In Vitro Anticancer Activity of Hexane and Ethyl acetate Extracts of Leaf, Petiole and Rhizome of Petasites japonicus

Abstract

The aim of this study was to ascertain the effect of different solvents on extraction yield, antioxidant activity and cytotoxicity of extracts from Petasites japonicus (Siebold & Zucc.) Maxim. P. japonicus, widely distributed in Korea, Japan and China, is used as a culinary vegetable and a traditional medicine for asthma, migraines and tension headaches, and allergic rhinitis. In this study, twelve extracts from leaves, petioles and rhizomes of P. japonicus were evaluated for their antiproliferative activity against AGS, HepG2, HCT-116, and MCF-7 cancer cell lines. Plant extracts were fractionated using hexane, EtOAc, BuOH and distilled water, after which the antiproliferative activity was measured by MTT assay. Cell viability decreased in a dose dependent manner in response to the hexane and ethyl acetate extracts for all cell lines, with a significant decrease in cell viability being observed for 100 μg/ml and 75 μg/ml in the AGS and HepG2 cancer cell lines (p < 0.05). The leaf extract appeared to be more potent against HepG2, as indicated by IC50 values of 4.31 μg/ml for approximately 2.5-and 6.3-fold compared with rhizome and petiole extracts, respectively. The IC50 values of hexane extracts from leaves and rhizomes against AGS, HepG2, and MCF-7 human cell lines were greater than those of other extracts, while ethyl acetate extracts from petioles showed higher significant effect for all cell lines. Hexane and EtOAc extracts exerted their antiproliferative activity in four cancer cell lines. Overall, the results indicate that P. japonicus has the potential to be developed into selective anticancer nutraceutical and/or pharmaceutical treatments with low cost.

Full text

Trop J Nat Prod Res, January 2023; 7(1):2213-2217 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
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© 2023 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
Tropical Journal of Natural Product Research
Available online at https://www.tjnpr.org
Original Research Article
In Vitro Anticancer Activity of Hexane and Ethyl acetate Extracts of Leaf, Petiole and
Rhizome of Petasites japonicus
Min H. Cho1, Byoung I, Je1, Seetharaman Rajasekar1, Hye M. Kang1, Sun Y. Park2, Young W. Choi1*
1Department of Horticultural Bioscience, Pusan National University, Miryang 627-706, Korea
2Bio-IT Fusion Technology Research Institute, Pusan National University, Busan 46241, Korea
Introduction
Petasites japonicus MAX is cultivated as a culinary vegetable
in Korea, Japan, and China, and Petasites species are used for the
treatment of asthma, migraines and tension headaches, and allergic
rhinitis in Europe.1,2 Additionally, the extract of P. hybridus root have
been used in the prophylactic treatment of migraines or gastric ulcers,
and as an antispasmodic agent for asthma.1-3 The bioactive compounds
of Petasites species roots are sesquiterpenes, such as petasin or
isopetasin.4-7 A disadvantage of the extract of the plant root is that it
contains a high level of toxic pyrrolizidine alkaloids.8,9 However, the
areal parts of Petasites species, which contain a negligible amount of
pyrrolizidine,8 have been reported to contain relatively lower polar
compounds such as petasitin, S-petasin, S-petasitin, petasinol, and S-
isopetasin.7 Ethnobotanical and ethnopharmacological data describing
plants with potential therapeutic activities are more economical and
beneficial for identifying potential anticancer molecules than mass
screening of plant species. Plants have long been used for their
therapeutic values, and 85,000 plants have been documented for
therapeutic use globally.10 The World Health Organization (WHO)
estimates that almost 80% of world’ population has therapeutic
experience with herbal drugs.11
*Corresponding author. E mail: ywchoi@pusan.ac.kr
Tel: 82-55-350-5522
Citation: Cho MH, Je BI, Rajasekar S, Kang HM, Park SY, Choi YW. In
Vitro Anticancer Activity of Hexane and Ethyl acetate Extracts of Leaf,
Petiole and Rhizome of Petasites japonicus. Trop J Nat Prod Res. 2023;
7(1): 2213-2217. http://www.doi.org/10.26538/tjnpr/v7i1.18.
Official Journal of Natural Product Research Group, Faculty of Pharmacy,
University of Benin, Benin City, Nigeria.
Cancer is one of the most dangerous diseases in humans; accordingly,
there is considerable demand for discovery of new anticancer agents
from natural products.12-14
The potential for using natural products as anticancer drugs was
recognized in 1950 by the United States Natural Cancer Institute
(NCI).15 Since then, many naturally occurring anticancer drugs have
been identified.16,17
Despite the recent dominance of synthetic chemistry as a method to
discover and produce drugs, there is still enormous potential for
bioactive plants or their extracts to provide new and novel products for
disease treatment and prevention.18,19 Plants have an almost unlimited
capacity to produce substances that attract researchers in the quest for
novel chemotherapeutics.18 The continuing search for new anticancer
compounds in plants and traditional foods is a realistic and promising
strategy for its prevention.20
The objective of this study was to investigate the anticancer activity of
the hexane, ethyl acetate, butanol and aqueous fractions of extract of
Petasites japonicus Max plant parts against four human cancer cell lines.
Materials and Methods
Plant materials
Wild Petasites japonicus Max was collected in September 2019 from
Chungdogun, Gyungbuk Province, Korea, and processed and divided
into leaves, petioles, and rhizomes. The plant species was verified by
Dr. Young Whan Choi, and a voucher specimen (No. PJR20190001)
and deposited in the Natural Product Research Laboratory, Department
of Horticultural Bioscience, College of Bioresources and Science,
Pusan National University, Korea.
Reagents
Hexane, dichloromethane, chloroform, ethyl acetate, butanol, methanol
and acetone were purchased from Fisher Scientific Korea, Ltd.
(Kangnam-gu, Seoul, Korea). Sodium bicarbonate and dimethyl
ART ICL E I NFO
ABSTRACT
Article history:
Received 13 October 2022
Revised 05 January 2023
Accepted 10 January 2023
Published online 01 February 2023
The aim of this study was to ascertain the effect of different solvents on extraction yield,
antioxidant activity and cytotoxicity of extracts from Petasites japonicus (Siebold & Zucc.)
Maxim. P. japonicus, widely distributed in Korea, Japan and China, is used as a culinary vegetable
and a traditional medicine for asthma, migraines and tension headaches, and allergic rhinitis. In
this study, twelve extracts from leaves, petioles and rhizomes of P. japonicus were evaluated for
their antiproliferative activity against AGS, HepG2, HCT-116, and MCF-7 cancer cell lines. Plant
extracts were fractionated using hexane, EtOAc, BuOH and distilled water, after which the
antiproliferative activity was measured by MTT assay. Cell viability decreased in a dose
dependent manner in response to the hexane and ethyl acetate extracts for all cell lines, with a
significant decrease in cell viability being observed for 100 μg/ml and 75 μg/ml in the AGS and
HepG2 cancer cell lines (p < 0.05). The leaf extract appeared to be more potent against HepG2,
as indicated by IC50 values of 4.31 μg/ml for approximately 2.5- and 6.3-fold compared with
rhizome and petiole extracts, respectively. The IC50 values of hexane extracts from leaves and
rhizomes against AGS, HepG2, and MCF-7 human cell lines were greater than those of other
extracts, while ethyl acetate extracts from petioles showed higher significant effect for all cell
lines. Hexane and EtOAc extracts exerted their antiproliferative activity in four cancer cell lines.
Overall, the results indicate that P. japonicus has the potential to be developed into selective
anticancer nutraceutical and/or pharmaceutical treatments with low cost.
Keywords: Anti-cancer activity, Petasites japonicus, MTT assays, Pharmaceutical vegetable.
Copyright: © 2023 Cho et al. This is an open-access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.

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sulfoxide (DMSO) were obtained from Merck (Darmstadt, Germany).
RPMI-1640 medium, polyoxyethylene sorbitan monooleate (Tween 80),
cell freezing medium-DMSO, trypsin-EDTA solution, and
penicillin/streptomycin (P/S) solution were acquired from Sigma-
Aldrich Co. (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) was obtained from Promega
Corporation (Madison, WI, USA). Fetal bovine serum (FBS) was
purchased from Invitrogen Canada Inc. (Burlington, Ontario, Canada).
Extract preparation
The collected leaves, petioles, and rhizomes of wild P. japonicus were
stored at -20°C until use. Briefly, 1 kg of frozen leaves, petioles and
rhizomes were placed into separate 5,000 ml Erlenmeyer flasks
containing 3,000 ml of 70 % ethanol (final concentration) and then
ground into a powder using a blender (Hanil, Bucheon city, Korea. The
flasks were then placed on a sonicator (Kodo, JAC-4020P, Hwaseong,
Gyeonggi-do, Korea) and extraction was carried out for 2 hours three
times. The 70 % ethanol extract of each plant part was then obtained by
recovering the ethanol with an evaporator (Heidolph, Rotavapor 4000,
91126 Schwabach, Germany) at 45°C. The total extracts of leaves,
petioles, and rhizomes were then successively fractionation with hexane,
ethyl acetate, butanol and distilled water and the average yields were
calculated on a fresh weight basis. Serial dilutions (3.125 μg/ml to 100
μg/ml) of the filtered crude extract stock were prepared in DMSO and
used in an MTT assay. The extraction flow chart is shown in Figure 1.
Cell culture
AGS (human gastric carcinoma), HepG2 (human hepato cellular
cancer), HCT-116 (human colorectal cancer), and MCF-7 (human
breast cancer) cell lines were cultured with RPMI-1640 plus 10 % FBS
and 1 % P/S. All cancer cell lines were obtained from the Korean Cell
Line Bank. These cell lines were maintained in RPMI-1640 media
supplemented with 10 % fetal bovine serum, 100 IU/ml penicillin and
100 μg/ml streptomycin (Invitrogen, Carlsbad, CA, USA). The cells
were grown in 25 cm2 tissue culture flasks (SPL Life Sciences,
Hwaseong, Gyenggi-do, Korea) and maintained in an incubator (Sanyo
Electric Co., Ltd., Moriguchi, Osaka, Japan) under a 95 % humidified
atmosphere and 5 % CO2 at 37°C.
Cell viability assay
Each plant fraction of wild P. japonicus was added to culture medium
in 96-well plates (SPL Life Sciences, Hwaseong, Gyenggi-do, Korea)
to give different final concentrations (3.125, 6.25, 12.5, 25, 50, 100
μg/ml). The cells were then seeded at 104 cells/100 μl/well. All samples
were analyzed in triplicate and the plates were incubated at 37°C for 24
hours, after which an MTT assay was performed according to the
manufacturer’s protocol. Briefly, 50 μl of MTT solution were added to
each well, after which the samples were incubated for an additional 4 h
and 100 μl of DMSO solution were added to each well. Finally, the
absorbance of each well was recorded at a wavelength of 540 nm using
a plate reader (BioTek, Epoch, Winooski, VT, USA). The percentage of
surviving cells based on the control treatment (untreated) was then
calculated and plotted against the extract concentrations and used to
determine the concentration of P. japonicus extracts that induced 50 %
inhibition of cancer cells (IC50 values). The values from three separate
experiments were averaged and the mean IC50 and the standard
deviation were determined for each extract.
Statistical analyses
All data were expressed as means ± standard deviation from three
replications. IC50 values were determined by a polynomial regression
equation composed of the logarithmic values of four graded
concentrations and the viabilities of cells induced by the plant extracts
of wild P. japonicus. Viability was calculated based on the formula: V
(%) = T / C x 100, where V represents the viability or survival rate, T
represents the absorbance of the extract-treated group, and C is the
absorbance of the non-treated negative control group. The background
MTT absorbance obtained from the blank control group was subtracted
from all test groups.
Figure 1: Systematic extraction of leaf, petiole and rhizome parts of Petasites japonicus Max with 70 % ethanol and successive
fractionation with hexane (H), ethyl acetate (E), butanol (B) and distilled water (W).

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Results and Discussion
Yield
The yields of the total extracts (weight of dried total extract/weight of
fresh plant parts) were 38.17 g, 33.55 g and 53.33 g for the leaf, petiole
and rhizome total extracts, respectively (Table 1). The yields of the LH
(leaf hexane fraction), PH (petiole hexane fraction) and RH (rhizome
hexane fraction) were 13.06 g/kg, 0.99 g and 1.03 g, respectively. The
yields of EtOAc extracts from leaves, petioles and rhizomes were
almost the same, whereas the BuOH and aqueous extracts showed
higher yields for rhizome extracts, followed by petiole and leaf extracts.
Cytotoxic effects of fractions
The IC50 values and viability curves of leaf, petiole and rhizome extracts
for AGS, HepG2, HCT-116 and MCF-7 cancer cells are presented in
Table 2 and Figure 2. Decreased cell viability was observed in response
to hexane and ethyl acetate extracts for all the cell lines in a dose
dependent manner, with a significant decrease in cell viability of AGS
and HepG2 being observed for 100 μg/ml and 75 μg/ml (p < 0.05). The
IC50 values for hexane and ethyl acetate extracts against four human
cancer cell lines were higher than those of the butanol and aqueous
extracts. When the hexane extracts of leaves, petioles and rhizomes
were compared, the leaf extract appeared to be more potent, as indicated
by IC50 values of 4.31 μg/ml for approximately 2.5-fold and 6.3-fold
compared with rhizome and petiole extracts for HepG2, respectively.
Among the four cancer lines, the IC50 values of hexane extracts from
leaves and rhizomes were higher for AGS, HepG2, and MCF-7, but the
IC50 values of the ethyl acetate extracts from petiole were higher than
hexane extracts for all cell lines (Table 2).
The top five fractions (LH, PH, PE, RH and TE) with the highest
cytotoxicities against AGS, HepG2 and MCF-7 cancer cells and IC50
values lower than 63.20 ± 0.38 μg/ml were selected from the 12
fractions by in vitro bioassay. All five fractions concentration-
dependently reduced the viabilities or survival rates of the four cancer
cell lines tested. The minimum survival rates of the AGS, HepG2, HCT-
116 and MCF-7 cancer cells induced by LH at 100 μg/ml were 7.25 ±
0.48, 2.45 ± 0.53, 75.35 ± 1.11 and 7.47 ± 1.04 % with IC50 values of
27.56 ± 0.36, 4.31 ± 0.15, >100 and 21.53 ± 1.05 μg/ml, respectively.
With PE at 100 μg/ml, the survival rates were 0.37 ± 0.28, 0.10 ± 1.03,
0.93 ± 0.21 and 1.26 ± 0.22 % for AGS, HepG2, HCT-116 and MCF-7
with IC50 values of 16.50 ± 0.57, 16.66 ± 0.88, 13.51 ± 0.62, and 14.06
± 0.23 μg/ml, respectively (Figure 2 and Table 2). Wild P. japonicus is
cultivated as a culinary vegetable in Korea, Japan and China2 and
extract from the roots of Petasites species has been used therapeutically
in Europe.1,2 This plant has also been screened for identification of
pharmaceutically important compounds.7
Table 1: Yields of hexane, ethyl acetate (EtOAc), butanol (BuOH), and aqueous extracts of leaves, petioles, and rhizomes of Petasites
japonicus Max.
Plant parts
Contents of extracts (g/fresh kg)
Plant parts
Hexane
EtOAc
BuOH
dH2O
Total
Leaf
13.06
1.90
4.61
18.60
38.17
Petiole
0.99
0.33
6.186
26.05
33.55
Rhizome
1.03
1.75
9.24
41.31
53.33
One kg aliquots of fresh leaves, petioles and rhizomes were extracted with 70 % ethanol for 2 hours three times. The total extracts of each part were
then further fractionated with hexane, ethyl acetate, butanol and water. Finally, the extracts were obtained by recovering the solvents in an evaporator
at 45°C.
Table 2: IC50 values of AGS, HepG2, HCT-116, and MCF-7 cancer cell lines induced by hexane, EtOAc, BuOH, and aqueous
extracts obtained from leaves, petioles, and rhizomes of P. japonicus Max.
Plant parts
Solvents
Abbreviation
IC50 (μg/ml)
AGS
HepG2
HCT-116
MCF-7
Leaf
Hexane
LH
27.56 ± 0.36
4.31 ± 0.15
>100
21.53 ± 1.05
EtOAc
LE
>100
94.57 ± 2.71
>100
99.36 ± 1.71
BuOH
LB
>100
>100
>100
>100
dH2O
LW
>100
>100
>100
>100
Petiole
Hexane
PH
30.94 ± 0.24
27.31 ± 1.42
39.67 ± 1.92
31.56 ± 0.81
EtOAc
PE
16.50 ± 0.57
16.66 ± 0.88
13.51 ± 0.62
14.06 ± 0.23
BuOH
PB
>100
>100
>100
>100
dH2O
PW
>100
>100
>100
>100
Rhizome
Hexane
RH
21.94 ± 0.30
10.69 ± 0.35
63.20 ± 0.38
20.47 ± 2.17
EtOAc
RE
44.89 ± 0.22
58.57 ± 2.81
61.39 ± 1.23
57.26 ± 1.48
BuOH
RB
>100
>100
>100
>100
dH2O
RW
>100
>100
>100
>100
The values from three experiments were averaged and the mean IC50 and standard deviation were calculated for each extract.

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Although many compounds have been identified as possessing
medicinal properties,3 the anticancer effects of extracts from the leaves,
petioles and rhizomes of P. japonicus have not yet been validated in
vitro. In this study, different fractions were extracted from wild P.
japonicus and their anticancer effects were tested on human cancer cell
lines. The results revealed that the hexane and ethyl acetate fractions
had the highest anticancer potency. The highest yield among these
fractions from each extract was obtained from LH.
A smaller IC50 value indicates a greater anticancer effect. Comparison
of the extracts revealed that PE had a greater effect against all four cell
lines investigated herein than PH, LH, LE, RH and RE. Moreover, the
most effective fraction was LH for anti-HepG2, PE for anti-AGS, PE
for anti-HCT-116, and PE for anti-MCF-7 cancer cells. Conversely, the
butanol and aqueous extracts from leaves, petioles and rhizomes exerted
no cytotoxicity against the four cancer cell lines at 100 μg/ml. These
findings agree with those of many previous studies that reported the
bioactivity of non-polar principles in plants such as Typhonium
flagelliforme and chisandra sphenanthera.21,22 The yield from the total
extract for LH was also about ten times higher than that for PH and RH
(Table 1).
This is the first study to report the antiproliferative activity of P.
japonicus. The hexane and ethyl acetate extract of P. japonicus plant
parts inhibited the proliferation of different cancer cell lines and showed
selective toxicity toward HepG2 cells (Table 2). Fractions of some parts
exhibited different activity against different cell lines. For example, the
IC50 (μg/ml)) values of the hexane fraction of the P. japonicus rhizome
were 21.94 ± 0.30 for AGS, 10.69 ± 0.35 for HpeG2, 63.20 ± 0.38 for
HCT-116 and 20.47 ± 2.17 for MCF-7 (Table 2), but the IC50 of hexane
and EtOAc from petioles and EtOAc from rhizomes were almost the
same. This selectivity could be due to sensitivity of the cell line to the
active compounds in the extract or to tissue specific response.23
The refined preparation (Ze 339) of the ethyl acetate or ethanol extract
of P. hybridus root has been used in prophylactic treatment of migraines
or gastric ulcers and as an antispasmodic agent for asthma.1 The main
bioactive constituents in the ethyl acetate extract of P. hybridus rhizome
are petasin derivative sesquiterpenes,7 eremophilane sesquiterpenes24
and bakkenolides sesquiterpenes.25,26 A disadvantage of the ethyl
acetate extract of the plant root is that it contains a high level of toxic
pyrrolizidine alkaloids.7 However, the leaves of Petasite species contain
a negligible level of pyrrolizidine.1,7 Based on our results, the
cytotoxicity estimates of leaf, petiole and rhizome extracts indicate that
P. japonicus is a repository for anticancer compounds, and the fact that
the IC50 values are significantly lower than the NCI criteria indicates its
potential for cancer drug discovery programs.27
As the global scenario is now changing towards the use of nontoxic
plant products with traditional medicinal uses, development of modern
vegetables from medicinal plants should be emphasized for the control
of various diseases, including cancer. The hexane and ethyl acetate
extracts from the leaves, petioles and rhizomes of P. japonicus have
high anticancer activity, and the procedure for LH extraction was simple
and afforded high yields.
Figure 2: Viabilities of AGS (human gastric carcinoma), HepG2 (human hepato cellular cancer), HCT-116 (human colorectal
cancer), and MCF-7 (human breast cancer) cell lines in the presence of hexane (A), ethyl acetate (B), butanol (C) and aqueous (D)
fractions of P. japonicus Max extract.

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Conclusion
From these results, we can conclude that hexane and ethyl acetate
extract of the roots of P. japonicus have potential anticancer activity.
The results of the present study clearly indicate the anticancer potential
of P. japonicus extract, validating its pharmaceutical vegetable use.
However, further study is necessary to elucidate the chemical nature and
active compounds of the hexane extract from leaves that are responsible
for the anticancer activity.
Conflict of Interest
The authors declare no conflict of interest.
Authors’ Declaration
The authors hereby declare that the work presented in this article is
original and that any liability for claims relating to the content of this
article will be borne by them.
Acknowledgements
This work was supported by a 2-Year Research Grant of Pusan National
University
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