Journal of Applied Science & Process Engineering

Vol. X, No. X, 20XX

Optimization and Kinetic Study of Plectranthus Amboinicus Essential Oil

Extraction

Siew Ming Sim1* and Soh Fong Lim2

1,2
Faculty of Engineering, Universiti Malaysia Sarawak, Kota Samarahan, Malaysia

Abstract

Plectranthus amboinicus leaves are the famous aromatic and medicinal herbs that widely used for
medicinal purposes because they show antimicrobial, anti-inflammatory and antitumor properties. P.
amboinicus essential oil plays an important role as naturally originated medicines that can be used as
remedy to cure illness and diseases without bring any side effects on the patients. Thus, the aim of this
project is to extract essential oil from P. amboinicus leaves by using microwave pre-treatment
followed by hydrodistillation (HD) extraction method. This method is proven can save heating and
extraction duration, cost and energy. The P. amboinicus essential oil extracted was characterized by
using Fourier Transform Infrared Spectrophotometer (FT-IR) and UV-Vis Spectrophotometer. From
the FTIR result obtained, at frequency of 3600-3106 cm -1, it shows the presence of phenolic –OH
group in the P. amboinicus essential oil where thymol and carvacrol as the major phenolic components
present in the essential oil. Besides, from Foulin- Ciocalteu method, the total phenolic content that
present in the essential oil was the highest which was 2.917mg/g for water-to-plant ratio of 20.
Moreover, a second order kinetic model proposed by Kusuma and Mahfud (2015) was fairly fitted to
the experimental data. This means that the performance of hydrodistillation of P. amboinicus essential
oil can be predicted by using this second order kinetic model. Apart from that, the yield of P.
amboinicus essential oil was optimised by using response surface methodology with the central
composite design. There were three independent variables - the water-to-plant ratio, microwave pre-
heating duration and microwave oven power with the yield of P. amboinicus essential oil as
responding variable. From ANOVA analysis, the optimal conditions that yielded the highest
production of P. amboinicus essential oil was microwave power of 260.86W, microwave pre-heating
duration of 3.83min and water-to-plant ratio of 18.10.

Keywords: Plectranthus amboinicus; microwave pre-treatment followed by hydrodistillation (HD);phenolic

components; Second Order Kinetic Model; ANOVA analysis

1. Introduction

Plectranthus amboinicus (Mexican mint) which is a well-known aromatic perennial herb is used
as medicines for centuries to cure asthma, coughs and diseases that caused by bacteria and virus [12].
Apart from that, P. amboinicus is found to be very useful in medical fields where it exhibits anti-
tumorigenic, anti-inflammatory, antimicrobial and antiviral properties against herpes virus-1 [5]. It
also can be used to treat digestive, urinary, skin and respiratory problems such as bronchitis. This
shows the importance of P. amboinicus essential oil towards human being in medicinal and
pharmaceutical fields.

* Corresponding author. Tel.: +019-836 8059

E-mail address: simsiewming@gmail.com

Manuscript History:
Received xx Month, 20xx, Revised xx Month, 20xx, Accepted xx Month, 20xx, Published xx Month, 20xx

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According to Fernandes et al. (2013), P. amboinicus exhibits anti-microbial, balsamic and anti-
inflammatory properties. The essential oil of P. amboinicus shows good inhibition activities on both
acute and chronic inflammatory processes and also not induce any side effects during the treatment
process [10]. Besides, this medicinal plant also contributes in medicinal fields where it possesses
bronchodilator, anti-bacterial, antiviral, anti-fungal and anti-tuberculous properties. Apart from that,
based on the study from Senthilkumar & Venkatesalu (2010), the essential oil from P. amboinicus was
proven can be used to reduce the population of malarial vector mosquito A. stephensi. This finding
benefits the countries of Africa and Asia that suffer from malaria disease which is known as a killer
disease within those countries because P. amboinicus essential oil is a natural and inexpensive
mosquito larvicidal agent that can be used to control the spread of malaria vector mosquito among
people [14]. Since the usage of modern medicines and drugs in the treatment of chronic diseases might
lead to various and severe side effects on the patient, thus the application of naturally originated
medicines are more favourable recently [12]. Hence, the essential oil from P. amboinicus plays an
important role as the remedy to cure illness and diseases nowadays.
In this study, the microwave pre-treatment of the leaves was suggested before the hydrodistillation
process. With the aid of microwave oven during pre-heating process, the extraction process will be
sped up. This is because the glands that contain essential oil will break during the microwave pre-
heating process and this will directly shorten the extraction time since the time for the release of
essential oil from the gland will be greatly decreased. Thus, this method will directly save both energy
and cost in extracting the P. amboinicus essential oil.
Hence, this research mainly studies the optimisation and kinetic modelling of the extraction of
essential oil from Plectranthus amboinicus leaves by using hydrodistillation method that is aided by
microwave pre-treatment.

2. Experimental

2.1 Preparation of Plectranthus Amboinicus Sample

The fresh Plectranthus amboinicus leaves were collected from the Stutong Community market
at the area of Kuching, Sarawak. The leaves were freshly cut and washed under running water to wash
off the sand and dust before drying the leaves. The leaves were air-dried under shade for two weeks
until they were crispy in texture. Before conducting the experiment, the plant samples were ground
into fine powder by using a grinder (MX-AC400, Panasonic).

2.2 Microwave Pre-Treatment of Plectranthus Amboinicus Leaves

25 g of dried P. amboinicus leaves was weighed and immersed in distilled water for 30
minutes before they were subjected to microwave pre-treatment process. The amount of distilled water
which was added was based on the water to plant ratio of 10:1, 20:1 and 30:1. After that, the P.
amboinicus leaves with water were pre-heated in a microwave oven (ME711K, Samsung) for 2 min, 4
min and 6 min, respectively. The power of the microwave oven was varied between 100 W and 300 W
for different microwave pre-heating conditions. During the microwave pre-heating process, the bottle
was loosely capped in order to prevent the evaporation of volatile oil to the microwave oven. After
pre-treatment in the microwave oven, the leaves and distilled water were subjected to hydrodistillation
for 90 minutes.

2.3 Hydrodistillation Extraction Process

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Prior to hydrodistillation extraction process, the P. amboinicus leaves that were pre-heated in
microwave oven were placed in a 1L three neck round bottom flask. The flask was then put into the
heating mantle (MS-E, Favorit). The neck of the round bottom flask was connected to the fractionating
column and then connected to the condenser in order to condense the volatile oil components. The
essential oil with hydrosol was collected in the separatory funnel.
Besides, for this experiment, the extraction duration was set to be 90 minutes with the water-
to-plant ratio varying between 10:1 and 30:1. In order to remove water component from the collected
essential oil, the extracted essential oil was then dried over anhydrous sodium sulphate, kept away
from light in vials that were covered with aluminium foil and stored at 4°C until they were used for
further analysis.

Figure 1. Experimental setup of the hydrodistillation method

2.4 Experimental Design

A 33 central composite design from Response Surface Methodology was used to determine the
optimum parameters to extract Plectranthus amboinicus essential oil by microwave aided
hydrodistillation method. There were three independent variables in which water-to-plant ratio (10:1-
30:1, A) was categorical variable while microwave pre-heating duration (2 min to 6 min, B) and
microwave oven power (100W to 300 W, C) were numeric variables (Refer to Table 1). The yield of
the P. amboinicus essential oil was calculated by using Equation 1 [13].
Yield of Essential Oil = Amount of Essential Oil Obtained, g/Amount of Raw Material Used, g (1)

Table 1. Experimental design summary

Factors Variable Name Units Low High Low High
Actual Actual Coded Coded
A Categoric Water-to-Plant - 10:1 30:1 -1 1
Ratio
B Numeric Microwave Pre- Minute 2 6 -1 1
Heating Duration
C Numeric Power of Watt 100 300 -1 1
Microwave Oven

2.5 Kinetic Experimental Run

The experiments were carried out for different water-to-plant ratio which is 10:1, 20:1, and
30:1 while the other two operating conditions which were microwave power and microwave pre-
heating duration were kept at constant values. The full extraction of the essential oil was 90 minutes.
Hence, the yield of P. amboinicus essential oil was taken from time, t=0 to t=90 min in an interval of
fifteen minutes each.

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2.6 Kinetic Model Study

There are three mathematical models used to predict the yield of P. amboinicus essential oil.
The kinetic models chosen are pseudo-first-order kinetic model and second-order kinetic models
proposed by Kusuma, H. S. & Mahfud, M. (2015) and Milojevic, Stojanovic, Palic, Lazic, &
Veljkovic (2008). The equations for each model are shown in Table 2.

Table 2. Kinetic models equation

Kinetic Model Equations Notes
Pseudo-First Cs = Equilibrium extraction capacity
Order Ct = Concentration of essential oil at
(2) any time
k1 = First-order extraction rate
Second Order h = Initial extraction rate
Kusuma & t = Time
Mahfud (2015) Ct = Concentration of essential oil at
(3)
any time
Cs = Extraction capacity
Second Order q0 = Essential oil content initially
Milojevic et al. contained in the leaves
(2008) (4) q = Essential oil yield at time, t
b = Relative amount of essential oil
extracted at t = 0
k= Kinetic constant

2.7 Characterization of Plectranthus Amboinicus Essential Oil

The characterization of P. amboinicus essential oil was conducted by using Fourier Transform
Infrared Spectroscopy (FT-IR) and UV-Vis Spectrophotometer. For FTIR analysis, the samples were
analysed by using Shimadzu Iraffinity-1 model from Japan with a scan range of 1000 to 4000
cm-1. On the other hand, Shimadzu UV-1800 UV-Vis Spectrophotometer was used to test the
total phenolic contents of the P. amboinicus essential oil by using Folin-Ciocalteu method.

3. Results and Discussion

3.1 Comparison between Traditional Hydrodistillation Method and Microwave Pre-Treatment

followed by Hydrodistillation Method

According to Hussein, Ahmed, & Algabali (2017), the traditional hydrodistillation method
usually required three to four hours for full extraction of the essential oil. Hence, in order to save time
and effort, microwave pre-treatment followed by hydrodistillation was used in the extraction of P.
amboinicus essential oil. According to the study by Azadmard-Damirchi, Alirezalu, & Achachlouei
Fathi (2011), microwave pre-treatment can increase the yield of essential oil. The microwave radiation
will delivered the energy directly and uniformly throughout the plant material. This will directly
rupture the cell membranes in a short period of time. By this way, the time for the essential oil to be
released from the glands will be greatly reduced [4].
From Figure 2, it clearly shows that the microwave pre-treatment with hydrodistillation method
produce better yield of P. amboinicus essential oil (0.36%) while the traditional hydrodistillation

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method yield only 0.20% of the P. amboinicus essential oil. Besides, for traditional hydrodistillation
method, the oil yield increased gradually at the beginning and it slowly approached equilibrium at 60
minutes. On the other hand, the equilibrium state of the oil yield for microwave pre-treatment with
hydrodistillation method reached 15 minutes earlier than the traditional method. This shows that the
released of the P. amboinicus essential oil was faster than in traditional method. Hence, it means that
microwave pre-treatment was important in destroying the structure of the P. amboinicus essential oil
gland and then decreased the essential oil releasing duration during extraction process.

Figure 2. Yield of P. amboinicus essential oil against the extraction duration for different
extraction method

3.2 Fourier Transform Infrared Spectroscopy (FT-IR)

Figure 3 illustrates the spectrum of P. amboinicus essential oil that extracted at 100, 200 and
300 W respectively with constant microwave heating duration of 4 minutes and water-to-plant ratio of
20:1. From the graph that for microwave power that ranged from 100W to 300W, the IR spectrums for
all of the P. amboinicus essential oil analysed were similar. This means that the microwave power did
not affect the presence of the same functional groups in the P. amboinicus essential oil produced.
Table 3 shows the IR spectral analysis results of P. amboinicus essential oil based on the
frequency at the peaks on Figure 3 with their shapes and respective functional group. In the IR
spectrums of P. amboinicus essential oil, there was a medium and slightly broad peak present at range
of 3106 cm-1 to 3600cm-1. This shows that there were phenolic –OH and alcoholic –OH groups present
in the essential oil. The main components of P. amboinicus essential oil are carvacrol and thymol
where they are monoterpenoid phenol with structure of –OH. Besides, the absorption band also shows
the presence of terpinen-4-ol in the P. amboinicus essential oil. Other examples of chemical
compounds that present in P. amboinicus essential oil at certain frequency are shown in Table 3.

P=300W
P=200W
P=100W

P=300W
P=200W
Figure 3. Spectrum of P. amboinicus essential oil produced at different microwave heating powe P=100W

Table 3. IR spectral analysis results of P. Amboinicus essential oil

% Transmittance Bands Functional Group [7] Examples of Chemical
Frequency (cm-1) Shape Compounds

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3600-3106 Medium and Phenolic –OH group and Thymol and Carvacrol
Slightly Broad alcoholic –OH group
2997-2829 Strong and Sharp Hydrogen bonded to sp2 p-cymene and ɣ-
and sp3 carbons terpinene
respectively
1621, 1589, 1548, Medium and Sharp Aromatic Compounds o-cymene,
1521,1505, thymolmethylether
1245, 1174, 1115, Medium and Sharp C-O of alkyl aryl ether Caryophyllene Oxide
1065

3.3 Total Phenolic Content by Folin-Ciocalteu Method

The total phenolic contents (Gallic acid equivalents) for water-to-plant ratio of 10, 20 and 30
were calculated to be 0.709 mg/g; 2.917 mg/g and 2.717 mg/g respectively. This means that under
same microwave pre-treatment condition but different water-to-plant ratio, the ratio of 20 have the
highest total phenolic content followed by water-to-plant ratio of 30 and then 10. This shows the
water-to-plant ratio of 20 was the most optimum to produce P. amboinicus essential oil with high total
phenolic content. In addition, the major phenolic compounds that can be found in P. amboinicus
essential oil are carvacrol and thymol [2]. Thus, high total phenolic contents shows that the P.
amboinicus essential oil extracted at water-to-plant ratio of 20 contained high concentration of thymol
and carvacrol.

3.4 Optimization by Using Response Surface Methodology (RSM)

Table 4 shows the experimental parameters such as microwave power, microwave pre-heating
duration and water-to-plant ratio and also the corresponding response which was the yield of P.
amboinicus essential oil. Figure 4 shows the result obtained from response surface methodology, in
which the optimum conditions to obtain high yield of essential oil were microwave power of the
microwave oven for pre-heating was 260.86 W; microwave pre-heating duration was 3.83 min and the
water-to-plant ratio preferable was 18.10. Based on these optimum conditions, the yield of essential oil
was expected to be 0.454227% with desirability of 0.944.

Table 4. Optimal design with uncoded independent variables and the corresponding response
Run Experimental Parameter Response
A: B: C: Yield (%)
Microwave Microwave Pre-Heating Water-to-Plant
Power (W) Duration (min) Ratio
1 100 2.00 30.00 0.05
2 200 6.00 20.00 0.16
3 200 4.00 20.00 0.38
4 300 2.00 30.00 0.28
5 100 4.00 20.00 0.36
6 100 6.00 30.00 0.02
7 200 4.00 20.00 0.44
8 100 6.00 10.00 0.26
9 200 4.00 20.00 0.41
10 100 2.00 10.00 0.12
11 200 4.00 20.00 0.48
12 300 6.00 10.00 0.22
13 200 4.00 20.00 0.48
14 200 2.00 20.00 0.32

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15 200 4.00 30.00 0.28

16 300 6.00 30.00 0.08
17 300 4.00 20.00 0.44
18 300 2.00 10.00 0.18
19 200 4.00 20.00 0.46
20 200 4.00 10.00 0.44

100.00 300.00 2.00 6.00

Microwave Power = 260.86 Microwave Heating Duration = 3.83

10.00 30.00 0.02 0.48

Water-to-Plant Ratio = 18.10 Yield of Essential Oil = 0.454227

Desirability = 0.944

Figure 4. Optimized parameters for hydrodistillation of Plectranthus amboinicus essential oil

3.5 Kinetic Studies of Plectranthus Amboinicus Hydrodistillation

3.5.1 Effect of Water-to-Plant Ratio on Yield

Figure 5 illustrates the yield of essential oil increased gradually at the beginning and then it
approached equilibrium at different time for different water-to-plant ratio. Besides, as the water-to-
plant ratio increased from 10 to 20, the yield of essential oil also increased from 0.32% to 0.36%. This
was due to the rise of driving force for mass transfer during the extraction of P. amboinicus essential
oil as the ratio of water increased from 10 to 20 [13]. However as the water-to-plant ratio was 30, the
yield decreased to 0.20%. In other words, the maximum oil yield was obtained from water-to-plant
ratio of 20, followed by ratio of 10 and then 30. Since distilled water was used as a carrier for the P.
amboinicus essential oil during the evaporation process, so the amount of distilled water used is not
necessarily too much. Besides, the usage of large volume of distilled water might increase the energy
and time needed in order to condense the extraction solution [8].

Figure 5. Cumulative oil yield against extraction duration for different water-to-plant ratio

3.5.2 Effect of Microwave Power on Yield

According to Jeyaratnam, Nour, & Akindoyo (2016), the microwave power that was used to
control the irradiation intensity of the microwave energy was very important in influencing the
efficiency of the extraction process. During the microwave pre-treatment process, the microwave
power supplied the energy to the P. amboinicus leaves powder that immersed in the water where the
energy was then converted into heat energy and thus the temperature of the sample was increased.

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Figure 6 shows the effect of different microwave power on the yield of P. amboinicus essential oil
hydrodistillation extraction. From Figure 6, it can be obviously seen that as the microwave power
increased from 100W to 200 W during microwave-pre-treatment process, the yield of the P.
amboinicus oil also increased. This shows that higher microwave power can lead to the rapid
generation of heat inside the immersed P. amboinicus sample with the absorption of microwave
energy [14]. This situation would directly increase the rate of releasing of the essential oil from the
glands as the glands broke under high temperature and high pressure gradients. However, as the
microwave power further increased, the yield of P. amboinicus essential oil decreased. This was
because the degradation of the components inside the P. amboinicus leaves occurred at high
temperature.

Figure 6. Essential oil yield against microwave power for different water-to-plant ratio

3.5.3 Effect of Microwave Pre-heating Duration on Yield

Figure 7 illustrates the effect of microwave pre-heating duration on the yield of P. amboinicus
essential oil. As being depicted in Figure 7, as the microwave pre-heating duration increased from 2
min to 4 min, the yield of essential oil also increased for the three water-to-plant ratio. This shows that
increasing microwave pre-heating time can increase the essential oil extraction yield [4]. For instance,
at water-to-plant ratio of 20, the essential oil yield increased from 0.32% to 0.38% as the microwave
pre-heating time increased from 2 min to 4 min. This is because the microwave radiation delivered
irradiation energy throughout the P. amboinicus leaves. By this way, the P. amboinicus leaves would
undergone rapid and uniform microwave treatment where the glands were ruptured and permanent
pores were generated and directly enabled the released of essential oil to move thorough the permeable
cell walls [4]. However, if the microwave pre-heating duration was prolonged to 6 min, the yield of
essential oil decreased. According to Azadmard-Damirchi, Alirezalu, & Achachlouei Fathi (2011),
longer microwave heating times will results in greater degree of essential oil deterioration. Hence, 4
min of microwave pre-heating duration was the best condition to obtain high yield of P. amboinicus
essential oil.

Figure 7. Essential oil yield against microwave pre-heating duration for different water-to-plant
ratio

3.6 Comparison between Three Kinetic Models

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Table 5 shows the statistical criteria result for all the kinetic models that are pseudo-first order,
Kusuma & Mahfud (2015) second order and Milojević et al. (2008) second order kinetic models. For
these three models, three criteria such as correlation coefficient (r), root mean square error (RMSE)
and mean relative deviation modulus (E) were compared in order to determine the whether the model
fit to the experimental data. Based on Amirah et al. (2016), the model fits the experimental data well
when RMSE 0, r
For water-to-plant ratio of 10, Kusuma & Mahfud (2015) second order kinetic model was the most
suitable mathematical model that best described the hydrodistillation behaviour at this condition. The
reason of choosing this model was that the value of root mean square error (RMSE) was the lowest -
0.01994 which was nearest to zero. Besides, the mean relative modulus (E) was 6.6062% which was
less than 10%. Since the value for mean relative modulus for another two kinetic models were more
than 10% which was not in acceptable range, thus Pseudo-first order and Milojević et al. (2008) were
not fitted to the experimental data in this case. On the other hand, for water-to-plant ratio of 20, even
though the value for correlation coefficient was 0.9272, which was lower than the other two models
with r = 0.9664. However, the root mean square error (RMSE) and the mean relative modulus (E) for
Kusuma & Mahfud (2015) was the lowest where the values were 0.01632 and 10.883% respectively.
This shows that this second order kinetic model best fit the experimental data. Same goes to water-to-
plant ratio of 30 with r value of 0.9799, RMSE of 0.00291 and lowest E of 1.1382%, Kusuma &
Mahfud (2015) was the best choice to fit the experimental data.
In a nutshell, Kusuma & Mahfud (2015) second order kinetic model fitted well with the
experimental data with different water-to-plant ratio. This means that the performance of the
hydrodistillation of P. amboinicus essential oil can be predicted by using this model.

Table 5: Statistical criteria result

W/P = 10 W/P = 20 W/P = 30
Model r RMSE E r RMSE E r RMSE E
(%) (%) (%)
Pseudo- 0.9820 0.03949 14.107 0.9664 0.02182 16.898 0.9942 0.00385 4.5486
First Order
Kusuma & 0.9872 0.01994 6.6062 0.9272 0.01632 10.883 0.9799 0.00291 1.1328
Mahfud
(2015)
Milojević 0.9820 0.03844 12.306 0.9664 0.03971 13.412 0.9942 0.01004 4.0363
et al.
(2008)
Best Model Kusuma & Mahfud (2015)

4.0 Conclusion

This study aimed to extract essential oil from Plectranthus Amboinicus. The maximum yield was
obtained when microwave pretreatment was applied before hydrodistillation process. Moreover, the
extraction time was reduced to only 45 min compared to conventional hydrodistillation. The results
obtained and the corresponding analysis confirmed the nature of the essential oil mainly determined as
thymol and carvacrol by FTIR analysis. To make efficient the extraction of 20:1 water-to-plant ratio
was found as fundamental if a heating process by microwave of 200W power of intensity during 4 min
as pretreatment was performed. On the other hand, the experimental data were fairly explained by
using a second order kinetic model when the hydrodistillation process was used. These data were
confirmed by optimization of the process by RSM where the variables were not significant different to
the experimental results. As a conclusion, in order to obtain a maximum amount of Plectranthus

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Amboinicus essential oil, a ratio of 18.10 (water-to-plant); microwave preheating of 3.8 min and
microwave oven power of 280.86 W must be used followed by hydrodistillation process.

Acknowledgements

The author would like to extend her sincerest gratitude to her final year project supervisor, Ir. Dr. Lim
Soh Fong for her guidance and supervision during entire project. Besides, the author would like to
express her appreciation to all the technical staffs of Department of Chemical Engineering who
assisted her throughout the research.

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