation and other biological activities
amount of phenolics of the two extracts expressed as (Sanadhya et al., 2013). They are very important in
Gallic Acid Equivalence (GAE). Apparently, the total plants because of their scavenging ability due to their
phenolics content of the aqueous extract is higher hydroxyl groups. In addition, the antioxidant ability
than the 80% methanol extract with GAE values of phenols is due to their redox properties which play
102.98± 0.003 and 12.49± 0.020, respectively. an important role in absorbing and neutralizing free
radicals, quenching singlet and triplet oxygen, or

28 Pizon et al.
 Int. J. Biosci. 2016

decomposing peroxides (Osawa, 1994). radical scavenging activity and showed considerably
higher amount of total phenolics than 80%
As observed, there is a positive correlation between methanolic extract. The result obtained is in contrast
DPPH radical scavenging activity and the total with the findings of Othman et al. (2014) that alcohol
phenolics content of aqueous leaf extracts of F. extracts exhibited higher total phenolics content
strobilifera. The aqueous extract exhibited higher compared to the water extracts.

Table 3. Compounds found in the 80% methanolic leaf extract of F. strobilifera.

Compound Similarity Index (%) Abundance (%)
Thiocyanic acid, ethyl ester 72 0.74
Limonene 60 0.41
Phenol, 3,5-bis (1,1-dimethylethyl) 74 0.92
Eicosane 78 0.73
Heptacosane 68 0.90
Hexanedioic acid, bis (2-ethylhexyl) ester 62 2.71
Hexacosane 94 3.50
Nonadecane 98 6.31

However, the finding in this study supports the work and total phenolics content might be due to the fact
of Vuong et al. (2013) where water was shown to be that total phenolics content does not necessarily
an effective solvent for extraction of polyphenols from incorporate all the antioxidants that may be present
papaya leaves contributing to the high total phenollics in an extract. These findings may help explain the
content of the extract. On the other hand, there is a negative correlation obtained between the DPPH
weak correlation between DPPH radical scavenging scavenging activity and the total phenolics content of
and total phenolics content of 80% methanol where the 80% methanol leaf extract observed in the present
the extract exhibits DPPH radical scavenging activity study.
but was found to contain relatively low amount of
phenolics. This might be due to the method used in GC-MS Analysis of Leaf Extract
the quantification of phenols. The Folin-Ciocalteu There were eight active compounds detected that
assay gives a crude estimate on the total phenolics could contribute to the medicinal property of the
content of an extract, whereas the DPPH free radical plant (Table 3). Among the eight compounds
scavenging assay is not only specific to polyphenols identified in this study, the major compounds are
(Prior et al., 2005). In addition, Folin-Ciocalteu hydrocarbons nonadecane (6.31%), hexacosane
reagent measures not only the total phenolics content (3.50%), and hexanedioic acid, bis (2-ethylhexyl)
but also other oxidation substrates. The other ester (2.71%) whereas thiocyanic acid, ethyl ester,
oxidation substrates present in a given extract sample limonene, phenol, 3, 5-bis (1, 1-dimethylethyl),
may interfere with the total phenolics measurement eicosane, and heptacosane are present in lesser
in an inhibitory, additive, or enhancing manner. The amount.
inhibitory effects could be due to the oxidants
competing with Folin-Ciocalteu reagent. Moreover, Although present in lesser quantity, the compounds
various phenolic compounds respond differently in limonene, eicosane, and hexanedioic acid, bis (2-
the DPPH assay, depending on the number of ethylhexyl) ester might also contribute to the
phenolic groups they have (Singleton and Rossi, antioxidant capacity of the 80% methanol extract
1965). Furthermore, Tahawa et al. (2007) suggested (Sermakanni and Thangapandian, 2012; Kazemi,
that negative correlation between antioxidant activity 2015). Nonadecane is reported to have high

29 Pizon et al.
 Int. J. Biosci. 2016

antioxidant activity (Dandekar et al., 2015) and Rao TR. 2009. Evaluation of antioxidant potential of
antidiabetic activity (Hamidi et al., 2012). Clitoria ternatea L. and Eclipta prostrata L.
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(Rukaiyat et al., 2015). Eicosane is found to have 46, 247-252.
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