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Research Report
Asia Pacific Publishing 2015 Sc i. Te chno l. Pr og ., 2015, 1, 10-1 3 | 10
Science
Technology Progress
www.apjor.com
Received 2nd December 2014
Accepted 31st May 2015
Keywords
Orthosiphon stamineus
Phenolic content
Flavonoid content
Extraction
Ultrasonic
Ultrasonic assisted extraction of phenolic and
flavonoid content from Orthosiphon stamineus leaves
Sook Fun Pang†1, Mashitah M. Yusoff2, L. Chuah Abdullah, Jolius Gimbun1,3
1Faculty of Chemical & Natural Resources Engineering, 2 Faculty of Industrial Science & Technology,
3CoE for Advanced Research in Fluid Flow (CARIFF), Universiti Malaysia Pahang, 26300 Gambang,
Pahang, Malaysia.
†E-mail: p.sookfun@gmail.com
This paper presents a combined effect of ultrasonic assisted extraction and solvents of varying
polarities on phenolic compound extraction from O. stamineus. The polyphenol content in the
plant extracts was analysed using Singl eton’ s met ho d and alumi ni um ch loride colorimetric
assay. The results suggest that the polyphenol extraction from O. stamineus is affected by the
solvent type. The highest phenolic content of 168.8 mg GAE/g DW was obtained from
ultrasonic assisted extraction (UAE) using 70% aqueous methanol and 70% aqueous propanol
solvent. The highest total flavonoid content of 185.3 mg QE/g DW was obtained using 70%
aqueous propanol. The phenolic acid and flavonoid yield increased with extraction time,
however, extraction beyond 120 min or at a temperature higher than 60°C induced degradation
and hence reducing extraction yield.
Introduction
Orthosiphon stamineus (vernacular name: ‘misai kucing’) is
traditionally used in Malaysia for treatment of bladder
inflammation, eruptive fever, edema, hypertension, diabetes
mellitus, rheumatism and diuretic
1
. Previous studies revealed
that extract of O. stamineus contained many useful bioactive
compounds such as terpenoids, polyphenols and sterols
2
leading
to various activities such as antibacterial, antifungal,
antimicrobial and antitumor.
The first step to recover and purify bioactive compounds
from plant materials involves extraction. The solvent used,
extraction method and condition affect the yield of bioactive
compound in the extract. Conventional method such as
maceration and soxhlet extraction is often performed at high
temperatures for several hours, and thus may cause thermal
degradation of polyphenols due to prolonged heat exposure.
Other methods have been developed such as the ultrasonic-
assisted extraction (UAE), accelerated solvent extraction and
supercritical fluid extraction. Supercritical method requires
higher capital and operating cost owing to its high pressure
requirement and hence less favourable. The UAE is an
inexpensive and efficient alternative compared with other
extraction techniques such as microwave-assisted extraction,
supercritical fluid extraction and conventional extraction
techniques. The UAE technique reduced the inner and external
mass transfer limitation and hence increases the yield of
extraction from plant material. Furthermore, ultrasonic wave
can break the cell membranes, which may enhance inner mass
transport
3
, thus UAE was employed in this work. Moreover,
limited comprehensive study on phenolic compound extraction
from O. stamineus leaves by UAE is available in the literature.
Although Pang and coworkers
4,5
recently performed an UAE of
O. stamineus as part of their work on phenolic compounds
microencapsulation, however, details study on the extraction
parameter is not presented.
It is known that phenolic and flavonoid content in the
extracts from the same plant material may vary widely
according to the polarity of the solvent used. A combined effect
of UAE and varying solvent polarity to the phenolic and
flavonoid extraction from O. staminues leaves has never been
studied previously, and hence this is the objective of this work.
Materials and methods
Plant material
Sodium nitrite, methanol, isopropanol, sodium hydroxide and
Folin–Ciocalteu reagent were obtained from Merck (Darmstadt,
Germany). Aluminium hexachloride was obtained from Sigma
Aldrich (St. Louis, MO). Leaves from a white-flowered O.
stamineus similar to one that has been deposited at the Forest
Science Technology Progress Research Report
Asia Pacific Publishing 2015 Sc i. Te chno l. Pr og ., 2015, 1, 10-1 3| 11
Research Institute, Malaysia (voucher no. ZAS1113) were
collected in Gambang, Pahang, Malaysia. The freshly collected
leaves were washed with deionised water and dried at 37°C for
3 days before crushed to powder. The powder was kept in an
air-tight plastic bag in a desiccator at room temperature to
prevent moisture absorption prior to experiment.
Ultrasonic assisted extraction
The powdered plant material was weighed (1 wt.%) and mixed with
100 ml of solvent in a 250 ml sealed Erlenmeyer flask. UAE was
carried out in an ultrasonic bath (CREST P1800D, United States)
filled with 12 litres of water at 45 kHz and 133.33 W for 90 min and
temperature was set at 50°C to study the effect of solvent type.
Various solvent with polarity index (PA) ranged from 3.9 to 9.0 such
as isopropanol (IPA) PA = 3.9, methanol PA = 5.1 and ultrapure
water PA = 9.0 were used for the extraction process. Aqueous
mixtures of IPA and methanol are also tested. The study was
performed in this way because solubility of methoxylated and
hydroxylated polyphenols in solvent is affected by its polarity. A
suitable solvent that enabled a simultaneous extraction of both
phenolic acid and flavonoid were chosen for the remainder of this
work. The study on extraction time (30 to 180 min) and temperature
(40 to 70°C) was performed using the selected solvent. The
parameters were determined based on literature and according to the
equipment limitation. The UAE supernatant was then separated from
the residue by filtration using 0.45 µm nylon membrane filter.
Total phenolic content
Total phenolic content (TPC) was assessed using the Folin–
Ciocalteu reagent, following Singleton’s method6. A sample aliquot
of 0.125 ml was added to a test tube containing 0.5 ml of ultrapure
water and 0.125 ml of the Folin–Ciocalteu reagent. After 3 min, 1.25
ml of 7% Na2CO3 solution was added, and the final volume was
made up to 3 ml with ultrapure water. The solution was mixed well
and incubated for 60 min in the dark. The absorbance was measured
against the prepared blank reagent at λ = 760 nm using a calibrated
ultraviolet–visible spectroscopy (Hitachi U-1800, Japan). TPC of the
leaves was expressed as mg gallic acid equivalents per gram dry
weight (mg GAE/g DW) by comparing with the calibration curve for
gallic acid.
Total flavonoid content
Total flavonoid content (TFC) was measured using the aluminium
chloride colorimetric assay7. A sample aliquot (0.2 ml) was added to
a 15 ml centrifuge tube containing ultrapure water (4.8 ml). NaNO2
(0.3 ml, 5%) was then added and mixed using a vortex mixer for 5
min. Subsequently, AlCl3 (0.3 ml, 10%) was added, followed by
addition of NaOH solution (2 ml, 1M), and the total volume adjusted
with ultrapure water to the final volume of 10 ml. The solution was
mixed well, and absorbance measured against a blank reagent at λ =
510 nm using a calibrated ultraviolet–visible spectroscopy (Hitachi
U-1800, Japan). The TFC of the sample solution was expressed as
mg quercetin equivalents per gram dry weight (mg QE/ g DW) by
comparing with the calibration curve for quercetin.
Statistical analysis
Each experiment was repeated in triplicates. Analysis of variance
(ANOVA) was performed by using the data analysis tools in
Microsoft Excel 2010, and a least significant difference (LSD) test
was used to compare the means with a confidence interval of 95%.
Results and discussion
Effect of solvent type on phenolic compound extraction
The solubility of the bioactive component in a different solvent was
directed by its structural characteristic. Highly methoxylated
compounds such as flavanoids, which are lipophilic were more
stable in lower polarity solvent such as methanol and isopropanol.
However, highly hydroxylated compound such as phenolic
compound is hydrophilic thus more soluble in water than in
isopropanol. Similar findings are also reported by Akowuah and co-
workers8 which found that the amounts of flavonoid are higher in
lower polarity solvent, i.e. chloroform extracts.
Fig. 1 Effect of solvent type of TPC obtained from UAE
Fig. 2 Effect of solvent type of TFC obtained from UAE
The results (Figs. 1 and 2) show that aqueous alcoholic solvent
has a significantly higher (> 30%) extracting capacity of total
flavonoid and total phenolic content compared to pure solvent
(100% methanol and 100% propanol). Similar findings are also
reported by Wach and co-workers9 who found that aqueous
methanol ranged from 40 to 80% is preferable for rutin and quercetin
extraction from Hypericum perforatum. The aqueous solvent has a
wider range of polarity as opposed to the pure solvent, and hence
enhances the simultaneous extraction of both lipophilic and
hydrophilic compounds, resulting in a better extraction. This
phenomenon can be seen clearly for the case of isopropanol and
aqueous isopropanol, which increases extraction of flavonoid
(methoxylated flavonoid) about10% from 167.7 to 185.3 mg QE/g
DW without adversely affecting the extraction of the hydroxylated
compound. Finding from this work suggests that aqueous alcoholic
solvent (i.e. 70% MeOH and 70% IPA) enables a simultaneous
extraction of hydrophilic component (phenolic) and lipophilic
component (flavonoids). The highest simultaneous extractions of
0
10
20
30
40
50
60
70
IPA MeOH Water 70% MeOH 70% IPA
TPC (mg GAE/g DW)
Solvent type
0
40
80
120
160
200
IPA MeOH Water 70% MeOH 70% IPA
TFC (mg QE/g DW)
Solvent type
Research Report Science Technology Progress
12 | Sci . Te chnol . Prog., 2015, 1, 10-13 Asia Pac ific Publishing 2015
both phenolic and flavonoid content were obtained using 70% IPA
and thus this solvent will be used for the remainder of this work.
Effect of extraction time on phenolic compound extraction
UAE extraction time is an important parameter for the
extraction process in order to maximize the phenolic and
flavonoid content obtained from the sample when the
equilibrium concentration reached before the apparent
reduction due to polyphenol degradation
10
. Extraction time was
studied by extracting O. stamineus dried leave powder at 50°C
with the extraction time ranging from 30 to 180 min. The
solvent, 70% propanol, was chosen due to its ability of
extracting simultaneously both phenolic and flavonoid content.
The effect of extraction time on the phenolic and flavonoid
content is shown in Fig. 3. TPC was significantly reduced after
120 min, however, TFC still increasing even after 120 min of
extraction. These dissimilarities might be due to differences in
solubility of phenolics and interaction of phenolics with other
components, which would lead to the difference in the times
needed to reach equilibrium between the solid matrix (O.
stamineus leaves) and the solvent (70% IPA). Similar findings
are also reported by Thoo and co-workers
11
, who studied
extraction of Morinda citrifolia; they reported a different
optimum extraction time for TPC and TFC.
Effect of temperature on phenolic compound extraction
Influence of temperature on polyphenol extraction for UAE
extraction was studied by extracting O. stamineus dried leaves
powder for 90 min using 70% aqueous propanol at temperature
ranging from 40°C to 70°C. It was understood from previous
finding, that extraction time longer than 90 min induces
significant degradation of rosmarinic acid and eupatorin, thus
time is fixed at 90 min for this study.
Fig. 3 Effect of UAE time on TPC and TFC at constant
temperature of 50°C
The relationship of extraction temperature phenolic and
flavonoid content are shown in Fig. 4. Extraction of both TPC
and TFC from O. stamineus leaves increases with increasing
temperature up to 60°C and followed by a slight decrease
afterwards due to thermal degradation of polyphenols. Higher
temperature may increase the diffusion rate and therefore,
increases extraction yield. In addition, mild heating softens the
plant tissue, weakening the cell wall integrity hence more
polyphenol diffuses to the solvent. However, increasing
temperature beyond a certain limit may induce polyphenol
degradation which can be observed for UAE above 60°C.
Similar findings are also reported by Tabaraki and Nateghi
12
, in
which was observed that TPC of rice bran extracts increases
with increasing temperature up to 54°C and followed by a slight
decrease thereafter. Similar findings are also reported by Ishak
et al.
13
, who studied the effect of temperature on extraction of
Habbatus sauda seeds.
Fig. 4 Effect of UAE temperature on TPC and TFC after 90
minutes
Conclusions
The highest phenolic content of 168.8 mg GAE/g DW was
obtained from UAE using 70% aqueous methanol solvent.
Similar TPC are also obtained using 70% aqueous propanol
solvent. The highest TFC of 185.3 mg QE/g DW was obtained
using 70% aqueous propanol. Aqueous solvent provides a
wider range of polarity as opposed to the pure solvent, and
hence enhances simultaneous extraction of both methoxylated
and hydroxylated compounds. Extraction beyond 120 min or at
a temperature higher than 60°C induced degradation and hence
reducing extraction yield. This work may be useful for
obtaining higher polyphenol extract from O. stamineus.
Acknowledgements
Research funding from the Ministry of Education Malaysia
(FRGS/2/2013/TK05/UMP/02/4 and RACE RDU121308) and
Universiti Malaysia Pahang (GRS140302) are gratefully
acknowledged. PSF is a recipient of MyPhD scholarship
through the MyBrain15 scheme by the Ministry of Education
Malaysia. The authors would like to thank Chau Ling Choy
(Waters), Siaw Jing Lau and Siew ling Lee (Faculty of
Chemical & Natural Resources Engineering, Universiti
Malaysia Pahang) for supporting the early stages of this project.
Notes and references
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90
100
110
120
130
140
150
70
75
80
85
90
95
100
050 100 150 200
TFC (mg QE/g DW)
TPC (mg GAE/g DW)
Time (min)
TPC TFC
90
100
110
120
130
140
150
70
75
80
85
90
95
100
105
30 40 50 60 70 80
TFC (mg QE/g DW)
TPC (mg GAE/g DW)
Temperature (°C)
TPC TFC
Science Technology Progress Research Report
Asia Pacific Publishing 2015 Sc i. Te chno l. Pr og ., 2015, 1, 10-1 3| 13
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