Difference of solvent polarity to phytochemical content and antioxidant activity of Pluchea indicia less leaves extracts

Abstract

Pluchea indica Less called local name Beluntas or Luntas, one of herb plants, is usually used as traditional medicine by people in Indonesia. Phytochemical content in this Pluchea leaves can reduce odor body and prevent many diseases. Phytochemical polarity in Pluchea leaves is various so that can be extracted by different solvents. The phytochemical contents determine their antioxidant capacity. The study was conducted to determine difference of solvent polarity (water, methanol, ethanol, ethyl acetate, and hexanes) to phytochemical contents and antioxidant activity of Pluchea leaves extracts. The results showed that major phytochemical in Pluchea leaves was polar properties extracted by water, methanol, and ethanol, including flavonoid, saponin, phenol hydroquinone, alkaloid, sterol, tannin, and reducing sugar. A part of them had semi polar properties, such as sterol, flavonoid, phenol hidroquinone, and alkaloid. Another of them had non polar properties, i.e. sterol, flavonoid, and phenol hydroquinone, and alkaloid. Phytochemical content was correlated with total phenolic and total flavonoid contents and antioxidant activity. Methanolic extract had the highest total phenol and total flavonoid, 1185.2 mg GAE/g samples dry base and 911.9 mg CE/g samples dry base, respectively, consequently it had the highest a DPPH free scavenging activity and iron ion reducing power, 794.9 mg GAE/g samples dry base and 2.14 mg GAE/g samples dry base, respectively. © 2014–2015, International Journal of Pharmacognosy and Phytochemical Research. All rights reserved.

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International Journal of Pharmacognosy and Phytochemical Research 2014-15; 6(4); 850-855
ISSN: 0975-4873
*Author for correspondence
Research Article
Difference of Solvent Polarity To Phytochemical Content and
Antioxidant Activity of Pluchea indicia Less Leaves Extracts
*Paini Sri Widyawati, Tarsisius Dwi Wibawa Budianta, Fenny Anggraeni Kusuma, Evelyn
Livia Wijaya
Study Programme of Food Technology, Faculty of Agricultural Technology, Surabaya Widya Mandala Catholic
University
Dinoyo Street Number 42-44 Surabaya 60265
Available Online: 22nd November, 2014
ABSTRACT
Pluchea indica Less called local name Beluntas or Luntas, one of herb plants, is usually used as traditional medicine by
people in Indonesia. Phytochemical content in this Pluchea leaves can reduce odor body and prevent many diseases.
Phytochemical polarity in Pluchea leaves is various so that can be extracted by different solvents. The phytochemical
contents determine their antioxidant capacity. The study was conducted to determine difference of solvent polarity (water,
methanol, ethanol, ethyl acetate, and hexanes) to phytochemical contents and antioxidant activity of Pluchea leaves
extracts. The results showed that major phytochemical in Pluchea leaves was polar properties extracted by water,
methanol, and ethanol, including flavonoid, saponin, phenol hydroquinone, alkaloid, sterol, tannin, and reducing sugar. A
part of them had semi polar properties, such as sterol, flavonoid, phenol hidroquinone, and alkaloid. Another of them had
non polar properties, i.e. sterol, flavonoid, and phenol hydroquinone, and alkaloid. Phytochemical content was correlated
with total phenolic and total flavonoid contents and antioxidant activity. Methanolic extract had the highest total phenol
and total flavonoid, 1185.2 mg GAE/g samples dry base and 911.9 mg CE/g samples dry base, respectively, consequently
it had the highest a DPPH free scavenging activity and iron ion reducing power, 794.9 mg GAE/g samples dry base and
2.14 mg GAE/g samples dry base, respectively.
Key words: Pluchea indicia Less, phytochemical, antioxidant activity.
INTRODUCTION
Pluchea Indica Less usually called as Beluntas or Luntas
is a one of herb plants that is used as a traditional medicine
to reduce body odor and prevent many diseases. This plant
is grouped in Asteraceae family. The plant is generally
grown as a wild plant in dry land with hard earth texture,
many stones, and need enough sunshine (Dalimarta, 2003;
Manan, 2002; Raharjo and Horsten, 2008).
Pluchea can use a traditional medicine because it contains
many phytochemical compounds. Many researchers have
identified that root and leaves of Pluchea have many
biological activities, such as antiinflammation, antiulcer,
antipyretic, hypoglicemic, diuretic, and many
pharmacological activities (Biswas et al., 2005; Biswas et
al., 2007; Widyawati et al., 2010; Widyawati et al., 2011;
Widyawati et al., 2012). This caused Pluchea contains
many phytochemical compounds, such as lignan, terpene,
phenylpropanoid, benzoid, alkanes (Luger, 2000), sterol,
2-(prop-1-unyl)-5-(5,6-dihydroxy hexa-1,3-diunyl)-
thiophene, (-)-catechin (Biswas et al. 2005), alkaloid
(Ardiansyah et al. 2003), saponin, tannin, phenol
hydroquinone, flavonoid (Widyawati et al. 2010;
Widyawati et al. 2011), flavonol (quercetin, kaempherol,
myricetin) (Andarwulan et al. 2010). Biswas et al. (2005)
said that Pluchea root methanolic extract contains
stigmasterol (+-sitosterol), stigmasterol glycoside (+-
sitosterol-glycoside), 2-(prop-1-unyl)-5-(5,6-dihydroxy
hexa-1,3-diunyl)-thiophene, and (-)-catechin. Traithip
(2005) also informated that essential oil of Pluchea leaves
is composed of boehmeryl acetate, HOP-17 (21)-ene- 3-
acetate, linaloil glucoside, linaloil apioxyl glucoside,
linaloil hidroxy glucoside, plucheoside C, quauhtermone,
3-(2’-3’-diacetoxy-2’-methyl-butyril), plucheol A,
plucheol B, plucheoside A, plucheoside B, plucheoside E,
and pterocarptriole. Widyawati et al. (2013) said that
essential oil of Pluchea leaves contains alcohols,
aldehydes, aliphatic unsaturated hydrocarbons, esters,
ketones, ethers, and sulfoxides. Cyclic unsaturated
hydrocarbons is the most numerous and (10S,11S)-
Himachala-3-(12)-4-diene (17,13%) is a volatile
compound with the highest proportion. Widyawati et al.
(2010) and Widyawati et al. (2011) also proved that
Pluchea leaves methanolic extract and its fractions (ethyl
acetate and n-butanol) are arranged tannin, sterol,
flavonoid, and phenol hydroquinone, but water fraction
doesn’t contain sterol.
Many researchers have found antioxidant activity of
Pluchea Leaves extract, such as ethanolic extract can

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Table 1: Yield of various extract of pluchea leaves
No
Extract
Yield (%)
1
Aquadest
40,65 ± 1,47d
2
Methanol
38.07 ± 2.08c
3
Ethyl acetate
32.97 ± 1.24b
4
Ethanol
31.09 ± 1.75ab
5
Hexanes
29.46 ± 1.47a
scavenge a DPPH free radical and inhibit β-carotene
linoleic acid (Widyawati, 2004; Andarwulan et al., 2010),
scavenge ABTS2+, reduce iron ion, and inhibit
malondialdehyde formation (Andarwulan et al., 2010) .
Widyawati et al. (2012) also explains that methanolic
extract has potency to scavenge a DPPH free radical,
reduce iron ion, inhibit β-carotene linoleic acid
peroxidation, and then ethyl acetate fraction has potency to
scavenge a DPPH and superoxide free radicals, reduce and
chelate iron ions. Phytochemicals of pluchea leaves have
different polarity so that they can be extracted by different
solvent polarity. Every phytochemical can contribute
antioxidant activity. Until now the study about effect of
different polarity solvent to phytochemical content and
antioxidant activity of pluchea indicia less leaves extracts
has not yet been done. The research was conducted to study
the different polarity solvent, including water, methanol,
ethanol, ethyl acetate, and hexanes to phytochemical
content and antioxidant activity of pluchea indicia less
leaves extracts.
MATERIALS AND METHODS
Plant material: Leaves of Pluchea indica Less at 1-6
segment levels were collected from areas at East Coast,
Bendul Merisi, Keputih, and Wiyung in Surabaya and
Kertosono, East Java.
Pluchea india Less leaves Extraction: 1-6 segment level of
Pluchea leaves from peak was used as sample (Widyawati
et al., 2011; Dorman and Hiltunen, 2004). These leaves
were dried at ambient temperature and grinded with 45
mesh size. Dried flour of Pluchea leaves was measured
moisture content. And then this flour was extracted by
different polarity solvent (water, methanol, ethanol, ethyl
acetate, and hexanes) with soxhlet extractor at a boiling
point for three hours. Extract was evaporated by rotary
evaporator. The extract was stored at 4oC in black glass
bottle until analysis further. Parameters were analyzed
including yield, phytochemical content, total phenol, total
flavonoid, iron ion reducing power, and DPPH free radical
scavenging capacity.
Moisture Content: Moisture content of dried flour of
Pluchea leaves is determined by gravimetry method
(AOAC, 1990). One gram of samples is measured moisture
content with vacuum oven at 70oC for 24 hours. Weight
difference of sample after heating was moisture content of
sample.
Yield Analysis: Yield of pluchea leaves extract was
determined by gravimetry method based on Ljubuncic et
al. (2005). Yield was measured with comparison between
weight of pluchea leaves extract and sample weight stated
by weight percentage (% w/w dry base).
Phytochemical Identification: Phytochemical assay was
done to determine existence of phytochemical specific in
sample, such as alkaloid, flavonoid, phenolic, sterol,
triterpenoid, phenol hidroquinone, saponin, tannin,
cyanogenic glycoside, cardiac glycoside, and reducing
sugar in pluchea leaves extract (Harborne, 1996).
Total Fenol Analysis: Total fenol of pluchea leaves extract
was determined by spectrometry method (Sahreen et al.
2010). Sample was added with potasium carbonate 75 g/L
and folin ciocalteus reagent and then sample was shaked.
And then sample was homogenous with aquadest. Solution
was be incubation in ambient temperature for one hour,
and absorbance of sample was measured at  760 nm. Total
fenol was stated by gallic acid equivalence (GAE)/g
sample dry base.
Total Flavonoid Analysis: Total flavonoid was determined
by colorimetry method based on aluminium chloride color
measurement (Sahreen et al. 2010). Pluchea leaves extract
was added to aquadest in flask bottle 10 mL. And then this
solution was added NaNO2 5 % (b/v), AlCl3 10 % (b/v),
and NaOH 1 M, respectively with shaked and dilluted until
volume 10 mL. Absorbance of solution was measured at 
510 nm. Total flavonoid was stated as mg catechin
equivalent (CE) /g sample dry base.
Iron Reducing Power Analysis: Iron reducing power was
determined based on modified of Oyaizu method (1986).
Various concentrations of pluchea leaves extract were
mixed with phosphate buffer 200 mM (pH 6,6) and
potasium ferricyanide 0,1 %, and then solution was
incubated at 50oC for 20 minutes. Chlorogenic acid 10%
was added to solution, shaked and filtered. Filtrate was
added aquadest and ferric chloride 0,1 %, and then
absorbance of sample was measured at  700 nm.
Increasing absorbance was indicated that iron reducing
power was increasing. Reducing power of sample was
determined as mg gallic acid equivalent (GAE)/g sample
dry base.
2,2-diphenyl-1-picrylhydrazyl radical (DPPH) Scaveging
Activity Analysis: Antioxidant activity of pluchea leaves
extract was determined based on modified of Sahreen et
al. (2010) method. Various concentrations of samples in
methanol were added DPPH (60 μM in methanol). When
DPPH free radical reacted with antioxidant compound,
capacity of compounds donating hydrogen atom was
reduced. Decreasing of DPPH scavenging activity could be
known based on absorbance of solution that measured at 
517 nm after 30 minutes incubation. DPPH free radical
scavenging activity was stated as % inhibition = [(A0-At)
/ A0] x 100%, A0 was control absorbance at t = 0 seconds
and At was antioxidant absorbance at t seconds.
RESULTS
Pluchea leaves used to get pluchea leaves extract had
moisture content around 14.19 ± 0.17%. This moisture
content of pluchea leaves is the same as Widyawati et al.
(2011) reported around 14.29%. The yield obtained from
solvent extraction with aquadest, ethanol, methanol, ethyl
acetate, and hexanes was showed at Table 1. Data showed
that aquadest extract had the highest yield (40,65 ± 1,47%).
And then methanolic, ethyl acetate, ethanolic and hexanes
extracts had yield 38.07 ± 2.08, 32.97 ± 1.24, 31.09 ± 1.75,
dan 29.46 ± 1.47 %, respectively. The yield of pluchea

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leaves extract contained phytochemical compounds that
were showed at Table 2. Data informed that methanol
could extract the most chemical compounds of pluchea
leaves including sterol, flavonoid, saponint, annin,
phenolic, alkaloid and glycoside compounds compared
with aquadest, ethanol, ethyl acetate, and hexanes. Total
phenol of methanolic extract (1185.2 mg GAE/g sample
Fig. 1: Total Phenol in Various Pluchea Leaves Extracts
Fig. 2: Total Flavonoid in Various Pluchea Leaves Extracts
Fig. 3: DPPH Scevenging Activity in Various Pluchea Leaves Extracts
Fig. 4: Reducing Power in Various Pluchea Leaves Extracts
369.4±29.9
1185.2±6.4
174.7±5.7
15.2±1.3 1.0±0.0
0.0
200.0
400.0
600.0
800.0
1000.0
1200.0
1400.0
Aquadest Methanol Ethanol Ethyl Acetate Hexanes
Total Phenol (mg GAE/g
Sample db)
Extract Type
81.3±1.2
911.9±65.4
93.1±2.1 7.8±0.3 0.9±0.3
0.0
200.0
400.0
600.0
800.0
1000.0
1200.0
Aquadest Methanol Ethanol Ethyl Acetate Hexanes
Total Flavonoid (mg CE/g
Sample db)
Extract Type
244.8±25.2
794.9±34.3
208.5±1.2
10.3±0.3 0.2±0.0
0.0
200.0
400.0
600.0
800.0
1000.0
Aquadest Methanol Ethanol Ethy Acetate Hexanes
DPPH Scavenging Activity
(mg GAE/g Sample db)
Extract Type
2.06±0.11 2.14±0.05 1.89±0.15
1.38±0.14
0.02±0.00
0.00
0.50
1.00
1.50
2.00
2.50
Aquadest Methanol Ethanol Ethyl acetate Hexanes
Reducing Power (mg
GAE/g Sample db)
Extract Type

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db) was the highest concentration compared with aquadest
(369.4 mg GAE/g sample db), ethanolic (174.7 mg GAE/g
sample db), ethyl acetate (15.2 mg GAE/g sample db), and
hexanes (1.0 mg GAE/g sample db) (Figure 1 and 2).
Consequently the methanolic extract of pluchea leaves
showed the biggest DPPH free radical scavenging activity
(794.9±34.3 mg GAE/g sample db) and reducing power
(2.14 ±0.05 mg GAE/g sample db) (Figure 3 and 4).
DISCUSSION
The different moisture content of leaves is determined by
environment, climate, earth texture, and weather. The
solvent difference used to extract pluchea leaves
determined yield of extract. Data also informed that
components contained in pluchea leaves were trended
polar and non polar components only gave the least
composition of pluchea leaves. Many researchers also
informed that the components arranged plants are largely
polar. Methanol is effective to extract leaves and flower of
Alphinia species (Wong, 2006), young leaves of Camelia
sinensis (Chan et al., 2007), Mulberry leaves (Yen et al.
1996), young leaves of Terminalia catappa (Chyau et al.,
2002). Dehkharghanian et al. (2010) also informed that
difference of polarity solvent determines difference of
type, composition, and antioxidant activity of
phytochemical.
Aquadest could dissolve alkaloid and glycoside
compounds, but ethanol was effective to extract sterol,
flavonoid, phenolic, and alkaloid. Ethyl acetate was
semipolar solvent that could dissolve sterol dan alkaloid.
There were less chemical compound of pluchea leaves that
had non polar properties. Previous research informed that
methanol and ethanol can dissolve polar compounds, such
as sugar, amino acid, glycoside compounds (Houghton and
Raman, 1998), phenolic compounds with low and medium
molecular weights and medium polarity (Yu Lin et al.,
2009), aglycon flavonoid (Dehkharghanian et al., 2010),
anthocyanin, terpenoid, saponin, tannin, xantoxilin,
totarol, quacinoid, lacton, flavone, phenone, and
polyphenol (Cowan, 1999). Whereas aquadest is effective
to extract glycoside compounds, amino acid, and sugar
(Houghton and Raman, 1998), aglycon compounds (Liu et
al., 2011; Dehkharghanian et al., 2010), vitamin C
(Dalimarta, 2003). Ethyl acetate is effective to extract
alkaloid, aglycon, and glycoside compounds (Houghton
and Raman, 1998), sterol, terpenoid, and flavonoid
(Cowan, 1999). Hexanes can solve non polar compounds,
such as lignin, wax, lipid, and aglycon (Houghton and
Raman, 1998), sterol, and terpenoid (Cowan, 1999).
Effectivity of methanol extracted phytochemical
compounds in pluchea leaves was supported by total
phenol and total flavonoid assays. Data also showed that
phenolic compounds in pluchea leaves had polar
properties. This was similar to yield assay that pluchea
leaves extract was dominant extracted by aquadest,
methanol, and ethanol. Phenolic compounds of methanolic
extract were effective to donating hydrogen atomic to
molybdenum ion in Folin ciocalteus phenol’s reagent so
that they resulted radical phenoxyl stabilized by resonansi
or delocalization. Effectivity of phenolic compounds was
depended on type, structure, number, and position of
hydroxyl group of benzene ring (Wong et al. 2006;
Widyawati et al. 2010; 2011; 2012).
Total flavonoid in methanolic extract also showed the
biggest concentration (119.9 ± 65.4 mg CE.g sample db)
compared with the other extracts (Figure 2). The
phenomena was similar to total phenolic assay because
flavonoid was major phenolic compounds in plants with
concentration around 80% (Aberoumand and Deokule,
2008). Phytochemical compounds in methanolic extract
were potential to donating hydrogen atom so that these
compounds could form complex compounds with
aluminium ion at total flavonoid assay. Effectivity of
flavonoid as radical scavenging and metal chelating
activities was determine by hydroxyl group of ortho
position in catechol structure (B ring), double bond at C2-3
conjugated with carbonyl group at C4 (C ring), and
hydroxyl group at C5 (A ring) (Tapas et al., 2008; Amic et
al., 2003). Based total flavonoid assay could be said that
flavonoid compounds dominant arraged of pluchea leaves
were polar properties.
Total phenol and total flavonoid of pluchea leaves could
be related with antioxidant activity. Capacity of
phytochemical compounds scavenging free radical
involved donating hydrogen atom or electron (Leopoldini
et al., 2011). Nakiboglu et al. (2007) said that capacity of
phytochemical compounds donating hydrogen
atom/electron could be criteria to measure radical
scavenging activity.
DPPH antioxidant assay is based on the ability of DPPH a
stable free radical, to decolorize in the presence of
antioxidants. The DPPH radical contains an odd electron,
which is responsible for the absorbance at 517 nm and also
for visible deep purple color. When DPPH accepts an
electron donated by an antioxidant compound, the DPPH
Table 2: Phytochemical compounds in various pluchea leaves extracts
Extract
types
Color intensitas of phytochemical compounds
Terpenoid
Sterol
Flavonoid
Saponin
Tannin
Phenolic
Alkaloid
Cardiac
glycoside
Aquadest
-
-
+++
++
+
+++
+++++++
+++++
Methanol
-
+++
+++++
++++
++++
+++++
++++++
+++
Ethanol
-
++++
++++
+
++
++++
++++
++
Ethyl
acetate
-
+++
++
+
-
++
+++
+
Hexanes
-
++
+
-
-
+
+
-
Note: + detected based on color intensity, - not detected based on color intensity

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is decolorized which can be quantitatively measured from
the changes in absorbance (Ara and Nur, 2009). The
reducing power of plants extract is determined on the basis
of the ability of antioxidant in extracts to reduce ferric (III)
iron to ferrous (II) iron based on decolorization of solution
from yellow to green-blue (Liu et al. 2011). Generally, the
antioxidant assay is used due to its simplicity and
reproducibility (Maizura et al., 2011). Zhang et al. (2009)
reported that reductone in extract can separate free radical
chain reaction with donating hydrogen atom or electron.
Capacity of phytochemical compounds in pluchea leaves,
especially phenolic and flavonoid, donating hydrogen
atom or electron determined antioxidant activity.
Antioxidant properties are determined for the phenolic
fractions in the extracts (Amarowicz et al., 2001). Maizura
et al. (2011) informed that phenolic compounds in spices
and herbs significantly contribute to their antioxidant
properties. Total phenolic content is correlated with a
DPPH free radical scavenging activity and reducing
power. The antioxidant activity of pluchea leaves extract
was contributed by phytochemical compounds content.
Sterol, flavonoid, saponin, tannin, phenol hydroquinone,
alkaloid, and cardiac glycoside have been proven to have
antioxidant activity (Nystrom et al., 2007; Li et al., 2007;
Tapas et al., 2008; Amic et al., 2003; Hagerman et
al.,.1998). Phenolic compounds that can donate hydrogen
atom or electron depend on structure, number, position,
and type of hydroxyl group in benzene ring. Potency of
phenolic compounds as antioxidant is determined by
stability of phenoxyl radical formation (Chludil et al.,
2008; Skerget et al., 2006).
CONCLUSION
The results obtained demonstrated that methanol was the
most effective solvent to extract phytochemical
compounds compared to aquadest, ethanol, ethyl acetate,
and hexanes. Phytochemical compounds identified
included sterol, flavonoid, saponin, tannin, phenolic,
alkaloid and glycoside compounds. Methanolic extract
obtained had the highest total phenolic content and
antioxidant activity (DPPH scavenging activity and
reducing power).
ACKNOWLEDGEMENT
The authors would like to thank the Higher Education
Directorate (DIKTI) of the Indonesian’s Government for
the financial support (The competitive research grant) and
Widya Mandala Catholic University in Surabaya for this
research.
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