food and bioproducts processing 9 1 ( 2 0 1 3 ) 1–6

Contents lists available at SciVerse ScienceDirect

Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Dynamic high pressure microfluidization-assisted

extraction and antioxidant activities of sweet potato
(Ipomoea batatas L.) leaves flavonoid

Xiaoqin Huang a , Zongcai Tu a,b,∗ , Hui Xiao c , Zhi Li a , Qiuting Zhang a , Hui Wang d ,
Yueming Hu a , Lan Zhang a
a State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China
b College of Life Science, Jiangxi Normal University, Nanchang, Jiangxi 330022, China
c Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, United States
d College of Life Science and Food Engineering, Nanchang University, Nanchang, Jiangxi 330031, China

a b s t r a c t

Dynamic high pressure microfluidization (DHPM) is an emerging technology utilizing the collective forces of high-
velocity impact, high-frequency vibration, instantaneous pressure drop, intense shear, cavitation, and ultra-high
pressures. DHPM technology was applied to improve the extraction of flavonoids from sweet potato leaves, and its
effect was evaluated using six samples under various conditions. Pretreatment with DHPM was found to strengthen
the antioxidant activities of the flavonoid, while no treatment or post-treatment with DHPM, produced weaker antiox-
idant activities. This suggests that the DHPM technology may provide a promising method of utilizing sweet potato
leaves as a natural antioxidant in food and pharmaceutical industry.
© 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

Keywords: Sweet potato leaves; Flavonoid; DHPM; Antioxidant

1. Introduction In 1999, sweet potato accounted for approximately 20% of

Sweet potato (Ipomoea batatas L.) is one of the most impor-
tant economic crops in the world (Bovell-Benjamin, 2007). It is
grown widely around the world and ranks fifth as a food crop
in developing countries after rice, wheat, maize and cassava
(Lin et al., 2007). The tubers of sweet potatoes are extensively
utilized both as food and as material for the production of bev-
erages, pasta, alcohol drink, and natural colorants (Yoshimoto
et al., 2002; Steed and Truong, 2008). In fact, the leaves of
the sweet potato contain many nutrients including protein,
dietary fiber, carotenoids, vitamins and minerals (Ishida et al.,
2000). In addition, they could be harvested several times each
year and their harvest is never worse than that of ordinary
leafy vegetables, therefore the annual yield is much higher
than other green vegetables. It has been reported that there is
a large amount of flavonoids with high bioactivity in the leaves
of sweet potato (Islam Shahidul et al., 2003).
the total world production of root and tuber crops (FAO, 1999).
Asia is the largest sweet potato-producing region in the world,
with an annual production of 125 million tons. China produces
roughly 65% of the world’s sweet potato, making it the lead-
ing supplier of sweet potatoes in the world (Hijmans et al.,
2002). However, the leaves of sweet potato have been neglected
except for a partial use as livestock feed in the countryside
(Xu et al., 2010), causing huge resource waste. So it is neces-
sary to study the processing and bioactivity of sweet potato
leaves cultivated in China to create value-added compounds
from a low-cost and underutilized raw material and upgrade
agricultural waste.
It has been reported that high pressure is an effective
method when applied in extracting the bioactive com-
pounds of natural resources, such as anthocyanins (Corrales
et al., 2009) and caffeine (Xi, 2009). Dynamic high pres-
sure microfluidization (DHPM) is an emerging dynamic high

∗
Corresponding author at: State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang, Jiangxi 330047, China.
Tel.: +86 791 88305938.
E-mail address: tuzc mail@yahoo.com.cn (Z. Tu).
Received 4 November 2011; Received in revised form 6 July 2012; Accepted 27 July 2012
0960-3085/$ – see front matter © 2012 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.fbp.2012.07.006
2 food and bioproducts processing 9 1 ( 2 0 1 3 ) 1–6
pressure technology applying the joint forces of high-velocity
impact, high-frequency vibration, instantaneous pressure
drop, intense shear, cavitation, and ultra-high pressures up
to 200 MPa (Liu et al., 2009). Recent studies of DHPM are
focused on the modification of biomacromolecule like protein
(Tu et al., 2009), dietary fiber (Wan et al., 2009), and enzyme
(Liu et al., 2010). Our research group applied DHPM to extract
bioactive compounds for the first time, and we found that
DHPM-assisted extraction could efficiently improve the yield
of flavonoids from sweet potato leaves. However, the bioactivi-
ties of flavonoid extracted using DHPM have not been studied.
Antioxidant activity is one of the most important properties of
flavonoid. The objective of this study was to prepare flavonoid
samples by different methods and investigate the antioxidant
activities of each sample.
2. Materials and methods
2.1. Materials
The leaves of the sweet potato were collected from the farm-
land of Nanchang suburbs. They were washed gently and
dried, and then grounded by an ultramicro pulverizer (Zhe-
jiang Wenling, China), and sieved through a 200 mesh screen,
then stored in a sealed polyethylene bags and kept in a drier
for use.
The reagents used in this study, such as rutin was obtained
from the National Pharmaceutical Engineering Center For
Solid Preparation in Chinese Herbal Medicine (Nanchang,
China), DPPH (1,1-diphenyl-2-picrylhydrazyl) was purchased
from Sigma Chemical Co. (St. Louis, USA). All other chemicals
were of analytical grade and obtained from Tianjin Reagent
Company (Tianjin, China).
2.2. Methods
2.2.1. Pretreatment of materials
The sweet potato leaves powder was pretreated by soxhlet
extraction with diethyl ether to remove the fat, pigments and
fat soluble impurities. Then it was dried in an oven at 50 ◦ C for
2 h, and stored in a drier for use.
2.2.2. Extraction of flavonoid
10.00 g of pretreated powder was immersed in 400 mL of
70%(v/v) ethanol for 12 h in a 4 ◦ C refrigerator, then homoge-
nized at 30 MPa by a valve homogenizer (GYB60-6S, Shanghai
Huadong homogenizer Co., China). The emulsion was treated
by a DHPM homogenizer (M-110EH, Microfluidics Company,
USA) at 100 MPa twice. Then it was extracted by refluxing at
75 ◦ C for 2 h twice, followed by cooling and centrifugation at
4000 rpm for 10 min. The supernatant was the crude extract
pretreated by DHPM (MC). Ethanol crude extract (EC) was pre-
pared using the same methods as above, however, without
pretreatment using DHPM. Ethanol crude extract with post-
treatment of DHPM (ECM) was obtained by applying 100 MPa
DHPM on EC twice.
2.2.3. Purification of sweet potato leaves flavonoid
The crude extract of flavonoid samples was purified by LK001
(Shandong LuKang Medicine group Co. Ltd., China) macro-
porous absorption resin in a chromatographic column (ϕ
16 mm × 400 mm). The samples were eluted using 70% (v/v)
ethanol as the eluent at a flow rate of 1.5 mL/min, and
the eluent was collected at the flow rate of 2 mL/min. The
corresponding purified samples of MC, EC, and ECM were MCP,
ECP and ECMP, respectively.
2.2.4. Total flavonoids determination
The content of the total flavonoid in each extract was mea-
sured by colorimetric aluminum chloride method as described
by Liu et al. (2011).
2.2.5. DPPH radical scavenging activity
The DPPH radical scavenging activity was measured using the
method of Wang et al. (2008) with modifications. Briefly, 2 mL
of 200 ␮g/mL DPPH was added to 2 mL various concentrations
(10–80 ␮g/mL) of flavonoid samples in 70% (v/v) ethanol. The
reaction mixture was incubated for 30 min at room temper-
ature in the dark. The DPPH radical scavenging activity was
determined by measuring the absorbance at 517 nm using a
spectrophotometer (TU-1900 PuXiTongYong, Beijing, China).
The DPPH radical scavenging activity (%) of the sample was
calculated as follows:
A0 − (As − Ac )
Scavenging activity = (1)
A0
where As is the absorbance with the presence of DPPH and
samples; A0 is the absorbance with the presence of DPPH and
70% (v/v) ethanol and Ac is the absorbance with the presence
of samples and 70% (v/v) ethanol.
2.2.6. Superoxide anion radical scavenging activity
The superoxide anion radical scavenging activity was evalu-
ated by the method of Li et al. (2011) with minor modifications.
Briefly, 5 mL of 50 mM Tris–HCl (pH 8.2) was mixed with 2 mL
samples of various concentrations, followed by incubation in
25 ◦ C water bath for 20 min. Then 50 ␮L of 5 mM pyrogallol
solution (preheated at 25 ◦ C) was quickly added to the above
mixture and shaken well. The absorbance of the mixture was
measured at 325 nm every 30 s within 5 min in a spectrometer.
The Tris–HCl (pH 8.2) solution of 50 mM was used as blank.
The superoxide anion radical scavenging activity was cal-
culated by the following formula:
(A0 /t) − (Ai /t)
Scavenging activity = (2)
A0 /t
where A0 /t was the auto-oxidation rate of the control group
(the ultrapure water instead of sample), Ai /t was the auto-
oxidation rate of samples.
2.2.7. Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity was measured using
the method of Su et al. (2009) with modifications. Briefly, 2 mL
sample was mixed with 2 mL of 2 mM freshly prepared ferrous
sulfate, 2 mL of 6 mM dinitrosalicylic acid–ethanol solution
was then added to the mixture. Finally, 2 mL of 6 mM hydrogen
peroxide was added to the mixture to initiate the reaction by
incubating at 37 ◦ C water bath for 30 min. The absorbance was
measured at 510 nm. Ultrapure water was used to substitute
hydrogen peroxide as the control. The scavenging activity was
calculated as the following equation:
(A1 − A2 )
Scavenging activity = 1 − (3)
A0
where A0 was the absorbance with the presence of ultrapure
water instead of sample; A1 was the absorbance of sample
 food and bioproducts processing 9 1 ( 2 0 1 3 ) 1–6 3

Table 1 – The contents of total flavonoid in different extracts.

Samples EC ECM MC ECP ECMP MCP

Concentration (mg/100 mL) 44.3 ± 0.1 a

42.8 ± 0.1 a
58.4 ± 0.1 b
316.2 ± 0.2 c
314.4 ± 0.2c
346.8 ± 0.3d

Values are mean ± standard deviation of three replicate analyses. Different letters indicate significant differences between mean values of
treatments (p < 0.05).

100 100 EC
EC
ECP
ECM
ECMP
MC
MCP
80 Rutin 80
Rutin
Scavenging activity (%)

Scavenging activity (%)

60 60

40 40

20 20

0 0
0 20 40 60 80 100 0 20 40 60 80
Sample concentration (μg/mL) Sample concentration (μg/mL)

Fig. 1 – Scavenging activities on DPPH of crude samples Fig. 2 – Scavenging activities on DPPH of flavonoids and
and rutin. rutin.

and reagents; and A2 was the absorbance with the presence
of ultrapure water instead of hydrogen peroxide.
2.2.8. Effect of DHPM on the cell structure of sweet potato
leaves
The cell structure of sweet potato leaves powder was exam-
ined using a scanning electron microscope (SEM). Samples
were dispersed on a double-sided adhesive tape mounted on
a circular aluminum stub. The aluminum stub was placed in
a vacuum chamber and coated with gold. The samples were
photographed in a SEM apparatus (Hitachi model S-570, Tokyo,
Japan) at an accelerating potential of 10 kV.
Each experiment was conducted in triplicate and the data
were expressed as means ± one standard deviation (SD). In
case of variation analysis, significance was p < 0.05 level of
Duncan test using analytical software SPSS statistics (Version
16.0, SPSS Inc., Chicago, IL, USA).
3. Results and discussion
3.1. The contents of total flavonoid in different extracts
The contents of total flavonoid in different extracts are dis-
played in Table 1. The MC presented higher flavonoid content
than the other two crude extracts, suggesting that the pre-
treatment with DHPM could accelerate the extraction of
flavonoids from sweet potato leaves. The EC and ECM had
no significant difference, indicating that DHPM showed lit-
tle influence on the flavonoid extract. Similarly, DHPM also
increased the content of falvonoid in the purified samples.
3.2. DPPH scavenging activity
Fig. 1 shows the DPPH scavenging activity of the crude samples
and rutin. The samples showed a concentration-dependent
DPPH scavenging activity, with significant stronger DPPH scav-
enging activity than rutin. This may be attributed to the
polyphenols extracted from sweet potato leaves. Xu et al.
(2010) extracted polyphenols from sweet potato leaves using
70% ethanol with strong DPPH scavenging activity. Among
the three crude samples, the one prepared by DHPM-assisted
extraction exhibited much higher scavenging activity than
the other two, while the one prepared by traditional ethanol
extraction and the traditional extract post-treated by DHPM
showed no significant difference.
Fig. 2 displays the scavenging activity of purified samples
and crude extract on DPPH radicals. The purified samples
showed much stronger scavenging activities than the crude
extract, probably because the antioxidant components, i.e.,
polyphenolic compounds were enhanced after purification.
Similarly, the purified sample assisted by DHPM treatment
exhibited higher scavenging activity than other samples. The
force of DHPM treatment may disrupt the cellular walls, lead-
ing to an accelerated mass transfer and enhanced solvent
penetration into the cells. Therefore, a higher content of
antioxidant can be obtained in the DHPM assisted extract,
resulting in a higher scavenging activity. Joo et al. (2012) also
reported that the mechanism of ultra high pressure extraction
was that the plant inner-membranes were destroyed, allow-
ing increased solvent influx and increased contact with the
solvent when the pressure was increasing.
3.3. Superoxide anion radical scavenging activity
The superoxide anion radical scavenging activities of crude
extract, purified samples and rutin are shown in Fig. 3. The
scavenging activity increased with the concentration. The
superoxide anion radical scavenging activity of MCP was close
to that of rutin. The MCP showed great scavenging activity
and, when the concentration of MCP reached 1 mg/mL, its
4 food and bioproducts processing 9 1 ( 2 0 1 3 ) 1–6

Rutin 100
100 MCP Rutin
ECP MCP
ECMP 80 ECP
80 MC ECMP
EC MC
Scavenging activity (%)

Scavenging activity (%)

ECM EC
60
60 ECM

40
40

20 20

0 0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Sample concentration (mg/mL) Sample concentration (mg/mL)

Fig. 3 – Superoxide anion radical scavenging activity of Fig. 4 – Hydroxyl radical scavenging activity of flavonoids
flavonoids prepared by different methods. prepared by different methods.

superoxide anion radical scavenging activity was 92 ± 5%. The
purified ones clearly exhibited much higher scavenging activ-
ity than the crude ones. Among the three purified samples,
MCP showed greater scavenging activity than the other two,
while the ECMP exhibited the weakest scavenging activity.
Similar trend was displayed in the crude ones. It has been
well documented that, for flavonoids, the keto group at C-4
and hydroxy group at C-3 or C-5 of the C-ring were effec-
tive in scavenging superoxide anions, and the greater the
number of hydroxy groups, the better is the scavenging activ-
ity (Wang et al., 2010). Therefore it can be concluded that
the DHPM-assisted extraction has changed the proportion of
components in the flavonoid and enhanced the antioxidant
compound dissolution. In contrast, the DHPM treatment on
ethanol extract may accelerate the oxidation of flavonoid,
resulting in a decrease of superoxide anion scavenging activ-
ity. It has been reported that different extraction methods led
to diverse components in the flavoniod from spearmint (Men-
tha spicata L.) leaves (Bimakr et al., 2011).
3.4. Hydroxyl radical scavenging activity
The hydroxyl radical scavenging activity of the samples and
rutin are shown in Fig. 4. When the sample concentration
was lower than 0.6 mg/mL, rutin had greater hydroxyl radi-
cal scavenging activity than MCP, but after that they became
very close to each other. Of all the treatments, the purified
DHPM-assisted extract showed the most distinct effect on

Fig. 5 – SEM images of sweet potato leaves powder by different treatment with 5000 enlargement. (A) before extraction; (B)
treated by valve homogenizer at 60 MPa; (C) treated by DHPM at 60 MPa; (D) treated by DHPM at 100 MPa; (E) unfolded cell
treated by DHPM at 140 MPa; and (F) folded cell treated by DHPM at 140 MPa.
 food and bioproducts processing 9 1 ( 2 0 1 3 ) 1–6
scavenging hydroxyl radicals. And its scavenging activity was
75 ± 3% when at the concentration of 1.0 mg/mL, indicating its
effective scavenging activity on hydroxyl radicals.
3.5. Effect of DHPM on the cellular structure of sweet
potato leaves
The SEM images of the sweet potato leaves powder untreated
and treated by different methods are shown in Fig. 5. Fig. 5(A)
was the raw material grounded before extraction. Fig. 5(B) indi-
cated the valve homogenizer had loosened the particle surface
of the materials to a certain extent. In Fig. 5(C), 60 MPa DHPM
treatment had disrupted the surface of the materials cellu-
lar structure, which is crumpled, and the integrity of the cell
wall was ruined. From Fig. 5(D) and (E), it could be found that
the cellular structure of the sweet potato leaves had been
destroyed, and the surface area of the cell was increased with
the pressure increasing.
Fig. 5(F) demonstrates that after treated by DHPM at
140 MPa, the cell structure was damaged, and the content
of the cell was released and was crushed into smaller gran-
ules. This may due to the instantaneous high pressure
promoting the solvent permeation into the cell, and sub-
sequently when the pressure was released instantaneously
the cellular content was carried by the solvent and released
from the cell membrane resulting in the rupture of the
cell (Prasada et al., 2009). Hence, it could be confirmed
that the DHPM treatment had accelerated the dissolution of
antioxidant compounds, leading to the rise of antioxidant
activity.
4. Conclusion
It was confirmed in this work that DHPM could acceler-
ate the extraction of sweet potato flavonoid. The crude and
purified flavonoid extracted from the sweet potato leaves
showed DPPH, superoxide anion and hydroxyl radical scaveng-
ing activity in a dose-dependent manner, and the scavenging
sequence was: DPPH scavenging activity > superoxide anion
scavenging activity > hydroxyl radical scavenging activity. The
purified sample showed stronger antioxidant activity than
the crude ones. The extract pretreated with DHPM exhibited
stronger radical scavenging activity than the ethanol solvent
extract and the extract post-treated by DHPM, suggesting that
the DHPM assisted extraction was a potential method for
preparing sweet potato leaves flavonoid with higher antiox-
idant activity.
Acknowledgements
The authors are grateful to the financial support of the
National Science Foundation of China (No. 20976078 and
No. 21276118), Program for State Key Laboratory of Food
Science and Technology (No. SKLF-TS-200923), Doctoral Fund
of Ministry of Education of China (No. 20093601110003)
and National Program on Key Basic Research Project
(No. 2012CB126314).
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