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Comparative Analysis of the Rats’ Gut Microbiota Composition in Animals with Different Ginsenosides Metabolizing Activity Wei-Wei Dong, Fang-Ling Xuan, Fei-Liang Zhong, Jun Jiang, Songquan Wu, Donghao Li, and Lin-Hu Quan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04848 • Publication Date (Web): 27 Dec 2016 Downloaded from http://pubs.acs.org on December 28, 2016

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Journal of Agricultural and Food Chemistry

Comparative Analysis of the Rats’ Gut Microbiota Composition in Animals with Different Ginsenosides Metabolizing Activity Wei-Wei Dong, Fang-Ling Xuan, Fei-Liang Zhong, Jun Jiang, Songquan Wu*, Donghao Li*, Lin-Hu Quan*

Key Laboratory of Natural Resource of the Changbai Mountain and Functional Molecular (Yanbian University), Ministry of Education, Park Road 977, Yanji City, Jilin Province 133002, China *Corresponding author Address: Yanbian University Park Road 977, Yanji City, Jilin Province 133002, China (Tel) 86-433-2436452 (Fax) 86-433-2432456 (E-mail)

[email protected]

(LH

Quan),

[email protected]

[email protected] (S Wu)

1

ACS Paragon Plus Environment

(D

Li),


1

ABSTRACT: Following oral intake of Panax ginseng, major ginsenosides are metabolized

2

to deglycosylated ginsenosides by gut microbiota before absorption into the blood. As the

3

composition of gut microbiota varies between individuals, metabolic activities are

4

significantly different. We selected 6 rats with low efficiency metabolism (LEM) and 6 rats

5

with high efficiency metabolism (HEM) from 60 rats following oral administration of Panax

6

ginseng extract, and analyzed their gut microbiota composition using Illumina HiSeq

7

sequencing of the 16S rRNA gene. The components of gut microbiota between the LEM and

8

HEM groups were significantly different. Between the 2 groups, S24-7, Alcaligenaceae, and

9

Erysipelotrichaceae occupied most OTUs of the HEM group, which was notably higher than

10

the LEM group. Furthermore, we isolated Bifidobacterium animalis GM1 that could convert

11

the ginsenoside Rb1 to Rd. The result implies that these specific intestinal bacteria may

12

dominate the metabolism of Panax ginseng.

13

KEYWORDS: ginsenoside, LC-MS/MS, metabolism, Panax ginseng, rats gut microbiota

2


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14


INTRODUCTION

15

In Asian countries, Traditional Chinese Medicines (TCMs) have been used to cure various

16

diseases for thousands of years,1 with oral administration being the most common intake

17

method. Therefore, contact inevitably occurs with the gastrointestinal tract, with the

18

majority of active components metabolized by gut bacteria.2 The gut microbiota, the

19

trillions of microbes residing in the human intestine, plays a crucial role in many biological

20

functions, including immunity, nutrition, and metabolism.3,4 As an important “microbial

21

organ”, they are nearly ten–fold greater than the total of our somatic and germ cells.

22

Additionally, they are also involved in diverse processes including the metabolic function

23

of TCMs.5

24

Panax ginseng is a type of TCM that is widely used as a functional food, and is found in tea,

25

powder, and capsules.6 The major pharmacological components in Panax ginseng are

26

ginsenosides,

27

immunomodulatory, anti-tumor, anti-aging, and anti-inflammatory activities.7-11 Following

28

oral intake of Panax ginseng, major ginsenosides are metabolized to deglycosylated

29

ginsenosides via intestinal microbiota before absorption into the blood.12,13 For example,

30

major ginsenosides Rb1, Rc, Rb2, Rb3, Rd, Rg1 and Re are mainly metabolized to

31

deglycosylated ginsenosides compound K, Rh1, and F1 by intestinal bacteria.14-18 The

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deglycosylated ginsenosides possess potent pharmacological activity compared with the

33

major ginsenosides. For example, the deglycosylated ginsenoside compound K displays

34

anti-cancer, anti-diabetic, and anti-obesity activities and the ginsenosides Rh1 and F1 exhibit

35

anti-aging, anti-oxidant, and anti-allergy function to a greater degree than major

which

possess

various

biological

functions

3


including

anti-diabetic,


36

ginsenosides.19-23 This indicates that gut microbiota play an important role in metabolizing

37

ginsenosides and producing bioactive metabolites. The conditions of the host, such as diet,

38

stress, and even environmental exposure could lead to the differences of gut microbiota

39

composition and function which affects the efficiency of metabolism and absorption of

40

ginsenosides.24 Therefore, studies on the relationship between the community structure of gut

41

microbiota and the metabolism of ginsenosides are significant.

42

In this study, we used LC-MS/MS to analyze the colonic content samples of 60 rats after

43

oral administration of Panax ginseng extract. From these, we selected 6 rats with low

44

efficiency metabolism (LEM) and 6 rats with high efficiency metabolism (HEM), and then

45

compared the gut microbiota composition of the LEM and HEM groups using Illumina

46

HiSeq sequencing of 16S rRNA gene.

47 48

MATERIALS AND METHODS

49 50

Chemicals and Materials. HPLC-grade deionized water, methanol (MeOH), and

51

acetonitrile (ACN) were obtained from Fisher Scientific (Pittsburgh, USA). LC-MS grade

52

formic acid was obtained from ROE Scientific Inc Co. (Dover, DE, USA). Other reagents

53

used were of analytical grade. Standard ginsenosides (Rg1, Re, Rf, Rb1, Rb2, Rd,

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20(S)-Rg2, Rc, 20(S)-Rh1, F1, F2, 20(S)-Rg3, 20(S)-PPT, compound K, and 20(S)-Rh2)

55

were purchased from the Chinese National Institute (Beijing, China). Fresh 4-year old

56

ginseng roots were supplied by JiAn, China.

57

Panax ginseng Extract Preparation. One-hundred grams of dried and pulverized Panax 4


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ginseng root powder was extracted with 1,000 mL 80% (v/v) ethanol solution at 82 °C

59

using reflux condensation. This process was repeated twice, and the combined extract was

60

evaporated to dryness at 40 °C in a rotary evaporator. The Panax ginseng extract was then

61

ready for the subsequent experiment.

62

Animal Experiments. Adult male Sprague-Dawley rats (7 weeks old, weight: 220 ± 20 g)

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were obtained from the Changchun Yisi Experimental Animal Research Center (Changchun,

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China). The rats were housed at a temperature of 24 ± 2 °C with a 12 h light/dark cycle.

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After a one-week acclimatization period at our facility, 60 rats were orally administered

66

100 mg/kg bodyweight of the total saponins in the Panax ginseng extract by gastric

67

intubation. After treatment, the colonic content samples were collected at 12 h. All samples

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were stored at -80 °C prior to analysis. Animal welfare standards and experimental

69

procedures were conducted according to the guidelines of Yanbian University’s Animal

70

Ethics.

71

LC–MS/MS Analysis of Metabolites. Colonic content samples (0.5 g) were mixed with

72

cold saline (5 mL) and then extracted 3 times by ultra-sonication with water-saturated

73

n-butanol for 30 min. The extracted solution was centrifuged at 5,000 × g for 10 min at

74

4 °C. The extract was concentrated by evaporation under vacuum. The resultant residue

75

was then removed with methanol solution. Samples were passed through 0.22-µm filters

76

and analyzed using LC–MS/MS.

77

HPLC separation was conducted on an Agilent 1260 series LC system (Agilent

78

Technologies, USA). A reversed-phase column was achieved with an Agilent Poroshell

79

ZORBAX SB-C18 column (4.6 mm × 150 mm, 5 µm) with a C18 guard column (4.6 mm × 5



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12.5 mm, 5 µm). The mobile phase consisted of (A) 0.1% formic acid in water, and (B)

81

0.1% formic acid in acetonitrile, with a gradient elution of 0–15 min (23–30% B), 15–34

82

min (30–44% B), 34–46 min (44–68% B), 46–61 min (68–85% B), 61–66 min (85–80% B),

83

and 66–73 min (80–23% B). Two microliters of the sample were injected at a flow rate of

84

0.5 mL/min, and measured at 203 nm. In this metabolism study, all mass spectrometric

85

experiments were conducted on an Agilent 6420 triple quadrupole mass spectrometer in

86

positive ion mode, and monitored by MS/MS detection in MRM mode, to detect the

87

metabolites of Panax ginseng saponins in colonic content samples. The parameters of the

88

acquisition system were set as follows: drying gas (N2) flow rate, 8 L/min; drying gas

89

temperature, 350 °C; nebulizer, 15 psi; capillary voltage, 4000 V; and scan range, m/z

90

100-1400. The dwell time was set at 100 µs. Data acquisition and qualitative analysis were

91

accomplished through Mass Hunter workstation software (version B.06.06). The

92

positive-ion fragments were detected to analyze ginsenosides.

93

Experimental Design. Ginsenosides in colonic content samples from 60 rats were

94

quantified by LC-MS/MS detection in MRM mode. The data indicated that metabolic

95

activities in individuals were significantly different (Figure 1A). Based on the level of

96

metabolism, we selected 6 rats with LEM (sample no. A7, A39, A28, A17, A36, and A53)

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and 6 rats with HEM (sample no. B12, B22, B23, B25, B35, and B49) from 60 rats to

98

analyze their gut microbiota composition.

99

Rats Gut Microbiota Analysis. Colonic content samples were snap-frozen in liquid

100

nitrogen and stored at -80 °C. Genomic DNA was extracted from the colonic content

101

samples (0.25 g) using a PowerSoil DNA Isolation Kit (MO BIO Laboratories, USA). Total 6


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genome DNA from samples was extracted via SDS method. The concentration and purity

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of the extracted DNA were measured using a Nanodrop spectrophotometer (ND-1000,

104

NanoDrop Technologies, USA). The V3-V4 region of the bacteria 16S rRNA gene was

105

amplified by PCR (95 °C for 5 min, followed by 25 cycles of 95 °C for 30 s, 50 °C for 30 s,

106

72

107

(5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT

108

-3′), where the barcode is an eight-base sequence unique to each sample. The PCR products

109

were mixed with the same volume of 2 × loading buffer and we operated electrophoresis on

110

1.8% agarose gel for detection. Samples with a bright main strip about 450 bp were chosen

111

and mixed in equidensity ratios. Then, a mixture of PCR products was purified using a

112

GeneJET Gel Extraction Kit (Thermo Scientific).

113

The 16S rRNA gene amplicons were used to determine the diversity and structural

114

comparisons of the bacterial species in rat intestinal microbiota using Illumina HiSeq

115

sequencing at Biomarker Bioinformatics Technology Co., Ltd, Beijing, China. The raw

116

reads have been submitted to the NCBI Sequence Read Archive (SRA) database

117

(Accession number SRP091529). Sequencing libraries were measured using an Agilent

118

2100 bioanalyzer following the manufacturer’s recommended protocol. The qualified

119

libraries were amplified on cBot to generate the cluster on the flow-cell.

°C

for

40

s,

and

72

°C

for

7

min)

using

the

primers

338F

120

Paired-end reads were allocated to samples based on their unique barcode. The

121

overlapping regions between the paired-end reads were merged using FLASH v1.2.7 and

122

raw reads were quality filtered under specific filtering conditions to obtain the high-quality

123

clean tags on the basis of the QIIME (V1.7.0), quality control process. The chimera 7



124

sequences were detected by comparing tags with the reference database (Gold database)

125

using the UCHIME algorithm and then removed. Bioinformatics analysis of sequences was

126

conducted using the QIIME software package. Sequences with ≥ 97% similarity were

127

grouped into the same operational taxonomic units (OTUs). Alpha (within sample), beta

128

(between sample) diversity, and Principal coordinate analysis (PCoA) based on unweighted

129

Unifrac distances were conducted by QIIME.

130

Screening of Metabolizing Ginsenosides by Rat Intestinal Bacteria. We collected 1 g of

131

rat colonic content samples and suspended in 9 mL of cold physiological saline. The

132

suspension was centrifuged at 500 × g for 5 min, and then, the supernatant was centrifuged at

133

10,000 × g for 20 min. The resulting precipitates were suspended in 15 mL of GAM broth and

134

cultivated under anaerobic conditions (85% N2, 10% H2, 5% CO2) and incubated at 37 °C for

135

48 h, and diluted to a concentration that ranged from 10-1 to 10-6. Two hundred microliters of

136

the diluted suspension were inoculated on GAM agar plates, and incubated anaerobically at

137

37 °C for 24 h.

138

We used Esculin-GAM agar to screen rat intestinal bacteria which generated

139

β-glucosidase.25 The growth medium (per 1 L) consists of 3 g of esculin and 0.2 g of ferric

140

citrate in GAM agar. Intestinal bacteria that produce β-glucosidase could hydrolyze esculin,

141

resulting in a reddish brown turning to dark-brown zone appeared around colonies on the

142

esculin-GAM agar plates. Ultimately, single colonies from those plates were anaerobically

143

cultured in the GAM broth at 37 °C until the absorbance at 600 nm reached 1.0. Ginsenoside

144

Rb1 (1.0 mg/mL) reacted with suspensions at 37 °C for 72 h under anaerobic conditions in

145

three replications. All of the samples were analyzed by LC–MS/MS. 8


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The

bacterial

16S

rRNA

was

amplified

with

the

universal

primers

27F

147

(5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGA

148

CTT-3′). After the PCR process, the production was sequenced by Shanghai Invitrogen

149

Biotechnology Co. Ltd., China and then we compared the sequence to the GenBank

150

databases using the BLAST algorithm. A phylogenetic tree was charted by the

151

neighbor-joining method via the MEGA 6.0 program with bootstrap values based on 1,000

152

replicates.

153

Statistics. Data obtained from 6 rats are shown as mean ± standard deviation. The

154

differences between treatment groups were analyzed by Student’s t-test. Significant

155

differences are indicated in the tables and figures by * p < 0.05, ** p < 0.01, *** p < 0.001.

156 157

RESULTS AND DISCUSSION

158 159

Characterization of Ginsenosides in the Panax ginseng Extract. A total of 15

160

reference standards of ginsenosides were analyzed in this work (Supplementary Figure 1A).

161

The precursor-product ion pairs used in the MRM mode were as follows: m/z 823.4➝643.6

162

for Rg1, 969.5➝789.2 for Re, 823.3➝481.0 for Rf, 1131.8➝789.8 for Rb1,

163

1101.7➝790.7 for Rc, 1101.5➝789.5 for Rb2, 969.5➝789.5 for Rd, 807.5➝481.0 for Rg2,

164

603.0➝423.3 for Rh1, 661.4➝481.2 for F1, 807.3➝627.2 for F2, 807.5➝465.0 for Rg3,

165

441.0➝423.3 for PPT, 645.6➝465.2 for compound K, and 587.0➝407.0 for Rh2. We

166

analyzed the Panax ginseng extract samples using LC-MS/MS. Supplementary Figure 1B

167

represents the typical total ion chromatogram (TIC) of the extract in the positive-ion mode. 9



168

Retention times, molecular mass, and characteristic MS/MS fragment ions were compared

169

to those of standard ginsenosides, and the 7 major ginsenosides were identified as Rg1, Re,

170

Rf, Rb1, Rc, Rb2, and Rd, respectively.

171

Characterization of Ginsenosides and their Metabolites in Colonic Content Samples.

172

Following the oral intake of Panax ginseng, ginsenosides are inevitably in contact with the

173

gastrointestinal tract, and most of the active components are metabolized by gut bacteria. In

174

this study, samples of colonic content from 60 rats were collected at 12 h after the oral

175

intake of Panax ginseng extract and analyzed by LC-MS/MS. The TICs of a blank colonic

176

content sample and that of a typical colonic content sample collected at 12 h are compared

177

in Supplementary Figure 1C and D. There were 14 ginsenosides that were identified as the

178

main components in colonic content, including 7 major ginsenosides (Rg1, Re, Rf, Rb1, Rc,

179

Rb2, and Rd) and 7 deglycosylated metabolites (Rg2, Rh1, F1, F2, Rg3, PPT, and

180

compound K). The average abundance of major ginsenosides in HEM was significantly

181

lower than in LEM, and deglycosylated metabolites in HEM were significantly higher than

182

in LEM (Figure 1B). In addition, we analyzed the amount of individual ginsenosides in

183

colonic content samples. In LEM, the amount of ginsenosides Re, Rg1, Rf, Rb1, Rc, Rb2,

184

and Rd was significantly higher than in HEM, whereas metabolites F2, PPT, and compound

185

K were significantly lower than in HEM (Figure 1C). Of the 14 ginsenosides in the colonic

186

content, the concentrations of PPT and compound K were significantly higher than the rest,

187

which indicates that ginsenosides PPT and compound K were the principal metabolites of

188

Panax ginseng extract after oral administration. Recently, research into the metabolism of

189

ginsenosides via gut microbiota has been reported. For example, after oral administration 10


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of single ginsenoside, the ginsenosides Rb1, Rb2, Rd, and Rc were primarily metabolized

191

to compound K, and ginsenosides Re and Rg1 were metabolized to Rh1 and PPT by gut

192

microbiota.16-17,26-30 In addition, compound K has been reported to be a primary metabolite

193

in the feces after oral administration of American ginseng extract.31 Research into the in

194

vitro bioconversion of American ginseng extract via human gut microbiota reveals that

195

ginsenosides Rg3 and compound K are the major metabolites.32

196

According to the metabolism of ginsenosides detected in this study and other reports of

197

metabolic pathways in previous studies, we speculated the pathway of ginsenosides by

198

intestinal microbiota (Figure 2). In the protopanaxadiol-type group, 2 metabolic pathways

199

can be charted. One way is that ginsenosides Rb1, Rc, Rb2, and Rd via selective

200

elimination of the C-20 and C-3 outer sugar moieties to moieties to produce F2 and then

201

compound K.33,34 Another way is to selectively eliminate the C-20 sugar chain to

202

ginsenoside Rg3 (Figure 2A).35 The metabolic pathways of the protopanaxatriol-type

203

ginsenosides via intestinal microbiota proposed in Figure 2B, illustrates that C-20 and C-6

204

sugar moieties of protopanaxatriol-type ginsenosides were hydrolyzed to transform PPT via

205

Rh1 or F1.36,37

206

Characterization of the Composition of Gut Microbiota in the LEM and HEM

207

Groups. To understand the correlation between the intestinal microbiota composition and

208

the gut microbiota metabolism of ginsenosides in individuals, we analyzed the gut

209

microbiota composition in colonic content of the LEM and HEM groups by performing

210

Illumina HiSeq sequencing-based analysis of bacterial 16S rRNA (V3–V4 region). A total

211

number of 1456961 of 16S rRNA valid sequence reads were obtained from intestinal flora 11



212

of 12 rats. An average of 121,413 sequence reads for each sample in the 2 groups was used

213

in this analysis. A total of 1,312,516 clean sequences were obtained after quality control,

214

containing 605,161,752 bp and the average length of the sequence reads was 461 bp from

215

12 rats. They were classified into different taxonomic categories by MGRAST. Alpha

216

diversity refers to the diversity or richness of the mean species diversity in a specific area.

217

The Chao and Ace were an estimator of phylotype richness, and Simpson and Shannon of

218

diversity reveal both the richness and community evenness (Table 1). These results

219

suggested that the abundance and diversity of the LEM and HEM groups have no

220

significant differences. Distinct clustering of microbiota composition between the LEM and

221

HEM groups was observed following UniFrac-based principal coordinates analysis (PCoA)

222

and is presented in Figure 3A. The multivariate analysis of the PCoA matrix scores show a

223

statistically notable separation between the microbiota of the 2 groups (Figure 3B). These

224

results suggested that the samples in the HEM group were well separated from the LEM

225

group.

226

Then, taxonomy-based analysis revealed the populations of the dominant intestinal

227

microbiota in the 2 groups. At the phylum level, 12 phylas were discovered in the LEM and

228

HEM groups. The major phylas were Firmicutes, Actinobacteria, Fusobacteria,

229

Spirochaetae, Bacteroidetes, and Proteobacteria, and the sequences of the 6 phylas

230

occupied more than 97% of the total amount (Table 2). Of these, the Actinobacteria

231

population in HEM was significantly higher than in LEM, while the level of Bacteroidetes

232

in HEM was significantly lower than in LEM, and there was no obvious difference in the

233

level of Firmicutes, Fusobacteria, Spirochaetae, and Proteobacteria between the LEM and 12


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HEM groups.

235

At the family level, 42 families were detected in all of the samples, and the populations

236

of Alcaligenaceae, Coriobacteriaceae, Bifidobacteriaceae, S24-7, Erysipelotrichaceae,

237

Peptostreptococcaceae and Campylobacteraceae were significantly higher in HEM than in

238

LEM, while the level of Lachnospiraceae, Prevotellaceae, Porphyromonadaceae,

239

Defluviitaleaceae, Lactobacillaceae and Veillonellaceae in HEM were significantly lower

240

than in LEM (Figure 3C). Kim et al. analyzed the different metabolic activity of FPG (with

241

metabolic activity) and FNG (non-metabolic activity) that metabolite ginsenoside Rb1 to

242

compound K from a pool of 100 subjects in vitro, and compared the fecal microbiota via

243

16s rRNA sequencing analysis.38 The result indicated that the population levels of

244

Clostridiales_uc,

245

Bifidobacteriaceae et al. in FPG were higher than in FNG, but those of Lachnospiraceae,

246

Erysipelotrichaceae, Peptostreptococcaceae, Streptococcaceae and Leuconostocaceae

247

were lower in FPG than in FNG.

Ruminococcaceae,

Bacteroidaceae,

Rikenellaceae

and

248

Between the 2 groups in this study, there are 114 significantly different operational

249

taxonomic units (OTUs), of which 75 OTUs are significantly higher and 39 OTUs in the

250

HEM group are significantly lower than the LEM group (Table 3 and Figure 3D). Of 75

251

OTUs, 51 OTUs, 10 OTUs and 8 OTUs belong to S24-7 (phylum Bacteroidetes),

252

Erysipelotrichaceae (phylum Firmicutes), and Alcaligenaceae (phylum Proteobacteria),

253

respectively. Of 39 OTUs, 9 OTUs and 9 OTUs belongs to Prevotella (phylum Bacteroidetes)

254

and Ruminococcaceae (phylum Firmicutes), respectively. Xiao et al. has reported that

255

Bacteroidetes plays a crucial role in the metabolism of Panax notoginseng saponins.39 In our 13



256

study, S24-7 (phylum Bacteroidetes) occupied most in OTUs of the HEM group, which was

257

significantly higher than the LEM group. In previous studies，the characterized metabolism of

258

ginsenosides intestinal bacteria primarily belonged to Bacteroidaceae, Bifidobacteriaceae,

259

Fusobacteriaceae, Propionibacteriaceae and Streptococcaceae families (Supplementary Table

260

1).21,28,40-43

261

Screening and Identification of Rat Intestinal Bacteria Metabolizing Ginsenoside

262

Rb1. A total of 200 microbes were isolated from the rat colonic content samples. Among

263

these, 112 β-glucosidase-producing isolates were screened using Esculin-GAM agar. Strain

264

GM1 showed the excellent activity of converting major ginsenoside Rb1. Phylogenetic

265

analysis based on 16S rRNA gene sequences indicated that GM1 belongs to the

266

Bifidobacterium animalis sp. Lactis AD011 (100% similarity). The relationships between

267

strain GM1 and other members of the genus Bifidobacterium were also evident in the

268

phylogenetic tree (Figure 4A).

269

The transformation of ginsenoside Rb1 via strain GM1 was analyzed by HPLC. (Figure

270

4B and C). The strain GM1 hydrolyzed the outer glucose moieties at the C-20 position of

271

ginsenoside Rb1 to transformed into ginsenoside Rd. The concentration of 1.0 mg/mL

272

ginsenoside Rb1 was transformed into 0.73 mg/mL ginsenoside Rd in 72 h, with a

273

corresponding molar conversion productivity of 86%. Ginsenosides are metabolized by

274

bifidobacteria, and it has been reported that Bifidobacterium K-50 and Bifidobacterium

275

K-103 converted ginsenoside Rc to compound K, Bifidobacterium. sp converted

276

ginsenoside Rb1 or Rb2 to compound K, Bifidobacterium longum H-1 converted

277

ginsenoside Rb1 to compound K via Rd, Bifidobacterium longum RD47 converted 14


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ginsenoside Rb2 or Rc to Rd, Bifidobacterium breve K-110 converted ginsenoside Ra1 to

279

Rb2, and Bifidobacterium. sp converted ginsenoside Rg3 to Rh2 and PPD.21,28,40-43

280

In conclusion, we quantitatively analyzed ginsenosides in rat colonic content samples,

281

and compared the levels of metabolized ginsenosides in individuals. The results showed

282

that ginsenoside metabolic activity was significantly different in individuals. We compared

283

the intestinal microbiota composition in colonic content samples of the LEM and HEM

284

groups using Illumina HiSeq sequencing of 16S rRNA gene, which noted significant

285

differences. Between the 2 groups, S24-7 (phylum Bacteroidetes), Alcaligenaceae (phylum

286

Proteobacteria), and Erysipelotrichaceae (phylum Firmicutes) occupied most OTUs of the

287

HEM group which was significantly higher than the LEM group. In addition, we isolated

288

Bifidobacterium animalis GM1 that could convert the ginsenoside Rb1 to Rd. The result

289

indicates that these specific intestinal bacteria may dominate the metabolism of Panax

290

ginseng.

291 292

Supporting Information

293

Ginsenosides detected by LC-MS/MS in the positive-ion mode (Supplementary Figure 1)

294

and biotransformation of ginsenosides by intestinal microbiota (Supplementary Table 1).

295

15



296

REFERENCES

297

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Funding

436

This study was supported by a grant from the National Natural Science Foundation of

437

China (No. 81660643; 81603365).

22


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438

FIGURE CAPTIONS

439

Figure 1. (A) Comparison of deglycosylated metabolites in 60 rats were collected in

440

colonic contents sample at 12 h following the oral administration of Panax ginseng extract.

441

(B) Average abundances of major ginsenosides and their deglycosylated metabolites in the

442

LEM and HEM groups. (C) The amount of individual ginsenosides in colonic content

443

samples in the LEM and HEM groups. The results shown represent the mean ± SD (n = 6

444

for each group; * p < 0.05, ** p < 0.01, *** p < 0.001).

445 446

Figure 2. The deglycosylated metabolic pathway of ginsenosides by gut microbiota were

447

speculated following oral administration of Panax ginseng extract. (A) The metabolic

448

pathways of the PPD-type ginsenosides and (B) the metabolic pathways of the PPT-type

449

ginsenosides.

450 451

Figure 3. Microbiota composition in the colonic content sample of the LEM and HEM

452

groups were analysed using Illumina HiSeq sequencing of the 16S rRNA gene. (A) Plots

453

shown were generated using the weighted version of the UniFrac-based PCoA. (B)

454

Multivariate analysis of variance from PCoA matrix scores between the LEM and HEM

455

groups. (C) Significant differences in family. (D) Heatmap showing the abundance of 114

456

OTUs significantly different in the LEM and HEM groups. The results shown represent the

457

mean ± SD (n = 6 for each group; * p < 0.05, ** p < 0.01, *** p < 0.001).

458 459

Figure 4. (A) The relationship between our isolates and the validated Bifidobacterium was 23



460

supported by a high bootstrap value in the constructed phylogenetic tree based on the 16S

461

rRNA gene. (B) HPLC analysis of the time course of the biotransformation of ginsenoside

462

Rb1 by Bifidobacterium animalis GM1. (C) The conversion of ginsenosides Rb1 via

463

Bifidobacterium animalis GM1was quantified via HPLC analysis. Data represent the means

464

of 3 experiments, and error bars represent the standard deviation.

24


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Page 25 of 36


Table 1. Number of Sequence Analyzed, Operational Taxonomic Unit (OTUs), Estimated OTU Richness (ACE and Chao1) and Diversity (Simpson, and Shannon) Phylotype Group Total reads OTU Number ACE Chao1 Simpson Shannon LEM1 A17 119766 852 883.79 884.01 0.02 4.92 A28 114629 799 843.69 862.86 0.06 4.46 A36 139885 792 837.89 840.61 0.07 4.17 A39 117838 799 847.27 853.44 0.03 4.71 A53 127430 905 929.25 946.59 0.02 5.00 A7 104506 792 843.05 860.71 0.05 4.34 Mean±SD 120675±12360 823±42 864.00±32.82 874.7±34.65 0.04±0.02 4.62±0.31 HEM2 B12 148407 783 823.51 828.64 0.03 4.58 B22 136281 850 893.34 901.74 0.03 4.48 B23 123435 817 896.43 910.67 0.04 4.53 B25 99307 854 900.71 910.28 0.02 4.74 B35 119538 816 886.98 911.39 0.07 4.09 B49 105939 831 860.51 858.24 0.02 4.97 Mean±SD 122151±16547 825±26 876.91±29.79 886.82±35.06 0.04±0.02 4.57±0.29

1

LEM, 6 rats with low efficiency metabolism; 2HEM, 6 rats with high efficiency

metabolism

25



Page 26 of 36

Table 2. The Differences Between HEM and LEM in the Composition (percent of total sequences) of Gut Bacterial Phyla Phylum Bacteroidetes Firmicutes Fusobacteria Proteobacteria Spirochaetae Actinobacteria

Population (% of total sequences) LEM

HEM

64.61±4.75 19.21±1.82 0.06±0.05 13.82±4.21 1.03±0.41 0.23±0.04

50.11±5.07 27.62±4.36 1.15±0.69 18.32±3.17 0.68±0.49 1.32±0.30

26


p value 0.04 0.08 0.14 0.41 0.56 0.002

Page 27 of 36


Table 3. Represented Bacterial Taxa Information (Genus, Family, Orders, Class, and Phylum) of Significantly Different OTUs Between the HEM and LEM Groups. Taxon OTU ID

Phylum

Class

Orders

Family

Genus

OTU15005

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7

uncultured_bacterium

OTU90456

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU94519

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU55556

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU146757

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU48187

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU96852

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU63427

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU166478

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU54087

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU164801

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU112204

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU86539

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU106824

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU29983

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU166641

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU79384

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU110231

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU43316

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU77640

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU45435

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU127275

Bacteroidetes

Bacteroidia

_Bacteroidales

S24-7


OTU92552

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU109618

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU30945

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU44759

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU8281

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU13240

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU7369

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU86203

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU86161

Proteobacteria

Betaproteobacteria

Burkholderiales

Alcaligenaceae

Parasutterella Allobaculum

OTU3398

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae

OTU91713

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU736

Actinobacteria

Actinobacteria

Bifidobacteriales

Bifidobacteriaceae

Bifidobacterium

OTU11533

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae

Allobaculum

OTU187

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae

Allobaculum Allobaculum

OTU2684

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae

OTU130125

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU120446

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


Unknown_Order

OTU21139

Cyanobacteria

Chloroplast

Unknown_Family


OTU57959

Proteobacteria

Gammaproteobacteria Enterobacteriales

Enterobacteriaceae

Buchnera

OTU39722

Proteobacteria

Betaproteobacteria

Burkholderiales

Alcaligenaceae

Parasutterella

OTU115943

Proteobacteria

Betaproteobacteria

Burkholderiales

Alcaligenaceae

Parasutterella

OTU85263

Proteobacteria

Betaproteobacteria

Burkholderiales

Alcaligenaceae

Parasutterella

OTU121775

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae


OTU16810

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae

OTU46037

Proteobacteria

Betaproteobacteria

Burkholderiales

Alcaligenaceae

Parasutterella

OTU18548

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae


OTU52565

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae

OTU24474

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU101237

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU71507

Proteobacteria

Betaproteobacteria

Burkholderiales

Alcaligenaceae

Parasutterella

OTU118120

Actinobacteria

Coriobacteriia

_Coriobacteriales

Coriobacteriaceae


OTU150781

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU26524

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU102138

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


27



Table 3. Continued. OTU23470

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7

OTU127270

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7

uncultured_bacterium uncultured_bacterium

OTU53163

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae

Allobaculum Collinsella

OTU158304

Actinobacteria

Coriobacteriia

Coriobacteriales

Coriobacteriaceae

OTU7977

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU17495

Proteobacteria

Betaproteobacteria

Burkholderiales

Alcaligenaceae

Parasutterella

OTU42598

Firmicutes

Erysipelotrichia

Erysipelotrichales

Erysipelotrichaceae

Allobaculum

OTU140630

Proteobacteria

Betaproteobacteria

Burkholderiales

Alcaligenaceae

Parasutterella

OTU17146

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU59779

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU68999

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU48097

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU57669

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU70947

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU135288

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU113806

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU28206

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU377

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU107292

Proteobacteria

Deltaproteobacteria

Desulfovibrionales

Desulfovibrionaceae

Desulfovibrio

OTU61594

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae

uncultured_bacterium Bacteroides

OTU11348

Bacteroidetes

Bacteroidia

Bacteroidales

Bacteroidaceae

OTU23163

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae


OTU2736

Firmicutes

Clostridia

Clostridiales

Defluviitaleaceae


OTU115059

Firmicutes

Clostridia

Clostridiales

Lachnospiraceae

OTU43911

Bacteroidetes

Bacteroidia

Bacteroidales

Rikenellaceae


OTU40651

Bacteroidetes

Bacteroidia

Bacteroidales

Rikenellaceae

RC9_gut_group RC9_gut_group

OTU116446

Bacteroidetes

Bacteroidia

_Bacteroidales

Rikenellaceae

OTU38144

Bacteroidetes

Bacteroidia

Bacteroidales

Porphyromonadaceae

Parabacteroides

OTU91992

Bacteroidetes

Bacteroidia

Bacteroidales

Rikenellaceae

RC9_gut_group

OTU153783

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae


OTU127622

Firmicutes

Clostridia

_Clostridiales

Ruminococcaceae

Incertae_Sedis

OTU89622

Proteobacteria

Gammaproteobacteria Enterobacteriales

Enterobacteriaceae

Buchnera

OTU134838

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU81500

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU90048

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae

OTU51872

Firmicutes

Clostridia

Clostridiales

Christensenellaceae


OTU38796

Bacteroidetes

Bacteroidia

Bacteroidales

Bacteroidaceae


OTU162980

Bacteroidetes

Bacteroidia

Bacteroidales

Porphyromonadaceae

Parabacteroides

OTU76633

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae

Incertae_Sedis

OTU107932

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7


OTU70273

Bacteroidetes

Bacteroidia

Bacteroidales

S24-7

RC9_gut_group

OTU105055

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae

Incertae_Sedis

OTU135090

Proteobacteria

Deltaproteobacteria

Desulfovibrionales

Desulfovibrionaceae


OTU44327

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae

Prevotella

OTU53682

Proteobacteria

Alphaproteobacteria

Rhodospirillales

Rhodospirillaceae

Thalassospira

OTU123062

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae


OTU110455

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae

Alloprevotella

OTU142438

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae

Prevotella

OTU67198

Candidate_division_TM7 Unknown_Class

Unknown_Order

Unknown_Family

Candidatus_Saccharimonas

OTU74712

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae

Incertae_Sedis

OTU109280

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae


OTU693

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae


OTU78381

Bacteroidetes

Bacteroidia

Bacteroidales

Bacteroidaceae

Bacteroides

OTU71345

Bacteroidetes

Bacteroidia

Bacteroidales

Prevotellaceae

Alloprevotella

OTU113741

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae


OTU70205

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae


OTU88733

Firmicutes

Clostridia

Clostridiales

Ruminococcaceae


OTU156566

Spirochaetae

Spirochaetes

Spirochaetales

Spirochaetaceae

Treponema

28


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Figure 1

29



Figure 2

30


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Figure 3

31



32


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33



Figure 4

34


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35



TOC

36


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Comparative Analysis of the Rats' Gut Microbiota ... - ACS Publications

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