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Extraction of Bioactives from Orthosiphon stamineus using Microwave and

Ultrasound-Assisted Techniques: Process Optimization and Scale Up

Article  in  Food Chemistry · November 2016

DOI: 10.1016/j.foodchem.2016.11.016

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This is an author generated postprint of the article: Chan, C.-H., See, T.-Y., Yusoff, R., Ngoh, G.-
C., & Kow, K.-W. (2017). Extraction of bioactives from Orthosiphon stamineus using microwave
and ultrasound-assisted techniques: Process optimization and scale up. Food Chemistry, 221,
1382-1387. doi: 10.1016/j.foodchem.2016.11.016

Extraction of Bioactives from Orthosiphon stamineus using Microwave and

Ultrasound-Assisted Techniques: Process Optimization and Scale Up

Chung-Hung Chan
Please email to rykenz87@yahoo.com for any inquiries

ABSTRACT
This work demonstrated the optimization and scale up of microwave-assisted extraction (MAE)
and ultrasonic-assisted extraction (UAE) of bioactive compounds from Orthosiphon stamineus
using energy-based parameters such as absorbed power density and absorbed energy density
(APD-AED) and response surface methodology (RSM). The intensive optimum conditions of
MAE obtained at 80% EtOH, 50 mL/g, APD of 0.35W/mL, AED of 250 J/mL can be used to
determine the optimum conditions of the scale-dependent parameters i.e. microwave power and
treatment time at various extraction scales (100-300 mL solvent loading). The yields of the up
scaled conditions were consistent with less than 8% discrepancy and they were about 91-98% of
the Soxhlet extraction yield. By adapting APD-AED method in the case of UAE, the intensive
optimum conditions of the extraction, i.e. 70% EtOH, 30 mL/g, APD of 0.22 W/mL, AED of 450
J/mL are able to achieve similar scale up results.

The published version is available on http://dx.doi.org/10.1016/j.foodchem.2016.11.016

Keywords: microwave extraction, ultrasonic extraction, bioactive compounds, Orthosiphon
stamineus, scale up

1. Introduction
Orthosiphon stamineus or known as Java tea is a traditional herbal plant that used as tea beverage
and herb medicine in Asia Pacific region mainly Malaysia and Indonesia (Akowuah et al., 2004).
It contains bioactive compounds such as sinensetin, 3’-hydroxy-5,6,7,4’-tetramethoxyflavone,
rosmarinic acid and eupatorin, which are good for the treatment of kidney problem, gout and
diabetes (Hossain & Rahman, 2015; Sriplang, Adisakwattana, Rungsipipat, & Yibchok-anun,
2007). Recent studies reveal that about 116 compounds can be isolated from this plants to provide
anti-oxidative, anti-tumor, diuretic and nephron-protective, anti-diabetic, anti-hypertensive, anti-
inflammation, anti-microbial, anti-obesity, anti-osteoporosis, vasorelaxative and hepatoprotective
effects (Adnyana, Setiawan, & Insanu, 2013; Bokhari, Mohamed, & Lau, 2016; Meilis, et al., 2016;
Yam, Tan, Ahmad, & Ruan, 2016). Due to its high therapeutic values, these bioactive compounds
are in great interest for the production of relevant natural product.

Conventional solvent extraction technique often associates with long heating time, which risks the
degradation of bioactive compounds. This gives rise to advanced techniques such as microwave-
assisted extraction (MAE) and ultrasonic-assisted extraction (UAE) that are efficient in terms of
extraction time and solvent consumption. In view of MAE, microwave heating is able to disrupt
plant structure via the increase of internal pressure of the cell (Chen & Spiro, 1995), and then to
release the bioactive compounds. On the other hand, ultrasonic cavitation in UAE produces
shockwaves that is capable of disrupting external structure of the plant sample, releasing the plant
bioactives effectively (Budynas & Nisbett, 2008; Floros & Liang, 1994). This work focuses on the
employment of both MAE and UAE techniques for the extraction in the aspect of optimization and
scale up.

Response surface methodology (RSM) is commonly used to optimize the operating parameters of
MAE and UAE based on experimentally-fitted empirical model that described the interactive
effects of the influencing parameters. Despite its ease of application and wide use in the literature,
the optimum conditions from RSM often restricted to a specific scale of extraction and a particular

2
microwave or ultrasonic system. This is because larger extraction scale requires higher power input
to sustain the process. Besides, different instrumental setups have different power efficiencies and
so their extraction performances. The above problems are alleviated in MAE process, through an
alternative optimization method based on absorbed power density (APD) and absorbed energy
density (AED) parameters (Chan, Yusoff, & Ngoh, 2014). This method not only capable to
optimizes MAE process, but also can scale up and reproduce the process at other system
consistently (Chan, Lim, Yusoff, & Ngoh, 2015; Chan, Yusoff, & Ngoh, 2014). APD and AED
parameters indicate the amount of power and energy absorbed by a unit volume solvent,
respectively. As such, they are able to characterize the heating efficiency of microwave system
and the input energy required by the system to drive the extraction to reach equilibrium yield (Chan,
Yusoff, & Ngoh, 2013). The above concept could be applicable in UAE process since ultrasonic
cavitation also provides thermal effect to the extraction solvent (Frederick, 1965).

Therefore, this work explores the application of APD and AED in UAE process for the first time.
In this study, optimization of MAE and UAE of bioactive compounds from Orthosiphon stamineus
were performed based on APD-AED method and RSM, respectively. Following that, scale up of
the optimized MAE and UAE were performed based on APD-AED method. The scale up method
involves the determination of optimum APD and AED at the optimum conditions and also the
calibration of nominal power or percentage amplitude and treatment time for microwave or
ultrasonic systems. Finally, the extraction performances of the up scaled MAE and UAE were
evaluated and assessed by comparing with the conventional Soxhlet extraction.

2. Materials and Methods

2.1. Materials and reagents

Standards of sinensetin and 3´-hydroxy-5,6,7,4´-tetramethoxyflavone were from Sigma-Aldrich
co. (USA). Acetonitrile and ethanol for chromatography analysis were purchased from Merck co.
(Germany). The extraction solvent, denatured alcohol (EtOH) was obtained from R&M chemicals
(Malaysia).

3
Fresh samples of Orthosiphon Stamineus leaves were obtained from local plantation in Negeri
Sembilan, Malaysia. The leaves obtained were washed and dried at 45 °C for 24 hr and achieved
a moisture content of below 15 %. The dried leaves were powdered to 250-500 μm and stored in
air-tight container at 4 °C until further use.

2.2. Extraction procedure

MAE was performed using domestic microwave oven (Samsung MW718). 2 g of sample was
mixed with extraction solvent at certain solvent-sample ratio in a closed Duran bottle. The mixture
was irradiated at constant nominal microwave power for certain duration. Upon extraction, the
extract was filtered using fine cloth and RC syringe filter before subjected to HPLC analysis.

UAE was performed using sonicator (Qsonica Q500, power 500 W, 20 kHz, 25 mm probe, max
amplitude 120 μm) with air cooling system. The air cooling system is to cool the ultrasonic
converter during use. 2 g of sample was mixed with extraction solvent at certain solvent-sample
ratio in a container. The mixture was sonicated at certain percentage amplitude (which is
corresponding to a specific applied power) for a predetermined extraction time under intermittency
ratio, α of 4/5 in which α is defined as the fraction of a cycle when sonication was on, i.e.
α=τon/(τon+τoff) where τon and τoff are the times in seconds when the sonication was turned on and
off, respectively. This intermittent ratio is to prevent overheating of the sonotrode. The subsequent
analysis procedure was similar to the MAE as previously described.

Soxhlet extraction was conducted using 2 g of sample and 200 mL of ethanol for 6 hours. After
extraction, the extract was subjected to HPLC analysis after filtered through the fine cloth and RC
syringe filter.

2.3. HPLC analysis

The bioactive compounds were quantified using Agilent 1200 Series HPLC system (USA)
equipped with Agilent ZORBAX Eclipse Plus C18 column, 5 μm (4.6 mm x 250 mm). The
following HPLC method (Bonaccorsi, Caristi, Gargiulli, & Leuzzi, 2008) was employed. The
mobile phase was linear gradient of acetonitrile in water: 5-20% (0-15 min), 20-30% (15-20 min),
30-50% (20-30 min), 50-100% (30-35 min), 100% (35-40 min), and 100-5% (40-50 min) at flow

4
rate of 1.0 mL/min. The injection volume of sample was 10 μL and the separation is detected by
UV-DAD at wavelength 340 nm using Agilent 1260 Infinity Diode Array Detector (USA). The
retention times of sinensetin and 3´-hydroxy-5,6,7,4´-tetramethoxyflavone were and 33.30 and
32.28 minutes, respectively. The extraction yields of the compounds were reported as the mass of
extracted bioactive compounds per unit mass of plant sample (mg/g). The total extraction yield of
sinensetin and 3´-hydroxy-5,6,7,4´-tetramethoxyflavone were the response of the optimization
study.

2.4. Determination of APD and AED

Absorbed power density (APD) parameter indicates the heating power experienced by a unit
volume of extraction solvent under microwave heating or ultrasonication (W/mL). The procedure
to calculate APD has been detailed in the previous work (Chan, Yusoff, & Ngoh., 2013). In this
study, the APD values of blank extraction solvent under various nominal power or percentage
amplitude (microwave or ultrasonication treatment) and solvent loading can be determined
experimentally based on Eq. (1).
Q
APD  Eq. (1)
V  tH

where Q is the amount of heat absorbed by solvent during treatment (J), V is the solvent loading
(mL) and tH is the treatment time (min). The heat absorbed (Q), can be easily determined based on
the specific heat capacity of the solvent and the temperature rise gradient. A representative value
of APD of blank solvent under various power or percentage amplitude and solvent loading of UAE
and MAE were obtained by averaging the APD values calculated for each conditions at different
heating time (tH) using Eq. (1). The averaged APD values are tabulated in Table 1. On the other
hand, absorbed energy density (AED) is related to APD and the extraction time as follows:
AEDt  APD  t Eq. (2)
where AEDt is the total heating energy experienced by a unit volume of extraction solvent under
microwave heating or ultrasonic cavitation (J/mL) and t is the extraction time (min).

5
 Table 1: APD values of MAE and UAE at different power and solvent loading

Nominal
microwave
Solvent
Extraction power (W) or Absorbed power
loading
system percentage density, APD (W/mL)
(mL)
amplitude of
ultrasound (%)
100 0.20 ± 0.01
150 0.35 ± 0.03
200 0.47 ± 0.02
100
250 0.96 ± 0.03
MAE 300 1.10 ± 0.00
350 1.56 ± 0.09
200 220 0.35 ± 0.01
300 300 0.35 ± 0.01
60 45 0.22 ± 0.00
UAE 180 78 0.20 ± 0.00
240 100 0.21 ± 0.00
Solvents used in the MAE and UAE are 80% and 70% v/v EtOH respectively

2.5. Optimization of MAE

The optimization of MAE was conducted using the energy-based optimization method (Chan,
Yusoff, & Ngoh., 2014). This method uses sequential single factor experiment to optimize the
solvent concentration, solvent to sample ratio (constant volume), AED and APD. The experiments
were performed using fresh sample in duplicate except for the determination of AED. This is
because the optimum AED can be observed from the overall trend of MAE extraction curve (yield
vs. time). In this study, the effect of solvent concentration (60-100% v/v aqueous ethanol) was
studied at arbitrary condition (150 W, 50 mL/g, 20 min) and the optimum value was obtained.
Then, the following optimization of solvent to sample ratio (20-80 mL/g at constant volume) was
performed based on the optimum solvent concentration at arbitrary condition (150 W, 100 mL, 15
min). In the subsequent experiment, the effect of AED at the extraction conditions of 150 W, 100
mL and the optimum solvent to sample ratio were studied by conducting the extraction at different

6
extraction time (2-20 min) using a fresh sample. The optimum extraction time obtained facilitates
the determination of optimum AED using Eq. (2). The same strategy was selected for the
subsequent experiments. Finally, the effect of APD at the optimum solvent to sample ratio and
AED was investigated at the specific solvent loading (100 mL) with varying microwave powers
of 100-350 W. To execute MAE at one specific AED implies that the extraction time at each
microwave power level must be determined based on their APD values using Eq. (2). The
microwave power and extraction time corresponded to the optimum APD and AED are the
optimum operating conditions of MAE at the specific solvent loading.

2.6. Optimization of UAE

Similar with optimization of MAE, non-interacting parameters such as extraction solvent (60-100%
v/v aqueous ethanol) and solvent-sample ratio at constant solvent volume (10-70 mL/g) in UAE
were first optimized using single factor experiment at arbitrary condition (percentage amplitude of
60 %, 30 min, 60 mL). Then, RSM with three level factorial design was employed to optimize
extraction time (X1) of 20-40 min and percentage amplitude (X2) of 40-80 % in 13 set of
experiments as tabulated in Table 2. The result from RSM optimization study was fitted into
second-order polynomial model as follows:
2 2 2
Y  Bo   Bi X i   Bii X i 2   Bij X i X j Eq. (3)
i 1 i 1 i j

where Y represent the predicted response; Xi was the actual value of an independent variable, B0
denotes the model intercept; Bi, Bii, and Bij are the coefficients of the linear, quadratic, and
interactive effects, respectively. Analysis of variance (ANOVA) was used to determine the
regression coefficients and the predicted model was verified by carried out the experiment in
triplicate under optimum extraction conditions.

7
Table 2: Optimization of percentage amplitude and extraction time of UAE based on RSM

Extraction time, Percentage Total extraction

Standard
X1 amplitude, X2 yields, Y
order
(min) (%) (mg/g)

1 20 40 1.51
2 30 40 1.70
3 40 40 1.67
4 20 60 1.56
5 30 60 1.68
6 40 60 1.79
7 20 80 1.67
8 30 80 1.67
9 40 80 1.27
10 30 60 1.74
11 30 60 1.72
12 30 60 1.78
13 30 60 1.84
Experiments were performed by using 500 W sonicator with intermittency ratio (ratio of turn on
time to total time) of 4/5, extraction solvent of 70% v/v EtOH and solvent to sample ratio of 30
mL/g (60 mL)

2.7. Scale up of MAE and UAE

This section elucidates the procedure to scale up MAE and UAE, via the determination of optimum
conditions of the scale-dependent parameters i.e. microwave power or percentage amplitude of
ultrasound and extraction time based on APD and AED. First, the optimum APD and AED of an
assisted extraction were determined by measuring the power and the energy absorbed of a blank
solvent at the optimum operating conditions using the procedure described in Section 2.4. This
step is not required in the case of MAE since they can be taken from its optimization result.
Following that, the optimum power and extraction time at larger scale extraction can be determined
such that the optimum power was selected based on the optimum APD and the optimum extraction

8
time was determined based on the optimum AED via Eq. (2). This step involves adjusting the
power and extraction time of extraction system to meet the desired APD and AED values for each
solvent loading. When the desirable APD is difficult to achieve due to power setting for certain
extraction system, the best tuning that gives to the nearest desired APD value for each solvent
loading is considered. Finally, the extraction results at these large scales MAE and UAE were
evaluated and compared.

3. Results and Discussion

3.1. Optimization of general extraction parameters

The general extraction parameters such as solvent concentration and solvent to sample ratio of
MAE and UAE were first optimized prior to the optimization of the scale dependent and interacting
parameters, i.e. extraction time and nominal power of microwave or percentage amplitude of
ultrasound. The solvent used in MAE and UAE was ethanol (EtOH) due to its non-toxicity, good
polarity for microwave absorbing properties and it is able to dissolve the bioactive compounds of
interest (Xiao, Han, & Shi, 2008). With the addition of water in the extraction solvent, Fig. 1a and
1b showed that the extraction yields of MAE and UAE were enhanced. This is because the
presence of water enhances the microwave heating efficiency due to its high dielectric constant
(Sato & Buchner, 2004), and increases the permeability of plant matrices to promote mass transfer
and diffusion of bioactive compounds (Boeing et al., 2014). Nevertheless, addition of water
beyond certain optimum concentration reduces the solvation power to dissolve bioactive
compounds. As the effects of aqueous ethanol have different impacts on MAE and UAE, the
optimum solvent concentration of the techniques are different, which were found to be 80% and
70% (v/v) EtOH respectively.

Solvent to sample ratio is another important parameters which when applied correctly could
decrease the mass transfer barrier and subsequently enhance the extraction yield. It can be
optimized using single factor experiment if the solvent volume is kept as constant. This is because
the change in solvent volume due to different ratio would affect the absorption of microwave
energy in MAE and the dissipation of cavitation energy in UAE, making the basis of comparison

9
inaccurate (Chan, Yusoff, & Ngoh., 2014; Sun et al., 2011). Fig. 1c and 1d illustrates that the
increase of solvent to sample ratio at constant solvent volume improves the extraction yields of
MAE and UAE. Nevertheless, the improvement was not significant once the ratio was increased
beyond its optimum value. Similar trend was also reported in the extraction of phenols from black
tea (Spigno & De Faveri, 2009). To avoid solvent wastage, the optimum solvent to sample ratio at
constant volume for MAE and UAE were selected to be 50 and 30 mL/g respectively.

Fig. 1: Effects of general extraction parameters on MAE and UAE of bioactive compounds from
Orthosiphon stamineus: Effect of solvent concentration on a) MAE (150 W, 50 mL/g, 20 min)
and b) UAE (60 % amplitude, 30 mL/g, 30 min); Effect of solvent to sample ratio on c) MAE
(100 mL of 80% v/v EtOH, 150 W, 15 min) and d) UAE (60 mL of 70% v/v ethanol, 60%
amplitude, 30 min)

10
3.2. Optimization of scale-dependent extraction parameters
Upon specifying the general extraction parameters, the scale-dependent extraction parameters such
as microwave power or percentage amplitude of ultrasound, and extraction time will be optimized
in this section. These parameters are crucial as they control the input power and energy for an
assisted extraction, characterizing the kinetic and the extraction yield. In this section, the
optimization of power and time of the MAE was performed using energy-based method, by
studying the individual effects of APD and AED on the extraction yields (Chan, Yusoff, & Ngoh.,
2014). The effect of AED on the extraction yield was investigated by profiling MAE extraction
yield using AED as a basis in Fig. 2a. The result shows that the extraction achieves equilibrium
yield at 12 min extraction time, which is equivalent to about 250 J/mL. This AED value gives
implication that the extraction solvent requires 250 J/mL to drive the MAE to reach equilibrium
yield, regardless of microwave power employed (Chan, Yusoff, & Ngoh., 2013). The optimum
AED value obtained, i.e. 250 J/mL, will be used to investigate the optimum APD for the MAE.
The effect of APD were studied by conducting the extraction using different microwave power but
at the same heating energy (AED) to ensure that the extractions could reach the same degree of
completion (Chan, Lim, Yusoff, & Ngoh., 2015; Chan, Yusoff, & Ngoh., 2014). As such, Fig. 2a
depicted the effect of APD corresponding to 100 to 350 W microwave power, at AED of 250 J/mL.
The result shows that the extraction favors low heating power (APD < 0.5 W/mL). High APD
conditions (> 1.0 W/mL) gave low extraction yield, suggesting the occurrence of thermal
degradation of bioactive compounds. APD of 0.35 W/mL (150 W, 100 mL) was taken as the
optimum condition for the MAE since there is no obvious optimum point in this case and any value
in the range of 0.2-0.5 W/mL is suitable for the extraction. Overall, the intensive heating conditions
of the MAE are APD of 0.35 W/mL and AED of 250 W/mL, which is equivalent to typical
operating conditions of 150 W and 12 min for 100 mL solvent loading.

11
 Fig. 2: Optimization of APD and AED parameter for the MAE of bioactive compounds from
Orthosiphon stamineus at solvent to sample ratio of 50 mL/g (100 mL) and extraction solvent of
80% v/v EtOH. a) Single factor experiment to optimize AED at specific APD of 0.35 W/mL
(150 W) by changing the extraction time (2-20 min). b) Single factor experiment to optimize
APD at specific AED (260 J/mL) by changing the microwave power. The specific AED is met
by changing the extraction time via Eq. (2)

12
The optimization of percentage amplitude and extraction time of UAE were performed using RSM
and the empirical relationship of the extraction yield (mg/g) was generated as follows:
Y  1.34  1.13x101 X1  9.88x103 X 2 1.18 x103 X12 1.02 x105 X 22 1.40 x10 4 X1 X 2 Eq. (4)
where X1 is the time (min) and X2 is the percentage amplitude of ultrasound (%) respectively. This
model is significant (P< 0.05) with insignificant lack of fit. Its contour plot is showed in Fig. 3.
The result shows that the optimum region was in between percentage amplitude of 40-65 % and
extraction time of 27-37 min. Note that the extraction yields was increased with the extraction time
but it was decreased when the extraction time is prolonged beyond 35 min especially at high
percentage amplitude (> 70 %). The latter is caused by the reduction in solvation power to dissolve
the bioactive compounds as more than 50% (vol.) of solvent were evaporated. Since a milder
heating condition is sufficient for the extraction, the optimum condition was taken as percentage
amplitude of 45 % and extraction time of 34 min as marked in Fig. 3 in this study.

Fig. 3: Response surface curve for the optimization of percentage amplitude of ultrasound and
treatment time in UAE of bioactive compounds from Orthosiphon stamineus at solvent to sample
ratio of 30 mL/g (60 mL) and extraction solvent of 70% v/v EtOH

13
3.3. Scale up of MAE and UAE based on APD and AED
The MAE and the UAE at their respective optimum conditions, i.e. 80% EtOH, 50 mL/g, 150 W,
12.2 min and 70% EtOH, 30 mL/g, percentage amplitude of ultrasound of 45 %, 34 min are
applicable at solvent loading of 100 mL and 60 mL respectively. To scale up this extraction,
calibration of APD and AED values at these conditions are required to obtain the intensive
optimum conditions in terms of type of solvent, solvent to sample ratio, APD and AED. The related
APD and AED values of the MAE and the UAE were tabulated in Table 1. In this study, the
intensive optimum conditions such as 80% EtOH, 50 mL/g, APD of 0.35W/mL, AED of 250 J/mL
for the MAE and 70% EtOH, 30 mL/g, APD of 0.22 W/mL, AED of 450 J/mL for the UAE were
used to determine the optimum operating conditions at various extraction scales as tabulated in
Table 3. As the first attempt to apply APD and AED in UAE, the results shows that the APD and
AED are able to reproduce and to scale up the optimum extraction as the yields were consistent
with less than 8% discrepancy and about 91-98% of the Soxhlet extraction yield. The consistency
is close to the previously conducted MAE of bioactive compounds from cocoa leaves (Chan,
Yusoff, & Ngoh., 2014). This implicitly suggests that APD and AED can be used to optimize UAE
and to develop predictive model for simulating the extraction profile using the methods (Chan,
Lim, Yusoff, & Ngoh., 2015; Chan, Yusoff, & Ngoh., 2013, 2014, 2015) established for MAE
process.

In this scale up study, APD and AED parameters serve as reference to ensure that the up scaled
conditions would have the optimum input of power and energy in the microwave and the ultrasonic
system, by tuning the nominal power or percentage amplitude and the treatment time. These
energy-based parameters are not related to the activation energy of the extraction process. Note
that the activation energy refers to a specific portion of the total energy that delivered to the
extraction mixtures to increase the internal pressure of the plant sample during microwave heating
(Zhou & Liu, 2006) or to impact the external plant structure during ultrasonic cavitation (Floros
& Liang, 1994) to disrupt the plant sample. This energy-based parameters, on the other hand, are
merely the respective power and energy that provide thermal effect to the extraction mixture.
Nevertheless, they are reliable calibration parameters to indicate the input power and energy
required to be set in microwave and ultrasonic system to complete an extraction.

14
 Table 3: Scale up of MAE and UAE using energy-based parameters

Extraction yields (mg/g)

Intensive Sample Optimum
Techniques optimum mass operating 3´-hydroxy
conditions (g) conditions -5,6,7,4´-
sinensetin Total
tetrametho-
xyflavone
80% EtOH, 150 W,
2 0.47 ± 0.01 1.11 ± 0.02 1.58 ± 0.02
50 mL/g, 12.2 min
APD of 220 W,
MAE 4 0.50 ± 0.01 1.10 ± 0.02 1.60 ± 0.03
0.35W/mL, 12.3 min
AED of 250 300 W,
6 0.52 ± 0.01 1.18 ± 0.02 1.70 ± 0.04
J/mL 12.5 min
45 %,
70% EtOH, 2 0.47 ± 0.01 1.11 ± 0.01 1.58 ± 0.03
34.0 min
30 mL/g,
78 %,
UAE APD of 0.22 6 0.48 ± 0.00 1.17 ± 0.01 1.65 ± 0.02
37.8 min
W/mL, AED
100 %,
of 450 J/mL 8 0.47 ± 0.01 1.11 ± 0.01 1.58 ± 0.01
36.3 min
EtOH, 100
Soxhlet 2 / 0.54 ± 0.02 1.19 ± 0.02 1.73 ± 0.05
mL/g, 6 hr

4. Conclusion
The MAE and UAE of bioactives compounds from Orthosiphon stamineus were successfully
optimized using established methods and scaled up based on the intensive optimum conditions in
terms of type of solvent, solvent to sample ratio, absorbed power density (APD) and absorbed
energy density (AED). The APD and AED parameters serve as reference to calibrate the optimum
nominal power or percentage amplitude and treatment time of microwave or ultrasonic system for
various scale of extraction. This scale up method gave consistent extraction yields at the
investigated scales hence feasible and applicable for MAE and UAE processes. Future work should
continue to delve into the optimization, predictive modeling and scale up involving the energy-
based parameters to commercialize the assisted techniques.

5. Acknowledgements
This work was performed under the Centre for Separation Science and Technology (CSST),
University of Malaya and financially supported through Fundamental Research Grant Scheme
(FRGS: FP076-2015A) and University of Malaya Research Grant (UMRG: RP002A-13AET).

15
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