Optimal Extraction of Phenolic Compounds from Ampelopsis cantoniensis

VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY
INTERNATIONAL UNIVERSITY
TWO-STEP OPTIMIZATION OF MICROWAVE-ULTRASOUND-ASSISTED

EXTRACTION OF PHENOLIC COMPOUNDS FROM AMPELOPSIS
CANTONIENSIS USING RESPONSE SURFACE METHODOLOGY (RSM)
A thesis submitted to
The School of Biotechnology, International University
In partial fulfillment of the requirements for the degree of
B.S. in Food Technology
Student name: Tran Bao Uyen - BTFTIU17007
Supervisor: Dr. Le Ngoc Lieu (Ph.D)
October 2021
ACKNOWLEDGEMENT
First of all, I am grateful to my university – International University for organizing the

Thesis course for us. This experience is great and essential for our future career.
I would like to express my deepest and heartiest thanks to Dr. Le Ngoc Lieu – my
devoted supervisor. It is my honor to be instructed by Dr. Lieu. During my thesis, I
was guided by her with dedication, patience, and kindness. She has given me precious
advice and have greatly encouraged me to overcome the obstacles as well. Without
her generous counselling and helps, this thesis would not have been possible. I have
learned a lot from her and now become more courage to try new things. Once again,
thank you so much, Dr. Lieu.
I want to send special thanks to the Proposal Committee for the valuable comments
and suggestions; and Dr. Dang Quoc Tuan for allowing me to work overtime for times.
Also, I feel proud and grateful for being taught by all lecturers of the Department of
Food Technology, School of Biotechnology, International University.
I love to convey my acknowledgement to all the lab technicians (Ms. Yen, Ms. Nguyen,
and Ms. An) for their kindness and enthusiastic supports during my lab work. They
have instructed me passionately on how to use the equipment and instruments that
I needed for my thesis.
Furthermore, I am thankful to have all my best friends Minh Hieu, Que Trinh, Yen Chi,
Lan Chi, Minh Han, Kim Ngan, Huynh Mai, Uyen Uyen, Diem Phuc and specially Ms.
Ha Thanh with me, who have not only helped me through the laboratory work but
also given me encouragements and advice. Special thanks go to my volunteer – Kim
Trinh, who has helped me a lot, both as a volunteer and a friend. Thanks to this
thesis, I made friends with new people, and I am glad to have this experience.
Finally, I would like to show my deepest appreciation, special gratitude and thanks to
my beloved parents for their heartfelt supports and belief in me. They always stand
behind me to encourage me to overcome so many difficulties in my life. Thanks to
their huge love, I can try and do new things. They are also my motivations to move
forward, too. Thank you so much for always beside me, Mom and Dad!
ii
TABLE OF CONTENT
ABSTRACT……………………..…………………………………………………………………..2
1. INTRODUCTION: ................................................................................. 3
2. MATERIAL AND METHODS: ................................................................. 6
2.1. Material and chemicals: .................................................................................... 6
2.1.1. Material: .......................................................................................................... 6
2.1.2. Chemicals and reagents: .......................................................................... 6
2.2. Methods: ................................................................................................................. 7
2.2.1. Sample preparation: .................................................................................. 7
2.2.2. Microwave-ultrasound-assisted extraction: ...................................... 7
2.2.3. Traditional tea infusion: ............................................................................ 8
2.2.4. Screening of variables by Plackett-Burman design: ...................... 8
2.2.5. Level screening of experimental factors: ........................................... 9
2.2.6. Box-Behnken design:................................................................................. 9
2.2.7. Regression analysis: .................................................................................. 9
2.2.8. Optimization: .............................................................................................. 10
2.3. Determination of total phenolic content (TPC): .................................... 10
2.4. Statistical analysis: ........................................................................................... 10
3. RESULTS AND DISCUSSION: ............................................................. 10
3.1. Screening by Plackett-Burman design (PBD): ....................................... 10
3.2. Level screening of experimental factors: ................................................. 13
3.2.1. Effect of solvent type on total phenolic content: .......................... 14
3.2.2. Effect of microwave time on total phenolic content:................... 15
3.2.3. Effect of liquid-to-solid ratio on total phenolic content: ............ 16
3.3. Optimization by response surface methodology (RSM): ................... 19
3.3.1. Model fitting: ............................................................................................... 19
3.3.2. Optimization of total phenolic content: ............................................ 22
3.4. Comparison with traditional tea infusion:................................................ 26
4. CONCLUSION: ................................................................................... 26
5. RECOMMENDATIONS: ....................................................................... 27
iii
LIST OF TABLES
Table 1. Summary of factor levels set in PBD……………………………………………………………8

Table 2. Result of Plackett-Burman screening…………………………………………………………..11
Table 3. Result analysis of Plackett-Burman design for experimental results for
extraction of total phenolic content…………………………………………………………………………….12
Table 4. Factor levels used in Box-Behnken design…………………………………………………..19
Table 5. Result of experiments following Box-Behnken design………..................... 20
Table 6. Result analysis of Box-Behnken design……………………………………………………….21
Table 7. Comparison between TPC of sample extracted in approximately optimal
conditions and sample infused traditionally……………………………………………………………….26
iv
LIST OF FIGURES
Figure 1. Vine tea product in Viet Nam………………………………………………………………………4

Figure 2. Pareto chart of factors influence on the yield of total phenolic content of
MUAE from Ampelopsis cantoniensis………………………………………………………………………….13
Figure 3. Effect of different solvent types on TPC……………………………………………………15
Figure 4. Effect of different microwave times on TPC……………………………………………..16
Figure 5. Effect of different liquid-to-solid ratios on TPC…………………………………………17
Figure 6. Effect of different ultrasound times on TPC………………………………………………18
Figure 7. Response surface plots indicating the effects of the process variables on
TPC of Ampelopsis cantoniensis………………………………………………………………………………….24
v
LIST OF ABBREVIATIONS
BBD: Box-Behnken design
DB: dry basis
FFD: full factorial design
GAE: gallic acid equivalent
HAE: heat-assisted extraction
L-S R: liquid-to-solid ratio
MA: maceration
MAE: microwave-assisted extraction
min: minute(s)
mL/g: milliliter per gram
MUAE: microwave-ultrasound-assisted extraction
M.P: microwave power
M.t: microwave time
PBD: Plackett-Burman design
P.S: particle size
RSM: response surface methodology
s: second(s)
S.T: solvent type
TPC: total phenolic content
UAE: ultrasound-assisted extraction
U.Tp: ultrasound temperature
U.t: ultrasonic time
v/v: volume/volume
W: watts
vi
TWO-STEP OPTIMIZATION OF MICROWAVE-ULTRASOUND-ASSISTED
EXTRACTION OF PHENOLIC COMPOUNDS FROM AMPELOPSIS
CANTONIENSIS USING RESPONSE SURFACE METHODOLOGY (RSM)
Uyen B. Trana, Lieu N. Lea,*
a
Department of Food Technology, School of Biotechnology, International University,
Vietnam National University, Ho Chi Minh City, Viet Nam
*
Corresponding author’s email address: lnlieu@hcmiu.edu.vn
1
ABSTRACT
Ampelopsis cantoniensis is a highly functional herbal ingredient of Vietnam. In this

study, two-step optimization of microwave-ultrasound-assisted extraction of phenolic
compounds from Ampelopsis cantoniensis was conducted. The sample was irradiated
intermittently prior to being treated with ultrasound wave at 40 kHz. After that,
centrifugation was carried out to collect clear extract for analysis of total phenolic
content. Plackett-Burman design was applied to choose four most significant variables
among seven independent studied factors. Then, response surface methodology, with
Box-Behnken design was employed to optimize and investigate the effects of four
chosen variables, including solvent type (50 – 90% ethanol in water (v/v)), irradiation
time (90 – 150 s), liquid-to-solid ratio (20 - 40 mL/g), and ultrasonic time (20 – 40
min). The data was fitted to a second-order polynomial equation, and the optimal
conditions included the solvent of 72.3% of ethanol in water (v/v), irradiation time of
122.9 s, liquid-to-solid ratio of 31 mL/g, ultrasonic time of 31.2 min. Under these
conditions, the predicted TPC was 264.03 mg GAE/g dry basis, which showed that
Ampelopsis cantoniensis has high phenolic content. Compared to sample traditionally
infused in hot water for 10 min, TPC of sample extracted in approximately optimal
conditions was approximately doubled. The combination of microwave and ultrasound
treatment is a new extraction technology. Plackett-Burman and Box-Behnken design
can reduce the number of experiments while providing the optimal parameters for the
system. The results of this study could provide useful data and confirmed that
Ampelopsis cantoniensis has great potentials for exploitation in pharmaceutical fields,
chemical, material and food industry.
Key words: two-step optimization; Ampelopsis cantoniensis; vine tea; microwave-

ultrasound-assisted extraction; phenolic content; antioxidants
2
1. INTRODUCTION:
Ampelopsis cantoniensis (Hook. Et Arn) Planch, which belongs to the family Vitaceae,
is widely distributed in Indonesia, China, Taiwan, India, Japan and Vietnam (Nguyen
et al., 2020; Van Thu et al., 2015). In Vietnam, this plant is commonly known as “Chè
dây”, “Thau rả” or vine tea, and is mainly found in mountainous areas such as Thai
Nguyen, Sapa, Cao Bang, Lam Dong, Da Nang, Gia Lai, etc. This fresh and long-lived
herb has the specific characteristics of vine (Figure 1). After harvesting, Ampelopsis
cantoniensis is dried and consumed as herbal tea or is processed to powder for other
applications (Nguyen Thi Diu et al., 2020).
Ampelopsis cantoniensis was proved to have high content of flavonoids (Sơn et al.,
2017; Tran et al., 2019) and phenolic compounds (Ánh et al., 2018) - plant secondary
metabolites that can protect human health (Khoddami et al., 2013), or be employed
as preservatives and additives in the food industry (Dahmoune et al.,2015). Ánh et
al. (2018) conducted research to evaluate and compare the total phenolic content
(TPC) among different herbs. At the end, Ampelopsis cantoniensis was found to have
higher TPC than other herbs such as Codonopsis javanica (Blume) Hook and Piper
siriboa L. Besides that, this herbal material also contains anthranoid, saponin, organic
acids and invert sugars (Nguyen Thi Diu et al., 2020), as well as flavone, protein,
macro-nutrients, vitamins, and minerals (Zhihong et al., 2000).
Due to its compositions, vine tea can perform strong functionality, great antioxidant,
and antimicrobial activities (Sơn et al., 2017; Van Thu et al., 2015) while producing
no acute toxicity, as well as no harmful effect on the biochemical and hematological
index of consumer (Nguyễn, 2014). Neither side effects such as vomiting, bloating,
headache, tiredness, nor other allergic reactions, were observed in people consuming
vine tea or compounds extracted from this plant (Hoàng Bảo Châu, 2008; Nam, 2008;
Nguyen Khanh Trach, 1994). Moreover, since Ampelopsis cantoniensis successfully
acts against LDL oxidation (Thuong & Thuan, 2007) and H.pylori (Nguyen Khanh
Trach, 1994), this plant is employed as traditional treatment for inflammatory and
gastric diseases (Pham et al., 2020; Thuong & Thuan, 2007). The flavonoids of vine
tea have been used in some medicines such as Ampelop and Cantonin (Nam, 2008).
However, other phenolic compounds have not been studied and exploited yet. Since
these features showed great potentials of this plant’s phenolic compounds in
pharmaceutical fields, it is one of the biggest motivations for this project to be carried
out.
3
Extraction is a crucial step for isolation, identification and quantification of phenolic
compounds (Lapornik et al., 2005). Though there are various methods to obtain
phenolics from leaves and herbal teas, such as maceration (MA), heat-assisted
extraction (HAE) (Ueda et al., 2019), microwave-assisted extraction (MAE) (Spigno &
De Faveri, 2009), ultrasound-assisted extraction (UAE) (Irakli et al., 2018) and
supercritical fluid extraction (Şahin & Bilgin, 2012), UAE and MAE are the two most
employed techniques (Dahmoune et al., 2015; Jovanović et al., 2017; Lee et al.,
2013).
(a) (b)
Figure 1. Vine tea product in Viet Nam: (a) Fresh plant; (b) Dried product
MAE uses irradiation energy to extract targeted compounds from various matrices
(Spigno & De Faveri, 2009). Water inside cells is heated and evaporated, exerting
pressure on cell wall, leading to cell wall disruption and release of bioactive
compounds (Zia et al., 2020). Meanwhile, UAE – a green extraction technique, uses
high-frequency soundwave (>20 kHz) to interrupt the cell wall structure, resulting in
better penetration of interested compounds into solvent (Zia et al., 2020). Thus, UAE
can increase the yield of the desired substances via the cavitation bubble collapses
(Deng et al., 2017). Both techniques use less solvent, power, and extraction time but
have higher rate than conventional techniques (Caleja et al., 2017; Zia et al., 2020),
thus minimizes losses and optimize the yields (Ferreira et al., 2019). MAE and UAE
are frequently used to extract antioxidants from plant materials and food waste
(Ballard et al., 2010; Caleja et al., 2017; Dahmoune et al., 2015; Irakli et al., 2018;
Lee et al., 2013).
Microwave-ultrasound-assisted extraction (MUAE), which combines two treatments -

microwave first and then ultrasonication, was applied to this research. Though both
MAE and UAE have many benefits, MAE sometimes could not homogeneously heat
the sample, proposing greater risk of hydrolysis and oxidation of bioactive compounds
(C.-H. Chan et al., 2011; Guglielmetti et al., 2017), while UAE could only generate
4
fairly low thermal effect. Thus, these weaknesses could limit the wider applications of
MAE and UAE. For that reason, MUAE, which can be considered as a complementary
extraction method, possessing benefits of both MAE and UAE, has been recently used
in many research (Y. Wang et al., 2018). Due to the synergetic effect of both heat
(microwave) and mass (ultrasound) transfer, MUAE was proved to be more efficient
than individual treatments in both accelerating extraction process and increasing the
yield of targeted compounds (Sillero et al., 2020). Recently, MUAE has been applied
to extract bioactive compounds from plant sources such as bark (Sillero et al., 2020),
piperine from black pepper (Gorgani et al., 2017), quercetin from red onion (Allium
cepa L.) skin wastes (Velisdeh et al., 2021). However, MUAE has not been applied to
optimize the phenolic extraction from Ampelopsis cantoniensis yet. Since MUAE was
proved to have more advantages than MAE and UAE by Sillero et al. (2020), MUAE
was applied as a new extraction technique with the expectation that it could increase
and maximize the yield of phenolic compounds from Ampelopsis cantoniensis.
When applying both irradiation and ultrasonic treatment for extraction, there were
seven factors that could affect the yields of phenolic compounds, including particle
size, solvent type, liquid-to-solid ratio, microwave power, irradiation time, ultrasonic
temperature and time (Dahmoune et al., 2015; Dahmoune et al., 2014; Irakli et al.,
2018; Jovanović et al., 2017; Rafiee et al., 2011; Sơn et al., 2017; Zia et al., 2020).
However, since the number of parameters is numerous, resulting in time-consuming
and ineffective experiments, conventional techniques should not be applied
(Jovanović et al., 2017). Thus, two-step optimization was utilized instead, which
applied two experimental designs, namely Plackett-Burman design (PBD) to screen
for four most important among seven chosen factors, and Box-Behnken design (BBD)
for optimization of four selected factors by RSM.
PBD is a useful mathematical tool to detect the most critical factors that drastically
influence the result of experiments with just a small number of experimental trials
(Anastácio & Carvalho, 2013). Meanwhile, RSM - a helpful arithmetic tool to develop
polynomial relationship between a response of interest and independent variables
(Lee et al., 2013), was utilized to optimize the extraction conditions. BBD with its
minimal number of experiments, simple structure (Tu et al., 2005), and higher
efficiency (Ferreira et al., 2019), was chosen for optimizing process parameters to
obtain ideal yields of bioactive compounds by RSM (Dahmoune et al., 2015; Jovanović
et al., 2017).
5
Two-step optimization was successfully employed in previous studies to optimize
phenolic content of Thymus serpyllum L. herbs (Jovanović et al., 2017); flavonols and
anthocyanins of black currant marc (Li et al., 2016); and flavonoids from hawthorn
seeds (Pan et al., 2012). Since all data mentioned in previous research fitted well to
the model, this experimental design can be considered as an efficient and good model
for optimization (Bengardino et al., 2019).
Recently, there have been several research conducted to optimize the extraction of
phenolic compounds from several types of leaves and herbal ingredients such as
Myrtus L. leaves (Dahmoune et al., 2015), Thymus serpyllum L. herb (Dahmoune et
al., 2015), olive leaf (Rafiee et al., 2011) and green tea (Lee et al., 2013). Up to now,
there has been no research carried out to optimize conditions for extracting phenolic
compounds from Ampelopsis cantoniensis. Using MUAE, combining with two-step
optimization was also new and advantageous for researcher as discussed above.
Thus, this project was carried out, aiming at obtaining the optimal conditions for
extracting phenolic compounds from Ampelopsis cantoniensis using MUAE and two-
step optimization. Moreover, as this material is highly functional but less popular, the
researcher hopes that its potential will be known and fully exploited in the future not
only by pharmaceutical fields, but also by chemical, material, and food industry (He
et al., 2015; Ouyang et al., 2019). Furthermore, this study also aims at elevating the
values and recognition of Ampelopsis cantoniensis in Vietnam, especially those were
encouraged to grow more in Quang Nam province (Nguyễn, 2014).
2. MATERIAL AND METHODS:

2.1.Material and chemicals:
2.1.1. Material:
Dried Ampelopsis cantoniensis was purchased from Tam Duc Ltd. Company, located
in district 6, Ho Chi Minh city. The company ensured that fresh plant was dried under
50oC until reaching a desirable moisture content. The purchased material was checked
for closure of package, expired date, and other damages before use. Only high-quality
material was used for experiments.
2.1.2. Chemicals and reagents:
All chemicals and reagents such as Folin-Cioalteau reagent, sodium bicarbonate, gallic
acid, concentrated ethanol, etc. were of analytical grade and were purchased from
certified suppliers.
6
2.2.Methods:
2.2.1. Sample preparation:
After receiving five kilograms of dried vine tea, the sample was stored at ambient
temperature in closed zipper bags. Then, all the stone, dirt or foreign materials were
removed from the material prior to moisture content measurement. Next, the satisfied
vine tea was ground by milling machine and sieved by sieving machine to achieve
required particle size. The resulting powder was stored in separated zipper bags, with
no air inside and was placed in desiccator after use.
2.2.2. Microwave-ultrasound-assisted extraction:
Phenolic compounds from Ampelopsis cantoniensis powder were extracted using a

domestic microwave oven system (Sharp R-21A1(S)VN, Vietnam) and sonicator of
frequency of 40 kHz. This process was carried out based on the methodology of
Dahmoune et al. (2015) and Irakli et al. (2018) with slight modifications.
First, a certain weight of sample was transferred to a 250 mL Duran bottle. Then, a
specific amount of solvent was added, following the experimental design. Next, the
bottle containing sample and solvent was irradiated intermittently (10 s power on,
then another 10 s power off before continuing irradiation treatment) for a particular
period under a certain microwave power to avoid overboiling and destruction of
sample. The cap of bottle was loosened every 30 s to avoid explosion of container.
When the irradiation finished, the sample was cooled down to room temperature in 5
min prior to ultrasonic extraction.
After being cooled down, the sample was then treated with ultrasound wave at 40
kHz. The lid of both container and sonicator were closed to avoid sample from direct
exposure to light. Ultrasonic time and temperature were set on the control panel of
the equipment. However, due to the properties of ultrasound wave, temperature was
checked every 5 min to prevent unexpected rise in temperature, and adjustments
were made if necessary. When the treatment ended, the sample was cooled down to
ambient temperature before being centrifuged at 7000 rpm for 10 min at 25oC. At the
end, the clear extract was obtained for analysis of total phenolic content (TPC). All
the experiments were triplicated.
7
2.2.3. Traditional tea infusion:
One gram of Ampelopsis cantoniensis powder was infused in hot water (90oC) for 10
min (Irakli et al., 2018). Then, the solution was cooled down to room temperature
and was centrifuged at 7000 rpm for 10 min at room temperature to collect the clear
solution. The solution was analyzed for TPC.
2.2.4. Screening of variables by Plackett-Burman design:
The extraction of phenolic compounds from Ampelopsis cantoniensis was significantly

influenced by seven factors such as particle size, type of solvent, liquid-to-solid ratio,
microwave power, microwave time, ultrasonic time, and ultrasonic temperature. In
total, PBD requires only 12 experiment runs, and each was triplicated following Table
1, and Table 2. Each factor was screened at two levels: low (-) and high (+) (Hassan
et al., 2016), and the obtained results was analyzed by Minitab software (Version 18,
Minitab, LLC, USA) to determine the parameters that significantly affected TPC of
Ampelopsis cantoniensis.
Table 1. Summary of factor levels set in PBD1
Factor levels
Factor
Low (-1) High (+1)
Particle size [µm] 125 500
Solvent type [%EthOH] 0 70
Liquid-to-solid ratio [mL/g] 10 200
Microwave power [W] 80 800
Microwave time [s] 3 180
Ultrasound temperature [ C]o
30 70
Ultrasonic time [min] 0 30
%EthOH: ratio of ethanol in water (v/v)

1
Based on the result analysis of PBD, factors with p-value<0.05 was indicated as
significant factors. Meanwhile, in the Pareto chart, parameters that had t-value
exceeded the limit of t-value were also indicated as significant. By combining these
results, four most crucial factors were selected for optimization by RSM with Box-
Behnken design. Then, they were screened by singled-factor screening methodology
to choose suitable levels for optimization by RSM following BBD.
8
2.2.5. Level screening of experimental factors:
After conducting initial screening stage following Plackett-Burman design, level

screening of the four selected factors was carried out to determine the factor levels
for optimization by Box-Behnken design. This step was based on the methodology of
Liu et al. (2021) with slight modifications (Liu et al., 2021). Six in seven factors were
fixed at a certain value, while the remaining factor was varied in the studied range.
The samples were analyzed for total phenolic content. All experiments were triplicated
and analyzed by analysis of variance (ANOVA).
2.2.6. Box-Behnken design:
In the experiment, the extraction conditions were optimized by RSM using BBD. The
model of BBD is based on the three-level incomplete factorial design, comprising
center points and middle points of the edges from a cube (Zolgharnein et al., 2013).
BBD was employed to evaluate the main and interaction effects of four selected
variables chosen from result of screening by PBD (based on result analysis and Pareto
chart of PBD). Factor levels were decided based on the results of level screening of
experimental factors. The values of these parameters are coded into three levels, as
-1, 0, +1 for low, middle, and high values, respectively. There were 27 runs to obtain
the results.
By following Eq. 1, the regression analysis of the data was fitted to second-order
polynomial equation (quadratic model), which was then applied to forecast the
optimum conditions of extraction process:
𝒀 = 𝑩𝟎 + ∑𝒌𝒊=𝟏 𝑩𝒊 ∗ 𝒙𝒊 + ∑𝒌𝒊=𝟏 𝑩𝒊𝒊 ∗ 𝒙𝟐 + ∑𝒌𝒊=𝟏 𝑩𝒊𝒋 ∗ 𝒙𝒊 ∗ 𝒙𝒋 + 𝑬 (1)
Where Y represents the response function (in our case the TPC yield); B 0 is a constant
coefficient; Bi, Bii and Bij are the coefficients of the linear, quadratic, and interactive
terms, respectively, and xi and xj represent the coded independent variables
2.2.7. Regression analysis:
The statistical package Design-Expert software (Version 12.0, Stat-Ease Inc., USA)
was used to fit the mathematical model. The equation describes a quadratic model
including linear, squared and interaction terms. In this model, significant terms were
revealed by analysis of variance (ANOVA) and statistical significance was evaluated
based on F-statistic calculation. Various descriptive statistical analyses, namely, p-
value, F-value, determination coefficient (R2), predicted determination coefficient
9
(R2pred), adjusted determination coefficient (R2adj) and coefficient of variance (C.V)
was used to evaluate the statistical significance of the developed second-order
mathematical model. The data was then used to plot the 3D response surface.
2.2.8. Optimization:
The Design Expert software (version 12.0, Stat-Ease Inc., USA) was used to
determine the optimized extraction conditions by desirability function methodology.
2.3. Determination of total phenolic content (TPC):
TPC of the extract were determined by Folin-Ciocalteau reagent methodology, based

on the research of Chan et al. (2010) with slight modifications. First, 0.3 mL of sample
was taken, then 1.5 mL of Folin-Ciocalteau 10% reagent was added to the test tube
covered with aluminum foil, which helped to avoid direct exposure to light. The
mixture was let to stand for 5 min before 1.2 mL of Na2CO3 7.5% was inserted. Then,
the mixture was vortexed, and incubated in dark for 30 min. Finally, absorbance was
measured by spectrophotometer at 765 nm (Chan et al., 2010). The concentration of
total phenolic content in sample was calculated following the equation of calibration
curve (R2 = 0.9996) Eq.2:
Y = 0.0163*X + 0.0088 (2)
Where Y is the absorbance of sample measured at 765 nm, X is the concentration of

phenolic content in sample (µg GAE/mL).
Then, TPC was calculated following Eq. 3:
𝑚𝑔 𝐺𝐴𝐸
𝑚𝑔 𝐺𝐴𝐸 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑝ℎ𝑒𝑛𝑜𝑙𝑖𝑐 𝑐𝑜𝑚𝑝𝑜𝑢𝑛𝑑𝑠 ( )∗𝑣𝑜𝑙𝑢𝑚𝑒 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑒𝑑 (𝑚𝐿)∗𝐷𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑇𝑃𝐶 ( )= 𝑚𝐿
(3)
𝑔 𝑑𝑟𝑦 𝑏𝑎𝑠𝑖𝑠 𝑀𝑎𝑠𝑠 𝑜𝑓 𝑡ℎ𝑒 𝑑𝑟𝑦 𝑚𝑎𝑡𝑡𝑒𝑟 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑢𝑠𝑒𝑑 (𝑔)
2.4. Statistical analysis:
All the experiments were carried out in triplicates. Mean and standard deviations of
the data were calculated, and analysis of variance (ANOVA) was carried out to
determine any significant difference (p<0.05).
3. RESULTS AND DISCUSSION:

3.1. Screening by Plackett-Burman design (PBD):
In the first stage of the research, all seven factors that may have significant effects
on the yield of bioactive compounds, including particle size, solvent type, liquid-to-
solid ratio, microwave power, microwave time, ultrasonic temperature, and ultrasonic
10
time were screened, following the study of Zia et al, (2020). In two-step optimization,
full factorial design (FFD) and PBD were usually employed for screening. However,
since the number of factors was commonly huge, it would be inefficient to screen
them by FFD (as FFD requires 27 runs to completely screen all variables). Thus, PBD,
which just needs 12 experimental trials, was employed to screen the significant
variables from a multivariable system. The aim of this step was to set a foundation
for sequential optimization (Li et al., 2016).
Table 2 presents the records of screening by Plackett-Burman design, while Table 3
and Figure 2 illustrates the result of analysis of variance (ANOVA) and the Pareto
chart. The model F-value of 15.55 shows that the model is significant. R2 value of
more than 75% indicates the aptness of data to the model (Niladevi et al., 2009;
Reddy et al., 2008; Xie, 2015). In this study, R2 value of 79.54% was enough to
indicate the fitness of model. In addition, there was also reasonable agreement
between the adjusted R2 (0.7442) and predicted R2 (0.6617), which implies that the
model was significant and adequate (Granato & Ares, 2014). Thus, this model can be
used to navigate the design space. According to the model, terms with p-value less
than 0.05 illustrate that the model term is significant. In contrast, those with p-value
more than 0.05 are considered as nonsignificant.
Table 2. Result of Plackett-Burman screening1,2,3
Std Run P.S S.T L-S R M.P M.t U.Tp U.t TPC
Order Order [µm] [%Eth] [mL/g] [W] [s] [OC] [min] [mg GAE/g DB]
4 1 500 0 10 800 3 70 0 12.32 ± 0.43
12 2 125 0 200 80 3 30 0 50.99 ± 1.27
3 3 125 70 10 80 180 30 0 131.61 ± 3.31
2 4 500 70 200 800 3 30 0 51.64 ± 1.19
1 5 500 0 10 80 3 30 30 10.81 ± 0.41
9 6 125 0 200 800 180 70 0 129.05 ± 6.92
6 7 500 70 10 80 180 70 0 88.80 ± 2.42
11 8 125 70 200 80 3 70 30 153.97 ± 5.08
7 9 125 70 10 800 3 70 30 145.58 ± 2.94
5 10 500 70 200 800 180 30 30 248.61 ± 2.59
8 11 125 0 10 800 180 30 30 51.14 ± 2.37
10 12 500 0 200 80 180 70 30 69.60 ± 0.82
1
P.S: particle size [µm], S.T: solvent type [%EthOH], L-S R: liquid-to-solid ratio
[mL/g], M.P: microwave power [W], M.t: microwave time [s], U.Tp: ultrasound
temperature [oC], U.t: ultrasonic time [min]
2
%Eth: ratio of ethanol in water (v/v), DB: dry basis
3
TPC: total phenolic content [mg GAE/g dry basis]
From Table 3 and Figure 2, factors including particle size, solvent concentration,
liquid-to-solid ratio, microwave time and ultrasound time were found to be significant
11
model terms. Though there were five factors considerably influence TPC of Ampelopsis
cantoniensis, four most significant factors including solvent type, irradiation time,
liquid-to-solid ratio and ultrasonic time were chosen, as indicated by the Pareto charts
(Figure 2).
Table 3. Result analysis of Plackett-Burman design for experimental results for

extraction of total phenolic content1,2,3,4
Analysis of variance
TPC
Source
F-value p-value Inference
Model 15.55 <0.0001 Significant
P.S 7.02 0.013 Significant
S.T 53.59 <0.0001 Significant
L-S R 15 0.001 Significant
M.P 3.77 0.062 Not significant
M.t 18.78 <0.0001 Significant
U.Tp 0.66 0.423 Not significant
U.t 10 0.004 Significant
Credibility of regression equation of Plackett-Burman design
R2 Adjusted R2 Predicted R2
79.54% 74.42% 66.17%
1
Results were obtained with Minitab 18.0 software
2
P.S: particle size [µm], S.T: solvent type [%EtOH], L-S R: liquid-to-solid ratio
3
%Eth: ratio of ethanol in water (v/v)
4
TPC: Total phenolic content [mg GAE/g dry basis]
This result agreed with the research of Zia et al. (2020), which suggested that solvent
type, liquid-to-solid ratio, irradiation time and ultrasonic time were the most
important factors that influence the irradiation and ultrasonic treatments. Likewise,
the result of PBD screening was relevant to the work of Dahmoune et al. (2013), since
these researchers considered solvent type, solid-liquid ratio, irradiation time as the
most critical factors that affects the yield of phenolics from Myrtus L. leaves. Caleja
et al. (2017) also revealed that ultrasonic time performed a strong influence on the
final yields of phenolic antioxidants from green tea, which was related to the result of
screening by PBD.
12
A P.S
7.3 B S.T
C L-S R
2 D M.P
4.3 E M.t
3 F U.Tp
3.8 G U.t
7
3.1
6
2.6
5
1.9
4
0.8
1
1
2
P.S: particle size [µm], S.T: solvent type [%EthOH], L-S R: liquid-to-solid ratio
3
Figure 2. Pareto chart of factors influence on the yield of total phenolic content of
MUAE from Ampelopsis cantoniensis1,2,3
3.2. Level screening of experimental factors:
In the next screening stage, the suitable levels of four factors were screened by
singled-factor screening methodology. 250 µm was chosen as ideal sample size for
experiments, as this sample size was found to be efficient for extraction by many
previous studies (Ansari & Goodarznia, 2012; Hemwimol et al., 2006; Rafiee et al.,
2011). Jovanovic et al. (2016) also found that sample size of 300 µm was optimal for
extracting polyphenols from Thymus serpyllum L. herb. Thus, 250 µm was chosen as
the particle size for both singled-factor screening and optimization stages. For other
non-significant factors, 800 W was selected as fixed value for microwave power as
this value can help to maximize the total phenolic content from material (Švarc-Gajić
et al., 2013). This is also relevant to the result obtained by Quintero et al. (2019),
when they set the microwave power at 700 W to obtain the optimal conditions for
extracting bioactive compounds from Annatto seeds (Quintero Quiroz et al., 2019).
In the research of Aybastier et al. (2013), the optimum temperature for obtaining
highest content of antioxidant from blackberry leaves was 66 to 68 degrees Celsius
(Aybastıer et al., 2013). Likewise, 70oC was recorded as the optimal temperature for
13
extracting phenolic compounds from Aronia melanocarpa (black chokeberry) by-
products (Ramić et al., 2015). Therefore, 70oC was fixed for ultrasonic extraction.
Other significant parameters were fixed at: solvent of 70% ethanol (v/v) (Sun et al.,
2011), liquid-to-solid ratio of 30 mL/g (Dahmoune et al., 2015; Jovanović et al.,
2017), microwave time of 60 s (Ayouaz et al., 2020; Dahmoune et al., 2015),
ultrasonic time of 30 min (Lee et al., 2013). When each factor was screened, only
different levels of that factor were varied, while the other factors were fixed at the
chosen value.
3.2.1. Effect of solvent type on total phenolic content:
From the results of PBD, solvent type was the factor that shows most influence on
TPC. In this screening process, effect of solvent type on TPC was evaluated. Since
ethanol - a green solvent for extraction, which can be degraded biologically, is secure
and appropriate for using in organic products (Ngan et al., 2017), this solvent was
selected for extraction in this research. One gram of sample of particle size 250 µm
was extracted in different percentage of ethanol/water (v/v) solutions (varying from
10% to 90% ethanol in water (v/v)) at a ratio of 1 g per 30 mL solvent, with a
treatment of microwave at 800 W for 60 s first. Then, the sample was treated with
ultrasound at 70oC for 30 min.
Figure 3 indicates that the concentration of ethanol in water performed a strong

influence on the total phenolic content yielded from Ampelopsis cantoniensis. The
content of phenolic compounds increased with the increasing concentration of ethanol
until reaching a peak at 70% ethanol in water (v/v) (211.13 ± 9.34 mg GAE/g dry
basis), after which TPC started to drop. This result correlated to the polarity of ethanol
and the solubility of phenolic compounds in Ampelopsis cantoniensis. Since ethanol is
low polar, water is strong polar, and they can be dissolved well in each other at
different ratios (Z.-S. Zhang et al., 2007), and the polarity of the resulting solvent
rises continuously with the increase in ethanol concentration. Phenolic compounds are
polar as well, thus the content of total phenolic substances increased gradually
according to the ‘‘like dissolves like’’ principle (Zhang et al., 2008). However, if the
concentration of ethanol in solution is too high, protein denaturation can occur,
leading to reduction in the extraction rate (Yang et al., 2010). For this reason, when
Ampelopsis cantoniensis was extracted in solution containing 90% ethanol, the TPC
yielded was lower, which is just nearly 87% of TPC found in 70% ethanol solvent.
14
250
TPC [mg GAE/g dry basis]

211.13a
200 183.64b
157.06c 159.55c
150
113.92d
100
50
0
10 30 50 70 90
Type of solvent [%EthOH]

1
Data (mean ± SD) with same letters indicates that there is no significant difference
2
Figure 3. Effect of different solvent types on TPC1,2
From the result of ANOVA, significant statistical differences were observed among
samples extracted in solvents containing 10%, 50%, 70%, and 90% ethanol in water
(v/v) (p-value<0.05). However, there was no statistically significant difference
between TPC of samples extracted in 30% and 50% ethanol in water (v/v) solution
(p-value>0.05). In this experiment, least TPC was observed from sample extracted
in 10% ethanol, which stood at a low of 113.92 ± 1.42 mg GAE/g dry basis. Based
on the obtained results, solutions ranging from 50% to 90% ethanol in water (v/v)
were chosen for optimization by Box-Behnken design, with 70% ethanol in water (v/v)
selected as center point for RSM experiments. This result agreed with the conclusion
of Sun et al. (2011), when they found that 70% ethanol was desirable for extracting
phenolic compounds from kudingcha (Sun et al., 2011).
3.2.2. Effect of microwave time on total phenolic content:
Microwave time is a factor that strongly influenced the yield of targeted compounds
from plant materials (Dahmoune et al., 2015). Since microwave treatment is based
on heat transfer principles (Zia et al., 2020), excessive irradiation time can lead to
potential thermal degradation of targeted compounds in the extract (Švarc-Gajić et
al., 2013). Thus, choosing optimal irradiation time is essential.
The sample of 250 µm was extracted in 70% ethanol/water (v/v) solution at a ratio
of 30 mL solvent per gram sample, with a treatment of microwave at 800 W first. The
15
irradiation time was varied in range from 30 s to 180 s. Sequentially, the sample was
treated with ultrasound at 70oC for 30 min.
300

256.92a
245.44b
250 225.49c 228.33c
214.94d
200
150
102.48e
100
50
0
30 60 90 120 150 180
Microwave time [s]

1
2
Figure 4. Effect of different microwave times on TPC1,2
Figure 4 illustrates the effects of microwave time on TPC. The TPC obtained from
sample increased gradually when the microwave time increased from 30 s to 120 s
and reached a peak at 256.92 ± 3.58 mg GAE/g dry basis when the sample was
irradiated for 120 s. Once the extraction time was greater than 120 s, the yields of
TPC reduced gradually. This result could link to the thermal degradation of phenolic
compounds (Švarc-Gajić et al., 2013) caused by overboiling of sample (Dahmoune et
al., 2014). TPC obtained at 120 s irradiation was significantly different from that value
of sample irradiated for 90 and 150 s, which was found to possess TPC of 225.49 mg
GAE/g dry basis and 245.44 ± 5.92 mg GAE/g dry basis, respectively. From these
results, microwave time in range of 90 s to 150 s was selected for optimization by
BBD, with 120 s selected as center point for RSM experiments.
3.2.3. Effect of liquid-to-solid ratio on total phenolic content:
In almost all extraction techniques, liquid-to-solid ratio is a critical parameter that

impacts on the recovery of phenolic compounds, as all researchers hope to boost the
extraction yield to maximum value, while minimizing the amount of solvent used
(Spigno & De Faveri, 2009). Similarly, excess solvent also displays no significant
effect on the equilibrium extraction yields, which can lead to solvent waste (Chiang
16
et al., 2017). Thus, the effects of solid-to-liquid ratio on total phenolic content needs
to be studied and chosen carefully.
In the extraction process, 1 g of Ampelopsis cantoniensis powder of 250 µm was

extracted in 70% ethanol in water (v/v) solution at different liquid-to-solid ratios
(varying from 10mL to 50mL solvent per gram sample). The sample was then treated
with microwave at 800 W for 60 s prior to being sonicated at 70oC for 30 min.
Figure 5 demonstrates the effects of liquid-to-solid ratio on the yields of phenolic

compounds. Overall, the higher the amount of solvent, the lower the values of TPC
were. Total phenolic content of sample extracted with a liquid/solid ratio of 30/1 was
highest, with a value of 208.77 ± 7.09 mg GAE/g dry basis, while the sample
extracted with 50 mL obtained the lowest content of phenolic compounds at 114.26
± 2.70 mg GAE/g dry basis.
250
208.77a
200
164.01b
150 134.41c 133.26c
114.26d
100
50
0
10 20 30 40 50
Liquid-to-solid ratio [mL/g]

1
2
Figure 5. Effect of different liquid-to-solid ratios on TPC1,2
These results were in accordance with other studies (Elboughdiri et al., 2020; Ince et
al., 2014; Jovanović et al., 2017). High liquid-to-solid ratio provides more solvent to
enter the cell, combining with the extraction time, to completely enhance the
dispersion of phenolic compounds to solvent (Sousa et al., 2016). Thus, the content
of total soluble phenolic compounds rises with increasing amount of solvent used (Bi
et al., 2011; Cacace & Mazza, 2003). However, using excessive quantity of solvent
can lead to decline in phenolic content (Elboughdiri et al., 2020). Since too high liquid-
to-solid ratio can result in improper microwave heating (Zia et al., 2020), as well as
17
can restrain the generation of aerosol and the cavitation effect of ultrasonic treatment
(Sheng et al., 2018), the TPC obtained would be lowered. This effect thus could be
used to explain for Figure 5, when TPC declines gradually at liquid-to-solid ratios
greater than 30 mL/g.
The TPC of sample extracted with ratio of 30 mL per gram showed great differences
with the samples extracted in 20 mL and 40 mL solvent. Thus, liquid-to-solid ratio in
range of 20 to 40 mL/g was chosen for optimization by Box-Behnken design, with the
center point was 30 mL/g.
3.2.4. Effect of ultrasound time on total phenolic content:
In extraction process, extraction duration is an important factor that can change the
operation cost, efficiency, and time consumption (Hemwimol et al., 2006). Meanwhile,
extraction time can also influence the yields and quality of targeted compounds
(Tomšik et al., 2016). Thus, its effects need to be evaluated.
In this study, one gram of sample of particle size 250 µm was extracted in 30 mL of
solvent containing 70% ethanol in water (v/v). The sample was then irradiated at 800
W for 60 s prior to being sonicated at 70oC for a certain time (ranging from 10 to 40
min).
280
240 219.92a
200.36b 201.59b 197.22b
200
153.41c
160 142.48d
125.63e
120
80
40
0
10 15 20 25 30 35 40
Ultrasound time [min]

1
2
Figure 6. Effect of different ultrasound times on TPC1,2
Figure 6 reveals the effects of ultrasonic time on TPC yielded from Ampelopsis
cantoniensis. Total phenolic content of sample increased gradually from 10 to 30 min
18
of extraction. This tendency can be explained by Fick’s second law of diffusion, which
stated that the longer the extraction time, the higher quantity of extracted phenolic
compounds is obtained (Nayak et al., 2015). Prolonged extraction time also improves
extraction efficiency, as the solvent completely interrupt cell wall structure, enabling
the phenolic compounds to be dissolved (Irakli et al., 2018). However, long extraction
can also lead to unexpected reduction in TPC, as the bioactive compounds might be
degraded, due to exposure to high heat, light and oxygen (Naczk & Shahidi, 2006;
Tomšik et al., 2016). The explanation is relevant with our result since the value of
TPC started to drop when the ultrasonic treatment was longer than 30 min. Among
the samples, the highest content of total phenolic compounds was observed in the
sample treated with ultrasonic wave for 30 min, with a high quantity of 219.92 ± 6.19
mg GAE/g dry basis. This value also showed significant differences with the sample
extracted in 20 and 40 min of ultrasonic treatment. Meanwhile, there was no
statistical difference among sample treated with ultrasound for 25, 35, and 40 min.
Thus, ultrasonic time in range of 20 to 40 min was chosen as values for optimization
by Box-Behnken design, with the center point of 30 min of ultrasonic treatment.
3.3. Optimization by response surface methodology (RSM):

3.3.1. Model fitting:
To evaluate the combined effects of independent variables (solvent type, microwave
time, liquid-to-solid ratio, ultrasonic time) on TPC of Ampelopsis cantoniensis,
experiments were carried out following BBD – the design that comprises the linear
and quadratic impacts of each parameter as well as their interactions. The selected
levels for BBD were illustrated in Table 4, and the result of experiments was recorded
in Table 5, where 27 runs of experiments were involved. All the experiments were
duplicated, following the experimental strategy produced by Design Expert software
(Version 12.0, Stat-Ease Inc., USA).
Table 4. Factor levels used in Box-Behnken design1
Factor Low (-1) Middle (0) High (+1)

Solvent type [%EthOH] 50 70 90
Microwave time [s] 90 120 150
Liquid-to-solid ratio [mL/g] 20 30 40
Ultrasound time [min] 20 30 40

1
After obtaining the results from experimental activities, result analysis was
conducted, which are given in Table 6. The model with an F-value of 93.12 and p-
19
value of less than 0.0001 indicated that the model is highly significant. Thus, this
quadratic model was suitable to be applied. The lack-of-fit F-value of 1.82 with p>0.05
implies that the lack-of-fit is not significant relative to pure error. Insignificant lack-
of-fit value is desirable.
The fitness of the model can also be expressed by the value of coefficient
determination (R2). R2 value demonstrates the degree of fitness by being the
proportion of the variances in the response values, which can be forecast from the
independent variables and can be used to explain and justify the regression analysis
(Nagelkerke, 1991). Meanwhile, the adjusted R2 value adjusts the R2 for the sample
size and number of variables in regression model, thus this value is usually used for
comparison (Ratner, 2009).
Table 5. Result of experiments following Box-Behnken design1,2,3
Run Pt S.T M.t L-S R U.t TPC

Blocks
Order Type [%Eth] [s] [mL/g] [min] [mg GAE/g dry basis]
1 2 1 50 120 30 20 178.31 ± 4.12
2 2 1 90 120 20 30 165.02 ± 5.35
3 2 1 50 90 30 30 176.66 ± 6.31
4 0 1 70 120 30 30 267.70 ± 3.22
5 2 1 90 90 30 30 176.17 ± 7.00
6 2 1 70 120 40 40 198.74 ± 5.03
7 2 1 70 150 40 30 190.92 ± 5.49
8 2 1 70 120 20 20 156.38 ± 6.86
9 2 1 50 120 40 30 168.41 ± 2.37
10 2 1 90 120 40 30 195.38 ± 7.41
11 2 1 90 150 30 30 199.99 ± 6.11
12 2 1 50 120 20 30 158.10 ± 1.23
13 0 1 70 120 30 30 263.34 ± 0.75
14 2 1 70 90 20 30 161.52 ± 1.05
15 2 1 70 90 40 30 166.54 ± 1.74
16 2 1 70 90 30 40 185.10 ± 4.80
17 2 1 70 120 20 40 171.06 ± 6.86
18 2 1 70 150 30 40 196.64 ± 9.19
19 2 1 70 150 20 30 176.82 ± 3.66
20 2 1 90 120 30 20 162.30 ± 2.88
21 2 1 70 120 40 20 170.29 ± 5.76
22 2 1 90 120 30 40 199.16 ± 4.12
23 2 1 50 150 30 30 169.87 ± 2.33
24 2 1 70 90 30 20 169.33 ± 2.81
25 2 1 70 150 30 20 180.25 ± 8.51
26 2 1 50 120 30 40 162.30 ± 1.37
27 0 1 70 120 30 30 256.16 ± 9.26
1
The design was obtained with Design Expert 12.0 software
2
S.T: solvent type [%EtOH], L-S R: liquid-to-solid ratio [mL/g], M.t: microwave time
[s], U.t: ultrasonic time [min], %Eth: ratio of ethanol in water (v/v)
20
3
TPC: Total phenolic content [mg GAE/g dry basis]
In this study, the adjusted R2 of 0.9605 was found to be in reasonable agreement

with the predicted R2 of 0.9447. These values indicate that only approximately 5% of
the total variations could not be explained by the model.
Adequacy precision measures the signal to noise ratio. A ratio of more than 4 is
desirable (Maran, Manikandan et al., 2013). The adequacy precision of 32.0702
confirms that an adequate signal was observed. Thus, this model can be used to
navigate the design space. Low C.V. value of 3.25% also implied that low deviations
were observed between the predicted and experimental values of TPC, thus ensuring
the precision and reliability of the experiments.
Table 6. Result analysis of Box-Behnken design1,2,3
Analysis of variance
Sum of Mean
Source df F-value p-value Inference
Squares Square
Model 47717.9 14 3408.42 93.12 < 0.0001 Significant
X1 1200.3 1 1200.29 32.79 < 0.0001 Significant
X2 1031.9 1 1031.85 28.19 < 0.0001 Significant

X3 1712.7 1 1712.68 46.79 < 0.0001 Significant
X4 1540.9 1 1540.85 42.1 < 0.0001 Significant
X1X2 453.6 1 453.61 12.39 0.0011 Significant
X1X3 201 1 200.98 5.49 0.0243 Significant
X1X4 1397.5 1 1397.46 38.18 < 0.0001 Significant
X2X3 41.3 1 41.28 1.13 0.2948 Not significant
X2X4 0.2 1 0.1981 0.0054 0.9417 Not significant
X3X4 95 1 95 2.6 0.1152 Not significant
X1² 20342.5 1 20342.5 555.76 < 0.0001 Significant
X2² 16143 1 16143 441.03 < 0.0001 Significant
X3² 24362.3 1 24362.3 665.58 < 0.0001 Significant
X4² 18331.4 1 18331.4 500.82 < 0.0001 Significant
Residual 1427.5 39 36.6
Lack of Fit 549.64 10 54.96 1.82 0.1023 Not significant
Pure Error 877.88 29 30.27
Cor Total 49145.4 53
21
Credibility of regression equation
Standard Adequacy
Mean C.V. % R² Adjusted R² Predicted R²
Deviation Precision
6.05 186.03 3.25 0.971 0.9605 0.9447 32.0702
1
Results were obtained with Design Expert 12.0 software
2
X1: solvent type [%EthOH]; X2: Microwave time [s]; X3: Liquid-to-solid ratio [mL/g];
X4: Ultrasound time [min]
3
As high dependability was established, the prediction of TPC in vine tea could be
obtained through the reduced second-order polynomial equation with coded variables
shown in Eq. 4:
TPC = 262.40 + 7.07X1 + 6.56X2 + 8.45X3 + 8.01X4 + 7.53X1X2 + 5.01X1X3 +
13.22X1X4 – 43.67X12 -38.90X22 – 47.79X32 – 41.46X42 (4)
3.3.2. Optimization of total phenolic content:
From Table 6, model terms with p-value less than 0.05 indicates that the term is
significant; thus, the linear and quadratic model terms of solvent type (X1),
microwave time (X2), liquid-to-solid ratio (X3), ultrasound time (X4) were significant
(p-value<0.0001), which reconfirmed the screening result. Furthermore, the
interaction between solvent type and microwave time (X1X2), solvent type and liquid-
to-solid ratio (X1X3), solvent type and ultrasound time (X1X4) were significant, as their
p-values were equivalent to 0.0011, 0.0243, and 0.0001, respectively. However,
interaction between microwave time and liquid-to-solid ratio (X2X3), microwave time
and ultrasound time (X2X4), and the interaction between liquid-to-solid ratio and
ultrasound time (X3X4) were not significant. From these results, it can be concluded
that solvent type was the most important variable, which shows strongest influence
on TPC yielded from Ampelopsis cantoniensis. Similar trends have been observed
from previous research (Cacace & Mazza, 2003; Wang et al., 2013), which reported
that solvent type was a significant factor impacting on the total phenolic content of
extracts from milled berry and Sparganii rhizome, respectively.
Figure 7 shows the response surface plots for TPC. Figure 7a shows the interaction
of solvent type (X1) and microwave time (X2), while Figure 7b illustrates the
interaction of solvent type (X1) and solid-liquid ratio (X3), and Figure 7c presents the
interaction of solvent type (X1) and ultrasonic time (X4). In this research, solvent type
was the most influencing variables on TPC of Ampelopsis cantoniensis. As discussed
above, since the polarity of the solvent containing ethanol and water rises
continuously with the increase in ethanol concentration, the content of total phenolic
22
substances also increased gradually (Zhang et al., 2008). Due to that fact, solvent
type can interact with microwave time and ultrasound time to show strong influence
on TPC of Ampelopsis cantoniensis as similarly reported in other studies (Dahmoune
et al., 2015; Irakli et al., 2018).
Illustrated in Figure 7a, the maximum point of TPC is obtained within the solvent
ranging from 50% to 90% ethanol in water (v/v) and irradiation time ranging from
90 s to 150 s. Similarly, Sun et al. (2011) also reported a considerable increase in
TPC when the ethanol concentration ranges from 60% to 80% ethanol in water, with
70% ethanol solution as center point. Likewise, irradiation time ranging from 90 s to
150 s was chosen as experimental range for optimizing polyphenols from Citrus
sinensis peels by BBD (Nayak et al., 2015). Long microwave time, accompanied with
extremely high ethanol concentration can result in reduction in TPC, as the heat-
sensitive phenolic compounds are destroyed by high heat generated from long
microwave treatment (Mandal et al., 2007). On the other hand, if the ethanol
concentration is low, the ability of solvent to make cell swell to extract more
compounds is reduced (Dahmoune et al., 2015), thus less compounds are extracted.
According to Figure 7b, the interaction between liquid-to-solid ratio and solvent is
demonstrated when microwave time was fixed at 120 s and ultrasonic time was set
30 min. The maximum point of TPC was found when liquid-to-solid ratio was varied
from 20 to 40 mL per 1 g of dry sample, while ethanol concentration fluctuated from
50% to 90%. Similar ratio of solvent to sample was chosen to study the optimal
conditions for extracting phenolic compounds from Azadirachta indica – a naturally
occurring biopesticide in margosa species (Patil et al., 2021). At low liquid-to-solid
ratios, accompanied with lower polarity of solvent containing low ethanol
concentration, the mass-transfer of tissue-bound phenolic compounds into solvent is
reduced (Cussler & Cussler, 2009). Meanwhile, high liquid-to-solid ratio provides
more solvent to enter the cell (Sousa et al., 2016), interacting with increasing polarity
of solvent having high concentration of ethanol, can increase TPC of sample, as the
content of total soluble phenolic compounds rises with increasing amount of solvent
used (Bi et al., 2011; Cacace & Mazza, 2003). However, excessive quantity of solvent
and too high ethanol concentration can lead to decline in phenolic content (Elboughdiri
et al., 2020). Since too high liquid-to-solid ratio can result in improper microwave
heating (Zia et al., 2020), and restraint of aerosol generation and cavitation effect of
ultrasonic treatment (Sheng et al., 2018), the TPC obtained would be lowered.
23
Similarly, ethanol solvent of extremely high concentration can lead to reduction in
diffusion of phenolic compounds to solvent (Sant’Anna et al., 2012).
90
90
80
80
70 X3:
X1: 70 X1:
X2: 60 Liquid - 60 Solvent
Microwave 50 Solvent to-solid
50
time ratio
(a) (b)
90
80
X4: 70 X1: Solvent

Ultrasound 60
time 50
(c)
Figure 7. Response surface plots indicating the effects of the process variables on
TPC of Ampelopsis cantoniensis (a) Interaction of microwave time and solvent type;
(b) Liquid-to-solid ratio and solvent type; (c) Ultrasound time and solvent type
In Figure 7c, TPC of Ampelopsis cantoniensis increases gradually when the

concentration of ethanol and ultrasonic time increase, which shows similar pattern to
the interaction between solvent and microwave time. This phenomenon can originate
from the fact that longer ultrasonic treatment can break down cell wall more
24
thoroughly, resulting in more phenolic compounds released to the solvent (Martínez-
Ramos et al., 2020). As the ethanol solvent has higher polarity than water individually
(Ngan et al., 2017), more phenolic compounds can be removed from the plant matrix
to ethanol solvent, thus increase the yield of TPC. Furthermore, since the microwave
treatment prior to ultrasonic treatment can perform synergetic effects on cell wall,
this partly contributed to the rise of TPC extracted from sample (Sillero et al., 2020).
For these reasons, the maximum point was observed when the ultrasound time was
set from 20 to 40 min, while ethanol concentration varied from 50 to 90%. Similar
conditions of ultrasonic time range were also studied by Nipornram et al. (2018),
when these researchers choose this range for optimizing phenolic compounds from
mandarin (Nipornram et al., 2018). However, since ultrasound treatment was longer
than 30 min and ethanol concentration was higher than 70%, the value of TPC started
to drop gradually. This pattern was in accordance with the conclusion of Naczk &
Shahidi (2006) that long ultrasonic time can negatively destroy cell wall structure,
which reduced the extraction rate.
Based on the yields of TPC, the optimum extraction conditions (solvent containing
72.3% of ethanol in water (v/v), irradiation time of 122.9s, liquid-to-solid ratio of 31
mL/g, ultrasonic time of 31.2 min) were estimated using the model equation and
solving the regression equation. The theoretical total yield of phenolic content from
the optimum conditions was 264.03 mg GAE/g dry basis (with desirability of 0.950),
which is equivalent to 250.46 mg GAE/g sample. This value was much higher than
other tropical and temperate herbal teas such as Lemon myrtle (nearly 75.6 mg
GAE/g sample), oregano (58.6 mg GAE/g sample), green tea (141.2 mg GAE/g
sample), and Ti Kuan Yin (90.9 mg GAE/g sample), as reported by Chan et al. (2010).
These newly found optimal conditions were in accordance Nayak et al. (2015), when
the optimal microwave time was reported at 122s and a liquid-to-solid ratio of 25
mL/g. The result of this study was also comparable to the work of Gorgani et al.
(2017), when researchers found 30 min of ultrasonic treatment was of optimal
conditions for extracting piperine – a natural bioactive compounds from black pepper.
In addition, the optimal liquid-to-solid ratio was also approximate to the result of
Dahmoune et al. (2013), when 28 mL/g was recorded as the best ratio for obtaining
antioxidants from Citrus limon residues (Dahmoune et al., 2013), whereas Sun et al.
(2011) chose 70% ethanol as optimal solvent for extracting antioxidants from
kudingcha.
25
3.4. Comparison with traditional tea infusion:
The sample of Ampelopsis cantoniensis was infused in hot water, following the
research of Irakli et al. (2018), and the result was recorded in Table 7. From the
result, it can be concluded that sample extracted in approximately optimal conditions
yielded nearly twice total phenolic content than the vine tea that was traditionally
infused in hot water for 10 min. This result might relate to the solubility of solvent,
as discussed before. Ethanol solution has higher solubility and polarity, thus can
extract more phenolic compounds from Ampelopsis cantoniensis. Moreover, if the
infusion time was longer (up to 30 min), the value of TPC might be different from the
current value.
Table 7. Comparison between TPC of the sample extracted in approximately

optimal conditions and the sample infused traditionally 1
TPC
Sample
[mg GAE/g dry basis]
Sample extracted in approximately optimal
261.48 ± 7.04
conditions*
Sample infused traditionally 137.52 ± 3.04
*
Approximately optimal conditions: Solvent of 70% ethanol in water (v/v), liquid-to-
solid ratio of 30 mL/g, microwave time of 120 s, and ultrasonic time of 30 min.
1
TPC: Total phenolic content [mg GAE/g dry basis].
4. CONCLUSION:
In this study, optimization of microwave-ultrasound assisted extraction conditions for

phenolic compounds from Ampelopsis cantoniensis was successfully carried out. The
results showed that Ampelopsis cantoniensis is rich in the extractives. Thus, it can be
considered as an interesting herbal ingredient with high functionality and high
potential for valorization.
On the other hand, the results of screening by PBD successfully proved that solvent
type (in this case is ethanol in water (v/v) solution), microwave time, liquid-to-solid
ratio and ultrasonic time were the most important variables that affect the total
phenolic content of vine tea. Optimization by Box-Behnken design also confirmed that
these individual parameters, especially solvent type, have significant impacts on final
yields of TPC. Meanwhile, since ethanol concentration was the most influencing
parameter, it showed strongest interactions with the three remaining variables. PBD
and BBD was also found to be effective mathematical tools in this study, since the
number of experiments was more than 50% less than the combination of full factorial
26
design and BBD (FFD requires 128 runs for screening seven factors at a time). Thus,
it can be concluded that this two-step optimization was cost-effective and less time-
consuming.
Optimal conditions for obtaining maximum content of phenolic compounds were as

follow: solvent containing 72.3% of ethanol in water (v/v), irradiation time of 122.9s,
liquid-to-solid ratio of 31 mL/g, ultrasonic time of 31.2 min. Furthermore, TPC of
sample extracted under approximately optimal conditions was nearly twice that value
of traditionally infused vine tea in hot water for 10 min.
Taking all above into account, it can be concluded that MUAE is a good extraction
methodology, not only for the good-quality extracts, but also for its efficiency. The
results obtained in this research confirmed the potential of Ampelopsis cantoniensis
as a natural source of phenolic compounds, which can be further studied to employ
in fields as varied as pharmaceuticals, chemistry, material, and food industry.
5. RECOMMENDATIONS:
Due to the outbreak of Covid-19 pandemic, Ho Chi Minh City was locked down. For
that reason, this project could not be completed as planned due to the limited time
for lab work, thus hoping to have sympathy from the Thesis Committee for these
shortcomings.
Moreover, in this research, though there were strong interactions between solvent
and other variables such as liquid-to-solid ratio, microwave time and ultrasonic time,
the relationship between microwave time and ultrasound time was not as clear and
significant as expected. Thus, further studies could be carried out in the future to
evaluate the impact and interaction of these factors and other parameters such as
microwave power, ultrasound amplitude, ultrasonic temperature and particle size on
TPC of Ampelopsis cantoniensis extract.
27
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Date of approval: 11/12/2021
Supervisor’s signature Lê Ngọc Liễu

Optimal Extraction of Phenolic Compounds from Ampelopsis cantoniensis

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Optimal Extraction of Phenolic Compounds from Ampelopsis cantoniensis

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VIETNAM NATIONAL UNIVERSITY – HO CHI MINH CITY

TWO-STEP OPTIMIZATION OF MICROWAVE-ULTRASOUND-ASSISTED

The School of Biotechnology, International University

In partial fulfillment of the requirements for the degree of

B.S. in Food Technology

Student name: Tran Bao Uyen - BTFTIU17007

Supervisor: Dr. Le Ngoc Lieu (Ph.D)

First of all, I am grateful to my university – International University for organizing the

Table 1. Summary of factor levels set in PBD……………………………………………………………8

Figure 1. Vine tea product in Viet Nam………………………………………………………………………4

Uyen B. Trana, Lieu N. Lea,*

Ampelopsis cantoniensis is a highly functional herbal ingredient of Vietnam. In this

Key words: two-step optimization; Ampelopsis cantoniensis; vine tea; microwave-

Microwave-ultrasound-assisted extraction (MUAE), which combines two treatments -

2. MATERIAL AND METHODS:

2.1.2. Chemicals and reagents:

2.2.2. Microwave-ultrasound-assisted extraction:

Phenolic compounds from Ampelopsis cantoniensis powder were extracted using a

2.2.4. Screening of variables by Plackett-Burman design:

The extraction of phenolic compounds from Ampelopsis cantoniensis was significantly

Table 1. Summary of factor levels set in PBD1

%EthOH: ratio of ethanol in water (v/v)

After conducting initial screening stage following Plackett-Burman design, level

2.2.6. Box-Behnken design:

𝒀 = 𝑩𝟎 + ∑𝒌𝒊=𝟏 𝑩𝒊 ∗ 𝒙𝒊 + ∑𝒌𝒊=𝟏 𝑩𝒊𝒊 ∗ 𝒙𝟐 + ∑𝒌𝒊=𝟏 𝑩𝒊𝒋 ∗ 𝒙𝒊 ∗ 𝒙𝒋 + 𝑬 (1)

2.2.7. Regression analysis:

2.3. Determination of total phenolic content (TPC):

TPC of the extract were determined by Folin-Ciocalteau reagent methodology, based

Y = 0.0163*X + 0.0088 (2)

Where Y is the absorbance of sample measured at 765 nm, X is the concentration of

Then, TPC was calculated following Eq. 3:

2.4. Statistical analysis:

3. RESULTS AND DISCUSSION:

Table 3. Result analysis of Plackett-Burman design for experimental results for

Credibility of regression equation of Plackett-Burman design

3.2. Level screening of experimental factors:

3.2.1. Effect of solvent type on total phenolic content:

Figure 3 indicates that the concentration of ethanol in water performed a strong

TPC [mg GAE/g dry basis]

Results were obtained with Minitab 18.0 software

Figure 3. Effect of different solvent types on TPC1,2

3.2.2. Effect of microwave time on total phenolic content:

TPC [mg GAE/g dry basis]

Results were obtained with Minitab 18.0 software

Figure 4. Effect of different microwave times on TPC1,2

3.2.3. Effect of liquid-to-solid ratio on total phenolic content:

In almost all extraction techniques, liquid-to-solid ratio is a critical parameter that

In the extraction process, 1 g of Ampelopsis cantoniensis powder of 250 µm was

Figure 5 demonstrates the effects of liquid-to-solid ratio on the yields of phenolic

Results were obtained with Minitab 18.0 software

Figure 5. Effect of different liquid-to-solid ratios on TPC1,2

3.2.4. Effect of ultrasound time on total phenolic content:

Results were obtained with Minitab 18.0 software

Figure 6. Effect of different ultrasound times on TPC1,2

3.3. Optimization by response surface methodology (RSM):

Factor Low (-1) Middle (0) High (+1)

%EthOH: ratio of ethanol in water (v/v)

Run Pt S.T M.t L-S R U.t TPC

In this study, the adjusted R2 of 0.9605 was found to be in reasonable agreement

Table 6. Result analysis of Box-Behnken design1,2,3

X1 1200.3 1 1200.29 32.79 < 0.0001 Significant

X2 1031.9 1 1031.85 28.19 < 0.0001 Significant

3.3.2. Optimization of total phenolic content: