Key words
fungal endophytes - Lasiodiplodia theobromae - Botryosphaeriaceae - anti-trypanosomal
activity - HR-LCMS - NMR - metabolomics - chemometrics
Introduction
Despite the increasing disinclination of the pharmaceutical industry to pursue natural
products in their pipelines, statistical findings show that natural products still
play a major role in drug discovery with more than 50 % of FDA-approved drugs were
derived from natural products [1]. However, natural products research has been found to be too laborious, time-consuming,
and uneconomical, which may have led to the declining trend. Nevertheless, with the
emergence of new and more advanced technologies such as genomics, transcriptomics,
proteomics, metabolomics, and bioinformatics, natural products research has become
more competent in finding promising novel drugs for the pipeline [1], [2], [3], [4].
The utilization of metabolomics in natural products research is increasingly powerful
in several perspectives. Metabolomics is defined as a global study of all or a subset
of chemical entities including either or both primary and secondary metabolites that
are present in living organisms (cells or tissues) under certain processes [5], [6], [7]. Metabolomics, or metabolome mining, in natural products research has been used
for dereplication studies of both known and new compounds in crude plant, marine,
or microbial extracts [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], in differentiating biologically active natural products (NPs) from non-active fractions
[19], [20], [21], [22], [23], optimizing the production of bioactive secondary metabolites, as well as in developing
cultivation processes for large-scale fermentation and understanding their biosynthetic
pathways [7], [24].
Endophytic fungi are microorganisms that mutually live inside plant tissues without
causing any immediate negative effects towards the host plant for at least a part
of the fungal life cycle [25]. The total population of endophytic fungi species has been estimated to be up to
1.3 million [26]. Another study places this estimate at approximately 1.5 million [27]. However, as of the year 2000, only 75 000 fungal species have been identified;
the remainder are still untapped and unexplored [28]. Bioactive NPs derived from endophytic fungi display wide ranges of activities,
such as (−)-oxysporidinone, (2,6-dihydroxyphenyl)pentan-1-one, and (Z)-1-(2-(2-butyryl-3-hydroxyphenoxy)-6-hydroxyphenyl)-3-hydroxybut-2-en-1-one, which
exhibited antimicrobial activity [29], [30], pullularin A and hinnuliquinone displayed antiviral activity [31], [32], and spiropreussione A and 9-deacetoxyfumigaclavine C showed anticancer activity
[33], [34]. Compounds like cochlioquinone A, isocochlioquinone A, and cercosporin exhibited
activity against neglected tropical diseases [35], [36].
Human African trypanosomiasis, or sleeping sickness, is a fatal vector-borne parasitic
disease caused by Trypanosoma brucei brucei transmitted by the tsetse fly (Glossina spp.). This neglected tropical disease occurs only in rural areas of sub-Saharan
Africa [37]. To date, only a few drugs have been approved for the treatment of human African
trypanosomiasis. These include suramin, pentamidine, melarsoprol, eflornithine, and
the combination of nifurtomox/eflornithine. Most of the drugs are old, having been
discovered in the 1940s and 1950s, and have adverse effects such as nausea, vomiting,
fatigue, seizures, fever, diarrhea, hypoglycemia, abdominal cramping, peripheral neuropathy,
hypertension, heart damage, and neutropenia on the patients. For this reason, mining
and developing new human African trypanosomiasis drugs from natural products is crucial
and essential because various natural sources including plants, microorganisms, animals,
and marine organisms offer a high number of NPs with diverse chemical structures and
novel pharmacological mechanism of action [38].
The aim of this study is to adopt an untargeted HR-LCMS and NMR-based metabolomics
approach to determine the optimal fermentation conditions of Lasiodiplodia theobromae for medium scale-up, and also to capture and trace the production of active anti-trypanosomal
metabolites by using statistical multivariate data analysis of generated HR-LCMS data,
such as principal component analysis (PCA) and orthogonal partial least squares-discriminant
and analysis (OPLS-DA). To verify the dereplication results obtained from HR-LCMS
data, 1D and 2D 1H NMR data were utilized. Finally, the isolation of active metabolites was performed
based on the outcome of HR-LCMS and NMR-based metabolomics profile data.
Results and Discussion
In search of the best condition for scaling up the culture of the endophyte L. theobromae obtained from the leaves of Vitex pinnata, HR-LCMS and NMR-based metabolomics along with the bioassay data were utilized. The
fungus was grown in solid rice cultures and liquid Wickerham cultures for 7, 15, and
30 days, after which the metabolites were extracted. Three different incubation times
were chosen based on the fungal life cycle [39]. In this case, the first 7 days represent the germination phase, while the 15-day
and 30-day cultures cover the hyphal growth stage and sporing phase, respectively,
of L. theobromae. The production of secondary metabolites was monitored using HR-LCMS and NMR at each
of the growth phases parallel to the bioassay results. Based on the bioassay results
([Table 1]), the 30-day rice culture extract exhibited the strongest activity against T. b. brucei with a minimum inhibitory concentration (MIC) of less than 25 µg/mL. The HR-LCMS
raw data was processed using MZMine 2.10 [40]. The results of the assay were reproducible between the scale-up batches. Metabolite
production and distribution between cultures were analyzed through ion peak scatter
plots ([Fig. 1]). Based on the MS data, the occurrences of the metabolites on the 7th and 15th days
were similar, while a decrease in metabolite production was observed on the 30th day.
However, the ion chromatogram both in positive and negative modes revealed a different
set of metabolites on the 30th day to those of the 7th and 15th day extracts ([Fig. 1]). Moreover, the 1H NMR data revealed findings complementary to the MS data ([Fig. 2]). Therefore, the bioactive 30-day rice culture condition was chosen for scale-up
and further isolation work.
Fig. 1 Scatter plot of the ion peaks of the L. theobromae extracts from different days (A) in positive ionization and (B) in negative ionization. (Color figure available online only.)
Fig. 2 The 1H NMR data of L. theobromae extracts obtained from solid rice culture at three different incubation periods (solvent
a is DMSO-d
6; b is chloroform-d). The 30-day rice culture extract could only be fully dissolved in chloroform, indicating
the compounds occurring in this extract are semi-nonpolar. (Color figure available
online only.)
Table 1 Anti-trypanosomal activity of L. theobromae extracts derived from V. pinnata in different types of media and incubation periods. MIC was only determined for the
bioactive extracts.
|
Sample
|
T. b. brucei 20 µg/mL % of viability
|
T. b. brucei MIC average ± SD (n = 4)
|
|
LT = L. theobromae.
|
|
LT-LC-7
|
112.3
|
Not tested
|
|
LT-LC-15
|
106.3
|
Not tested
|
|
LT-LC-30
|
126.0
|
Not tested
|
|
LT-RC-7
|
103.9
|
Not tested
|
|
LT-RC-15
|
94.3
|
Not tested
|
|
LT-RC-30
|
1.4
|
25 ± 1.3 µg/mL
|
|
Suramin
|
Not tested
|
0.1 ± 0 µM
|
The medium-scale 30-day rice culture extract of L. theobromae was fractionated yielding 19 fractions (LT-1 until LT-19). These were submitted for
the anti-trypanosomal activity and subjected to MS-1H NMR-based metabolomics data profiling. The nonpolar fractions LT-2 to LT-8 exhibited
strong bioactivity, except for LT-4, which showed only moderate activity against T. b. brucei ([Fig. 3]). LT-1 was excluded from the bioassay screening because it contained only fatty
acids, as indicated by its 1H NMR data ([Fig. 4]). The 1H NMR data of the 19 fractions ([Fig. 4]) were analyzed, and unique chemical fingerprints of the active fractions were detected.
Among these active fractions, two distinctive subgroups, LT-2 to LT-4 and LT-6 to
LT-8, clustered together, as they shared similar spectral data. Fraction LT-5 was
a mixture of both groups. The 1H NMR spectra of fractions LT-2 to LT-5 displayed a pair of meta-coupled aromatic protons at δ
H 6.23 and 6.28 (J = 2.7 Hz) and a multiplet peak at δ
H 5.15, which may be an olefinic or oxygenated methine. The upfield shift of the meta-coupled aromatic protons at the 6 ppm region suggested the presence of an electron
withdrawing group such as a hydroxyl or halogen substituent. In fractions LT-5 to
LT-8, two meta-coupled doublets at δ
H 6.18 and 6.25 (J = 2.6 Hz) were observed (designated by red arrows on [Fig. 4]). The proton signals between 4.2–4.8 ppm revealed the presence of oxygenated methines
while proton signals between 6.6 to 7.7 ppm indicated the presence of aromatic compounds
in fractions LT 6 to LT8.
Fig. 3 Anti-trypanosomal activity of L. theobromae fractions against T. b. brucei. LT: L. theobromae extract as the positive control; LT2-LT19: L. theobromae fractions. (Color figure available online only.)
Fig. 4 A The 1H NMR spectra of the 19 fractions. B The expansion of the 1H NMR data of active anti-trypanosome fractions highlighting several unique chemical
fingerprints found only in these fractions. (Color figure available online only.)
Supervised methods of multivariate data analysis were used to analyze the similarity
and differences of the data sets between 19 samples. PCA was used in an earlier step
to observe an overview of variance between the fractions and metabolites generated
from MS data and also to identify any outliers. The distribution difference of the
type of metabolites between active vs. inactive fractions of L. theobromae against T. b. brucei was analyzed by subjecting the data to OPLS-DA. The results of the analysis led to
the prediction of compounds that contribute towards the anti-trypanosomal activity
of the fractions. For the OPLS-DA model ([Fig. 5 A]), the MS-based metabolomics data set was assigned as the X independent variable,
while the fractionsʼ anti-trypanosomal response was the Y dependent variable. The
quality of the OPLS-DA model was measured by two parameters, R2 (goodness of fit)
and Q2 (predictability), and the modelʼs R2X was 0.261, R2Y was 0.955, and Q2 was
0.733. These results were interpreted that 26.1 % of the X variables could be used
to describe 95.5 % of the variation between active fractions and inactive fractions,
while this model has 73.3 % of the average predictability. The value of R2Y and Q2
was greater than 50 %, indicating a well-fitted model exhibiting good prediction [41]. The quality and robustness of the OPLS-DA model was validated by a permutation
test (n = 100). The Q2 intercept value was − 0.368 (below 0.05), showing that the
original model is statistically effective ([Fig. 5 B]) [42]. For the OPLS-DA scores plot ([Fig. 5 A]), the active fractions were grouped together versus the inactive ones. Under the
active group, fractions LT-2 to LT-8 were clustered together, indicating a shared
set of metabolites, while fractions LT-9 to LT-19 were observed as the inactive group.
The generated S-plot ([Fig. 5 D]) determined the “end point” or unique compounds for each of the respective groups,
indicating the metabolites that are potentially responsible for the bioactivity against
T. b. brucei, which discriminated the active from the inactive fractions of L. theobromae. Eight metabolites were identified from Antibase as shown in [Table 2]. The end point compounds were targeted for bioassay-guided isolation work ([Fig. 6]).
Fig. 5 Multivariate analysis of L. theobromae fractions and anti-trypanosomal activity data correlation. A The score scatter plot of OPLS-DA shows the samples were grouped based on their bioactivity.
B The permutation test result of the OPLS-DA model. (C) The loading scatter plot of OPLS-DA shows the m/z values of active metabolites. (D) The S-plot generated from the OPLS-DA model shows the end point compounds that are
the predicted metabolites responsible for the bioactivity (highlighted in red). (Color
figure available online only.)
Fig. 6 Chemical structures of unique metabolites (1–8) predicted from the S-plot and further secondary metabolites (6, 8, 9) isolated from an active fraction of L. theobromae (Group 1).
Table 2 Putatively identified unique metabolites of L. theobromae active fractions obtained from S-plot “end point” data as shown on [Fig. 5 D]. (P = positive mode; N = negative mode).
|
Ionization mode
|
MS m/z
|
Rt (min)
|
Chemical formula
|
Name
|
|
N
|
191.035
|
11.32
|
C10H8O4
|
6,8-Dihydroxy-3-methylisocoumarin(1)
|
|
N
|
291.124
|
13.17
|
C16H20O5
|
6-Oxo-de-O-methyllasiodiplodin (2)
|
|
P
|
293.139
|
13.71
|
C16H20O5
|
6-Oxo-de-O-methyllasiodiplodin (2)
|
|
N
|
395.077
|
13.60
|
C21H16O8
|
Preussomerin-C (3)
|
|
N
|
363.051
|
14.61
|
C20H12O7
|
Preussomerin-H (4)
|
|
N
|
333.077
|
17.61
|
C20H14O5
|
Palmarumycin CP17 (5)
|
|
N
|
349.072
|
13.71
|
C20H14O6
|
Cladospirone B (6)
|
|
P
|
321.170
|
19.14
|
C18H24O5
|
Phomopsin B (7)
|
|
N
|
319.155
|
19.16
|
C18H24O5
|
Phomopsin B (7)
|
|
N
|
277.144
|
21.79
|
C16H22O4
|
Desmethyl-lasiodiplodin (8)
|
|
P
|
279.159
|
21.80
|
C16H22O4
|
Desmethyl-lasiodiplodin (8)
|
|
N
|
555.296
|
21.79
|
|
Complex of 277.144
|
The active metabolites listed from the dereplication step of the HR-LCMS data were
identified as palmarumycin CP17 (5), cladospirone B (6), and desmethyl-lasiodiplodin (8). Further analysis of the 1H-1H COSY NMR spectrum of fraction LT-3 revealed substructures belonging to the predicted
active metabolites. For example, the correlation of the meta-coupled aromatic protons at δ
H 6.23 and 6.28 as well as that of the methyl doublet at δ
H 1.35 with one oxygenated methine proton at δ
H 5.15, which further correlated with the aliphatic chain, was characteristic of the
desmethyl-lasiodiplodin structure (8) (Table S1, Fig. S1, Supporting Information). Substructures of palmarumycin CP17 and cladospirone B were
elucidated in the same manner.
Fraction LT-7, an outlier in the active group, was also selected based on the OPLS-DA
results, which indicated the bioactive metabolite. Among these metabolites, the structure
of the compound 6-oxo-de-O-methyllasiodiplodin (2) was confirmed by its COSY spectral data, which exhibited correlations similar to
desmethyl-lasiodiplodin (8), as shown in Fig. S2 A, Supporting Information. In the aromatic region, the COSY spectrum revealed correlations
as in preussomerin-C (Fig. S2 B, Supporting Information).
Isolation and purification of the compounds in the active group was performed by high-throughput
medium pressure liquid chromatography (MPLC). Three known compounds were isolated
and elucidated based on their NMR and MS data as cladospirone B (6) [43], desmethyl-lasiodiplodin (8) [44], and R-(−)-mellein (9) [45] (chemical structures shown in [Fig. 6]). The isolation of cladospirone B (6) and desmethyl-lasiodiplodin (8) confirmed the putative identification of the bioactive metabolites predicted from
the S-plot of the OPLS-DA model. R-(−)-mellein (9) is a structurally close analogue of 6,8-dihydroxy-3-methylisocoumarin (1). All isolated compounds were tested against T. b. brucei, cladospirone B (6) and desmethyl-lasiodiplodin (8) had MICs of 17.8 and 22.5 µM, respectively. All three metabolites were checked again
in the S-plot and R-(−)-mellein was located in the middle of the plot, suggesting that the anti-trypanosomal
activity would be less ([Fig. 7]); this is indeed the case as confirmed by the bioassay results ([Table 3]). Due to low concentration, palmarumycin CP17 was not isolated. Desmethyl-lasiodiplodin
(8) has been known to exhibit anticancer activity against MCF-7 via apoptosis with an
IC50 sevenfold more potent than its toxicity on normal cells [46]. On the other hand, cladospirone B (6) has been reported to be inactive in antibacterial and antifungal assays [45].
Fig. 7 Three isolated compounds from L. theobromae labelled in the S-plot. R-(−)-mellein was in the middle of the plot, suggesting less anti-trypanosomal activity
for this compound. (Color figure available online only.)
Table 3 Anti-trypanosomal activity of isolated compounds obtained from L. theobromae fermented in solid rice culture for 30 days.
|
Compound
|
Anti-trypanosomal activity (T. b. brucei) MIC average ± SD (n = 4) (µM)
|
|
Cladospirone-B (6)
|
17.8 ± 0
|
|
Desmethyl-lasiodiplodin (8)
|
22.5 ± 1.50
|
|
R-(−)-mellein (9)
|
> 100 ± 2.75
|
|
Suramin
|
0.1 ± 0
|
In our screening program, we observed that the crude extract of L. theobromae from agar plates showed anti-trypanosomal activity against T. b. brucei. In order to find the optimal conditions to grow L. theobromae, HR-LCMS-based metabolomics was applied, which resulted in the selection of solid
rice culture for 30 days as the best conditions for medium-scale fermentation. Fractionation
was performed on the crude extract, and based on the 1D 1H NMR data comparison of 19 fractions, several unique chemical fingerprints in the
active fractions were highlighted. Furthermore, by utilizing the HR-LCMS data for
multivariate analysis such as OPLS-DA, consequently, a set of the metabolites that
were predicted to be active, was generated. All predicted metabolites were easily
identified with the aid of AntiBase coupled to MZMine. The application of 1H NMR and COSY allowed for the detection of the predicted metabolites in the active
fractions as well as the confirmation of the dereplication results obtained from the
HR-LCMS data. Three known compounds were isolated and identified as cladospirone B
(6), desmethyl-lasiodiplodin (8), and R-(−)-mellein (9). To the best of our knowledge, this is the first report of isolation of cladospirone
B (6) from L. theobromae. It is also the first report to indicate the good anti-trypanosomal activity of cladospirone
B (6) and desmethyl-lasiodiplodin (8) against T. b. brucei in comparison with suramin (MIC value of 0.1 ± 0 µM). Our strategy of emphasizing
HR-LCMS and NMR-based metabolomics to search for active anti-trypanosomal compounds
has therefore been proven to be effective. In conclusion, this study determined that
the combination of HR-LCMS and NMR-based metabolomics is a powerful and advantageous
decision-making tool in mining active metabolites of L. theobromae against T. b. brucei and is also promising for implementation in other drug discovery programs elsewhere.
Materials and Methods
Fungal sampling
The fungus L. theobromae was isolated from fresh healthy leaves of V. pinnata collected in April 2011 near Kuala Terengganu, Malaysia. The plant was identified
by Dr. Nashriyah Mat from the Faculty of Bioresources and Food Industry, Universiti
Sultan Zainal Abidin, and a voucher specimen was deposited (collection number VP 01).
Samples were kept in zip lock bags and stored at 4 °C until the isolation of endophytic
fungi was performed 4 days later upon arrival at the University of Strathclyde in
Glasgow. The surfaces of the leaves and stems were sterilized with 70 % iso-propanol
for 2 min and subsequently rinsed in sterile water. Small tissue samples from inside
the leaves and stems were cut aseptically and pressed onto agar plates (composition
of isolation medium: 15 g/L malt extract, 15 g/L agar, and 0.2 g/L chloramphenicol
in distilled water, pH 7.4–7.8, adjusted with 10 % NaOH or 36.5 % HCl). Chloramphenicol
(≥ 98 %; purity Sigma-Aldrich) was added to inhibit bacterial growth. The plates were
left for a few days until fungal growth was observed. Reinoculation onto new malt
agar plates was repeated several times until pure strains were attained.
Identification of fungal strains
The fungal strain was identified using DNA amplification and sequencing of the internal
transcribed spacer (ITS) region as described previously [47]. The sequence data has been submitted to GenBank with accession number KC960898.
The fungal strain was identified as L. theobromae. A voucher strain was submitted and is kept at the Natural Product Metabolomics Laboratory,
SIPBS.
Small- and medium-scale fermentation
The fungal strain was cultivated on malt agar plates for 7 days at 30 °C. The colonies
and agar were cut into small pieces and were placed in liquid or solid media. The
liquid medium used was Wickerham medium, consisting of 3 g yeast extract, 3 g malt
extract, 5 g peptone, 10 g glucose, and distilled water adding up to 1000 mL in 2 L
Erlenmeyer flasks, pH 7.2–7.4, adjusted with 10 % NaOH or 36.5 % HCl. The solid medium
used was rice medium, with 100 g of long grain rice and 100 mL of distilled water
autoclaved together in 1 L Erlenmeyer flasks. The strain was grown for 3 different
incubation times: 7, 15, and 30 days, under static conditions at room temperature.
For medium-scale fermentation, L. theobromae was cultivated in 1 L flasks using the optimal conditions as determined by the small-scale
cultures. In this case, the growth of the fungus on rice medium for 30 days under
static conditions was determined to be the most favorable condition for the production
of anti-trypanosomal metabolites.
Extraction and isolation of pure compounds
The medium-scale rice cultures were extracted with ethyl acetate and homogenized as
finely as possible using a T18 Basic Ultra-Turrax (IKA) at maximum speed. These were
then kept overnight. The extract was subsequently dried under vacuo using a rotary evaporator (Buchi Labortechnik AG). The crude extract (5.0 g) was
then partitioned with 10 % n-hexane and 90 % methanol to remove fatty acids. The extract containing methanol-soluble
compounds (2.0 g) was collected for further isolation work. The fractionation of the
methanol extract was accomplished using MPLC. Linear gradient elution was employed
with hexane (A) and ethyl acetate (B) as the mobile phase at a flow rate of 20 mL/min.
A prepacked silica column (20–45 µm, 23 × 110 mm, Silica VersaPak cartridge) was used.
It was connected to a Buchi Pump Manager C-615 coupled to binary pumps (Buchi Modules
C-601). 100 % A was run for 5 min, followed by 100 % A to 100 % B for 20 min, and
finished with 100 % B for the last 5 min. The total run time was 30 min. Fractions
were collected in collection tubes automatically every 2 mL using a fraction collector
Frac 920 (GE Healthcare Bio-Sciences AB). Fractions with similar TLC profiles were
pooled together, yielding a total of 19 fractions. Fraction LT-2 (435 mg) was further
subjected to MPLC on a prepacked silica column (20–45 µm, 23 × 53 mm, Silica VersaPak
cartridge) utilizing an isocratic gradient system (80 % n-hexane: 20 % ethyl acetate) for 30 min at a flow rate of 20 mL/min. In total, 300
collection tubes of fractions (2 mL in each tube) were collected automatically using
their TLC profiles. Similar fractions were pooled, resulting in 17 fractions and giving
3 known compounds, 6 (3 mg), 8 (73 mg), and 9 (11 mg) with a purity > 95 % (determined by HPLC).
In vitro anti-trypanosomal assay
The samples were prepared to a final concentration of 10 mg/mL (stock solution) by
being dissolved in the appropriate amount of DMSO. To screen for in vitro activity, a concentration of 200 µg/mL was used. This was achieved by diluting the
stock solution 1 in 10 with HMI-9 (drug solution). Four µL of drug solution were transferred
to 96 µL of HMI-9 in the 96-well plate. One hundred µL of trypanosome suspension,
consisting of T. b. brucei S427 blood stream form at 3 × 104 trypanosomes/mL, were then added to the 96-well plate to make the final concentration
of the compounds range from 100 µg/mL to 0.17 µg/mL. DMSO was used as the negative
control (concentration of 1 to 0.002 %) and suramin (Calbiochem-Novabiochem Co., purity
> 98 % by HPLC) was selected as the positive control (concentration of 1 to 0.008 µM).
The plate was incubated for 48 h at 37 °C, 5 % CO2 with a humidified atmosphere, after which 20 µL of Alamar blue were added. The plate
was again incubated for another 24 h under the same conditions. The fluorescence was
measured using a Wallac Victor microplate reader (PerkinElmer) with excitation at
530 nm and emission at 590 nm. The results were calculated as percentages of control
values. All samples that exhibited > 90 % inhibition were selected for the MIC assay
to determine the MIC value.
Nuclear magnetic resonance instrumentation
One- and two-dimensional 1H and 13C NMR spectra were recorded at 400 MHz on a JEOL-LA400 FT-NMR spectrometer system
with a 40TH5AT/FG probe (JEOL LTD.). Compounds 6 and 8 were reconstituted in deuterated chloroform (CDCl3) while compound 9 was reconstituted in deuterated DMSO (DMSO-d
6).
High-resolution-liquid chromatography mass spectrometry procedure
HR-LCMS was measured using an Accela 600 HPLC pump with Accela autosampler and UV/Vis
detector (Thermo Scientific) and an Orbitrap Exactive mass spectrometer (Thermo Fisher
Scientific, Inc.). Analysis of samples was done using similar protocols described
previously [23], [47], [48].
High-resolution-liquid chromatography mass spectrometry data processing
Initially the raw HR-LCMS data were sliced into two data sets according to ionization
mode using the MassConvert tool from ProteoWizard (http://proteowizard.sourceforge.net/).
The sliced data were imported to MZMine 2.10 (http://sourceforge.net/projects/mzmine/),
software developed for the differential analysis of mass spectrometry data. The data
processing step was performed in the same manner as explained previously [23], albeit with slightly modified parameters. In this analysis, the data set was crop
filtered from 0.1 to 35 min and the retention time normalizer was not applied because
only one batch of data was used.
Statistical analysis
MS spectral data were converted to an ASCII text file and imported to MS Excel. The
data was sorted to exclude background peaks that belonged to the MeOH blank. The sorted
data were then exported to the SIMCA-P software 14.0 version (Umetrics). Pareto scaling
was employed on the MS data set. Finally, PCA, OPLS-DA, and S-plot were performed.
