microbial biotransformation of gentiopicroside by...

1

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

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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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of a novel steroid (PM-9) on the inhibition of 5alpha-reductase present in Penicillium 536

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45:1486-1490. 547

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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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