International Journal of Chemical &

Petrochemical Technology (IJCPT)

ISSN(P): 2277-4807; ISSN(E): 2319-4464
Vol. 6, Issue 2, Apr 2016, 1-10
TJPRC Pvt. Ltd.

INFLUENCE OF EXTRACTING SOLVENTS ON ITS ANTIOXIDANT

PROPERTIES OF BAWANG DAYAK (ELEUTHERINE PALMIFOLIA L. MERR)
ANGGI REGIANA AGUSTIN, SITTI FAIKA & YI-HSU JU
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan
ABSTRACT
Bawang dayak (Eleutherine palmifolia L. Merr) is an indigenous plant in Borneo, Indonesia, traditionally used
by Dayak tribes to treat degenerative diseases such as diabetes mellitus and cancer. In this study, 2.5 gram of sample
powdered was extracted using 100 ml of deionized water, methanol in various concentrations (30%, 50%, 70%, 90%, and
100% v/v), absolute ethanol, acetone, and ethyl acetate at 40oC for 6 h. The antioxidant activity, total phenolic content
(TPC) and total flavonoid content (TFC) of the freeze-dried E. palmifolia extracts were investigated using
various in vitro assays. The extract obtained by 70% methanol in deionized water showed the strongest antioxidant activity
(IC50 39.062.33 g/mL) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, while absolute acetone
extract showed the weakest antioxidant activity (IC50 235.902.12 g/mL). Aqueous methanol (70% methanol) also was

and flavonoid content of 15.030.11 mg quercetin equivalent/g of dry sample weight), while absolute ethanol, acetone,
ethyl acetate and water were less effective in extracting polyphenol. The highest extraction yield was obtained by using
water (21.400.04 %). These results suggest that E. palmifolia is a potential source of natural antioxidant for food and
dietary applications with a potential to reduce oxidative stress.

Original Article

most effective in extracting polyphenol (phenolic content of 20.050.06 mg gallic acid equivalent/g of dry sample weight

Received: Mar 04, 2016; Accepted: Mar 17, 2016; Published: Mar 28, 2016; Paper Id.: IJCPTAPR20161

INTRODUCTION
In the past few years, there has been a growing interest in phytochemicals as new sources of natural
antioxidant and antimicrobial agents [1]. This fact has encouraged researches on vegetables and fruits as
polyphenolic and natural antioxidant sources. Further studies have shown that polyphenolics have the ability to
reduce cellular damage and therefore may be beneficial in promoting human health and protecting against numerous
diseases linked to oxidative events such as cardiovascular and respiratory disorders, cancers and diabetes. Many
studies have shown a strong relationship between polyphenolic compounds and reduced risk of various diseases [2].
Natural antioxidants are critical for human health because they reduce risk of chronic diseases, diabetes,
cancer, and cardiovascular and neurodegenerative diseases. Accordingly, natural sources of antioxidants have
nowadays attracted considerable amount of attention and growing studies have been carried out to find natural
substitutes for synthetic antioxidant suspected to have potentially toxic effect [3]. Antioxidants can be extracted by
various solvents and extraction methods. Solvent extraction is the most common method used for isolating natural
antioxidant. Solvent properties will undoubtedly play a key role in the extraction of antioxidative compounds.
Therefore, it is difficult to develop a unified standard method for the extraction of antioxidant from all plant
materials [4].

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Anggi Regiana Agustin, Sitti Faika & Yi-Hsu Ju

Bawang Dayak is a well known plant among Dayak tribe living in Kalimantan, Indonesia. This plat is originated
from South America. Other species from this genus for example are E. americana, E. bulbosa, E. elicata and E. latifolia.
They are cultivated and naturalize in Africa, Malaysia, Indonesia (Kalimantan and West Java) and Philippines. The plant
has a good adaptation capability to grow on various types of climate and soil. Dayak tribe uses the plant to cure various
type of illness such as cancer, high blood pressure, diabetes mellitus, cholesterol, and ulcers [5].
There is only a few studies regarding E. palmifolia bioactivities and its chemical constituents. Febrianda et al. [6]
reported that aqueous and ethanolic extracts of E. palmifolia were able to prevent diabetes complications through its antihyperlipidemic activities. Bulbs of the plant have been used against heart diseases and function as immonostimulant, antiinflamatory, anti-tumor, anti-bleeding agent [7]. Studies demonstrated that bulbs of Eleutherine contain naptoquinones
(elecanacine, eleutherine, eleutherol, eleutherinone) [8]. Napthoquinones were recognized to exhibit antimicrobial,
antifungal, antiviral, and antiparasitic properties, and to demonstrate bioactivity as anti-cancer and antioxidant, are usually
located within cell vacuoles in the form of glycosides [9].
This study aimed to obtain a crude extract from E. palmifolia that could be used as a new source of antioxidants.
The influence of the solvent on the properties of extracts was investigated. The major phenolic compound were quantified
by spectrophotometer. In addition, the radical scavenging activities of different extracts were quantified using DPPH.

MATERIALS AND METHODS

Materials
Fresh E. palmifolia plants were purchased in the traditional market of Bontang, East Kalimantan. The bulbs of E.

palmifolia were peeled and dried at room temperature for 48 h. After freeze dried for 24 h, the sample was ground into
powder and sieved through a 25 mesh screen. The obtained powdered sample was kept in a sealed polyethylene bottle at 25oC before use.
Methanol, ethanol, acetone and ethyl acetate were obtained from Echo Chemical (Miao Li, Taiwan). Aluminium
chloride, sodium bicarbonate, gallic acid, quarcetine, Follin-Ciocalteu reagent and DPPH-a free radical 2,2 diphenyl 1pycrylhydrazyl were purchased from Sigma Aldrich (St. Louis, MO).

Preparation of Extracts
Extracts were obtained using deionized water, methanol-water (30, 50, 70, 90, and 100% methanol), absolute

ethanol, acetone and ethyl acetate as solvent. Briefly, 2.5 g of sample was placed in a 200 mL glass flask. Then 50 mL of
solvent were added into the flask. The flask was placed in a water bath 40oC for 3 h at 500 rpm stirring. After that, the
filtrate was separated from the solid residue by filtering through Advantech no. 2 filter paper. The process was repeated two
times and the obtained extracts were pooled. The solvent was removed under vacuum at 40oC using a vacuum rotary
evavorator. Crude concentrated extract was weighed to calculate the yield and stored at -15oC.

Determination of Total Phenolic Content (TPC)

The concentration of phenolics in the extracts was determined using spectrophotometric method [10]. Methanolic

solution of the extract (1 mg/mL) was used in the analysis. The reaction mixture was prepared by mixing 0.5 mL of
methanolic solution extracts, 2.5 mL of 10% Follin-Ciocalteus reagent dissolved in water and 2.5 mL 7.5% natrium
bicarbonate. Blank was concomitantly prepared containing 0.5 mL methanol, 2.5 mL 10% Follin-Ciocalteus reagent
Impact Factor (JCC): 3.5367

Index Copernicus Value (ICV): 6.1

Influence of Extracting Solvents on its Antioxidant Properties

of Bawang Dayak (Eleutherine palmifolia L. merr)

dissolved in water and 2.5 mL 7.5% natrium bicarbonate. The sample was thereafter incubated at room temperature for 30
min. Finally the absorbance was determined using spectrophotometer at 760 nm. The sample was prepared in triplicate for
each analysis and the mean value of absorbance was obtained. The procedure was repeated for the standard solution of
gallic acid with concentration from 10 to 70 g/mL and calibration line was constructed (Figure 1). The results were
expressed as mg galic acid equivalent (GAE) per gram of dry matter.

Figure 1: Calibration Curve of Gallic Acid

Figure 2: Calibration Curve of Quarcetine

Determination of Total Flavonoid Content (TFC)

The content of flavonoid in the extract was determined using spectrophotometric method [11]. The sample

contained 1 mL of methanol solution of the extract in the concentration of 1 mg/mL and 1 mL of 2% aluminium chloride
solution dissolved in methanol. The sample was incubated for 30 min at room temperature. Finally the absorbance was
determined using spectrophotometer at 410 nm. The samples were prepared in triplicate for each analysis and the mean
value of absorbance was obtained. The procedure was repeated for the standard solution of quarcetine with concentration
from 10 to 70 ppm and calibration line was constructed (Figure 2). The results were expressed as mg quarcetine equivalent
(QE) per gram of dry matter.

Determination of Total Antioxidant Activity Using DPPH Test

2,2-Diphenil-1-picrylhydrazyl radical (DPPH) assay was carried out to measure free radical scavenging activity as

described by Husain et al. [12]. Extract in methanol (25-400 g/mL) was mixed with 100 L of methanol solution of
DPPH (1000 g/mL). After 30 min incubation at room temperature in dark, the absorbance was read at 517 nm,
scavenging was calculated by the following formula:
Scavenging (%) = 100 (Ablank Asample/Ablank)
Where Ablank is the absorbance of DPPH solution and Asample is the absorbance of the extract solution. Extract
concentration providing 50% scavenging (IC50) was calculated from the graph of plotted inhibition percentage against
extract concentration.
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Statistical Analysis
All data were presented as mean values standard deviation of triplicate determination. Statistical analysis of data

was performed by analysis of variance using MINITAB 14 software and probability value of p0.05 was considered to
denoted a statistical significance difference.

RESULTS AND DISCUSSIONS

Effect of Solvent Extraction on Extract Yield
Solvent extraction was the most frequently used technique for isolation of plant antioxidant compounds. Both
extraction yield and antioxidant activity of extract are strongly dependent on the nature of extracting solvent, due to the
different polarities of plant compounds [13].
Table 1: Yield of E. palmifolia Extracts Using Different Solvents
Solvents a
Yield of Extract (%)b
Water (W)
21.400.04 i
Ethanol (E)
11.260.11 c
Methanol
100% (M)
13.230.11 d
90% (90M)
14.490.21 e
70% (70M)
18.680.16 h
50% (50M)
17.500.09 g
30% (30M)
17.150.10 f
Acetone (A)
8.710.18 a
Ethyl acetate (EA)
10.550.22 b
a
Expressed as % Solvent in Solvent-Water.
b
Expressed as 100 X (G Dry Extract/ G Dry Sample).
Values Are Mean Standard Deviation. Different Alphabetic
Uppercase Letter Indicate Significant Differences among
Extraction Protocols.
As indicated in Table 1, the solvents used for the extraction of phenolic compounds from E. palmifolia show
significantly different extraction yields (P < 0.05). Thus yield of extract is sensitive to extraction solvent used. For pure
solvents, water gave the highest yield followed by methanol and ethanol. The yield of extract by various solvents decreases
in the following order: water > 70% methanol > 50% methanol > 30% methanol > 90% methanol > 100% methanol >
ethanol > ethyl acetate > acetone. The highest extraction yield was obtained by using aqueous solution which may be
ascribed to the increase of polarity of the solvents by adding water [14].
Our result is in good agreement with that of Fatma et al. [15] who reported that highest amount of extract was
obtained from garlic husk waste by using aqueous solvent. Sultana et al. [16] reported that the amount of antioxidant
extracted from different barks with different solvents varied widely. Differences in the yield of extracts from plant
materials might be attributed to the availability of different extractable components, mechanism involved in the extraction
procedures and nature of soil and agro-climatic conditions of cultivation region [17].
Evaluation of Antioxidant Potential
The properties of phenolic compounds make it impossible to develop an exhaustive method for their extraction
[18]. The solubility of a phenolic compound is influenced by the nature of solvent used and its polarity [19]. Phenolic
compounds are generally combined with other substances (protein, polysaccharides, terpenes, chlorophyl, lipids, inorganic
compounds) [20].
Impact Factor (JCC): 3.5367

Index Copernicus Value (ICV): 6.1

Influence of Extracting Solvents on its Antioxidant Properties

of Bawang Dayak (Eleutherine palmifolia L. merr)

Table 2: Total Phenolic Contents, Total Flavonoid Contents of E. palmifolia Extracts Using Different Solvents
Solventsa

TPC (Mg GAE/ G

DM)B
9.420.46 e
5.950.10 a

TFC (Mg QE/ G

DM)C
3.760.26 b
2.790.32 a

Water (W)
Ethanol (E)
Methanol
100% (M)
10.170.19 f
4.950.11 d
g
90% (90M)
11.500.07
7.500.78 f
i
70% (70M)
20.050.06
15.030.11 i
h
50% (50M)
13.440.29
8.580.30 h
c
30% (30M)
7.700.08
4.390.03 c
b
Acetone (A)
6.830.06
5.140.27 e
d
Ethyl acetate (EA)
9.200.31
8.540.03 g
a
Expressed as % Solvent in Solvent-Water.
b
Expressed as Miligram Gallic Acid Equivalent Per Gram Dry Matter
c
Expressed as Miligram Quarcetine Equivalent Per Gram Dry Matter
Values Are Mean Standard Deviation. Different Alphabetic Uppercase
Letter Indicate Significant Differences among Extraction Protocols.

Table 2 shows the effect of extraction solvent on TPC in the extract. TPC of extracts ranged from 5.950.10 mg
GAE/g DM for 100% ethanol to 20.050.06 mg GAE/g DM for 70% methanol. Solvent has significant effect (p0.05) on
TPC of extract. It seems that more TPC can be extracted from plant materials using more polar solvent. It is considered that
phenolic compounds contribute to overall antioxidant activity of extract [16]. Several studies showed excellent linear
correlation between TPC and antioxidant activity [21, 22, 23]. Methanol is efficient and the most widely used solvent to
prepare phenolic/antioxidant extract because methanolwater mixture has high dielectric constant/polarity and can be
categorized as the most suitable extracting solvent [24,25, 26]. The extraction protocol for the preparation of antioxidant
extract has been found to be the key factor toward the antioxidant performance of extracts.
Table 2 also shows the effect of extraction solvent on TFC in the extract. TFC of extract obtained was from
2.790.32 mg QE/ g DM for 100% ethanol to 15.030.11 mg QE/ g DM for 70% methanol. As with TPC, the maximum
TFC was found in 70% methanolic extract and the lowest in 100% ethanolic extract. Again, solvent has significant effect
(p0.05) on TFC of extract. Comparing the results of TPC anf TFC in ethanol and acetone extracts, ethyl acetate contained
more TPC and TPC. This may be atrributed to the solubility of some polyphenol compounds in semipolar sovent.
There is no report available regarding TFC of E. palmifolia extract obtained by using different solvents. Our
findings on TFC are in agreement with the results of Sultana et al. (2007) who reported more TFC in 80% methanol extract
of bark than that in 80% ethanol extract. The correlation between TPC and TFC was found to be 0.9695. This indicates that
flavonoids are the dominating phenolic groups in E. palmifolia. The results is similar to the extraction of phenolics from
pink flesh guava [27] and L. aromatica [28].

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Figure 3: DPPH (2, 2-Diphenyl-1-Picrylhydrazyl) Radical Scavenging Activity of E. palmifolia

Extracts Obtained By Using Different Solvents. 30M: 30% Methanol,
50M: 50% Methanol, 70M: 70% Methanol, 90M: 90% Methanol
Table 3: DPPH Scavenging Activity of E. palmifolia Extracts Using Different Solvents
Solvent Systema
IC50 (g/Ml) B
Water (W)
108.320.48
Ethanol (E)
233.171.00
Methanol
100% (M)
116.360.48
90% (90M)
103.792.42
70% (70M)
39.062.33
50% (50M)
63.292.12
30% (30M)
108.250.59
Acetone (A)
235.902.12
Ethyl acetate (EA)
152.490.78
a
Expressed as Fraction of Solvent to Water.
b
Expressed as Inhibition at 50 %
Concentration (g/Ml).
Values Are Mean Standard Deviation.
Different Alphabetic Uppercase Letter Indicate
Significant Differences among Extraction
Protocols.
Solvent Effect on Antioxidant Activity
DPPH radical is a stable organic free radical with an absorption band at 517nm. It loses this absorption when
accepting an electron or free radical species and results in visually noticeable discoloration from purple to yellow. It can
accommodate many samples in a short period and is sensitive enough to detect active ingredients at low concentration [29].
Free radical scavenging activities of the E. palmifolia extracts were measured and the results are presented in
Figure 3. The results show that DPPH free radical scavenging activity of methanol extracts were significantly higher
(P<0.05) than those of extracts obtained using other solvents. The extract obtained by 70% methanol yielded the highest
DPPH radical scavenging activity.
The IC50 of a compound is inversely related to its antioxidant capacity, as it express the amount of antioxidant
required to decrease the DPPH concentration by 50%, which is obtained by interpolation from linear regression analysis
[30]. Lower IC50 indicates higher antioxidant activity of a compound. Table 3 shows IC50 values in the DPPH radical
Impact Factor (JCC): 3.5367

Index Copernicus Value (ICV): 6.1

Influence of Extracting Solvents on its Antioxidant Properties

of Bawang Dayak (Eleutherine palmifolia L. merr)

scavenging activity assay of extracts. It was found that 70% methanol extracts possesses the strongest DPPH radical
activity (IC50 = 39.062.33 g/mL) followed by 50% methanol (IC50 = 63.292.12 g/mL) and 90% methanol (IC50 =
103.792.42 g/mL).
Table 4: Polarity Index of Organic Solvent-Water Mixture
Solvent
Water
Concentration %
Polarity Index
10
Calculated from Eq.1

30
8.53

Methanol
50
70
90
7.55 6.57 5.59

100
5.10

Ethanol

Acetone

Ethyl Acetate

5.20

5.40

4.40

Effect of Solvent Polarity on Antioxidant Activity of Extract

Variations in the value of antioxidant activities of different extracts might be attributed to the change in relative
polarity of different solvents used. As found in this investigation, by using pure solvent extraction efficiency was poor. The
addition of some water enhances extraction efficiency. The polarity index of a mixture of two solvents, Pm, can be
calculated from Eq. 1 [31].
P m = 1P 1 + 2P 2

(1)

where 1 and 2 are the volume fractions of solvents 1, and solvent 2, respectively, and P1 and P2 are polarity
index of solvent 1 and solvent 2, respectively. Table 3 shows the polarity of solvent used in this study. As can be seen from
the table, polarity index of the mixture increases with incresing water amount in mixture. Each solvent seems to have
distinct specificity in the extraction of antioxidant. This fact is in accordance with polarity of the solvent used for the
extraction and its solubility and content of antioxidants in fruit [32]. From the results shown in Table 2 along with Table 3,
it may be suggested that a certain degree of increase in solvent polarity (up to 50% water) could enhance the solubility of
antioxidant compounds in solvent mixture.

CONCLUSIONS
The results obtained in our work showed that E. palmifolia can be used as an easily accessible source of natural
bioactive compounds. We have demonstrated for the first time, as far as we know, that solvent-water mixure extracts of E.
palmifolia possess strong antioxidant activity. The antioxidant activity of the extract can probably be attributed to its
phenolic compounds. Antioxidant extraction depends on the solubility of antioxidant compounds of plant material in the
extraction solvent. In this study, 70% methanol was found to be the best solvent. The addition of water to organic solvent
incrased the effectiveness of extraction.

ACNOWLEDGEMENTS
This work was supported by a grant from National Taiwan University of Science and Technology. The authors
wish to tank to Indonesian Ministry of Research and Technology and Higher Education.
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