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Microbial biotransformation of gentiopicroside by 1
endophytic fungus Penicillium crustosum 2T01Y01 2
Wenliang Zeng1, 2
, Wankui Li1, Han Han
1, Yanyan Tao
3, Li Yang
1, Zhengtao 3
Wang1 *
and Kaixian Chen1
4
1 The MOE Key Laboratory for Standardization of Chinese Medicines and the SATCM Key 5
Laboratory for New Resources and Quality Evaluation of Chinese Medicines, Institute of Chinese 6
Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China. 7
2 Shanghai PharmExplorer Co., Ltd., Shanghai 201203, China. 8
3 Institute of Liver Diseases, ShuGuang Hospital affiliated to Shanghai University of Traditional 9
Chinese Medicine, Shanghai 201203, China 10
Correspondence to: Zhengtao Wang, Institute of Chinese Materia Medica, Shanghai University of 11
Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201210, China. Telephone: 86 21 12
51322507, Fax: 86 21 51322519. 13
E-mail address: [email protected](Z. Wang) 14
15
AEM Accepts, published online ahead of print on 18 October 2013Appl. Environ. Microbiol. doi:10.1128/AEM.02309-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.
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Abstract 16
Endophytic fungi are symbiotic with plants and possess multi-enzyme systems 17
showing promising metabolite potency with region- and stereo-selectivities. The aim 18
of this study was to utilize these special microorganisms as an in vitro model to mimic 19
the potential mammalian metabolites of a natural iridoid gentiopicroside (GPS, 1). 20
The fungi isolated from a medicinal plant Dendrobium candidum Wall.et Lindl were 21
screened for their biotransformation abilities using GPS as the substrate and one strain 22
with high converting potency was identified as Penicillium crustosum 2T01Y01 based 23
on the sequence of the internal transcribed spacer of ribosomal DNA (rDNA-ITS) 24
region. On an optimized incubation of P. crustosum 2T01Y01 with the substrate, 25
seven deglycosylated metabolites were detected using an ultra performance liquid 26
chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS). A 27
preparative-scaled biotransformation with whole-cells of the endophytic fungus 28
resulted in the production of five metabolites including three novel ones as 29
5g-(hydroxymethyl)-6く-methyl-3,4,5,6-tetrahydropyrano[3,4-c]pyran-1(8H)-one (2), 30
(Z)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (3) and (E)-4- 31
(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one (4), along with two known ones, 32
5g-(hydroxymethyl)- 6く-methyl-1H,3H-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (5) 33
and 5g-(hydroxymethyl)-6g-methyl-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (6), 34
aided by the nuclear magnetic resonance and high-resolution mass spectral analyses. 35
The other two metabolites were tentatively identified by on-line UPLC/Q-TOF-MS as 36
5-hydroxymethyl-5,6-dihydroisochromen-1-one (7) and 5-hydroxymethyl-3,4,5,6- 37
tetrahydroisochromen-1-one (8), among them 8 is a new metabolite. To test the 38
metabolic mechanism, the ȕ-glucosidase activity of the fungus P. crustosum 2T01Y01 39
was assayed using と-nitrophenyl-く-D-glucopyranoside (とNPG) as a probe substrate 40
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and the biotransformation pathways for GPS by the strain 2T01Y01 was proposed. In 41
addition, the hepatoprotective activities of GPS and the metabolites 2, 5 and 6 against 42
human hepatocyte cells line HL-7702 injury induced by hydrogen peroxide (H2O2) 43
were evaluated. 44
Keywords: Microbial biotransformation, Gentiopicroside, Endophytic fungi, 45
Penicillium crustosum 2T01Y01, Hepatoprotective activity 46
47
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1. Introduction 48
Microorganisms have been used to produce chemicals, pharmaceuticals and 49
perfumes for decades and also for pollutants degradation and recovery of the 50
environment contaminated by chemicals (1). Another interesting use of 51
microorganisms is for studying metabolism of drugs and other chemicals. Smith and 52
Rosazza, in the early 1970’s, established the use of microbial models for mammalian 53
metabolism (2, 3). It has been demonstrated that microbial biotransformation system 54
is very similar to the mammalian phase I metabolic reactions. Therefore, this in vitro 55
biotransformation can be an attractive alternative for metabolism of new drugs, 56
making possible scale-production of metabolites, facilitating the structural elucidation 57
and toxicological tests (4). Other advantages of using microorganisms for drug 58
metabolism studies include the low cost and the ease of experimental design in 59
microbial transformation (5, 6, 7, 8). 60
Endophytes are bacterial or fungal microorganisms which colonize living internal 61
tissues of plants without causing any disease symptoms (9). Endophytes can produce a 62
great number of novel compounds with broad spectral biological activities, such as 63
antifungal, antibacterial, immunosuppressive and antineoplastic activities (10). 64
Endophytic fungi extensively transformed 2-hydroxy-1,4-benzoxazin-3(2H)-one 65
(HBOA) and 2-hydroxy-7-methoxy-1,4-benzoxazin-3(2H)-one (HMBOA) to less 66
toxic metabolites probably by their oxidase and reductases. Agusta et al. reported the 67
stereoselective oxidation at C-4 of flavans by the endophytic fungus Diaporthe sp. 68
isolated from a tea plant Camelia sinensis (11). It has been documented that 69
Penicillium crustosum could metabolize enantioselectively albendazole to albendazole 70
sulfoxide (12) and biotransform testosterone into five reduction products of 71
5g-dihydrotestosterone, dihydrotestosterone, 3g-hydroxy-5く-androstan-17-one, 3g- 72
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hydroy-5g-androstan-17-one, 4-androstene-3,17-dione, and 5g-androstane-3,17-dione 73
(13). 74
Therefore, endophytes attracted more and more attention not only for producing 75
novel compounds but also for transforming natural products to change their structures 76
and bioactivities. 77
Gentiopicroside, or 5-ethenyl-6-(beta-D-glucopyranosyloxy)-5,6-dihydro-1H,3H- 78
pyrano[3,4-c]pyran-1-one (GPS, 1), a secoiridoid-glucoside, is a principal bitter 79
substance found in many gentianaceous plants, such as Gentiana scabra Gbe., 80
Gentiana lutea L.襯Swertia pseudochinensis Hara. and Swertia mussotii Franch that 81
are widely used as medicinal herbs in China and Europe (14, 15). GPS has been 82
shown to exhibit a variety of pharmacological properties including anti-bacterial, 83
anti-apoptotic, bitter stomachic, cholagogue and hepatoprotective activities (16,17). 84
Nevertheless, like other iridoid glycosides, GPS normally acts as a prodrug, and its 85
activities are induced when the compound is activated by enzymes or 86
non-enzymatically by acid-hydrolysis. The hydrolytic β-glucosidases (EC 3.2.1.21) 87
have been shown to convert the non-reactive iridoid glycosides into highly reactive 88
aglycones (18, 19). As GPS belongs to the subclass of secoirioids, the aglycone is not 89
stable after hydrolysis, and as a result, no one has prepared its aglycone by enzymes 90
or acid-hydrolysis up to date. 91
Biotransformation of GPS by a strain of human intestinal bacteria, Veillonella 92
parvula ss parvula, produced five metabolites: erythrocentaurin, gentiopicral, 93
5-hydroxymethylisochroman-1-one, 5-hydroxymethylisochromen-1-one and 5,6- 94
dihydro-5-hydroxymethyl-6-methyl-1H,3H-pyrano[3,4-c]pyran-1-one (20). Wang 95
et al. reported the biotransformation of GPS by asexual mycelia of Cordyceps 96
sinensis yielding a new pyridine monoterpene alkaloid, (Z)-5-ethylidene 97
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-8-hydroxy-3,4,5,6,7,8-hexahydropyrano [3,4-c]pyridine-1-one (21). 98
In the present study, the biotransformation of GPS by an endophytic fungus isolated 99
from a Orchid medicinal plant, Dendrobium candidum Wall.et Lindl. was carried 100
aiming at discovery of the reactive pharmacophore and the metabolic pathway was 101
proposed. 102
2. Materials and Methods 103
2.1 Media and chemicals 104
The potato dextrose agar slant and preparation of the preculture were previously 105
described (22). Liquid seed medium: glucose at 20 g/liter and 200 g potato 106
water-boiled for 30 min, filtrated and diluted to one liter by deionized water at, pH 6.2. 107
The biotransformation experiment was carried out with 100 ml of liquid seed medium 108
and 1 ml substrate of GPS at 30 mg/ml. GPS (HPLC purity, 98.5%) was purchased 109
from Shanghai R&D Center for Standardization of Chinese Medicine. Acetonitrile 110
and methanol were high-performance liquid chromatography (HPLC) grade and were 111
purchased from Sigma-Aldrich (St, Louis, MO, USA). All other chemicals used for 112
extraction and isolation were analysis grade and commercially available. Deionized 113
water was used throughout the study. 114
2.2 Analytical and instrumental methods 115
During the biotransformation process, 5 ml culture broth from the flask were each 116
taken at 1, 2, 4 and 6 days, and an equal volume of acetonitrile was then added to the 117
broth. The diluted solution was centrifuged at 12000 × g for 30 min to remove 118
proteins. The supernatant was filtered with a 0.45-ȝm-micropore filter and transferred 119
into a sampling vial for HPLC analysis. HPLC analysis was carried out with an 120
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Agilent 1200 series HPLC system equipped with a UV detector (Agilent Technologies, 121
USA). A Zorbax Bonus-RP (3.5 µm, 75 mm × 4.6 mm i.d., Agilent Technologies, 122
USA) was used. The mobile phase consisted of water with 0.1% formic acid (A) and 123
acetonitrile (B) with a flow rate of 1.5 ml/min. The gradient condition of mobile phase 124
was: 5%-20% B from 0 to 10 min, 20%-95% B from 10 to 13 min, and 95% B from 125
13 to 15 min. The HPLC oven temperature was maintained at 40oC, and the detection 126
wavelength was 225 nm. 127
UPLC/Q-TOF MS analysis was carried out on a Waters ACQUITYTM
Synapt G2 128
system (Waters Corp., Manchester, UK). The column effluent was monitored by a 129
quadrupole time-of-flight (Q/TOF) tandem mass spectrometer (Waters Co., UK) 130
equipped with a LockSpray and ESI interface. High-purity nitrogen was used as the 131
nebulizer and auxiliary gas. ESI–MS/MS experiment was performed in the positive 132
mode under the following operating parameters: capillary voltage was set at 3.0 kV; 133
The sample cone voltage was set at 45 V; and extracting cone was set at 4 V; The 134
source and desolvation temperatures were set at 150oC and 450
oC , respectively. The 135
cone and desolvation gas flow rates were set at 50 and 850 L/h, respectively. 136
MassLynx 4.1 software (Waters Co., USA) was used to control the 137
UPLC–ESI–MS/MS system, as well as for data acquisition and processing. The 138
chromatographic separations were achieved on a Waters Acquity UPLCTM
T3 column 139
(100 × 2.1 mm i.d., 1.8 たm particle size; Waters Corporation, Milford, MA, USA) 140
kept at 40 °C. The mobile phase consisted of A (0.1% aqueous formic acid) and B 141
(acetonitrile) with a flow rate of 0.5 mL/min. The gradient elution procedure was: 5% 142
B from 0 to 12 min, 5%-90% B from 12 to 15 min, 90% B from 15 to 17 min, 143
90%-5% B from 17 to 18 min. 144
1H and
13C nuclear magnetic resonance (NMR) spectra were run on a Bruker 145
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AVANCE 400 FT-NMR spectrometer operating at 400 MHz for 1H and 100 MHz for 146
13C, respectively, with deuterated dimethyl sulphoxide (DMSO-d6, St, Louis, MO, 147
USA) as solvent. Coupling constants were expressed in Hertz and chemical shifts 148
were reported on a ppm scale with tetramethylsilane (TMS) as an internal standard. 149
2.3 Microorganisms 150
2.3.1 Sampling Dendrobium candidum Wall.et Lindl. plants were gathered from 151
the Nanhui Dendrobium candidum artificial cultivation base, Shanghai, China. 152
Dendrobium candidum Wall. et Lindl. plants with bulk soil and fresh humus soil were 153
carefully packed and transformed to the laboratory within 48 hours. 154
2.3.2 Isolation and growth The endophytes were isolated from the healthy stems 155
of Dendrobium candidum Wall.et Lindl. The stem was cut into pieces at about 1 cm of 156
length and thoroughly washed using distilled water, followed by 75% (vol/vol) 157
ethanol for 1 min and 5% sodium hypochlorite for 5 min to accomplish surface 158
sterilization. The pieces were then rinsed in sterile demineralized water three times for 159
1 min. Small pieces of the inner tissue of the stems were placed on potato dextrose 160
agar petriplates pretreated with 0.1% chloramphenicol and incubated at 28oC ± 2
oC 161
until fungal growth was initiated. The tips of the fungal hyphae were then removed 162
from the aqueous agar and inoculated onto the mycological medium. A similar 163
procedure, but without surface sterilization, was used as a negative control to check 164
the surface-contaminated fungi. In total, 39 pure culture isolates were obtained. Each 165
strain was aseptically transferred onto agar slants and allowed to grow for 4 days at 28
166
oC, and three tips of the slant endophytic fungi were subsequently inoculated into a 167
250-ml shake flask containing 100 ml of liquid seed medium, and the culture was 168
incubated for 3 days at 28oC on a rotary shaker at 120 rmp. A 10-ml aliquot of liquid 169
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culture was then used for inoculation in the microorganism screening experiment as 170
described below. 171
2.3.3 Fermentation procedures Microbial metabolism studies were carried out 172
by incubating cultures on a incubator shaker (ZHWY-211, Zhicheng Instrument 173
Manufacture Co., Shanghai) operated at 120 rpm and 28oC. The medium was 174
sterilized at 121oC and 18 Ib/in
2 for 20 min. Fermentations were carried out according 175
to a standard two-stage protocol (23). Endophytic fungus stock inoculums were first 176
prepared by suspending the fungus from one agar slant in 1 ml of sterile distilled 177
water. Submerged stage I cultures were then initiated by adding 0.1 ml of the 178
endophytic fungus stock inoculums to a 250 ml flask containing 50 ml of liquid 179
medium. Following incubation of stage I cultures for 2 days on the shaker, stage II 180
cultures were initiated by inoculating 50 ml of fresh, sterile liquid medium with 1 ml 181
of stage I culture broth. After incubation of stage II cultures for 2 days, the complex 182
medium was used for biotransformation of substrates. 183
2.3.4 Microorganisms screening In order to screen the microorganism with the 184
biotransformation ability of GPS, a biotransformation experiment, a culture control, 185
and a substrate control were run to identify the substrate metabolites, microorganism 186
metabolites, and chemical degradation of substrates by chromatographic analysis. The 187
biotransformation experiment was run through the substrates with the inoculation of 188
microorganisms in a 250-ml shake flask containing 90 ml of the above mentioned 189
liquid medium and 10 ml of the above mentioned liquid cultures. The 190
above-mentioned experiments were allowed to proceed for 6 days at 28oC. 191
Periodically, three shake flasks were taken out at each sampling time. 192
Twelve strains of endophytes were selected to incubate with the substrate according 193
to above procedures, respectively. The GPS biotransformation abilities of the tested 194
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fungus strains were evaluated by the consumption of GPS, and the appearance of new 195
products, aided by HPLC and UPLC-Q TOF-MS/MS analyses. 196
2.4 Preparative-scale biotransformation 197
The preparative-scaled biotransformation of GPS was carried out with 50 250-ml 198
shake flasks and each contained 100 ml stage II of cultures with 1 ml of GPS solution. 199
In total, 1.5 g of GPS was used to prepare the biotransformed products. The 200
incubation was continued for 6 additional days. The other procedures were the same 201
as those described previously in the strain screening experiments. 202
2.5 Extraction, isolation and purification 203
The cultures after incubation were filtered through 4 layers of gauze and washed 204
with distilled water, then the filtrations and washings (calcd. 5 liters) were combined 205
and extracted with three 5-liters of n-butyl alcohol. The combined organic layers were 206
concentrated under reduced pressure to yield 2.6 g of residue. The residue was first 207
chromatographed on a MCI column (Mitsubishi Chemical Corporation, Japan) (60 by 208
4 cm, 50 g of MCI gel) and eluted with water, water-methanol (50 : 50, vol/vol) and 209
water-methanol (20 : 80, vol/vol) to obtain three fractions, respectively. The fraction 210
of (water-methanol (20 : 80, vol/vol)) was subjected to a Gilson 215 prep-HPLC 211
system consisting of a Gilson 811D dynamic mixer, UV detector (Gilson Corporation, 212
USA). The separations were run on an YMC-Pack ODS-A column (10 たm, 250 × 20 213
mm I.D., 12 nm, YMC, Japan). The mobile phase consisted of A (0.1% aqueous 214
trifluoroacetic acid, v/v), and B (0.1% trifluoroacetic acid in acetonitrile). The 215
gradient elution procedure was as follows: 0-25 min, 8% B; 25-30 min, 8%-95% B; 216
30-35 min, 95% B; Flow rate used was 10 ml/min and monitored at 225 nm. In total 217
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five metabolites were prepared and purified: metabolite 2, brown powder with a 218
HPLC purity of 98.5% (8.0 mg, 0.5% yield); metabolites 3 and 4, yellow powder with 219
a HPLC purity of 97.0% (16.0 mg, 1.1% yield); metabolite 5, brown powder with a 220
HPLC purity of 99.0% (19.2 mg, 1.3% yield); metabolite 6, brown powder with a 221
HPLC purity of 98.0% (8 mg, 0.5% yield); 222
Structural elucidation of metabolites 2 to 6 was based on one-dimensional and 223
two-dimensional NMR and high-resolution mass spectral analyses. 224
2.6 Human hepatocytes protective effect of GPS, metabolites 2, 5 and 6 225
Human hepatocyte line (HL-7702) was maintained in RPMI 1640 medium 226
supplemented with 10% (v/v) heat-inactivited fetal bovine serum, 100 U/mL 227
penicillin and 100 たg/mL streptomycin, 2 mM of glutamine, and 10 mM of Hepes 228
buffer at 37oC in a humid atmosphere (5% CO2, 95% air). HL-7702 cells were 229
pretreated with culture medium containing different concentrations of GPS, 230
metabolites 2, 5 and 6 (5.0 µM, 10.0 µM and 20 µM) for 24 h, respectively, and 231
subsequently, the cells were exposed to H2O2 (2.0 mM) diluted in the culture medium 232
for 1 h at 37oC (24, 25). Then, cell counting kit-8 (CCK-8) was added into each cell 233
culture and maintained in incubator for 2.5 h before analysis. Five replicate wells 234
were used for each concentration of GPS, metabolites 2, 5 and 6 in the experiments. 235
The cell viability was measured spectrophotometrically at 450 nm by using an ELISA 236
reader (26). 237
2.7 Assay for ȕ-glucosidase activity 238
The determination of ȕ-glucosidase activity of the fungus was conducted according 239
to the method of Otieno et al (27) with modification. The organism was activated first 240
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according to 2.3.3 Fermentation procedures. Subsequently, 5 ml of activated culture 241
was inoculated into a 1000 ml flask containing 500 ml of liquid medium and 242
incubated at 28oC. After incubation of stage II cultures for 2 days, 50 ml of aliquots 243
were taken aseptically from the liquid medium at 1, 2, 4, 5, 6, 7 and 8 days and the 244
enzyme activity was determined immediately. The ȕ-glucosidase activity was 245
determined by measuring the rate of hydrolysis of と-nitropheyl ȕ-D-glucopyranoside 246
(と-NPG). 1 ml of 5 mM と-NPG prepared in 100 mM sodium phosphate buffer (pH 7.0) 247
was added to 10 ml of each aliquot and incubated at 37oC for 24 h, and 0.5 ml of 1 M 248
cold sodium carbonate (4oC) were added to stop the reaction. The absorbance of each 249
mixture was measured using a spectrophotometer at 420 nm. The absorbance of a 250
series of dilutions of と-nitrophenol was used to calculate the enzymatic activity. 251
2.8 Fungal 5.8 S rDNA amplification, sequencing, phylogenetic analysis and 252
nucleotide sequence accession numbers 253
The identity of the organism was determined based on partial or nearly full-length 254
5.8 S rDNA gene sequence analysis. Fungal DNA was extracted from pure cultures by 255
using a genomic DNA miniprep kit (Generay Biotechnology Corporation, China) 256
according to the manufacturer’s instructions. Primers ITS1 (5ガ- 257
AACTCGGCCATTTAGAGGAAGT-3ガ) and ITS4 (5ガ-TCCTCCGCTTATTGATAT 258
GC-3ガ) were used for the amplification of P. crustosum 2T01Y01 5.8 S rDNA. The 259
PCR mixture (total volume, 50 µl) contained 5 µl 10× PCR buffer, 4µl 25 mM Mg2+
, 260
2 µl 10 mM deoxynucleoside triphosphates (dNTPs), 1 µl of each primer (10 µM), 2 261
µl original template, 1 µl Taq polymerase, and double-distilled water (dd H2O) (34 µl). 262
Thirty-four cycles were run, with each cycle consisting of a denaturation step at 94oC 263
(60 s), an annealing step at 53oC (45 s), and an extension step at 72
oC (90 s). After the 264
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34th cycle, a final 10-min extension step at 72oC was performed. The reaction 265
products were separated on a 1.0% (wt/vol) agarose gel, and the amplicons were 266
purified by using a gel band purification kit (Generay Biotechnology Corporation, 267
China). 268
The final sequence sets were then submitted to BLAST analysis, and identities of 269
≥99% were considered conspecific. To verify the phylogenetic positions of genotypes, 270
the sequences were aligned with Clustal X 2.0.1 multiple-sequence alignment 271
software and imported into MEGA 4.1. The evolutionary history was inferred by 272
using the neighborjoining method. The bootstrap consensus tree inferred from 1,000 273
replicates is taken to represent the evolutionary history of the taxa analyzed.Branches 274
corresponding to partitioins reproduced in fewer than 50% bootstrap replicates are 275
collapsed. The robustness of the tree topology was tested by bootstrap analysis (1,000 276
replicates). 277
The 5.8 S rDNA gene sequences of P. crustosum 2T01Y01 has been deposited in 278
GenBank database under accession number KC193255. 279
3. Results 280
3.1 Screening and identification of the microorganism strain 281
In total, 39 endophytic fungi were isolated from the healthy stems of Dendrobium 282
candidum Wall.et Lindl. On the basis of morphological features and genotypes of 283
these fungi, twelve strains were screened and three strains showed the ability to 284
metabolize GPS, among which one strain (P. crustosum 2T01Y01) indicated the 285
highest transformation rate. 286
The 5.8S rDNA gene sequence of the strain 2T01Y01was determined and classified 287
into the genus Penicillium in its phylogenetic affiliation. The length of the PCR 288
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product of the strain 2T01Y01 was 507 bp. The G + C content of the DNA of the 289
strain 2T01Y01 was 58.2 %. A Basic Local Alignment Search Tool (BLAST) search 290
for the 5.8S rDNA sequence from the strain 2T01Y01 revealed the highest degree of 291
similarity with a Penicillium crustosum strain reported under GenBank accession 292
number KC193255 (Fig.1). As a result, the strain 2T01Y01 is named as P. crustosum 293
2T01Y01. 294
3.2 Identification of metabolites of GPS 295
Seven metabolites (2 to 8) of GPS were detected by HPLC and UPLC-Q 296
TOF-MS/MS (Fig. 2)., of which five metabolites (2 to 6) were isolated by repeated 297
chromatographic separation and structurally elucidated by 1H- and
13C-NMR and MS 298
spectral data. The 1H and
13C-NMR data for metabolites 2 to 6 are summarized in 299
Table 1. The structures of metabolites 7 and 8 were tentatively identified by online 300
Q-TOF MS/MS analyses (positive ion). The retention times, the maximum ultraviolet 301
absorption wavelength and accurate measurements of metabolites 2 to 8 are listed in 302
Table 2. 303
Metabolite 2 was obtained as a powder. Its molecular formula C10H15O4 was 304
deduced from the HR-ESI-MS at m/z 199.0984 (calcd. for [M+H]+: 199.0965) and 305
confirmed by the 13
C NMR data, indicating four degrees of unsaturation. The 1H-,
13C 306
NMR and HSQC spectral data revealed ten carbon signals consisting of one methyl 307
group, four methylenes (including three oxygen – bearing carbons), two methines 308
(including one oxygen – bearing carbon), two olefinic quaternary carbons, and one 309
carbonyl quaternary carbon. There was no olefinic proton or aromatic proton signals 310
at low field of the 1H NMR spectrum of metabolite 2, implying the olefinic bond 311
existing between C-11 and C-12, not between C-8 and C-11 or between C-4 and C-12 312
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(Fig. 3). The 1H,
1H-COSY spectrum of metabolite 2 (Fig. 4 (A)) implied 313
connectivities of CH2(3) to CH2(4), and of H-C(5) to H-C(6). The HMBC spectrum 314
(Fig. 4 (A)) showed correlations between CH2(3) and C(1) and C(12), between H-C(6) 315
and C(8), between H-C(8) and C(11) and C(12), between H-C(9) and C(6) and C(12), 316
between methyl and C(6) and C(5), respectively. With these correlations, the 317
constitution of metabolite 2 could be deduced. The relative configuration of 318
metabolite 2 was determined by NOESY experiments. The coupling constant between 319
H-C(5) and H-C(6) was too small to be measured, indicating the ee coupling between 320
these two protons. The NOESY spectrum of metabolite 2 revealed enhancements 321
between H-C(5) and methyl proton, H-6 and H-9, which demonstrated a trans 322
configuration between the methyl at C-6 and side chain at C-5. Based on all the 323
evidence, metabolite 2 was identified as 5g-(hydroxymethyl)-6く-methyl-3,4,5,6- 324
tetrahydropyrano[3,4-c]pyran-1(8H)-one. 325
Metabolites 3 and 4 gave the same retention time and the same molecular formula 326
of C9H13O3, by positive HR-ESI-MS (m/z 169.0882, calcd. for [M+H]+: 169.0859) 327
(Table 2). Both metabolites showed closely similar 1H-,
13C NMR spectral features 328
(Table 1). Compared with the NMR spectral data of metabolite 2, each compound 329
exhibited a carbonyl carbon (3, h(C-1) 163.94; 4, h(C-1) 169.66), and two methylene 330
(3, h(H-3) 4.29 (t, J= 6.0 Hz), h(H-4) 2.41 (t, J= 6.0 Hz); 4, h(H-3) 4.80, h(H-4) 3.01), 331
which suggested a similar lactone ring was present in the two compounds. 332
Furthermore in the 1H NMR spectra, a couple of olefinic signals (3, h(H-6) 5.79, 333
h(H-8) 5.84, h(H-9) 5.17; 4, h(H-6) 5.76, h(H-8) 5.83, h(H-9) 5.08) were observed in 334
the two metabolites. These results suggested that the pyran ring from its parent 335
compound (1) was cleaved and further decarboxylated. Thus a hydroxyl group in each 336
metabolite was formed and the resonance of hydroxymethylene at h(H-10) 3.57(d, J = 337
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6.4 Hz), h(C-10) 61.97 for metabolite 3 and h(H-10) 3.51(d, J = 6.4 Hz), h(C-10) 338
62.14 for metabolite 4 were displayed. The 1H,
1H-COSY spectrum of metabolites 3 339
and 4 (Fig. 3 (B, C)) implied connectivities of CH2(3) to CH2(4), of H-C(7) to 340
CH2(10), of H-C(8) to CH2(9). The HMBC spectrum of metabolites 3 and 4 (Fig. 3 (B, 341
C)) show correlations between CH2(3) and C(5), between CH2(4) and C(6), between 342
CH(7) and C(6) between CH2(9) and C(7), between CH2(10) and C(5) and C(8). From 343
these data, the constitutional formulae of metabolites 3 and 4 could be deduced. 344
The relative configuration of metabolites 3 and 4 was determined as follows. In the 345
NOESY spectrum of metabolite 4, correlations of H-C(6) with H-C(9) was observed, 346
which indicated a Z-stereochemistry for metabolite 4. Whereas correlations of H-C(6) 347
and H-C(9) was not displayed in the NOESY spectrum of metabolite 3, revealing an 348
E-stereochemistry for metabolite 3. Based on all the evidence, metabolite 3 and 4 349
were determined as (E)-4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one and (Z)- 350
4-(1-hydroxybut-3-en-2-yl)-5,6-dihydropyran-2-one, respectively. 351
Metabolite 5 was obtained as brown powder and the HR-ESI-MS showed its 352
molecular ion at m/z 197.0832 (calcd. for [M+H]+: 197.0808) (Table 2), 353
corresponding to a molecular formula of C10H13O4. The structure of metabolite 5 354
was identified on the basis of their NMR and MS data as 355
5g-(hydroxymethyl)-6く-methyl-1H,3H-5,6-dihydropyrano [3,4-c]pyran-1(3H)-one 356
(Table 1), which was described in previous papers ( 20, 21 ). 357
Metabolite 6 gave the same molecular ion at m/z 197.0832 and exhibited closely 358
similar 1H-,
13C-NMR spectra patterns with metabolite 5, suggesting the two 359
compounds were a pair of geometric isomers. The relative configuration of metabolite 360
6 was determined as follows. In the 1H-NMR spectrum, the coupling constant 361
between H-C(5) and H-C(6) was too small to be measured, suggesting the ee coupling 362
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between these two protons. Furthermore in the NOESY spectrum, correlations 363
between H-C(5) and H-C(6), between H-C(9) and methyl proton (H-10) were 364
observed, confirming a cis nature between the methyl at C-6 and side chain at C-5 for 365
metabolite 6. Based on these observations, metabolite 6 were identified as 366
5g-(hydroxymethyl)-6g-methyl-5,6-dihydropyrano[3,4-c]pyran-1(3H)-one (20, 21). 367
Metabolites 7 and 8 were eluted at the retention time of 6.96 min and 4.14 min, and 368
gave a positive HR-ESI-MS at m/z 179.0729 (calcd. for [M+H]+: 179.0708) and m/z 369
181.0883 (calcd. for [M+H]+: 181.0865) (Table 2), respectively. The CID 370
(collision-induced dissociation)-MS/MS spectrum of the precursor ion extracted from 371
7 gave three main product ions [M+H-CO]+ at m/z 151.0775, [M+H-CH2O2]
+ at m/z 372
133.0670 and [M+H-CH2O2-CO]+ at m/z 105.0720 (Fig. 4 (A)). The MS/MS spectrum 373
of metabolite 8 gave the product ions [M+H-CH2O]+ at m/z 151.0773, 374
[M+H-CH2O-H2O]+ at m/z 133.0676, and [M+H-CH2O-CH2O2]
+ at m/z 105.0718 (Fig. 375
4 (B)). Compared with the MS/MS spectrum of parent drug (1), metabolites 7 and 8 376
were tentatively identified as 5-(hydroxymethyl)-5,6-dihydroisochromen-1-one and 377
5-(hydroxymethyl)-3,4,5,6-tetrahydroisochromen-1-one, respectively(20). 378
3.3 Hepatoprotective effects of GPS metabolites 379
The protective effects of GPS and its three available metabolites (2, 5 and 6) on the 380
survival ability of HL-7702 cells were examined. The CCK-8 assay showed that the 381
viability of HL-7702 cells was decreased by H2O2 remarkablely. However, after 382
pretreatment with GPS and the three metabolites 2, 5 and 6, the metabolites (2, 5, 6) 383
could restore the cell viability at the concentration of 20, 10, and 5 µM respectively, 384
while GPS showed no activity at that concentration, which indicated that the 385
biotransformation products exhibiting more potent protective effects than the substrate 386
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GPS. 387
4. Discussion 388
Penicillium crustosum has been found commonly in food, feed and plants (28-30) 389
and had a wide range of biological functions and possessed multi-enzyme systems 390
with significant region- and stereo-selectivities (31, 32), which had already been 391
utilized to biotransform natural products (33, 34), chemicals (12, 35, 36) and 392
endogenous materials (32) to change their structures and bioactivities. 393
Gentiopicroside (GPS, 1), a secoiridoid-glucoside, has been found as principal 394
component in many gentianaceous medicinal plants and some of the species, such as 395
Gentiana scabra Gbe., and G. lutea L. are recorded in official Pharmacopoeias of 396
China, Britain and Europe. GPS, like other iridoidal glycosides, acts as a prodrug 397
which need to be activated by gut microbes to exert its biological activities. Yet the 398
biotransformation pathway and the activity-responsible pharmacophore remain to be 399
evaluated. . 400
In the present study, a strain of Penicillium fungi isolated from a medicinal herb 401
was detected to show high transforming ability of GPS, and identified as 402
Penicillium crustosum 2T01Y01 based on the internal transcribed spacer of ribosomal 403
DNA (rDNA-ITS) region. A preparative-scaled whole cells incubation of GPS with 404
this fungus resulted in the isolation and structural elucidation of five metabolites (2 to 405
6). While the other two metabolites (7 and 8) were identified tentatively by online 406
UPLC-MS technology because of their limited concentration in the incubation system. 407
A time course analysis by HPLC revealed that the metabolites were detected on the 408
second day and the substrate GPS was nearly completely consumed on the 6th day of 409
the incubation. 410
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The biotransformations involved in drug metabolism in microorganisms are often 411
the reactions of hydrolysis, reduction, oxidation and isomerization (37). For the 412
biotransformation of GPS by P. crustosum 2T01Y01, the deglycosylation by 413
ȕ-glucosidase existing in the fungus might be the initiation step. In this study, the 414
ȕ-glucosidase activity in P. crustosum 2T01Y01 was determined using 415
と-nitrophenyl-く-D-glucopyranoside (とNPG) as the probe substrate and the result 416
showed that the enzymatic activity of the fungus was activated in the second day and 417
reached the highest level on the seventh day of incubation (Fig. 5). 418
Therefore, the metabolic pathway of GPS in P. crustosum 2T01Y01 can be 419
proposed as shown in Fig. 6. GPS was firstly hydrolyzed by the fungal ȕ-glucosidase 420
to form an unstable hemiacetal aglycone, which was readily converted to the reactive 421
intermediate aldehyde alcohol (Ia) or dialdehyde (Ib). Subsequently Ia and Ib 422
underwent through intramolecular cyclization to produce an pyrano[3,4-c]pyran (Ic), 423
and an isocoumarine (Id) derivatives, respectively. Ic was further converted by 424
reduction and hydrogenation to produce compounds 2, 5 and 6, or alternatively by 425
oxidation and decarboxylation to gave two pyran ring-opening products, 3 and 4, a 426
pair of cis-trans isomers. Simultaneously, Id was subjected to reduction to produce 427
compound 7, or further hydrogenated to form 8. 428
It was obvious that the fungus is a multi-enzyme system and showed more potent 429
biotransformation activity than the single enzyme, as indicated in our previous paper 430
(38), in which a single glycoside hydrolase was used for biotransformation of GPS 431
and only four metabolites including the two intermediates Ic and Id were identified. 432
The in vitro bioassay indicated that the three available metabolites (2, 5 and 6) 433
showed potent protective effects against HL-7702 cells injury induced by H2O2, while 434
the substrate GPS exhibited no activity at the tested concentration襯which gave 435
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evidence that iridoid glycosides could only be biotransformed by enzymes into their 436
aglycon derivatives to exert their broad spectral bioactivities. 437
The function of the metabolites of GPS for the fungus remains to be extensively 438
investigated in the future. 439
Acknowledgements 440
We thank the National Natural Science Foundation of China (No. 81073027), the 441
Program for Changjiang Scholars and Innovative Research Team in University 442
(IRT1071), the Shanghai Rising-Star Program (12QH1402200) and the Shanghai 443
Municipal Health Bureau Program (XYQ2011061) for their financial support of this 444
work. 445
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reduction of ketones. Angew Chem Int Ed Engl. 23:570– 578. 552
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556
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Figure Captions 557
Fig. 1. Phylogenetic trees of an endophyte strain (Penicillium crustosum 2T01Y01) 558
inferred based on 5.8s rDNA sequences. Maxium-parsimony bootstrap values of 50% 559
are indicated above branch nodes. The number of bootstrap replicates was 1,000. 560
GenBank accession numbers are in parenthese. 561
Fig. 2. Total ion chromatograms (TICs) by UPLC/QTOF-MS in positive ion mode of 562
the incubation solution with substrate (A) and without substrate (B). 563
Fig. 3. Key 1H,
1H-COSY and HMBC correlations for metabolite 2 (A); and 564
metabolites 3 (B) and 4 (C). 565
Fig. 4. MS/MS spectra of the [M+H]+ ion: (A) at m/z 179.0729 for metabolite 7; (B) 566
at m/z 181.0883 for metabolite 8. 567
Fig.5. Time-dependent release of PNP after incubation of PNP く-D-glu with 568
Penicillium crustosum 2T01Y01 (n=3, mean±SD) 569
Fig. 6. Proposed metabolic pathway for gentiopicroside in Penicillium crustosum 570
2T01Y01 571
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Table 1. 1H and 13C NMR data for metabolites of gentiopicroside
Position Compound 2 Compound 3 Compound 4 Compound 5 Compound 6
13C (ppm)
a
1H (ppm), J
(Hz)b
13C (ppm) 1H (ppm), J (Hz) 13C (ppm) 1H (ppm), J (Hz) 13C (ppm) 1H (ppm), J
(Hz)
13C (ppm) 1H (ppm), J (Hz)
1 163.08 163.94 169.66 163.17 163.30
3 65.69 4.38 (2H, m) 65.86 4.29 (2H, t, J= 6.0 Hz) 67.71 4.80 (2H, br,) 68.81 5.00 (2H, m) 68.81 4.95 (2H, m )
4 25.38 2.35,2.65 (2H,
m)
25.57 2.41 (2H, t, J= 6.0 Hz) 32.39 3.01 (2H, br) 114.45 5.62 (1H, m) 113.02 5.53 (1H, br)
5 46.73 2.12 (1H, br) 162.19 134.74 43.96 2.39 (1H, br) 41.79 2.64 (1H, br, 5-H),
6 69.80 3.73 (1H, m) 116.13 5.79 (1H, s) 117.80 5.76 (1 H, s) 72.94 4.60 (1H, m) 74.82 4.44 (1H, m)
7 52.28 3.12 (1H, dd, J= 6.4
Hz, 6.8 Hz)
50.85 2.91 (1H, dd, J =
6.4 Hz, 6.8 Hz)
8 62.05 4.15 (2H, m) 135.67 5.84 (1H, m) 136.93 5.83 (1H, m) 150.61 7.46 (1H, s) 152.24 7.53 (1H, s)
9 58.21 3.55 (2H, m) 117.62 5.17 (2H, m), 116.57 5.08 (2H, m) 60.52 3.40 (2H, m) 58.02 3.53 (2H, m)
10 19.03 1.26 (3H, dd,
J= 6.4 Hz, 8.4
Hz)
61.97 3.57 (2H, d, J = 6.4
Hz)
62.14 3.51(2H, d, J = 6.4
Hz)
18.25 1.22 (3H,d, J
= 6.6 Hz)
15.53 1.25 (3H, d, J = 6.6
Hz)
11 123.30 101.80 102.40
12 152.04 124.81 126.97
a 1H and
13C NMR spectra were obtained with deuterated dimethyl sulfoxide (DMSO-d6).
b Abbreviations for NMR signals are as follows: s, singlet; d, doublet; t, triplet; m, multiplet; br, broad.
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Table 2. Retention time, the maximum UV absorption wavelength, accurate
measurements of elemental formulas of protonated molecules and product ions by
Q-TOF MS/MS analysis of parent drug and metabolites
Compound tR
(min)
そmax
(nm)
Fragment ions,
Da (relative intensity)
Elemental
composition
1 12.56 205, 235, 270 [M+H]+ 357.1190 (23) C16H21O9
[M+H-Glc]+ 195.0670 (66) C10H11O4
[M+H-Glc-H2O]+ 177.0565 (100) C10H9O3
[M+H-Glc-H2O-CO]+ 149.0612 (89) C9H9O2
[M+H-Glc-H2O-C2O2]+ 121.0666 (44) C8H9O
2 3.52 230 [M+H]+ 199.0984 (52) C10H15O4
[M+H- C2O2]+ 155.0723 (100) C8H11O3
[M+H- C2O2-H2O]+ 137.0618 (18) C8H9O2
[M+H-C2O2- 2H2O]+ 119.0511 (17) C8H7O
[M+H-C3O3- 2H2O]+ 91.0548 (37) C7H7
3, 4 5.72 225 [M+H]+
169.0882 (100) C9H13O3
[M+H- H2O]+
151.0759 (6) C9H11O2
[M+H- CH2O]+
139.0778 (43) C8H11O2
[M+H- C2H4O3]+
93.0720 (24) C7H9
5 7.75 214, 245, 285 [M+H]+ 197.0832 (52) C10H13O4
[M+H-H2O]+
179.0732 (17) C10H11O3
[M+H-CH2O]+
167.0726 (27) C9H11O3
[M+H-CH2O-H2O]+
149.0617 (100) C9H9O2
[M+H-CH2O-H2O-CO]+
121.0617 (50) C8H9O
6 8.44 214, 245, 285 [M+H]+
197.0836 (80) C10H13O4
[M+H-H2O]+
179.0725 (12) C10H11O3
[M+H-CH2O]+
167.0726 (23) C9H11O3
[M+H-CH2O-H2O]+
149.0621 (100) C9H9O2
[M+H-CH2O-H2O-CO]+
121.0617 (45) C8H9O
7 6.96 220, 285 [M+H]+179.0729 (89) C10H11O3
[M+H-CO]+151.0775 (100) C9H11O2
[M+H-CO-H2O]+
133.0672(12) C9H9O
[M+H-C2O2-H2O]+105.0718(19) C8H9
8 4.14 230 [M+H]+
181.0883(52) C10H13O3
[M+H-CH2O]+
151.0773(100) C9H11O2
[M+H-CH2O-H2O]+
133.0670 (16) C9H9O
[M+H-CH2O-CH2O2]+105.0720 (65) C8H9
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