Compositional variation of atranorin-related components of lichen Myelochroa leucotyliza dependent on extraction solvent and their quantitative analysis by qHNMR

Article abstract of DOI:10.1002/pca.3048

Introduction: Quantitative nuclear magnetic resonance (qNMR) is one of the effective and reliable quantification tools for natural product research. Myelochroa leucotyliza belongs to the genus Myelochroa, a common foliose lichen genus found in the Korean Peninsula, and has not been quantitatively analysed using NMR. Previous chemical studies on M. leucotyliza have been limited to the main components by traditional thin-layer chromatography (TLC) experiments. Objective: We explored the stability of atranorin, a major component of M. leucotyliza, in methanol and acetone using NMR and characterised the changes in the chemical profiles of the lichen extracts in methanol and acetone using qNMR. Methodology: Atranorin transformation in the presence of methanol was analysed using time-dependent proton (¹H)-NMR analysis (600 MHz NMR spectrometer). A ¹H qNMR (qHNMR) method was established using dimethyl sulfone as the internal standard for quantifying the selected components isolated from M. leucotyliza. Homogenous mixtures of the samples were dissolved in deuterated chloroform. Results: Time-dependent ¹H-NMR experiments revealed that atranorin (5) from lichen M. leucotyliza decomposed into atraric acid (1) and methyl haemmatommate (2) in methanol. Four components were identified from M. leucotyliza: 1, 2, usnic acid (4), and 5, and their respective contents were determined using qHNMR. The percentages (w/w) of 1, 2, and 4 in the methanol extract were calculated as 5.66%, 0.69%, and 0.90%, while those of 1, 4, and 5 in the acetone extract were 1.70%, 1.68%, and 19.11%, respectively. Conclusion: We used qHNMR to effectively analyse quantitative compositional variations in two different M. leucotyliza extracts and reliably determined the chemical conversion of the unstable compound atranorin.

Full text of DOI:10.1002/pca.3048

Received: 5 November 2020
DOI: 10.1002/pca.3048
Revised: 3 March 2021
Accepted: 15 March 2021
R E S E A R C H A R T I C L E
Compositional variation of atranorin-related components of
lichen Myelochroa leucotyliza dependent on extraction solvent
and their quantitative analysis by qHNMR
Manju Ojha1 | Yun-Seo Kil1 | Ui Joung Youn2,3 | Young Jun Ok4
Hyukjae Choi1,5 | Joo-Won Nam1
|
1College of Pharmacy, Yeungnam University,
Abstract
Gyeongsan-si, Gyeongsangbukdo, Republic of
Korea
Introduction: Quantitative nuclear magnetic resonance (qNMR) is one of the
effective and reliable quantification tools for natural product research. Myelochroa
leucotyliza belongs to the genus Myelochroa, a common foliose lichen genus found in
the Korean Peninsula, and has not been quantitatively analysed using NMR. Previous
chemical studies on M. leucotyliza have been limited to the main components by
traditional thin-layer chromatography (TLC) experiments.
2Division of Life Sciences, Korean Polar
Research Institute (KIOST), Incheon, Republic
of Korea
3Department of Polar Sciences, University of
Science and Technology, Incheon, Republic of
Korea
4Core Research Support Centre for Natural
Products and Medical Materials, Yeungnam
University, Gyeongsan-si, Gyeongsangbukdo,
Republic of Korea
Objective: We explored the stability of atranorin,
a major component of
M. leucotyliza, in methanol and acetone using NMR and characterised the changes in
the chemical profiles of the lichen extracts in methanol and acetone using qNMR.
Methodology: Atranorin transformation in the presence of methanol was analysed
using time-dependent proton (1H)-NMR analysis (600 MHz NMR spectrometer). A
1H qNMR (qHNMR) method was established using dimethyl sulfone as the internal
standard for quantifying the selected components isolated from M. leucotyliza.
Homogenous mixtures of the samples were dissolved in deuterated chloroform.
Results: Time-dependent 1H-NMR experiments revealed that atranorin (5) from
lichen M. leucotyliza decomposed into atraric acid (1) and methyl haemmatommate (2)
in methanol. Four components were identified from M. leucotyliza: 1, 2, usnic acid (4),
and 5, and their respective contents were determined using qHNMR. The percent-
ages (w/w) of 1, 2, and 4 in the methanol extract were calculated as 5.66%, 0.69%,
and 0.90%, while those of 1, 4, and 5 in the acetone extract were 1.70%, 1.68%, and
19.11%, respectively.
5Research Institute of Cell Culture, Yeungnam
University, Gyeongsan-si, Gyeongsangbukdo,
Republic of Korea
Correspondence
Dr Joo-Won Nam, College of Pharmacy,
Yeungnam University, Gyeongsan-si,
Gyeongsangbukdo 38541, Republic of Korea.
Email: jwnam@yu.ac.kr
Funding information
Yeungnam University Research Grant, Grant/
Award Number: the 2020 Yeungnam
University Research Grant
Conclusion: We used qHNMR to effectively analyse quantitative compositional
variations in two different M. leucotyliza extracts and reliably determined the
chemical conversion of the unstable compound atranorin.
K E Y W O R D S
atranorin, Myelochroa leucotyliza, qNMR
Phytochemical Analysis. 2021;1–7.
wileyonlinelibrary.com/journal/pca
© 2021 John Wiley & Sons, Ltd.
1

2
OJHA ET AL.
1
|
INTRODUCTION
metabolites in lichens, also reported transesterification into atraric
acid and methyl haemmatommate in the presence of methanol.15
Therefore, considering this crucial issue, the current quantitative
study on the lichen M. leucotyliza was performed on two different sol-
vent extracts. Herein, the qNMR technique was used for the first time
for the quantitative analysis of this lichen.
Lichens are formed via a symbiotic association between fungi and
algae or cyanobacteria.1 The association of their mutual relationship
and adaptive characteristics produces
a variety of chemical
substances, such as mono-phenolic compounds, quinones, depsides,
depsidones, dibenzofurans, amino acids, xanthones, and terpenoids.
According to previous studies, these substances exhibit anti-oxidant,2
antiproliferative,3 antibacterial (Burkholder, 1944 #51),4 and anti-HIV5
activities. Myelochroa is a genus of the family Parmeliaceae, compris-
ing common foliose lichen. Approximately 33 species of the genus
Myelochroa are widely distributed in East Asia, 11 of which are
dispersed on the Korean Peninsula.6 Among them, Myelochroa
leucotyliza (Nyl.) Elix & Hale, which has been documented in Korea,
Japan, Malaysia, China, and Nepal, is characterised by the presence of
closely attached thalli, 3–7 cm wide, with narrow lobes forming granu-
lated soredia. The lower surface of this species is composed of black
rhizines, and the medulla is pale yellow. Morphologically,
M. leucotyliza resembles M. aurulenta and M. hayachinensis; however,
it lacks galbinic acid.7,8
2
|
EXPERIMENTAL SECTION
| General experimental procedures
2.1
The NMR spectra for structure identification were recorded using a
Bruker 250 MHz AVANCE DPX spectrometer (Bruker, Billerica, MA,
USA). The quantitative proton nuclear magnetic resonance (qHNMR)
experiments were performed on a 600 MHz Varian NMR spectrome-
ter (VNS-600, Palo Alto, CA, USA) at the Core Research Support
Centre for Natural Products and Medical Materials (CRCNM). NMR
data were analysed and processed using Mnova 9.0.1 software from
Mestrelab Research S.L. (Santiago de Compostela, Spain). Optical rota-
tion was measured using a JASCO DIP-1000 polarimeter (Tokyo,
Japan). Low resolution electrospray ionisation mass spectrometry
(LR-ESI-MS) was performed using an Agilent 6120 series LC–MS sys-
tem (Agilent Technologies, Santa Clara, CA, USA). Medium pressure
liquid chromatography (MPLC) was conducted on a Biotage Isolera™
prime (Uppsala, Sweden) using silica gel SNAP cartridges KP-Sil
(100 g, 25 g). Semi preparative high-performance liquid chromatogra-
The chemical composition of M. leucotyliza was previously
explored in taxonomic studies of Myelochroa species, wherein thin-
layer chromatography (TLC) was used to identify atranorin, zeorin,
leucotylin, and secalonic acid A.7,8 TLC analysis has been widely
applied in chemical profiling studies owing to its fast and simple oper-
ation, as well as its various selective and sensitive detection methods.9
However, TLC experiments can be unreliable because they are con-
ducted in open systems and are easily influenced by environmental
conditions such as humidity or temperature. It is also sometimes diffi-
cult to find suitable solid components and developing systems for
these experiments, especially when separating isomeric mixtures.9,10
Nuclear magnetic resonance (NMR) spectroscopy is a crucial analytical
technique for natural product research because it provides tremen-
dous structural information. Recently, it has become a reliable tool for
quantitative studies. This is because the intensity of the NMR signals
is proportional to the concentration of samples corresponding to the
signals. Moreover, NMR spectroscopy is highly robust and non-
destructive. Quantitative NMR (qNMR) can be used for relative or
absolute quantitative measurements. Three major calibration methods
are applied when using qNMR for absolute quantitative analysis:
internal calibration (IC), external calibration (EC), and external calibra-
tion of the internal calibration signal (ECIC, a dual calibration method
using the residual solvent signal as an internal calibrant).11 In the pre-
sent study, for the quantitative analysis of M. leucotyliza, we used the
IC method, which has the advantage of minimising errors by obtaining
NMR data of the analyte and calibrant under the same conditions.
Extracting raw materials using the desired solvents is the first
important step in phytochemical research. However, various chemical
properties of the metabolites in natural sources can impede the selec-
tion of appropriate solvents for extraction.12,13 Alcohols, such as
methanol or ethanol, are typically introduced for initial extraction;
however, this commonly results in undesirable esterification
products.13,14 A previous study on atranorin, one of the major
phy (HPLC) was conducted using
a Waters system (Waters
Corporation, Milford, CT, USA) equipped with a YMC-Pack ODS-AQ
(10 mm × 250 mm, 5 μm). TLC was performed using silica gel 60F254
(0.2 mm thickness, Merck, Darmstadt, Germany) and RP-18F 254S
(Merck) plates by visualisation under ultraviolet (UV) light at 254 nm
and 10% (v/v) sulphuric acid in ethanol followed by heating.
2.2
|
Collection of the plant material
Myelochroa leucotyliza in this study was collected by one of the
authors, Dr Ui Joung Youn, at Heami-Myon, Seosan, South Korea, in
November 2017, and the collected sample was identified by Dr Ji Hee
Kim at the Life Science Department, Korean Polar Research Institute.
A voucher specimen (SHG7) was deposited at the Division of Life
Sciences, Korean Polar Research Institute.
2.3
|
Extraction and isolation
The dried material (124 g) was first extracted with 100% methanol
(500 mL × 3) for 48 h at room temperature. The solvent was evapo-
rated under reduced pressure to yield a concentrated methanol
extract (8 g). This extract was suspended in distilled water (500 mL)
and successively partitioned with hexanes (500 mL × 3), methylene
chloride (MC) (500 mL × 3), ethyl acetate (EtOAc) (500 mL × 3), and

OJHA ET AL.
3
n-butanol (n-BuOH) saturated with water (500 mL × 3), to afford
hexanes-soluble (4.0 g), MC-soluble (1.2 g), EtOAc-soluble (1.0 g), and
n-BuOH-soluble partitions (800 mg), and aqueous (500 mg) residue.
The hexanes partition (4 g) was subjected to MPLC (SNAP KP-Sil
100 g) using MC–methanol (95:5 to 0:100) as a gradient solvent
system to afford 12 fractions (H1–H12). Fraction H3 was selected for
isolation using semi-preparative reversed-phase HPLC (YMC-Pack
ODS-AQ, 10 mm × 250 mm, 5 μm) with an isocratic solvent system of
methanol–water (80:20, 2 mL/min) to obtain compound 1 [retention
to integral of target analyte, ms is the exact weight of sample, MWIC
the molecular weight of internal calibrant, mIC the exact weight of
calibrant in the sample, IntIC the integral value of internal calibrant, nIC
the number of protons giving rise to internal calibrant, PIC the purity
of internal calibrant.
3
|
RESULTS AND DISCUSSION
time (tR) 18 min, 16 mg], compound
compound 3 (tR 21 min, 0.7 mg).
2
(tR 20 min, 8 mg), and
3.1
|
Isolation and characterisation of the isolates
Similarly, a second crude material (2 g) was extracted with ace-
tone. The acetone extract (500 mg) was dissolved in deionised water
and sequentially partitioned with hexanes, MC, EtOAc, and water-
saturated n-BuOH, yielding hexanes-soluble (50 mg), MC-soluble
(200 mg), EtOAc-soluble (100 mg), and n-BuOH-soluble partitions
(30 mg), and aqueous (20 mg) residue. The MC fraction (200 mg) was
separated using MPLC (SNAP KP-Sil 25 g), employing hexanes–EtOAc
(95:5 to 0:100) as a step gradient solvent system to obtain 13 sub-
fractions (MC1–MC13). Among the resulting subfractions, MC3 was
Compounds 1–4 were identified as atraric acid (1),1,17 methyl
haemmatommate (2),17,18 methyl chlorohaemmatommate (3),19 and
(+)-usnic acid (4),1,20 based on comparison of their spectral properties
with the literature data (Figure 1, Figures S1–S10). The absolute con-
figuration of 4 was determined by comparing its optical rotation value,
[α]D31 + 13.005 (c 0.05, CHCl3), with the reported value.1
From reversed-phase isolation, an unidentified compound was
obtained together with atraric acid (1) and methyl haemmatommate
(2) (Figure 2A). However, after isolation, the unidentified compound
showed a chromatographic pattern identical to that of compound
2 (Figure 2B). For further confirmation, the 1H-NMR spectra of the
two compounds were acquired, resulting in identical spectral data
(Figure 2C). This observation suggests a possible chemical change
occurring in the unidentified compound.
identified as compound
4 (1.5 mg), and MC6 as compound
5 (10.0 mg). The EtOAc fraction was further purified by reversed-
phase HPLC (YMC-Pack ODS-AQ, 10 mm × 250 mm, 5 μm) with an
isocratic solvent system of acetonitrile–water (30:70, 2 mL/min) to
afford compound 6 (3.5 mg).
Reportedly, atranorin (5), which is present in abundance in
lichens, breaks down into atraric acid (1) and methyl haemmatommate
(2) in the presence of methanol through transesterification,15
suggesting that an analogous phenomenon may occur in isolated
unstable compounds related to atranorin (5). Therefore, the uni-
dentified compound was expected to be 5, the precursor of 2. Having
established that atranorin is more stable in acetonitrile and acetone,15
the second extraction was performed with acetone. Subsequently, the
HPLC profiles of the methanol and the acetone extracts were com-
pared, revealing compositional variations, as shown in Figure 3. To
investigate further, the MC fraction was selected for isolation and
subjected to MPLC; 5 was obtained and identified as atranorin. The
structure was confirmed on the basis of one-dimensional/two-
dimensional (1D/2D) NMR experiments and MS, and the obtained
data were in agreement with reported values (Figures S11 and
S12).18,20,21 According to the NMR data, 5 was the major component
of the acetone extract, while it was absent in the methanol extract.
Therefore, we concluded that 1 and 2 were likely formed via
alcoholysis of 5 during methanolic extraction. The EtOAc fraction of
the acetone extract was also investigated further, resulting in the iso-
lation of 6. The physical and spectral data of 6 (Figures S13–S15) were
identical to those reported for (+)-praesorediosic acid22; 6 was iso-
lated as white needles and its LR-ESI-MS spectrum displayed
molecular ion peaks at m/z 383.1 ([M + H]+), and 381.1 ([M − H]−),
which correspond to the molecular formula C21H34O6. The absolute
configuration of (+)-praesorediosic acid was determined by comparing
its measured optical rotation value, [α]D31 + 8.6 (c 0.2, CHCl3), with
that reported in the literature (Figure 1).22
2.4
|
Quantitative proton nuclear magnetic
resonance (qHNMR) experiments
Deuterated chloroform (CDCl3, 99.80%) and the internal calibrant,
dimethyl sulfone (DMSO2, 99.96%), were purchased from Sigma-
Aldrich (St Louis, MO, USA). DMSO2 was selected as an internal
calibrant fulfilling the ideal features of a standard calibrant for qNMR
analysis.16 For sample preparation, 10.04 mg of extract and 1.14 mg
of DMSO2 were dissolved in 700 μL of CDCl3. The solution containing
the extract and calibrant was transferred into a 5-mm NMR tube. The
qHNMR spectra of both methanol and acetone extracts of
M. leucotyliza were recorded using standard qHNMR conditions,11
which entailed a relaxation delay (D1) of 60 s, a calibrated 90ꢀ pulse
(P1), 32 number of scans (ns = 32), a spectral width of 12019.2 ppm,
and an acquisition time of 4.0 s. Post-acquisition processing included
zero filling to 256 K, a mild Lorentzian–Gaussian window function
(exponential factor −0.3, Gaussian factor 0.05 in GF mode), and base-
line correction (fifth-order polynomial). The residual solvent signal of
CDCl3 at 7.26 ppm was used for chemical shift referencing. The con-
tents were calculated using the following equation.11
n
IC:Intt:MWt:mIC
P½%ꢁ =
:PIC
nt:IntIC:MWIC:ms
where MWt is the molecular weight of a target compound, Intt is the
integral value of target analyte, nt is the number of protons giving rise

4
OJHA ET AL.
FIGURE 1 Chemical structures of compounds 1–6
FIGURE 2 (A) A HPLC chromatogram before isolation of 1, 2, and an unidentified compound (λ = 254 nm), and comparative (B) HPLC
chromatograms and (C) 1H-NMR spectra (CDCl3 at 250 MHz) after isolation of the two compounds [Colour figure can be viewed at
wileyonlinelibrary.com]
FIGURE 3 Comparison of HPLC profiles of
(A) the methanol and (B) the acetone extracts of
Myelochroa leucotyliza (λ = 254 nm) [Colour figure
can be viewed at wileyonlinelibrary.com]
The present study reports the isolation and structure identifica-
tion of three mono-phenolic compounds (1–3), a dibenzofuran (4), a
depside (5), and a γ-lactone carboxylic acid (6) derivative. Among
them, 1–3 and 6 were isolated from M. leucotyliza for the first time.
Although mono-phenolic compounds are commonly found in the
family Parmeliaceae, this is the first report of atraric acid (1), methyl
haemmatommate (2), and methyl chlorohaemmatommate (3) isolated
from this species. These mono-phenolic compounds play a remarkable
role in the formation of didepsides and tridepsides via esterification or
decarboxylation of their free carboxylic acid groups.23 Usnic acid (4) is
the most studied secondary metabolite in lichen, reportedly exhibiting
several biological activities, such as antimicrobial, antiproliferative,
and anti-inflammatory behaviours.24 Atranorin (5) is another charac-
teristic secondary metabolite in many lichens, especially in parmelioid

OJHA ET AL.
5
lichens.15 Previously, 5 was identified as the major component of the
crystalline material extracted from the thallus of M. leucotyliza.25 It is
biosynthesised by the acetyl polymalonyl pathway, and this was
known as a photo-buffer to reflect UV radiation on the thalamus
represent chemotaxonomic value. Therefore, the present study pro-
vides valuable information and insights for chemotaxonomic studies
of M. leucotyliza.
As demonstrated in our study, as well as in previous research,15
the choice of extraction solvent has a significant influence on the
content of atranorin (5) in the extract. Therefore, to increase the
atranorin content, acetone must be used as the extraction solvent
instead of methanol. To monitor the instability of atranorin in
methanol, a time-dependent 1H-NMR experiment was performed.
As the solubility of 5 in methanol is poor, 10 mL of methanol was
used to dissolve 2 mg of 5, followed by sonication to enhance solu-
bility. Initially, the 1H-NMR spectrum of isolated 5 was acquired
immediately after sample preparation, and no change was observed
during data acquisition (Figure 4A). Thereafter, it was dried on a
rotatory evaporator, re-dissolved in methanol, and stored in metha-
nol for 2 and 7 days; 1H-NMR spectra were acquired at these two
time points in CDCl3. As shown in the 1H-NMR spectrum obtained
after 2 days of methanol exposure (Figure 4B), the appearance of
signals at δH 2.46 and 3.92 ppm indicated that the chemical conver-
sion occurred slowly. The transformation of 5 into atraric acid (1)
and methyl haemmatommate (2) became evident after 7 days of
methanol exposure (Figure 4C).
surface.15,26 (+)-Praesorediosic acid (6) is
a
trisubstituted
γ-butyrolactone.27–29 This compound was first isolated from the
lichen Parmotrema praesorediosum.22 Methyl or methylene groups
substituted at the α-position of this class of compounds play an
important role in determining the inherent biological activities of the
compounds, such as antitumour and antimicrobial behaviours.28,30 In
addition, atraric acid (1), methyl haemmatommate (2), methyl
chlorohaemmatommate (3), usnic acid (4), and (+)-praesorediosic acid
(6), isolated for the first time from this genus, likewise appear to
The presence of singlet methyl peaks at δH 2.10 and 2.46 ppm, a
methoxy peak at δH 3.92 ppm, and an aromatic proton signal at δH
6.21 ppm indicated the formation of atraric acid (1). Similarly, the
presence of a methyl peak at δH 2.53 ppm, a methoxy peak at δH
3.96 ppm, an aromatic proton signal at δH 6.29 ppm, and an aldehydic
proton signal at δH 10.34 ppm, verified the formation of methyl
haemmatommate (2) arising from the transesterification process of
atranorin (5). The products remained in the equilibrium state after
7 days, with no further change observed after that period. Hence, we
concluded that atranorin decomposes into 1 and 2 in the methanol
extract of M. leucotyliza, which supports the previously proposed
mechanism of atranorin decomposition via transesterification in the
presence of methanol (Figure 5).15
FIGURE 4 1H-NMR spectra of atranorin (A) immediately after
sample preparation, (B) 2 days, and (C) 7 days after methanol
exposure (CDCl3 at 250 MHz) [Colour figure can be viewed at
wileyonlinelibrary.com]
FIGURE 5 1H-NMR spectrum of atranorin 7 days after methanol exposure in the state of equilibrium measured in CDCl3 at 250 MHz. The
numbers in black, blue, and red indicate assignments of atranorin (5), atraric acid (1), and methyl haemmatommate (2), respectively, at the state of
equilibrium [Colour figure can be viewed at wileyonlinelibrary.com]

6
OJHA ET AL.
FIGURE 6 Stacked qHNMR spectra (CDCl3 at 600 MHz) of the methanol and the acetone extracts of Myelochroa leucotyliza and 1H-NMR
spectra of atraric acid (1), methyl haemmatommate (2), usnic acid (4), and atranorin (5) (CDCl3 at 250 MHz) [Colour figure can be viewed at
wileyonlinelibrary.com]
3.2
|
qHNMR analysis of atranorin-related
atraric acid (1), methyl haemmatommate (2), usnic acid (4), and
atranorin (5), respectively as shown in Figure 6. The percentages (w/w)
of these compounds in the methanol extract were determined to be
5.66, 0.69, and 0.90 for 1, 2, and 4, respectively. Similarly, the
percentages (w/w) of these compounds in the acetone extract were
determined to be 1.70, 1.68, and 19.11 for 1, 4, and 5, respectively. The
presence of 5 was not detected in the methanol extract.
compounds in both methanol and acetone extracts
Atranorin (5) was the major constituent of the acetone extract,
although it was absent in the methanolic extract of M. leucotyliza.
Therefore, we were interested in measuring the compound concentra-
tions in both extracts. The poor solubility of the acetone extract in the
HPLC mobile phase and the non-polar nature of atranorin interfered
with quantification by HPLC, which is a commonly used approach.
Therefore, variations in the chemical compositions of the methanol and
acetone extracts of M. leucotyliza were analysed by qHNMR
(Figures S16 and S17). To the best of our knowledge, qHNMR has not
been utilised for the characterisation of compounds from the genus
Myelochroa in previous studies. In both extracts, the signals arising from
atraric acid (1), methyl haemmatommate (2), usnic acid (4), and atranorin
(5), were assigned according to the reference spectra of the isolated
compounds. The overlapped peaks were subjected to spectral
deconvolution (SD). SD was performed for all methyl and methoxy
singlets of isolated compounds in the qHNMR spectra, excluding the
methyl signals from the internal calibrant (DMSO2) located at δH
3.00 ppm. The SD process commenced by peak picking from the
spectrum and processing the peaks using a global SD function with five
fitting cycles. The areas of the deconvoluted signals were acquired from
the peak table obtained by applying the “edit fit” option in the peak
picking mode, which was employed for quantitation by 1H-NMR.
The signals appearing at δH 3.92 (-OCH3, s), 3.96 (-OCH3, s), 2.12
(-CH3, s), and 3.98 (-OCH3, s) ppm were used for the quantification of
The present study reports the isolation and structure identifica-
tion of atraric acid (1), methyl haemmatommate (2), methyl
chlorohaemmatommate (3), (+)-usnic acid (4), atranorin (5), and
(+)-praesorediosic acid (6). Among them, 1–3 and 6 were isolated from
M. leucotyliza for the first time. As demonstrated in our study, as well
as in previous research,15 the choice of extraction solvent has a signif-
icant influence on the content of this compound in the extract. There-
fore, to increase the atranorin content, it is essential to select acetone
as the extraction solvent instead of methanol.
ACKNOWLEDGEMENT
This research was supported by the 2020 Yeungnam University
Research Grant.
ORCID
Joo-Won Nam
https://orcid.org/0000-0001-5502-0736
REFERENCES
1. Huneck S, Yoshimura I. Identification of Lichen Substances. Berlin:
Springer Verlag; 1996:498.

OJHA ET AL.
7
2. Kosanic MM, Rankovic BR, Stanojkovic TP. Antioxidant, antimicrobial
and anticancer activities of three Parmelia species. J Sci Food Agric.
2012;92(9):1909-1916.
20. König GM, Wright AD. 1H and 13C-NMR and biological activity
investigations of four lichen-derived compounds. Phytochem Anal.
1999;10(5):279-284.
3. Bucar F, Schneider I, Ogmundsdottir H, Ingólfsdóttir K. Anti-
proliferative lichen compounds with inhibitory activity on 12(S)-HETE
production in human platelets. Phytomedicine. 2004;11(7-8):602-606.
4. Burkholder P, Evans AW, McVeigh I, Thornton H. Antibiotic activity
of lichens. Proc Natl Acad Sci USA. 1944;30(9):250-255.
21. De Carvalho MG, De Carvalho GJA, Braz-Filho R. Chemical constitu-
ents from Ouratea floribunda: Complete 1H and 13C NMR assignments
of atranorin and its new acetyl derivative. J Braz Chem Soc. 2000;11
(2):143-147.
22. David F, Elix J, Binsamsudin M. Two new aliphatic acids from the
5. Neamati N, Hong H, Mazumder A, et al. Depsides and depsidones as
inhibitors of HIV-1 integrase: Discovery of novel inhibitors through
3D database searching. J Med Chem. 1997;40:942-951.
lichen Parmotrema praesorediosum. Aust J Chem. 1990;43(7):
1297-1300.
ꢀ
23. Rankovic B. Lichen secondary metabolites: Bioactive properties and
pharmaceutical potential. Cham: Springer International Publishing;
2019.
6. Moon KH, Kashiwadani H, Kobayashic K.
A new species of
Myelochroa (Parmeliaceae, Lecanorales) from Shiga prefecture, Japan.
J Jap Bot. 2013;88:140-143.
24. Ingólfsdóttir K. Usnic acid. Phytochemistry. 2002;61(7):729-736.
25. Arakawa-Kobayashi S, Kobayashi T, Hasebe M, Kanaseki T. Identifica-
tion of crystalline material found in the thallus of the lichen,
Myelochroa leucotyliza. J Struct Biol. 2004;146(3):393-400.
7. Jayalal U, Joshi S, Oh S-O, Young JK, Hur J-S. A taxonomic study of
the genus myelochroa in South Korea. Mycobiology. 2012;40(4):
217-224.
ꢀ
8. Moon K, Ahn C, Kashiwadani H. Revision of the lichen genus
Myelochroa (Ascomycotina: Parmeliaceae) in Korea. J Species Res.
2015;4(1):23-32.
26. Studzinska-Sroka E, Galanty A, Bylka W. Atranorin – an interesting
lichen secondary metabolite. Mini Rev Med Chem. 2017;17:1-1.
27. Chhor RB, Nosse B, Sörgel S, Böhm C, Seitz M, Reiser O.
Enantioselective synthesis of paraconic acids. Chem A Eur J. 2003;9
(1):260-270.
9. Sherma J. Thin-layer chromatography in food and agricultural analysis.
J Chromatogr A. 2000;880(1-2):129-147.
10. Costanzo SJ. Optimization of mobile phase conditions for TLC
methods used in pharmaceutical analyses. J Chromatogr Sci. 1997;35
(4):156-160.
28. Comini A, Forzato C, Nitti P, Pitacco G, Valentin E. Chemoenzymatic
synthesis of enantioenriched 5-oxo-tetrahydro-3-furancarboxylic acid
derivatives. Tetrahedron. 2004;15(4):617-625.
11. Pauli GF, Chen S-N, Simmler C, et al. Importance of purity evaluation
and the potential of quantitative 1H NMR as a purity assay. J Med
Chem. 2014;57(22):9220-9231.
29. Huynh BL, Duong TH, Do TML, et al. New γ-lactone carboxylic acids
from the lichen Parmotrema praesorediosum (Nyl.) Hale, Parmeliaceae.
Rec Nat Prod. 2016;10:332-340.
12. Zhang Q-W, Lin L-G, Ye W-C. Techniques for extraction and isolation
of natural products: A comprehensive review. Chinas Med. 2018;13:
20/21-20/26.
30. Jacobi PA, Herradura P. Enantioselective syntheses of (+)- and
(−)-phaseolinic acid. Tetrahedron Lett. 1996;37(46):8297-8300.
13. Silva GL, Lee I-S, Kinghorn AD. Special problems with the extraction
of plants. Methods Biotechnol. 1998;4:343-363.
SUPPORTING INFORMATION
14. Maltese F, van der Kooy F, Verpoorte R. Solvent derived artifacts in
natural products chemistry. Nat Prod Commun. 2009;4:447-454.
15. Vos C, McKinney P, Pearson C, Heiny E, Gunawardena G, Holt E. The
optimal extraction and stability of atranorin from lichens, in relation
to solvent and pH. The Lichenologist. 2018;50(4):499-512.
16. Wells RJ, Cheung J, Hook JM. Dimethylsulfone as a universal stan-
dard for analysis of organics by QNMR. Accred Qual Assur. 2004;9:
450-456.
Additional supporting information may be found online in the
Supporting Information section at the end of this article.
How to cite this article: Ojha M, Kil Y-S, Youn UJ, Ok YJ,
Choi H, Nam J-W. Compositional variation of atranorin-
related components of lichen Myelochroa leucotyliza
dependent on extraction solvent and their quantitative
analysis by qHNMR. Phytochemical Analysis. 2021;1–7.
https://doi.org/10.1002/pca.3048
17. Carpentier C, Queiroz EF, Marcourt L, et al. Dibenzofurans and
pseudodepsidones from the lichen Stereocaulon paschale collected in
northern Quebec. J Nat Prod. 2017;80(1):210-214.
18. Youn UJ, So JE, Kim JH, et al. Chemical constituents from the
Antarctic lichen, Stereocaulon caespitosum. Biochem Syst Ecol. 2018;
80:73-75.
19. Azman AA, Khalid R, Abu Bakar M. A characterization NMR of sec-
ondary metabolites from lichen Parmotrema praesorediosum AIP Conf
Proc 2018;1940:020082/020081–020082/020085.