South African Journal of Botany 112 (2017) 376–382

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South African Journal of Botany

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

Antioxidant and hepatoprotective effects of different ethanol

concentrations in extraction from leaves of Sasa quelpaertensis Nakai
J.-H. Ra a,1, M. Nakamura a,1, K.H.I.N.M. Herath b, Y. Jee b, J.-S. Kim a,c,⁎
a
Majors in Plant Resource and Environment, College of Agriculture & Life Sciences, SARI, Jeju National University, Jeju 63243, Republic of Korea
b
Department of Veterinary Medicine and Veterinary Medical Research Institute, Jeju National University, Jeju 63243, Republic of Korea
c
The Research Institute for Subtropical Agriculture and Biotechnology, Jeju National University, Jeju 63243, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: Biological ability and optimal extraction yield of Sasa quelpaertensis Nakai leaf extract with different ethanol
Received 11 May 2017 (EtOH) concentrations (20, 40, 60, 80, and 100%) and distilled water were determined. The 60% EtOH extract ar-
Received in revised form 12 June 2017 chived the highest extraction yield and exhibited the highest rates of DPPH and ABTS radical scavenging, reduc-
Accepted 30 June 2017
ing power, and tyrosinase inhibitory activity. Also, when the various concentrations of EtOH (0–1600 mM) were
Available online 14 July 2017
applied, significant loss was observed at 400 mM and above. However, the 60% EtOH extract of S. quelpaertensis
Edited by V Steenkamp Nakai leaves induced cell viability by 7% and 9% in 400 and 800 μg/ml concentrations, respectively. Moreover, for
the combination of 60% EtOH extract from S. quelpaertensis Nakai leaves (0–800 μg/ml) and 400 mM EtOH, a 6.3%
Keywords: increase of viability was observed at 800 μg/ml as compared to no sample treatment. These results indicate that
Sasa quelpaertensis Nakai leaves S. quelpaertensis Nakai leaves could be good candidate as safe antioxidants and tyrosinase inhibitor.
Antioxidant activity © 2017 SAAB. Published by Elsevier B.V. All rights reserved.
EtOH-induced cell viability
Different EtOH concentrations
Tyrosinase inhibitory

1. Introduction (Jiménez-Zamora et al., 2016). Antioxidants, bioactive compounds in

Oxygen is the essential element of our lives. It is one of main
constituents of air, occupying 21% of total air mass, and it is also a signif-
icant component of many other fundamental substances such as water
and carbon dioxide (Wildman et al., 2004). If there was no oxygen
atom, these compounds simply would not exist. Oxygen is therefore
obviously deeply associated with our bodily functions. In the body,
reactive oxygen species, or ROS, takes on a positive role in fighting
back against intruding pathogens, helping us to survive harsh environ-
ments, or allowing us to adjust to difficult environmental changes.
ROS are oxygen-based molecules or compounds like the superoxide
anion (O−2 ), singlet oxygen (O2), hydrogen peroxide (H2O2), and the hy-
droxyl radical (•OH), which are spontaneously generated in the body
(Lee and Lim, 2008). However, because of our industrialized and social-
ized world, numerous extraneous influences (smoke, diet, alcohol, illicit
drugs, and many others) in addition to simple aging processes diminish
the body's ability to counteract excessive ROS (Jeong et al., 2011), induc-
ing oxidative stress and subsequently leading to attacks on DNA, pro-
teins, and lipids. Consequently, oxidative reactions could promote
cancer, neurodegenerative disorders, and cardiovascular diseases
⁎ Corresponding author at: Majors in Plant Resource and Environment, College of
Agriculture & Life Sciences, SARI, Jeju National University, Jeju 63243, Republic of Korea.
E-mail address: aha2011@jejunu.ac.kr (J.-S. Kim).
1
Jong-Hwan Ra and Masaya Nakamura are contributed equally to this work.
charge of maintaining the rigidity of cell structures, stable the free rad-
icals and prevent the spread of more oxidative damage, thus protecting
the body from endogenous collapse (Zou et al., 2016). Butylated
hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are the
two main synthesized antioxidants that have been broadly recognized
used from few decades ago, but because of some reports indicating
the possibility of carcinogenic effects and high dose toxicity to the
cells (Yu et al., 2000), governments in some countries have strictly
regulated their use in food products; still, the extent of prohibition is
generally different depending on the country (Ding and Zou, 2012). As
a result of such restrictions, natural antioxidants are now being given
significant attention because of their minimal side effects and limited
toxicity. For instance, a number of technical reports have been
published supporting the antioxidant properties of plants (Tauches
et al., 2015), vegetables (Nour et al., 2013), and even microorganisms
(Mohamed et al., 2014).
Sasa quelpaertensis Nakai is a perennial plant also called the “Jeju
dwarf bamboo”. S. quelpaertensis Nakai belongs to the Poaceae family.
Even though the genus Sasa, bamboo grass, is broadly distributed
throughout many Asian countries, S. quelpaertensis Nakai only grows
near Mount Halla on Jeju Island, South Korea (Kim et al., 2014). As a
local remedy, dry leaves of S. quelpaertensis Nakai have been used to
treat diabetes, hypertension, and gastritis, and they have also been
popular for making bamboo tea for many years (Sultana and Lee,
2010). Recent research has revealed the beneficial properties of these

http://dx.doi.org/10.1016/j.sajb.2017.06.027
0254-6299/© 2017 SAAB. Published by Elsevier B.V. All rights reserved.
 J.-H. Ra et al. / South African Journal of Botany 112 (2017) 376–382
leaves for their anticancer (Kim et al., 2013a), and anti-inflammatory
effects (Kim et al., 2015), among many others valuable uses. Also, the
researches in cosmetic field are prevailing. For instance, Sultana and
Lee (2010) isolated 10 new alkene glycosides from an ethyl acetate frac-
tion of S. quelpaertensis Nakai, and one of the 10 new alkene glycosides
(compound 13) showed strong 1,1-diphenly-2-picrylhydrazyl (DPPH)
radical scavenging activity with IC50 of 26.8 μg/ml. However,
S. quelpaertensis Nakai has been gathering attention not only for its
remarkable effects on the health of human beings, but also for its
threatening invasion of the habitat of local indigenous plants, given its
notable reproducing capability along with the effects of gradual climate
change. Therefore, efficient elimination of unnecessary plants and
research about their potential use for improving health is urgently
required.
In this study, in order to evaluate the natural source of antioxidants
and tyrosinase inhibitors, which block the melanin biosynthesis that
causes many dermal diseases such as melisma, we evaluated
antioxidant activity assays (DPPH, 2,2′-azino-di-2-ethyl-benzthiazoline
sulphonate (ABTS) radical scavenging assay, and reducing power
assay), tyrosinase inhibitory activity. In addition to these assays, the he-
patic cell protective effect of S. querpaeltensis Nakai leaves is performed
using 60% ethanol extract since some cytological researches reported
about S. querpaeltensis Nakai. We also conducted experiments with dif-
ferent concentrations of EtOH (20, 40, 60, 80, and 100%) and distilled
water to determine which solvent and concentrations are best suited
to extract the active compounds from S. quelpaertensis Nakai leaves.
2. Material and methods
2.1. Chemicals
Dulbecco's modified eagle medium (DMEM), fetal bovine serum
(FBS), and penicillin–streptomycin were purchased from Gibco BRL.
EtOH was purchased from EMD Millipore Co., USA. Trypan blue dye, L-
tyrosine, and arbutin were purchased from Sigma Chemical Co. in St.
Louis, MO, USA. Sodium dodecyl sulfate (SDS), dimethyl sulfoxide
(DMSO), and 2,2-azino-bis-(3-ethylbenzothiazothiazoline-6-sulfornic
acid (ABTS) were purchased from Life Sciences, Amresco, Solon. DPPH,
BHT, trichloroacetic acid, and potassium persulfate were purchased
from Wako Pure Chemical Industries Ltd. in Osaka, Japan. All reagents
were of analytical grade or better.
2.2. Plant collection and extraction
The S. quelpaertensis Nakai leaves were purchased from the Jeju Plant
Resource Lab in 2015 in dry condition. After being purchased, the leaves
were pulverized into 1 mm fine powder to increase the surface area.
Then, coarse powder (5 g) was extracted with the addition of 100 ml
of distilled water, 20% (v/v), 40% (v/v), 60% (v/v), 80% (v/v), and 100%
EtOH in an ultrasonic bath (Power Sonic 520, Hwashin Co., Korea) for
90 min below 25 °C. The extraction process was carried out in triplicate
to maximize the yield. The solution was filtered with Whatman No. 2
filtering paper (Whatman International Limited, Kent, England) and
concentrated into dry material by using a rotary vacuum evaporator
(Hei-VAP Precision 280 rpm, Heidolph, Germany). The sample was
stored at − 20 °C until further analysis. The extraction yield was
calculated with the equation below:
Extraction yield ð%Þ
¼ fweight of dry soluble solid ðgÞ=weight of sample ðgÞg  100 ð1Þ
2.3. Determination of DPPH radical scavenging activity
The DPPH radical scavenging activity assay was implemented
through the method of Blois (1958). In the process, 100 μl methanol
377
(MeOH) was put in to the first and third rows in a 96-well microtiter
plate, and 200 μl of an appropriate diluted sample was added to the
second row. The sample solution was then serially diluted to adjust to
the 100 μl final volume. After that, 100 μl of DPPH solution (0.15 mM)
was mixed with each reactant, except for the first row, which served
as a control. Then, the reactant was incubated for 30 min at room tem-
perature in a dark chamber. Finally, the absorbance was measured at
490 nm by using a microplate reader (iMark, Bio-Rad Laboratories,
Inc., USA). As positive controls, α-tocopherol and BHT were used, and
the DPPH radical scavenging activity was calculated by employing the
equation below:
DPPH radical scavenging ð%Þ ¼ ð1−AS =AC Þ  100 ð2Þ
where AC is the absorbance of the negative control (MeOH solution),
and AS is the actual absorbance value of each of the samples. The
lower the sample value, the higher the scavenging activity. The calculat-
ed value was expressed as RC50, which is the required amount of the
sample to inhibit 50% of the DPPH radical after 30 min.
2.4. Analysis of ABTS radical scavenging activity
To measure how much of the ABTS radical was scavenged by the
sample solution, the technique of Re et al. (1999) was implemented
with minor modification. To generate the ABTS radical, potassium
persulfate (2.45 mM) and ABTS (7 mM) were mixed together and
kept for 16 h in dark conditions. After that, the initial absorbance was
adjusted from 1.2 to 1.3 through dilution with a sodium phosphate buff-
er at 750 nm by UV-spectrophotometer. Next, 40 μl of each sample solu-
tion was allowed to react with the ABTS solution and incubated for
20 min at ambient temperature. Finally, the absorbance was measured
through a microplate reader at 750 nm. Scavenging of the ABTS radical
was calculated as follows:
ABTS radical scavenging ð%Þ ¼ ð1−AS =AC Þ  100 ð3Þ
where AC stands for absorbance of the negative control (only the
sodium phosphate buffer) and AS is the actual absorbance value of
each of the samples. The higher the sample value, the higher the scav-
enging activity. The calculated value was expressed as RC50, which is
the required amount of sample to inhibit 50% of the ABTS radical after
20 min.
2.5. Determination of reducing power assay
The reducing power assay was employed according to the meth-
od of Hyun et al. (2014) with subtle modification. First, the sample
solution (10, 20, 30 μl) was added to the test tube and adjusted up
to 100 μl with distilled water. Then, the 500 μl of sodium phosphate
buffer (pH 6.6, 0.2 M) and 1% of potassium ferricyanide were
mixed with the sample solution and incubated for 20 min at 50 °C.
After incubation, 2.5 ml of 10% trichloroacetic acid was added to
stop the reaction, and the aliquot of the mixture (500 μl) was
added to the microtube with 0.1% of ferric chloride. Subsequently,
absorbance was read at 700 nm with a UV-spectrophotometer (UV-
1800, Shimadzu Co., Japan).
2.6. Inhibition of tyrosinase activity
For tyrosinase inhibitory activity, L-tyrosine was used as a sub-
strate according to the method adopted by Mason and Peterson
(1965). For each sample solution, 1.66 mM L-tyrosine and a 0.1 mM
potassium phosphate buffer were mixed in before adding 40 μl tyros-
inase solution (125 U/ml). After that, the mixture was incubated for
30 min at 37 °C. The absorbance was read at 490 nm with a micro-
plate reader. Arbutin was also used as a positive control. The
378 J.-H. Ra et al. / South African Journal of Botany 112 (2017) 376–382
tyrosinase inhibitory activity was calculated by the following
equation:
Inhibition of tyrosinase ð%Þ ¼ ð1−ðAS =AbÞ=AcÞ  100 ð4Þ
The substance without the sample solution (Ac) served as the
control, and the substance without the tyrosinase solution (Ab) was
considered blank. The absorbance of the sample solution was expressed
as “AS”.
2.7. Cell culture
A human HepG2 cell line was obtained from the Korean cell line
bank. HepG2 cells were cultured in DMEM supplemented with 10%
FBS and 1% antibiotics (100 U/ml penicillin–streptomycin). Cells were
cultured in T-75 culture flasks (SPL Life Sciences, Korea) under a
humidified atmosphere of 5% CO2 at 37 °C. The medium was renewed
approximately three times a week and a subculture was extracted at
the 75–80% confluency stage at a density of 4 × 106/flask. HepG2 cells
with 70–80% confluency and cell viability higher than 95% were used
in this study. Cell counting was performed using trypan blue dye.
2.8. EtOH cytotoxicity and biosafety of S. quelpaertensis Nakai leaves
HepG2 cells (1 × 104/well) were seeded in a 96-well microplate
(Nunc, Copenhagen, Denmark) and cultured as described above, and
24 h was allotted for adherence to the cells. The HepG2 cells were
then exposed to varying concentrations (0, 100, 200, 400, 800,
1600 mM) of EtOH or different concentrations (0, 200, 400, 800 μg/
ml) of S. quelpaertensis Nakai leaf material for 24 h under the same con-
ditions in subsequent experiments in order to determine the cytotoxic-
ity of EtOH and the biosafety of S. quelpaertensis Nakai leaves on HepG2
cells, respectively.
Then, the effects of EtOH and 60% EtOH extract of S. quelpaertensis
Nakai leaves on percentage cell viability of the HepG2 cells were deter-
mined by a 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay (Farshori et al., 2015; Al-Oqail et al., 2017). In brief, 15
μl MTT (5 mg/ml) was added to each well and allowed to incubate for a
3 h period. Supernatant was discarded and 100 μl solubilization buffer
containing 10% SDS and 50% DMSO (pH 4.7) was added to each well
for the solubilization of formazan crystal. The developed purple color
was measured at 540 nm was measured by using an enzyme-linked im-
munosorbent assay (ELISA) plate reader. Optical density readings of the
untreated wells were considered as 100% viable and viable percentage
of cells was measured in respective treatments in compared to the un-
treated control.
2.9. Hepatoprotective effect of 60% EtOH extract of S. quelpaertensis Nakai
leaves against EtOH cytotoxicity
HepG2 cells were seeded in a 96-well plate at a density of 1 × 104/well
in three replicates as described above. After 24 h of seeding, varying con-
centrations (0–800 μg/ml) of S. quelpaertensis Nakai leaf extract were si-
multaneous exposed with 400 mM EtOH. After a 24 h incubation
period, cell viability was determined by MTT assay as described above.
2.10. Statistical analysis
Results were presented as the means ± SE for each group. A
Student's t-test was used to compare the values from the two experi-
mental conditions, and p values of less than 0.05 were considered as
significant.
3. Results and discussion
3.1. Extraction yield
For isolating active compounds such as phenol, a number of steps
were involved, including milling, grinding, homogenization, and
extraction. However, extraction is the main process of obtaining
phytochemical compounds. There are many factors affecting the
efficiency of extraction, such as storage time or the existence of interfer-
ing substances (Stalikas, 2007). Generally, if the extraction time and
temperature are constant, the solvent and intrinsic compounds are the
two main factors to be considered (Fatiha et al., 2012). Thus, extraction
recovery mostly depends on what kinds of processing methods are used
in the study. In our study, different concentrations of EtOH were used to
evaluate which concentrations have the highest extraction yield. Based
on 5 concentrations (20, 40, 60, 80, and 100%) and distilled water, 60%
EtOH extract demonstrated the highest extraction yield compare to
the other extracts (Table 1). The extraction yield of S. quelpaertensis
Nakai leaves ranged between 12.04 to 15.23 depending on the concen-
tration and the type of solvent in the following order: 60% EtOH extract
(15.23 ± 0.24%) N 20% EtOH extract (14.19 ± 0.16%) N 40% EtOH extract
(13.98 ± 0.03%) N 80% EtOH extract (13.64 ± 0.49%) N distilled water
extract (12.04 ± 1.28%) N 100% EtOH extract (5.58 ± 0.29%). The
hydroethanolic solution demonstrated a greater recovery of active or
non-active compounds than pure aqueous and EtOH solvents. The
results indicate that mixing ethanol with some amount of water actually
increases the extraction yield of S. quelpaertensis Nakai leaves. Accord-
ing to Spigno et al. (2007), the combination of distilled water and
EtOH actually increases the extraction yield, but too high of an amount
of distilled water quantitatively decreases the extraction recovery. This
partly applied to our study because the extraction yield decreased from
the peak of the 60% EtOH extract (15.23 ± 0.24%) down to the 100%
EtOH extract (5.58 ± 0.29%). A similar propensity was observed in the
study of Yang et al. (2011). In their examination of antioxidant, antimi-
crobial, and α-glucosidase inhibitory activity for Cortex Moutan, the 60%
EtOH extract of the entire dried plant showed a higher extraction yield
(26.28 ± 2.49%) than 80% (24.73 ± 0.39%) and 100% EtOH extract (6.03
± 0.16%). Furthermore, the distilled water extract yielded the lowest re-
covery of extraction. Therefore, the solubility of constituents in the
S. quelpaertensis Nakai leaves might affect the distilled water extraction
yield, indicating that S. quelpaertensis Nakai leaves may contain more
polar compounds than non-polar compounds (Kim et al., 2010).
3.2. DPPH radical scavenging activity
The DPPH radical scavenging activity assay has broadly been
accepted and used as a fast, simple, and accurate tool to measure
oxidant pressure. Free radicals are known for their instability in reacting
or binding with other compounds, but the DPPH radical is distinguished
as one of the stabilized radicals because of its structural components.
The DPPH radical lacks one electron in its outer shell, and if it receives
the electron from the antioxidants, its color is reduced from a deep (pur-
ple) shade to a light (yellow) color; determining the reduction of its ab-
sorbance is usually measured around 510 nm (Ashok Kumar et al.,
2011). The DPPH scavenging activity against the extract solution is
shown in Table 1. As can be seen, 60% EtOH extract exhibited the highest
DPPH radical scavenging activity (21.20 ± 0.42 μg/ml), followed by 80%
EtOH extract with the second highest ability (29.40 ± 0.94 μg/ml), and
100% EtOH extract (33.42 ± 1.87 μg/ml) and 20% EtOH extract (35.36 ±
2.02 μg/ml) with relatively high activity rates. The distilled water and
40% EtOH extract had almost the same value, but 40% EtOH extract dem-
onstrated slightly higher value than the distilled water extract (40.52 ±
0.42 μg/ml for distilled water extract and 40.21 ± 0.02 μg/ml for 40%
EtOH extract). Although all extracts did not promote higher scavenging
activity than α-tocopherol (4.03 ± 0.08 μg/ml), it was found that 60%,
80%, 100%, and 20% EtOH extracts possess greater DPPH radical
 J.-H. Ra et al. / South African Journal of Botany 112 (2017) 376–382
Table 1
The extraction yield and radical scavenging activity for both DPPH and ABTS of S. quelpaertensis Nakai leaves with the extract of different ethanol concentrations and distilled water.
Extracta Yield (%)b DPPH radical scavenging activity RC50 (μg/ml)c
H2O 12.04 ± 1.28 40.52 ± 0.42
20E 14.19 ± 0.16 35.36 ± 2.02
40E 13.98 ± 0.03 40.21 ± 0.02
60E 15.23 ± 0.24 21.20 ± 0.42
80E 13.64 ± 0.49 29.40 ± 0.94
100E 5.58 ± 0.29 33.42 ± 1.87
Toco 4.03 ± 0.08
BHT 39.23 ± 1.35
a
H20: distilled water, 20E: 20% EtOH extract, 40E: 40% EtOH extract, 60E: 60% EtOH extract, 80E: 80% EtOH extract, 100E: 100% EtOH extract, Toco: α-tocopherol, BHT: butylated
hydroxytoluene.
b
Extraction yield was calculated by extraction yield (%) = {weight of dry soluble solid (g) / weight of sample (g)} × 100 in triplicate.
c
RC50: The value of 50% DPPH radical reduction.
d
RC50: The value of 50% ABTS radical reduction.
scavenging activity than BHT (39.23 ± 1.35 μg/ml). Kim et al. (2013a)
implemented an experiment with antioxidant activity assays including
the DPPH radical scavenging activity of S. quelpaertensis Nakai leaves ex-
tracted by 70% EtOH and distilled water, which resulted in the 70% EtOH
extract showing higher DPPH radical scavenging activity (EC50 = 129.5
± 4.91 μg/ml for the 70% EtOH extract and 151.1 ± 5.92 μg/ml for the
distilled water extract). Furthermore, the 70% EtOH extract contained
much higher phenol (1362.67 mg/g for 70% EtOH extract and
622.67 mg/g for distilled water extract) and flavonoid content
(76.50 mg/g for 70% EtOH extract and 16.70 mg/g for distilled water ex-
tract). The phenol and flavonoid content in each extract might affect the
DPPH radical scavenging activity in this study. It is predicted that more
active phenol and flavonoid compounds would be extracted by 60%
EtOH, showing greater DPPH radical scavenging activity.
3.3. ABTS radical scavenging activity
The ABTS radical scavenging activity assay is one of the famous as-
says among other in vitro tests, which scavenges radical the similar
way with DPPH radical scavenging activity. For DPPH radical scavenging
activity, the DPPH radical is selective in its reaction with hydrogen, and
is more actively bound with water-soluble compounds. However, in
contrast to DPPH radical scavenging activity, the ABTS radical has less
selectivity, meaning that it reacts with both soluble and non-soluble
compounds energetically. Without phenolic compounds or other
antioxidants, the ABTS radical is quite stable, but if these compounds
meet with the ABTS radical, it is decolorized into a non-colored form.
The main principle involves measuring the decolorized form of the
ABTS radical with absorbance between the 600–750 nm range
(Roginsky and Lissi, 2005). The strength of ABTS radical scavenging
activity for the different EtOH concentrations and distilled water ex-
tracts of S. quelpaertensis Nakai is also shown in Table 1. Similar to the
case of DPPH radical scavenging activity, none of extracts showed
higher ABTS radical scavenging activity than α-tocopherol (4.99 ±
0.42 μg/ml), but the 60% EtOH extract demonstrated the strongest
ABTS radical scavenging activity (36.21 ± 1.96 μg/ml), followed by
80% EtOH extract (40.69 ± 0.27 μg/ml), 40% EtOH extract (48.77 ±
1.01 μg/ml), 100% EtOH extract (49.73 ± 0.13 μg/ml), 20% EtOH extract
(59.73 ± 0.28 μg/ml), and distilled water extract (59.90 ± 1.56 μg/ml).
All things considered, a lower EtOH concentration of the extract tended
to be associated with lower ABTS radical scavenging activity, whereas
crude or higher EtOH concentrations of the extract demonstrated higher
radical scavenging activity. Although all extracts were inferior to α-
tocopherol, ABTS radical scavenging activity was still much higher
than the rates reported in the study by Jananie et al. (2011), who inves-
tigated antioxidant activity (including ABTS radical scavenging activity)
of Cynodon dactylon, which is the same family as S. quelpaertensis Nakai
(Poaceae), extracted by 80% EtOH (IC50 = 710 μg/ml). In addition, our
379
ABTS radical scavenging activity RC50 (μg/ml)d
59.90 ± 1.56
59.73 ± 0.28
48.77 ± 1.01
36.21 ± 1.96
40.69 ± 0.27
49.73 ± 0.13
4.99 ± 0.42
values were higher than those in the study by Song et al. (2015). In
their study, 80% EtOH and boiled distilled water extracts of S. borealis,
which is a closely-related species to S. quelpaertensis Nakai, were inves-
tigated in terms of ABTS radical scavenging activity, and exhibited rates
of 300.2 μg/ml and 410.5 μg/ml, respectively. These results of these stud-
ies indicate that S. quelpaertensis Nakai leaf extract contains significant
ABTS radical scavenging agents.
3.4. Reducing power assay
The reducing power of the test sample is said to be a significant indi-
cator of potential antioxidant activity by allowing the hydrogen atom to
break the radical chain. Depending on the strength of the active com-
pounds, F3+, ferrous complex, is reduced into F2+ (ferrous form), turn-
ing the solution from a yellow to a green color. The reducing power is
usually monitored at the absorbance of 700 nm, which is directly related
to the reducing power of intrinsic active substances (Akanni et al.,
2014). The reducing power of S. quelpaertensis Nakai leaf extract is illus-
trated in Fig. 1. In all extracts, as described in another literature on this
topic (Kim et al., 2011), the reducing power was increased as the con-
centration became higher. Even if there is no significant difference be-
tween all extracts, 60% EtOH extract showed the greatest reducing
power for all concentrations (100, 200, and 300 μg/ml). An EtOH extract
of 80% in 300 μg/ml showed slightly higher reducing activity than α-
tocopherol in 200 μg/ml. Of the remaining extracts, which were distilled
water, 40%, 80%, and 100% EtOH extract, similar values were exhibited
in 300 μg/ml. Based on this result, the active compounds that effectively
reduce the free radical would be extracted by both distilled water and
high and low concentrations of hydroalcoholic solution. Saha and Paul
(2014) reported that the existence of different reductones is strongly
associated with the reducing power of the test sample, and its
reductones simultaneously react with specific precursors of peroxide
to prevent peroxide formation. The greater reducing power of the 60%
EtOH extract might rely on this kind of compound, and so higher antiox-
idant activity was therefore observed.
3.5. Tyrosinase inhibitory activity
Melanin is the pigment that can be found in many organisms, includ-
ing human beings. Melanin acts as a vital element in blocking or absorb-
ing the radiation that hits the skin, but mass production of melanin
becomes a menace, causing many conditions such as melasma, freckles,
ephelides, and senile lentigines. Melanin synthesis is a complex process.
Tyrosinase, which is copper-containing monooxygenase enzyme,
hydroxylates the tyrosine and creates 3,4-dihydroxyphenylalanine,
usually known as L-DOPA and which later becomes dopaquinone.
Dopaquinone has a high potential to react with other substances by it-
self, and eventually oxidizes into black melanin, especially that called
380 J.-H. Ra et al. / South African Journal of Botany 112 (2017) 376–382

Fig. 1. The reducing power of S. quelpaertensis Nakai leaf extract of different EtOH concentrations and distilled water. H20: distilled water extract, 20E: 20% EtOH extract, 40E: 40% EtOH
extract, 60E: 60% EtOH extract, 80E: 80% EtOH extract, 100E: 100% EtOH extract, Toco: α-tocopherol.

“eumelanin”. Thus, a substantial amount of research is being conducted
on tyrosinase inhibitors for moth clinical and cosmetic proposes.
Recently, natural tyrosinase inhibitors extracted from plants are getting
more attention (Uchida et al., 2014). All extracts increased in a dose-
dependent manner from 63 μg/ml to 500 μg/ml concentrations
(Fig. 2). Out of all concentrations, the 60% EtOH extract showed the
highest tyrosinase inhibitory activity and the 20% EtOH extract demon-
strated the lowest level. Following the 60% EtOH extract, the order was
80%, 100%, 40%, and distilled water. However, even the 20% EtOH
extract, which had the lowest inhibitory activity, exhibited stronger
tyrosinase inhibitory activity than arbutin, which was used as a positive
control. This indicates that the EtOH extract of S. quelpaertensis Nakai
leaves possesses a large amount of active compounds working against
tyrosinase activity. Moon et al. (2010) also performed a tyrosinase
inhibitory activity assay, subjecting 299 parts of 263 plants (extracted
by 80% EtOH) from Jeju Island. The 60% EtOH extract in our study
exhibited stronger tyrosinase inhibitory activity than any other test
samples they investigated (excluding Distylium racemosum Siebold &
Zucc. Leaves in 500 μg/ml (87.6 ± 0.3%)). Even if 20% EtOH extract in
our study showed the lowest inhibitory activity, its value was higher
than their results in 500 μg/ml except in the case of only 12 species. Fur-
thermore, there have been some reports describing one constituent of
S. quelpaertensis Nakai leaves, p-coumaric acid, as strongly inhibiting
cellular melanogenesis and in vitro tyrosinase inhibitory activity (An
et al., 2008). According to An et al. (2010), p-coumaric acid exhibited
slightly lower tyrosinase inhibitory activity than kojic acid, which is
mostly used as a whitening agent, in tyrosinase isolated from mush-
rooms, but it was much stronger than kojic acid when separated from
murine and human tissue. Strong tyrosinase inhibitory activity of
S. quelpaertensis Nakai leaves, especially for the 60% EtOH extract,
might therefore be based on the existence of p-coumaric acid in each
extract.
3.6. Cytotoxicity of EtOH, the effect of S. quelpaertensis Nakai leaf extract,
and combination of both on the viability of HepG2 cells
The cytotoxicity effects of varying concentrations of EtOH (0–
1600 mM) on HepG2 cells were determined by MTT assay. We observed
that cell viability decreased dose-dependently with EtOH treatment
(Fig. 3A). More specifically, significant cell viability loss was observed
when HepG2 cells were exposed to EtOH concentrations higher than
400 mM (p b 0.05 at 400 mM; p b 0.005 at 800 mM; p b 0.0005 at
1600 mM). According to recent studies on EtOH toxicity on HepG2
cells, it is documented that ethanol can cause pleiotropic effects at
cellular level such as formation of reactive oxygen species and DNA
damage even at lower concentrations (Kade et al., 2016). Considering
these points, 400 mM (17% viability loss) of EtOH was used for the
subsequent experiments to induce EtOH toxicity. In the meantime, we
assessed the toxicity of S. quelpaertensis Nakai leaf EtOH extract treat-
ment on HepG2 cells by MTT assay. Interestingly, it was revealed that
treatment of 60% EtOH extract of S. quelpaertensis Nakai leaves was
not toxic to HepG2 cells even at higher concentrations (800 μg/ml),
and cell viability increased in a concentration-dependent manner
(Fig. 3B). We observed 7% (p b 0.05) and 9% (p b 0.5) increases in cell
viability with respective sample treatments of 400 and 800 μg/ml.
When the cells exposed to EtOH, we observed decreased cell viability
compared to untreated control. However, treatment with 60% EtOH ex-
tract of S. quelpaertensis Nakai leaves exhibited an increasing pattern of
viable cell percentage compared to only EtOH exposed cells (Fig. 3C)
signifying the biosafety of 60% EtOH extract of S. quelpaertensis Nakai
leaves against EtOH induced toxicity. Lee et al. (2012) reported similar
outcomes to our results. They performed a cell viability test with
Duchesnea chrysantha on a lipopolysaccharide (LPS) stimulated RAW
264.7 cell with and without sample treatment, and concluded that sam-
ple treatment enhances cell viability. According to Lee et al. (2000), the

Fig. 2. Tyrosinase inhibitory activity using L-tyrosine as substrate for the S. quelpaertensis Nakai leaf extract with different EtOH concentrations and distilled water. H20: distilled water
extract, 20E: 20% EtOH extract, 40E: 40% EtOH extract, 60E: 60% EtOH extract, 80E: 80% EtOH extract, 100E: 100% EtOH extract.
 J.-H. Ra et al. / South African Journal of Botany 112 (2017) 376–382 381

Fig. 3. Effects of (A) EtOH exposure, (B) 60% EtOH extract of S. quelpaertensis Nakai leaf, and (C) simultaneous exposure for 400 mM EtOH + 60% EtOH extract of S. quelpaertensis Nakai leaf
on cell viability of HepG2 cells. HepG2 cells were exposed to varying concentrations of EtOH (0–1600 mM) or 60% ethanol extract of S. quelpaertensis Nakai leaves (0–800 μg/ml) for 24 h.
Cytotoxicity was determined by MTT assay. Experiments were carried out in triplicates and each bar represents the mean ± SE. * denotes p b 0.05, ** denotes p b 0.005 and *** denotes p b
0.0005 compared to untreated control.

remarkable effect of natural resources for cell viability proceeds from
flavonoid or phenol compounds. For practical proposes, p-coumaric
acid found in S. quelpaertensis Nakai leaves inhibited adipogenesis in
3T3-L1 cells (Kang et al., 2013) and Oleic acid-induced lipid accumula-
tion (Kim et al., 2013b). This lends support to the idea that phenolic
compounds, including p-coumaric acid, work tremendously not only
as tyrosinase inhibitors but also as cellular viability agents.
4. Conclusion
The results of this study indicate that S. quelpaertensis Nakai leaves
are excellent source of antioxidants, and function as a tyrosinase inhib-
itor and cellular viability agent, which may help to develop the new safe
antioxidant food and tyrosinase inhibitor in cosmetic field. Our study
also provided support for the idea that bioactive compounds are more
efficiently extracted by 60% and 80% EtOH. In all assays, 60% EtOH ex-
tract demonstrated the strongest effect and 80% EtOH extract also
showed good radical scavenging and tyrosinase inhibitory activity. For
further investigation, we performed a cell viability test for the 60%
EtOH extract via the employment of an MTT assay, which revealed
that EtOH treatment on HepG2 cells dramatically reduced the cell via-
bility, but the sample treatment gained more cell viability. Similar to
the case of the sample treatment, the combination of S. quelpaertensis
Nakai leaf extract and EtOH treatment showed increased cell viability.
This clearly indicates that S. quelpaertensis Nakai leaf extract does not
harm the cell, but instead induces cell viability. Further studies are
now required to identify the active compounds and their benefits
when isolated from S. quelpaertensis Nakai leaves.
382 J.-H. Ra et al. / South African Journal of Botany 112 (2017) 376–382
Acknowledgments
This research was financially supported by the Ministry of Trade, In-
dustry and Energy (MOTIE) and Korean Institute for Advancement of
Technology (KIAT) through Promoting Regional specialized Industry
(Grant Number: R0003895).
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