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Anti-oxidation activity of ethanol extracts from natural thalli of lichens

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Mycosystema
菌 物 学 报 15 November 2011, 30(6): 950-954

jwxt@im.ac.cn
ISSN1672-6472 CN11-5180Q
©2011 Institute of Microbiology, CAS, all rights reserved.

Anti-oxidation activity of ethanol extracts from natural thalli of

lichens
Kojiro HARA Marie ENDO Hiroko KAWAKAMI Masashi KOMINE Yoshikazu YAMAMOTO*

Faculty of Bioresource Sciences, Akita Prefectural University, 241-438 Kaidobata-Nishi, Shimoshinjo-Nakano, Akita City 010-0195, Japan

Abstract: Screening test on anti-oxidation activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH) was performed for 99 ethanol
extracts of 85 species of natural thalli of lichens in order to find novel anti-oxidation compounds. The 17 extracts of natural thalli
showed high anti-oxidation activity. Among them, the activities of extracts from Hypogymnia vittata, Peltigera aphthosa,
Nephromopsis ornata, Pseudevernia furfuracea, Cladonia vulcani and Peltigera elizabethae were higher. Extracts of Peltigera
spp. showed higher activity than those of other genera. The ethanol extract of P. aphthosa had been separated into ethyl
acetate-soluble and water-soluble fractions. Two anti-oxidative spots were found only in the water-soluble fractions by thin-layer
chromatography. The compound in the lower spot had the same Rf value, UV spectrum, and color as authentic solorinine that was
previously found as a unique quaternary ammonium compound from Peltigera spp. We now report that the hydrophilic lichen
substance, solorinine showed a nearly same anti-oxidation activity (EC50=120µmol/Lol/L) as standard antioxidant Trolox
(EC50=150µmol/L).
Key words: anti-oxidation activity, natural thalli, lichen, screening

INTRODUCTION enzymes (Kinoshita et al. 1994; Proska et al. 1994;

Lichens are symbiotic organisms, which are Takahashi et al. 2005) and photosynthesis (Endo et al.
consisted of fungal and algal partners, and have been 1998), virus activation (Yamamoto et al. 1995),
used as medicines from ancient times all over the world. nematocidal growth (Ahad et al. 1991) and bacterial
Their secondary metabolites are known as “lichen growth (Yamamoto et al. 1998).
substances” and contain many peculiar phenolics such as Reactive oxygen species (ROS) such as superoxide
depsides, depsidones, dibenzofurans and pulvinic acids. anion are produced in human cells by extracellular
In last two decade, they have shown several processes such as ultraviolet rays. They cause lipid
pharmacological activities: for examples, inhibitions of peroxidation to damage cell membranes and lead to

*
Corresponding author. E-mail: yyamamoto@akita-pu.ac.jp
Received: 10-08-2011, accepted: 12-09-2011
Vol.30 No.6
promote ageing and tumor. Normally ROS are scavenged
by enzymes such as superoxide dismutase (SOD). Plants
have an anti-oxidation system to protect themselves;
therefore, in recent studies metabolites showing a
potential property for anti-oxidation have been
investigated in plants by using various anti-oxidation
systems. There was a first report in 1993 (Yamamoto et
al. 1993) on anti-oxidation activity in lichens by the
method using SOD. Thereafter, Korean groups (Bhattarai
et al. 2008; Paudel et al. 2008; Luo et al. 2009) have
developed this research and published several papers.
They have isolated hydrophobic depsides showing anti-
oxidation activity.
This paper shows the screening results of anti-
oxidation activity in natural thalli of lichens and isolation
of hydrophilic and anti-oxidative compounds from them.
1 MATERIALS AND METHODS
1.1 Extraction with ethanol from natural thalli of
lichens
Ninety-nine natural thalli of 85 species of 41 genera
shown in Table 1 were collected in Japan and other
countries. These voucher specimens are deposited at the
Akita Prefectural University and were stored at -30℃ in
a freezer until extraction. Each crashed sample (ca. 0.2g)
was soaked in ethanol (10mL) by starring at room
temperature for 24h and filtered, and then the filtrate was
evaporated under reduced pressure at less than 40℃. The
extracts were dissolved in 80% ethanol to prepare a
1mg/mL solution for the assay.
1.2 Assay for anti-oxidation activity of extracts by
DPPH method
The anti-oxidation activity of ethanolic solution of
ninety-nine extracts was determined using 1,1-Diphenyl-
2-picrylhydrazyl (DPPH) method (Suda 2000). All
ethanolic solution (1mg/mL) was diluted to tenth and
hundredth concentration by 80% ethanol. 50µL DPPH
(800µmol/L), 50µL MES buffer (0.2mol/L, pH=6.8) and
50µL ethanol (20%) were dispensed to the 96-well
micro plate. Each 50µL of sample was added to the
mixture, and 50µL ethanol (80%) was added to the
other wells as control. After 30min, absorbance at 520nm
951
was measured using a micro plate reader (BIO-RAD
Model 680).
The antioxidant activity was represented as the
6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid
(Trolox) equivalent value which was calculated by the
formula as described below:
Trolox equivalent (TE) value=[the absorbance of the
control] − [the absorbance of the sample] / [the absorbance of
the control] − [the absorbance of Trolox]
The calibration curve of Trolox was made to
measure the absorbance of different concentrations
(200µmol/L, 50µmol/L, 12.5µmol/L, 3.125µmol/L,
0.78µmol/L and 0.18µmol/L) of Trolox.
1.3 Assay for anti-oxidation activity of solorinine by
DPPH method
The anti-oxidation activity of solorinine was
determined using the DPPH method. Solorinine was
dissolved in ethanol (3mmol/L) and made a half dilution
series. 50µL DPPH (400µmol/L), 50µL MES buffer
(0.2mol/L, pH=6.8) and 50µL ethanol (20%) were
dispensed to the 96-well micro plate. Each 50µL sample
was added to the mixture, and 50µL ethanol (80%) was
added to the other wells as the control. After 20min,
absorbance at 520nm was measured using a micro plate
reader (BIO-RAD Model 680). The calibration curve of
solorinine was made from the absorbance, and EC50 was
calculated.
1.4 Separation of anti-oxidation products from
natural thalli of Peltigera aphthosa
The dried natural thalli (21.5g) of Peltigera
aphthosa collected in British Columbia, Canada were
submerged three times in fresh ethanol over night and
filtered. Combined filtrate was concentrated under
reduced pressure to obtain the ethanol extract (1.04g).
The extract was dissolved in methanol (50mL) and water
(150mL) was added to the methanolic solution. Aqueous
solution was extracted three times with ethyl acetate
(200mL). Combined ethyl acetate solution and water
solution were evaporated in vacuo to yield the ethyl
acetate extract (0.51g) and water extract (0.49g),
respectively.
菌物学报
952 Mycosystema

Table 1 Anti-oxidation activity of ethanol extracts of natural lichen thalli as Trolox equivalent (TE) value

TE TE TE
Speciesa Speciesa Speciesa
(μmol/L) (μmol/L) (μmol/L)

Alectoria ochroleuca 143 Coenogonium luteum 20 Parmelia submontana 136

Alectoria sarmentosa 22 Collema subflaccidum 0 Parmelia sulcata 144

Anzia opuntiella-1 48 Evernia divaricata 22 Parmotrema chinense-1 94

Anzia opuntiella-2 89 Evernia esorediosa 69 Parmotrema chinense-2 158

Anzia opuntiella-3 165 Evernia prunastri 76 Peltigera aphthosa-1 193

Arctoparmelia centrifuga 0 Flavoparmelia caperata 0 Peltigera aphthosa-2 267

Baeomyces placophyllus 104 Heterodermia diademata 12 Peltigera elizabethae 203

Bryoria fremontii 155 Heterodermia hypoleuca 58 Peltigera neopolydactyla 179

Bryoria nadvornikiana 75 Heterodermia isidiophora 0 Peltigera praetextata 149

Canoparmelia aptata 99 Heterodermia japonica-1 13 Peltigera pruinosa 83

Cetraria laeviganda 55 Heterodermia japonica-2 11 Peltigera rufescens 192

Cetrelia braunsiana-1 0 Hypogymnia hypotrypella 59 Pseudevernia furfuracea 235

Cetrelia braunsiana-2 39 Hypogymnia vittata 297 Pseudocyphellaria crocata 112

Cladia aggregate 0 Hypotrachyna pseudosinuosa 32 Punctelia rudecta 25

Cladina arbuscula 36 Leptogium pedicellatum 0 Pyxine limbulata 0

Cladina rangiferina-1 20 Letharia columbiana 114 Ramalina capitata 74

Cladina rangiferina-2 47 Letharia vulpine 70 Rimelia clavulifera 22

Cladina subrangiferina 52 Lobaria fuscotomentosa 179 Stereocaulon intermedium-1 86

Cladonia amaurocraea 93 Lobaria isidiophora 0 Stereocaulon intermedium-2 44

Cladonia arbuscula var. mitis 97 Lobaria linita 0 Stereocaulon intermedium-3 149

Cladonia convolute 71 Lobaria pulmonaria 0 Stereocaulon japonicum 107

Cladonia crispate 0 Lobaria scrobiculata 78 Stereocaulon sorediiferum 14

Cladonia furcata 135 Lobaria spathulata 22 Sticta nylanderiana-1 60

Cladonia gracilis 78 Menegazzia terebrata 102 Sticta nylanderiana-2 198

Cladonia krempelhuberi-1 29 Nephroma laevigatum 48 Sulcaria sulcata 108

Cladonia krempelhuberi-2 24 Nephromopsis nephromoides 110 Umbilicaria sp. 1

Cladonia macilenta 103 Nephromopsis ornate 254 Usnea bismolliuscula 63

Cladonia rangiferina-1 20 Ochrolechia yasudae 104 Usnea filipendula 174

Cladonia rangiferina-2 13 Pannoparmelia angustata 140 Usnea rubrotincta 84

Cladonia rangiferina-3 34 Parmelia adaugescens 152 Usnea trichodeoides 53

Cladonia scabriuscula 8 Parmelia laevior 165 Xanthoparmelia tuberculiformis 36

Cladonia sp. 42 Parmelia praesquarrosa 139

Cladonia vulcani-1 205 Parmelia sinanoana 99

Cladonia vulcani-2 49 Parmelia squarrosa 68

1.5 TLC analysis of extracts and solorinine hexane-methyl t-butyl ether-formic acid=140:72:18 and
Ethanol extracts of Peltigera spp., ethyl acetate and (B) chloroform-methanol-water=6:4:1, detection (A)
water extracts of Peltigera aphthosa and solorinine heating at 100℃ after spray of sulfuric acid, (B) 0.2%
were analyzed by TLCs. TLC conditions are as follows; DPPH spray and (C) 0.5% ninhydrin (2,2-
TLC silica gel 60F254. (Merck), solvent systems (A) Dihydroxyindane-1,3-dione) in butanol.

http://journals.im.ac.cn/jwxtcn
Vol.30 No.6
1.6 HPLC analysis of extracts from Peltigera aphthosa
and solorinine
Solorinine and water extract from P. aphthosa thalli
were analyzed by HPLC with a photodiode-array detector
(Shimadzu 10A-DP). HPLC conditions are as follows;
column YMC-Pack ODS-A, 150×4.6mm I. D., S-5μm,
12nm, solvent composition methanol:water:phosphoric
acid=80:20:1, solvent flow 1mL/min, column temp.
40℃, detection wave length 180－700nm.
2 RESULTS AND DISCUSSIONS
2.1 Anti-oxidation activity of extracts from natural
thalli
Ninety-nine ethanol extracts from natural thalli of
85 species were evaluated for the anti-oxidation activity
by the DPPH method. Table 1 shows that 17 extracts
were higher activity than 150 of Trolox equivalent (TE)
value and 6 extracts (Hypogymnia vittata, Peltigera
aphthosa, Nephromopsis ornata, Pseudevernia
furfuracea, Cladonia vulcani and Peltigera elizabethae)
were higher activity than 200 of TE value.
Except for Peltigera pruinosa, all extracts of
Peltigera spp. showed higher activities from 149 to 267
TE values than those of other genera. Their extracts were
analyzed by TLC with solvent A. All tested extracts of
Peltigera spp. had a spot at Rf 0 showing strong activity
(data not shown). This indicates that the substance(s)
showing anti-oxidation activity might be a common
metabolite among Peltigera spp.
2.2 Anti-oxidation activity of extracts from Peltigera
aphthosa
Ethanol extract from Peltigera aphthosa showed
higher activity of anti-oxidation and had an active spot at
Rf 0 on TLC with solvent A. Therefore, the ethanol
extract was separated to water soluble and ethyl acetate
soluble fractions. The water-soluble fraction had two
spots at Rf 0.28 and 0.35 on TLC with solvent B. These
spots showed strong (Rf 0.28) and weak (Rf 0.35)
anti-oxidation activity.
Matsubara et al. (1999) isolated a new amino acid
from methanol extract of natural thalli of Solorina crocea
and identified it as 2-trimethylamino-4-(3,5-dimethoxy-
953
4-hydroxyphenyl) butanoate (solorinine, Fig. 1-A) by a
single-crystal X-ray diffraction and other instrumental
analyses. They found that solorinine and a new amino
acid peltigerine (N-demethylsolorinine, Fig. 1-B) were
distributed among Peltigera genus and these compounds
showed Rf 0.27 and 0.35 on TLC with solvent B
(Matsubara et al. 1999).
Fig. 1 The structures of amino acid derivatives from lichens.
Authentic solorinine and extract from P. aphthosa
thalli were analyzed by TLC with solvent B (Fig. 2).
Solorinine was found to appear as a spot with
anti-oxidative activity at Rf 0.28 on TLC and the color of
this spot was changed to pale yellow with ninhydrin. On
the other hand, water extract had two spots (Rf 0.27 and
0.35) and the lower spot had the same Rf value and color
of solorinine. The color of upper and lower spots was
changed to reddish purple and slight pale yellow with
ninhydrin, respectively. The pale yellow color by
ninhydrin test of solorinine and the compound at the
lower spot is assumed to represent similar reaction as
proline, which has a unique status at its amino group.
In addition, solorinine and the water extract from P.
aphthosa thalli were analyzed by HPLC with a
photodiode detector. Water extract had two peaks (1.53
and 1.67min) and the earlier peak had the same Rt and
UV spectrum (λmax 210 and 271nm) compared with
authentic solorinine (data not shown). We conclude that
solorinine is a major active compound of Peltigera
aphthosa. Solorinine showed similar anti-oxidation
activity (EC50=120µmol/L) as Trolox (EC50=150µmol/L)
(Fig. 3) and stronger than ascorbic acid (888µmol/L)
as a water-soluble agent (data not shown). Solorinine
is a unique quaternary ammonium compound in nature
and has remarkable hydrophilic and anti-oxidative
properties.
菌物学报
 954
Fig. 2 TLC analysis of water extract from Peltigera aphthosa thalli.
The water extract from P. aphthosa (ext) and solorinine (Sol) were
separated and detected on TLC plate by heating after spray of sulfuric
acid (A), DPPH spray (B) and ninhydrin (C).
Fig. 3 Anti-oxidation activity of solorinine. Anti-oxidation activities of
solorinine (closed circle, solid line) and trolox (open circle, dashed line)
was measured from decrease of absorbance for 20min by DPPH method.
Acknowledgments: We thank Dr. Kaoru Kinoshita of Meiji
College of Pharmacy for the gift of solorinine.
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