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Journal of Advanced Research in Applied Sciences and Engineering Technology 27, Issue 1 (2022) 1-8
1
Journal of Advanced Research in Applied
Sciences and Engineering Technology
www.akademiabaru.com/ submit/index.php/araset/index
ISSN: 2462-1943
A Comparative Study on Zerumbone Concentration, Radical
Scavenging Activity and Total Phenolic Content of Zingiber
Zerumbet Extracted via Green and Conventional Extraction
Izzati Mohamad Abdul Wahab1,*, Mariam Firdhaus Mad Nordin1, Nabilah Zaini1, Kamyar Shameli1
Siti Nur Khairunisa Mohd Amir1, Nurul ‘Uyun Ahmad1,2, Norrashidah Mokhtar3
1
Malaysia Japan international Institute of Technology, Universiti Teknologi Malaysia, Jalan Sultan Yahya Petra, 54100, Malaysi a
2
School of Chemical Engineering, College of Engineering, Universiti Teknologi MARA, Cawangan Kampus Bukit Besi, 23200 Bukit Besi, Dungun,
Teregganu, Malaysia
3
AM Zaideen, 35E-G-05, Jalan Wangsa Delima 5, KLSC 2, Seksyen 5 Wangsa Maju, 53300 Kuala Lumpur, Malaysia
ABSTRACT
Extraction is crucial for herbal extraction to ensure high quality of bioactive compounds from natural herbs. In the current work,
Zingiber Zerumbet was extracted via subcritical water extraction (SWE) and is compared with the conventional solvent extraction,
Soxhlet. The quality of the extract was investigated in terms of zerumbone concentration, radical scavenging activity (RSA) and total
phenolic content (TPC). For zerumbone concentration, extraction via SWE gave 19.82 ±0.004 mg/g as compared with Soxhlet;
28.51±0.079 mg/g. However, the extraction time required for SWE to yield such concentration only required 40 minutes instead of
Soxhlet which took 480 minutes. The same trend was recorded for RSA, which yielded 60.70±0.070% inhibition for SWE and
68.81±0.024% inhibition from Soxhlet extraction. In contrast to TPC, SWE recorded a higher response than Soxhlet extraction, which
was 19.19±0.003 mgGAE/g DW, while Soxhlet; 8.30±0.019 mgGAE/g DW. Therefore, the SWE method is more favorable for obtaining
a higher value of TPC, slightly good in antioxidant properties but lower zerumbone concentration value than organic s olvent
extraction. However, the reduced extraction time was almost 12 times quicker for SWE when compared with Soxhlet extraction.
Overall, SWE is a promising alternative environmentally friendly since it only uses water as solvent and is co mparable to the
conventional method.
Keywords:
Subcritical water extraction, Zingiber
Zerumbet, zerumbone, radical scavenging
activity, total phenolic content
Received: 3 March 2022
Revised: 14 April 2022
Accepted: 25 May 2022
Published: 14 July 2022
1. Introduction
Medicinal plants have recently received significant attention due to their extraordinary
beneficial properties. For example, Z.zerumbet, which belongs to the ginger family, is also known as
bitter ginger, shampoo ginger, and pinecone ginger[1]. This plant has long been used as a whole or
by parts (leaves, rhizome, flower) as part of traditional medicine [2]–[4], food and beverage [5], [6] as
* Corresponding author.
E-mail address: mariamfirdhaus@utm.my
https://doi.org/10.37934/araset.27.1.18
Journal of Advanced Research in Applied Sciences and Engineering Technology
Volume 27, Issue 1 (2022) 1-8
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well as personal care [1], [7]. Previous studies [8], [9] have reported the presence of high content of
zerumbone (35.5-84.8%) found in Z.zerumbet extract, which is believed to be the significant
compound to exhibit antioxidant capabilities, antiproliferative, antibacterial, anticancer among the
medicinal benefit [4], [10]–[12].
However, to obtain the bioactive compound from Z.zerumbet, it is crucial to choose an
extraction process that is efficient and safe, and cost-effective. Several extraction methods can be
used, such as Soxhlet, maceration, boiling, supercritical fluid extraction, ultrasound-assisted
extraction, and subcritical water extraction. SWE is a promising candidate among these methods
since it only uses water as a solvent. This extraction method is different from the widely known
conventional extraction; Soxhlet [13], [14] employs organic solvent and requires extended extraction
time [15]. In addition, SWE has the advantage of shorter extraction time, thus reducing the overall
cost [16]. Therefore, the primary aim of this paper is to critically examine the quality of Z.zerumbet
extractant from the 1L SWE prototype and Soxhlet in terms of zerumbone concentration, radical
scavenging activity, and total phenolic content.
2. Methodology
2.1 Sample preparation
Z.zerumbet rhizomes used in this study were acquired from a local farmer from Kuala Krau,
Pahang. The rhizomes were cleaned, sliced, dried, ground, and sieved to the following sizes: 3.36,
2.00, 1.00 and 0.50 mm. The moisture content was also measured using a moisture analyzer (OHAUS,
MB 25, Switzerland) and is ensured to be lower than 10% in dry basis to prevent microbial growth
[17]
2.2 Extraction of Z.zerumbet using SWE
The schematic diagram of 1 L SWE prototype (CLEAR, UTM KL) was shown in Fig. 1. Briefly, 25
grams of the ground rhizome material were placed in the extraction vessel filled with 500 ml of water
and subjected to subcritical conditions. The processing parameters were optimized based on
previous study [18] (temperature: 170 , time: 40 minutes; mean particle size: 2.36 mm, pressure:
2.0 MPa; p-value<0.05) by employing Design Expert Software Version 12. The extraction time started
once the target temperature was reached, as indicated by the temperature indicator in the extraction
cell. The extract was immediately transferred to the cooling vessel when the extraction was
completed. Finally, the crude extract was collected and further subjected to analysis.
Fig. 1: Schematic diagram of 1L SWE
prototype
Fig. 2: Schematic diagram of soxhlet apparatus
Journal of Advanced Research in Applied Sciences and Engineering Technology
Volume 27, Issue 1 (2022) 1-8
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2.3 Soxhlet Extraction
25 g of dried Z.zerumbet with a mean particle size of 2.36 mm was weighed and extracted
with 500 ml ethanol for 8 hours. The best operating condition was based on the previous study[19],
[20]. The extraction temperature was kept constant at the boiling point of ethanol (78.10℃) and was
monitored using an infrared laser thermometer (AR300, China). As illustrated in Fig. 2, the sample
was gradually filled with condensed new ethanol from the distil lation flask. A siphon aspirates the
extracted sample from the sample matrix and unloads it into the distillation flask whenever the liquid
overflows [21]. This is a repetitive process until the extraction is completed. Afterward, the ethanol
was left to evaporate in the oven at 40℃ overnight before analysis. From the soxhlet extraction, the
percentage recovery of Z.zerumbet was calculated using Eq (1);
Percentage of recovery= Ci,μg of bioactive
g dried Z.zerumbet
Coi,μg of bioactive
g dried Z.zerumbet
×100 (1)
Whereby ci is the sample concentration in the bulk solution, coi is the initial sample concentration
from soxhlet extraction.
2.4 HPLC Analysis
The targeted marker compound for Z.zerumbet was zerumbone. HPLC analysis was conducted
using Waters (e2695, Waters, USA) with a photodiode array detector (PDA) on a C18 column
(Symmetry®). Before analysis, the extracted samples were homogeneously dissolved in 10mL of
methanol, followed by filtration through 0.45 µm membrane filter (Nylon, Waters Corporation). The
mobile phase was methanol: acetonitrile (35:65 v/v) and was carried out in isocratic elution. The total
running time was 10 minutes with a flow of 1 ml/min at the wavelength of 254 nm. This is a modified
method as conducted by [22]. The standard calibration curve was established by diluting the standard
zerumbone with methanol to six concentrations: 5 ppm, 10 ppm, 20 ppm, 50 ppm, 100 ppm, and 500
ppm. Then, the absorbance was plotted against the concentration to obtain the equation of a straight
line.
2.5 Radical Scavenging Activity
The RSA of the extract was analyzed against the stable DPPH (2,2-diphenyl-1-picrylhydrazyl,
Sigma Aldrich, Germany). According to previous literature[19], [23]. Initially, the samples were
diluted at a ratio of 10:40 of extract to 80% ethanol. Next, about 0.5 ml of each extract was added to
3.5 ml of prepared DPPH ethanolic solution (0.1 mM). The solution was incubated for 30 min at room
temperature in the dark and the discoloration was measured at 517 nm using the UV-VIS
spectrophotometer. The inhibition ability radical scavenging activity (RSA) was calculated using Eq
(2):
RSA (% inhibition)=Acontrol-Asample
Acontrol ×100 (2)
Acontrol is the absorbance of the control (DPPH+80%EtOH) at t=0 min and Asample was the absorbance
of sample at t=30 min.
Journal of Advanced Research in Applied Sciences and Engineering Technology
Volume 27, Issue 1 (2022) 1-8
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2.6 Total Phenolic Content
TPC was evaluated using Folin-Ciocalteu modified method [19], [24]. Initially, the extract was
diluted in distilled water with a ratio of 1:10 v/v. Afterward, 0.5 mL of diluted extract or standard
prepared was mixed with 2.5 mL of diluted Folin-Ciocalteu reagent in distilled water (1:10 v/v). The
mixture was hand-shaken vigorously. The mixture was left for 5 min rest before 2 mL of 7.5% (v/v)
sodium carbonate was added. Next, the mixture was incubated for about 2 hours and was measured
using UV/VIS Spectrometry at 750 nm. The TPC for each sample was determined from a standard
curve of gallic acid ranging from 10 to 100 mg/L solutions of gallic acid in water. The yield in total
polyphenol (YTP) was calculated using Eq (3).
Total phenolic content, (YTP)= CTP×V×d
m (3)
Where CTP is the concentration of gallic acid in water from the standard curve regression line
(y=mx+c) and is represented in mg/L, V is the volume of extraction solvent (L), d is the dilution factor
and m is the weight of dried rhizome used (g)
2.7 Statistical Analysis
The extraction process was conducted in triplicate and standard deviations were calculated.
Results were expressed in the form of mean, absolute average deviation (AAD) and percentage
calculated using Microsoft Excel 2021.
3. Results
The standard calibration curve of zerumbone concentration gave good R2 value of 0.9998 and
linear equation of y=17130x + 17719. The total phenolic content (TPC) evaluation was measured in
terms of gallic acid equivalent (GAE) based on the linear standard curve equation y=0.0078x - 0.0099
and R2 value of 0.9993.
3.1 Zerumbone Concentration
For zerumbone concentration, it is apparent that the SWE method reported significantly
lower than Soxhlet, which is depicted in Fig. 3 (a). This finding is consistent with that of Tzeng et al.
[25] who found the highest amount of zerumbone content from ethanol solvent. Zerumbone has
been confirmed to be a polar compound in a study conducted by Rosnani et al. [26]. Following the
“like dissolve like” principle [27], zerumbone is more likely to be extracted with a polar solvent. This
is reflected in the findings whereby the zerumbone content via SWE is comparable to that of Soxhlet
extraction by 70% recovery rate which is in the acceptable recovery rate of 60-140% based on [28],
[29]. This is a significant finding since Soxhlet has always been used as the benchmark for any
extraction methods. Under SWE condition, water can mimic the characteristic of organic solvent via
the decrease in dielectric constant [30]. Therefore, it is possible in this study that water in SWE
behaved like organic solvent from the result attained. In addition, it is worthy to point out that the
extraction time taken for SWE to complete one cycle was 12 times faster than Soxhlet. This is a
remarkable outcome of SWE as it could produce comparable extract properties with a significantly
reduced in time. Consequently, the operational expenses are greatly reduced due to the decreased
in the extraction time [31], [32].
Journal of Advanced Research in Applied Sciences and Engineering Technology
Volume 27, Issue 1 (2022) 1-8
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3.2 Radical Scavenging Activity
Zerumbone has been widely recognized to have a wide range of pharmaceutical activities
including antioxidant activity. The RSA results are in agree with the zerumbone content, whereby the
RSA % inhibition increases accordingly with the zerumbone content obtained vi a both extraction
methods. In general, the RSA from SWE was slightly lower than Soxhlet by 12%, as shown in Fig. 3
(b). The RSA was also compared with ultrasound-assisted extraction (UAE) from previous study which
suggested the frequency has little effect on the RSA value. By comparison, the RSA from UAE was
two times lower than SWE in this study. This suggests the efficiency of SWE 1L prototype in
comparison to Soxhlet method. Meanwhile, it is clearly elucidated that temperature and time played
a significant role in higher RSA value. From the results, the use of UAE at lower temperature and time
gave low RSA value in comparison to SWE and Soxhlet. The possible explanation on the narrow
margin between SWE and Soxhlet might be contributed by the high temperature of SWE (170 ).
Previous study by Plaza et al. [33] has suggested the formation of new compounds such as
hydroxymethylfurfural and melanoidin formed through hydrolyzation and Maillard reaction under
extreme temperature and long exposure. These new compounds might have an impact on the
increase in antioxidant activity.
Table 1
Zerumbone concentration, RSA and TPC of Z.zerumbet using SWE, Soxhlet and UAE extraction
SWE
Soxhlet
UAE[19]
Conditions
Solvent
Water
Ethanol
Water
Temperature
170
78.4
60
Time (min)
40
480
25
Pressure (MPa)
2.0
0.1
0.1
Frequency (kHz)
n.a
n.a
25
Response
Zerumbone
concentration
19.82 ± 0.004
28.51 ± 0.79
n.a
RSA
60.70 ± 0.070
68.81 ± 0.024
28.01 ±
0.05
TPC
19.19 ± 0.003
8.30 ± 0.019
2.48 ± 0.20
Fig. 3: Zerumbone concentration, RSA and TPC of SWE and Soxhlet in this study
0
5
10
15
20
25
30
35
SWE Soxhlet
Zerumbone concentration,
(mg/g)
0
10
20
30
40
50
60
70
80
90
100
SWE Soxhlet
RSA, (%inhibition)
b
0
5
10
15
20
25
SWE Soxhlet
TPC, mg GAE/g DW)
c
a
Journal of Advanced Research in Applied Sciences and Engineering Technology
Volume 27, Issue 1 (2022) 1-8
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3.3 Total Phenolic Content
The total phenolic content gave a surprising result whereby, a different trend was observed
for TPC. The SWE extract gave more than 100% which is over twofold compared to Soxhlet extraction.
Figure 3 (c) shows the TPC from SWE and Soxhlet showed immense difference. UAE in comparison
still gave lower value of TPC which was 9 times lower than SWE and almost four times lower
compared to Soxhlet. The high value of TPC from SWE may be explained by the softening of
polyphenol in water which was influenced by the increase in the diffusivity, solubility, and mass
transfer of compound rate [34]. However, this outcome is contrary to that of [35] which concludes
that the TPC decreases with the increase in water content. Various studies have demonstrated the
success of using SWE as an efficient method for extracting bioactive compound from various
resources. Previous study conducted by Vladić et al. [31] has found significant improvement on S.
montana extracts in terms of increasing antioxidant activity and TPC compared to soxhlet extraction.
Thus, the findings reported here suggest that SWE can be used to extract Z.zerumbet and has
improved TPC while having similar value of zerumbone concentration and RSA compared with solvent
extraction.
4. Conclusions
In conclusion, the present study has demonstrated the potential of SWE to produce a high
quality of Z.zerumbet extracts comparable with the established conventional extraction method;
Soxhlet. For the investigated response of zerumbone concentration, the value was 19.82±0.004 mg/g
and 28.51± 0.79 mg/g for SWE and soxhlet extraction, respectively. In terms of RSA, the value for
SWE and Soxhlet were somewhat close to one another with 60.70±0.070 and 68.81±0.024,
respectively. In contrast, SWE yielded the highest value of TPC compared to Soxhlet extraction with
19.19±0.003 and 8.30±0.019 mg GAE/gDW, respectively. Overall, SWE is a promising alternative
extraction method that can safely and efficiently extract high-quality bioactive compounds.
Acknowledgement
We would like to extend our gratitude and appreciation to the main sponsor company, AM Zaideen
Ventures Sdn. Bhd. under Contract Research DTD Scheme with Vote Number of
R.K130000.7643.4C365 for the financial support given in conducting this research.
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