An ecofriendly ultrasound – assisted extraction (UAE) method was developed for the
triterpenoids extraction from Vietnamese red Ganoderma lucidum (G. lucidum). The appropriate
kind of solvent for extraction procedure was selected as ethanol based on the yield of
triterpenoids obtained, followed by the investigation of four single factors including solvent-tomaterial ratio, extraction temperature, extraction time, and ultrasonic power. According to the
result of single factor experiments, optimal extraction conditions were determined with solventto-material ratio of 30:1 mL/g, extraction temperature of 55 oC, extraction time of 60 min, and
ultrasonic power of 480 W. Under these conditions, UAE produced a highest triterpenoids yield
of 7.479 0.033 mg/g by using ultraviolet-visible spectroscopy (UV-Vis) analysis. Extract
obtained from UAE method showed mild antioxidant activity compared to that of ascorbic acid
with the half-maximal inhibitory concentration values were of 29.778 and 6.714 µg/mL,
respectively. As a result, triterpenoids extracted from G. lucidum could be considered as a
promising antioxidant agent.
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Vietnam Journal of Science and Technology 58 (6A) (2020) 91-101
doi:10.15625/2525-2518/58/6A/15473
EXTRACTION OF TRITERPENOIDS FROM THE VIETNAMESE
RED GANODERMA LUCIDUM BY ULTRASOUND-ASSISTED
EXTRACTION METHOD AND ANTI-OXIDANT ACTIVITY OF
EXTRACT
Tran Do Dat
1
, Nguyen Tan Tai
2
, Phan Le Thao My
1
, Ngo Thi Thuy Linh
1
,
Nguyen Thi Kim Ngan
1
, Vuong Hoai Thanh
2
, Nguyen Duc Viet
2
,
Hoang Minh Nam
2, 3
, Mai Thanh Phong
2, 3
, Nguyen Huu Hieu
1, 2, 3, *
1
VNU-HCMC Key Laboratory of Chemical Engineering and Petroleum Processing (CEPP Lab),
Viet Nam, 70000
2
Faculty of Chemical Engineering, Viet Nam, 70000 Ho Chi Minh City University of
Technology, 268 Ly Thuong Kiet Street, Ward 14, District 10, Ho Chi Minh City, Viet Nam
3
Vietnam National University Ho Chi Minh City, Linh Trung Ward, Thu Duc District,
Ho Chi Minh City, Viet Nam
*
Email: nhhieubk@hcmut.edu.vn
Received: 9 September 2020; Accepted for publication: 27 January 2021
Abstract. An ecofriendly ultrasound – assisted extraction (UAE) method was developed for the
triterpenoids extraction from Vietnamese red Ganoderma lucidum (G. lucidum). The appropriate
kind of solvent for extraction procedure was selected as ethanol based on the yield of
triterpenoids obtained, followed by the investigation of four single factors including solvent-to-
material ratio, extraction temperature, extraction time, and ultrasonic power. According to the
result of single factor experiments, optimal extraction conditions were determined with solvent-
to-material ratio of 30:1 mL/g, extraction temperature of 55
o
C, extraction time of 60 min, and
ultrasonic power of 480 W. Under these conditions, UAE produced a highest triterpenoids yield
of 7.479 0.033 mg/g by using ultraviolet-visible spectroscopy (UV-Vis) analysis. Extract
obtained from UAE method showed mild antioxidant activity compared to that of ascorbic acid
with the half-maximal inhibitory concentration values were of 29.778 and 6.714 µg/mL,
respectively. As a result, triterpenoids extracted from G. lucidum could be considered as a
promising antioxidant agent.
Keywords: Ganoderma lucidum, ultrasound – assisted extraction, triterpenoids.
Classification numers: 1, 3.
1. INTRODUCTION
Belonging to the Ganodermataceae family, Ganoderma lucidum (G. lucidum) is one of
functional foods used to boost health and longevity, especially in China [1]. Various components
from G. lucidum including the fruiting body, spores, and mycelia have a long history of use for
Tran Do Dat, et al.
92
preventing and treating numerous diseases such as hepatitis, bronchitis, nephritis, hypertension,
and cancer [2 - 5]. It is reported that G. lucidum contains diverse phytochemicals including
polysaccharides, triterpenoids, oils, sterols, organic germanium, and inorganic ions [6].
Triterpenoids with over 400 compounds have been found and many more still being discovered,
are regarded as one of the crucial bioactive components exhibiting anticancer, anti-
inflammatory, antioxidant, anti-proliferation, antiangiogenesis, and anti-lymphatic effects [7 -
11]. Therefore, effective extraction methods for triterpenoids from G. lucidum needed to be
developed.
As new-type extraction technique, ultrasound – assisted extraction method (UAE) has
many advantages compared to conventional techniques including shorter extraction time, higher
extraction rate, reduction in the amount of solvent used, and increase in purity of product
obtained. In addition, the easy maintenance of the equipment area enhances the wide
applicability of UAE method [12 - 14]. The principle of UAE is based on the ability of
continuous compression generation and rarefaction with the assistance of an extraction solvent
leading to formation of micro-bubbles. As a result, substances could be released effectively from
plant materials [12,15]. However, extraction efficiency is affected by many process variables
such as solvent type, solvent-to-material ratio, extraction time, and extraction temperature [16].
The objective of the work was to determine the applicability of UAE method for a reliable and
reproducible extraction process of triterpenoids from G. lucidum as well as evaluate the anti-
oxidant activity of the extract obtained in comparison with ascorbic acid.
2. MATERIALS AND METHODS
2.1. Materials and chemicals
The fruiting body of Vietnamese G. lucidum was provided by National Institute of
Medicinal Materials with the moisture value of materials at 12 %, Viet Nam. 99.5 % ethanol
(C2H5OH) was purchased from ChemSol, Viet Nam. 99.0 % methanol (CH3OH), 70.0 - 72.0 %
perchloric acid (HClO4), acetic acid (CH3COOH), ascorbic acid, vanillin, and ursolic acid were
purchased from Xylong, China. Double-distilled water was used in all experiments. All
chemicals were used as received without further purification.
2.2. Selection of appropriate kind of solvent for extraction procedure
The influences of different kinds of solvents including ethanol, water, hexane, methanol,
and ethyl acetate on the yield of triterpenoids were examined while extraction conditions were
fixed as follows: solvent-to-material ratio of 30:1 mL/g, extraction time of 60 min, extraction
temperature of 55 , and ultrasonic power of 480 W.
2.3. Experiment procedure
After being dried at 50
o
C to remove moisture, 2.00 g of dried powder of Vietnamese G.
lucidum was accurately weighed and dispersed in the appropriate kind of extraction solvent with
the assistance of ultrasound wave. Then the samples were filtered and concentrated under
reduced pressure using the vacuum evaporation to obtain crude extracts.
2.4. Determination of the yield of triterpenoids
Extraction of triterpenoids from the Vietnamese red Ganoderma lucidum
93
The yield of triterpenoids was evaluated based on previous study [17]. Ursolic acid was
used as a standard and an adequate range of concentrations including 0.02, 0.03, 0.04, 0.05, and
0.06 mg/mL in ethyl acetate was used to create a calibration curve.
1.0 mL of perchloric acid solution and 0.4 mL of 5 % vanillin-glacial acetic acid reagent
were added to the extract sample. Reaction mixture was further heated for 60
o
C for 15 min and
then cooled in an ice-water bath. Thereafter 5.0 mL of glacial acetic acid solution was mixed
with it and the absorbance of the sample solutions was measured at 548 nm using ultraviolet-
visible spectroscopy analysis against a blank (ethyl acetate). All of determinations were made in
triplicate. The yield of triterpenoids (%) was calculated by the following equation:
( )
(1)
where Y is the yield of triterpenoids (%); C is the concentration of triterpenoids obtained from
the calibrated regression equation (mg/mL); V is the volume of triterpenoids solution (mL); n is
the dilution factor; and m is the dried sample mass (g).
2.5. Single factor experimental design
The effects of factors on the yield of triterpenoids, including solvent-to-material ratio (20:1,
25:1, 30:1, 35:1, and 40:1 mL/g), extraction time (30, 60, 90, 120, and 150 min), ultrasonic
power (240, 360, 480, 600, and 720 W), and extraction temperature (45, 50, 55, 60, and 65
o
C)
were determined based on the simultanous influences of the factors to select main factors.
Throughout the single-factor experiments, one variable was changed while the other variables
were kept constant.
2.6. Anti – oxidant activity investigation of the extract
The scavenging of DPPH radicals was assayed according to previous study [18]. 4.0 mL of
sample extract was added an equal volume of 6.0 mL of 1 mM DPPH solution with methanol.
The mixture was then mixed well and allowed in the dark for 30 min at room temperature before
the absorbance of the mixture was read at 517 nm. The anti-oxidation activity is proportional to
the disappearance of DPPH.
The ascorbic acid was used as a positive control sample in DPPH anti-oxidation activity
test. A different concentration of ascorbic acid (1.0, 2.0, 4.0, 6.0, 8.0, and 10.0 µg/mL) was
prepared and the procedure was carried out in the same manner as for the sample extract. All the
experiments were conducted in triplicate.
2.7. Statistical analysis
The one-way analysis of variance (ANOVA) test with least significant difference (LSD) was
used to statistically investigate the average yield of triterpenoids. The software package
Statgraphics Centurion 18 (Statgraphics Technologies, Inc., Warrenton, VA, USA) was
employed for the statistical data evaluation. The results were expressed as mean standard
deviation (SD) (n = 3). The p values less than 0.05 or less than 0.01 are considered significant or
highly significant, respectively. All graphs were plotted using OriginPro 8.5.1 (OriginLab
Corporation, Northampton, MA, USA).
Tran Do Dat, et al.
94
3. RESULTS AND DISCUSSION
3.1. Selection of appropriate kind of solvent for triterpenoids extraction procedure
Figure 1 shows the effect of different kinds of solvents including n-hexane, ethyl acetate,
water, ethanol, and methanol on the yield of triterpenoids under extraction conditions including
solvent-to-material ratio of 35:1 mL/g, extraction temperature of 55
o
C, extraction time of 60
min, and ultrasound power of 480 W.
Figure 1. Effect of different kinds of solvents on the yield of triterpenoids. Dissimilar letters in the
same column indicate significantly different at p < 0.05 using one-way ANOVA.
As can be seen from Figure 1, the highest yields of triterpenoids could be obtained when
methanol and ethanol were used as solvents, while the extraction yield obtained using water,
ethyl acetate, and n-hexane were 5.3568, 4.7730, and 2.3399 mg/g, respectively. This result
could be explained due to the fact that medium polar solvents could facilitate extraction
efficiency
[19]. However, methanol is regarded as a highly toxic solvent while ethanol is a safe
solvent with good yield of triterpenoids. Therefore, ethanol was selected as the most appropriate
solvent used for the extraction process. It is reported that ethanol was widely used as an efficient
solvent for triterpenoid extraction [20, 21].
3.2. Effect of single factors
3.2.1. Extraction temperature
Figure 2 illustrates the effect of extraction temperature on the yield of triterpenoids and
extraction conditions were set as solvent-to-material ratio of 30:1 mL/g, extraction time of 60
min, ultrasound power of 480 W with using ethanol as extraction solvent. As shown in Figure 3,
the yield of triterpenoids increased from 5.00 0.069 to 7.480 ± 0.234 mg/g when extraction
Extraction of triterpenoids from the Vietnamese red Ganoderma lucidum
95
temperature was increased from 45 to 55
o
C. After that, the yield of triterpenoids tended to
decrease although extraction time continued to rise. It was expected that enhancement of
extraction temperature would lead to higher extraction efficiency due to improvement in
desorption of analytes from the active sites in the matrix, solvent capacity of solubilizing
analyte, and sample penetration [22, 23]. When the extraction temperature of above 55 was
processed, high temperature could cause five – ring structures of triterpenoid molecules
destruction based on reported studies [24, 25]. A maximum yield of triterpenoids was achieved
when using an extraction temperature of 55
o
C and this value was chosen for subsequent
experiments.
Figure 2. Effect of extraction temperature on the yield of triterpenoids. Dissimilar letters in the same
column indicate significantly different at p < 0.05 using one-way ANOVA.
3.2.2. Extraction time
Figure 3. Effect of extraction time on the yield of triterpenoids. Dissimilar letters in the same
column indicate significantly different at p < 0.05 using one-way ANOVA.
Extraction temperature (
o
C)
Y
ie
ld
o
f
tr
it
er
p
en
o
id
s
(m
g
/g
)
Y
ie
ld
o
f
tr
it
er
p
en
o
id
s
(m
g
/g
)
Extraction time (min)
Tran Do Dat, et al.
96
Figure 3 demonstrates the effect of extraction time on the yield of triterpenoids when the
experimental conditions including solvent-to-material ratio of 30 mL/g, extraction temperature
of 55
o
C, extraction time of 60 min, and ultrasound power of 480 W. The result indicated that
with increasing extraction time until 60 min, the yield of triterpenoids increases sharply and
peaked at 7.472 0.024 mg/g, after which a slight reduction is observed. During the ultrasonic
process, it is reported that with longer extraction time as ultrasound wave could facilitate the
equilibrium establishment for the dissolution of triterpenoids between the plant cell wall and
extraction solvent in a short time [23, 26]. However, prolonged extraction time could exert an
adverse impact on the yield of triterpenoids as keeping at high temperature for a long time
period results in decomposition of triterpenoids [27]. This result is suitable with previous
research [25, 28]. Consequently, 60 min was chosen for further experiments.
3.2.3. Ultrasonic power
As shown in the Figure 4, the effect of ultrasonic power on the yield of triterpenoids was
investigated while the other extraction parameters were set as follows: solvent-to-material ratio
of 30 mL/g, extraction time of 60 min, extraction temperature of 55
o
C. When ultrasonic power
was varied from 200 to 500 W, the yield of triterpenoids was increased considerably and
obtained its maximum value of 7.479 0.033 mg/g at ultrasound power of 480 W, then sharply
reduced. It could be explained that greater ultrasound power would accelerate formation and
collapses of burble at the plant surface interface, thus drastically destroy cell walls and release
components within the cells. Nevertheless, thermal degradation resulted from local heating
effect induced by sonication. This result is in agreement with previous research [25]. Hence, 480
W was selected for the next study.
Figure 4. Effect of ultrasonic power on the yield of triterpenoids. Dissimilar letters in the same column
indicate significantly different at p < 0.05 using one-way ANOVA.
3.2.4. Solvent-to-material ratio
Ultrasonic power (W)
Y
ie
ld
o
f
tr
it
er
p
en
o
id
s
(m
g
/g
)
Extraction of triterpenoids from the Vietnamese red Ganoderma lucidum
97
Figure 5 presents the effect of solvent-to-material ratio on the yield of triterpenoids while
triterpenoids was extracted using ethanol with extraction temperature of 55 , extraction time of
60 min, and ultrasound power of 480 W. It is clearly that the yield of triterpenoids rose
dramatically when the solvent-to-material ratio increased. The highest yield of triterpenoids
obtained the value of 7.475 0.045 mg/g at the solvent-to-material ratio of 30:1 mL/g. It is
stated that the sufficient volume of solvent must be used for the immersion of entire sample
[22].
Generally, extraction yield improvement could be acquired when there is an increment of
solvent-to-material ratio as substances dissolve more effectively [23]. Nevertheless, large
solvent-to-material ratio would lead to a decrease in the yield of triterpenoids due to the negative
influence on the cell wall material break and mass transfer kinetics. This result is consistent with
previous studies
[24]. As a result, the solvent-to-material ratio of 30:1 mL/g was used to conduct
the following experiments.
Figure 5. Effect of solvent-to-material ratio on the yield of triterpenoids. Dissimilar letters in the same
column indicate significantly different at p < 0.05 using one-way ANOVA.
The influence of four factors on triterpenoid content was determined by ANOVA with the
use of LSD measuring the different meaning (p < 0.05) in order to compare the average
difference between four variables. Table 1 demonstrates the effects of four operating parameters
on the triterpenoid content. Fromt the result, four operating parameters namely extraction time,
extraction power, extraction temperature and solvent-to-material ratio were reported to illustrate
the same level of influence with solvent-to-material ratio.
Table 1. The effects of four operating parameters on the triterpenoid content.
Operating parameters Count Mean Homogeneous Groups
Extraction time 3 7.472 X
Ultrasonic power 3 7.479 X X
Extraction temperature 3 7.48 X
Solvent-to-material ratio 3 7.475 X
7.475 ± 0.045
c
4.980 ± 0.069
a
6.069 ±
0.109
b
6.480 ± 0.066
d
6.495± 0.044
d
Tran Do Dat, et al.
98
3.3. Antioxidant activity investigation of the extract
Figure 6 presents the anti-oxidant activity of different concentrations of ascorbic acid (a)
and the extract (b) in DPPH scavenging assay. As can be seen from Figure 6, the half-maximal
inhibitory concentration (IC50) value of the extract and ascorbic acid was 29.778 and 6.714
µg/mL, respectively. Thus, the IC50 value of the extract was 4.44 times higher than that of
ascorbic acid. In preliminary studies, the anti-oxidant activities of Vietnamese G.lucidum have
been in association with the presence of phenolic, triterpenoids, and polysaccharide compounds
[29,30]. This could be elucidated by several reasons. Firstly, the antioxidant activities of the
extract have been revealed by four isolated polysaccharides with different compositions in its
structures
[30]. In the second explanation, it might be due to reduction of molecular weight by
ultrasound waves. In addition, it was reported that the break of polysaccharides into
monosaccharides would horn the ability to scavenge DPPH
-
radicals according to previous
studies [31].
(a) (b)
Figure 6. Anti-oxidation activity of different concentrations of ascorbic acid (a) and the extract (b) in
DPPH scavenging assay.
4. CONCLUSIONS
In this study, triterpenoids were extracted from Vietnamese red G. lucidum by using UAE
method. The highest yield of triterpenoids obtained under the optimal extraction conditions
including solvent-to-material ratio of 30:1 mL/g, extraction temperature of 55 , extraction time
of 60 min, and ultrasonic power of 480 W was reported to be of 7.479 0.033 mg/g based on
ultraviolet-visible spectroscopy analysis. The extract obtained from UAE method showed low
antioxidant activity in comparison with that of ascorbic acid, with IC50 values were of 29.778
and 6.714 µg/mL, respectively. Therefore, UAE could be useful for extraction of triterpenoids
from Vietnamese red G. lucidum and further studies related to the application of this modern
extraction technique need to be conducted.
Acknowledgments. This research is funded by Vietnam National University Ho Chi Minh City (VNU-
HCM) under grant number B2019-20-02. We acknowledge the support from Ho Chi Minh City University
of Technology (HCMUT), VNU-HCM for this study.
Extraction of triterpenoids from the Vietnamese red Ganoderma lucidum
99
CRediT authorship contribution statement. TDD: Methodology, Investigation, Writing - original draft.
NTT: Methodology, Investigation, Writing - original draft. PLTM: Investigation, Writing - review &
editing. NTTL: Conceptualization, Methodology, Investigation, Writing - original draft. NTKN:
Investigation, Writing - review & editing. VHT: Writing - review & editing. NDV: Validation, Formal
analysis, Data curation, Supervision. HMN: Resources, Visualization, Project administration, Funding
acquisition. MTP: Resources, Visualization. NHH: Conceptualization, Resources, Writing - review &
editing, Visualization.
Declaration of competing interest. We confirm that this work is original and has not been published
elsewhere, nor is it currently under consideration for publication else