Chemicals and reagents
General anaerobic medium (GAM) broth was obtained from Nissui Pharmaceutical Inc. (Tokyo, Japan). Leibovitz’s L-15 medium was purchased from Life Technologies Co. (Grand Island, NY, USA). Brain heart infusion (BHI) broth was manufactured from Oxoid Ltd. (Basingstoke, England). Fetal bovine serum (FBS) was purchased from Gibco (Gaithersburg, MD, USA). HPLC-grade acetonitrile (ACN) was purchased from Merck (Darmstadt, Germany). Deionized water (18 MΩ cm−1) was purified using a Milli-Q Ultrapure water system (Milford, MA, USA). Ginsenoside F1, GRh2, and PTT were bought from Baoji Herbest Bio-Tech Co., Ltd. (Shaanxi, China). GC-K, PPD and digoxin (the internal standard, IS) were provided by Chengdu Push Bio-technology Co., Ltd. (Sichuan, China). The purity of all compounds was determined by HPLC (≥ 98%), and their chemical structures were shown in Fig. 1.
Bacteria genomic DNA extraction kit was purchased from Omega Bio-tek (Norcross, GA, USA). Mixture Polymerase Chain Reaction (PCR) product purification kit was purchased from Qiagen (Hilden, Germany). Sequencing library generation kit was purchased from Illumina (San Diego, USA).
Sample collection and gut microbiota preparation
Stool samples were collected from Chinese-diet and western-diet pattern healthy subjects. Inclusion criteria were following as, (i) age between 20 and 25 years; (ii) body mass index (BMI) between 19-24 kg/m2; (iii) absence of systemic and metabolic disease; (iv) no use of alcohol and tobacco; and (v) stable diet patterns in the last one year, pertinent to HF-HP and LF-PF diets. Exclusion criteria were defined as, (i) history of any antibiotics or probiotics medications in the last three months; (ii) history of drug allergies and highly sensitive to environmental; and (iii) mental illness rendering the participants unable to understand the nature, scope, and possible consequences of the study. All individuals provided written informed consent prior to participating in the study.
According to our previous study, 1 g of fresh fecal sample was suspended in 20 mL of cold physiological saline and then centrifuged to collect the resultant fecal supernatant. The precipitation was re-suspended with Leibovitz’s L-15 medium containing glycerol as the gut microbiota solution stored in −80 ℃ freeze.
PNS preparation and incubation
The air-dried root of P. notoginseng was purchased from Wenshan city (Yunnan, China) and extracted by heat-refluxing with 70% ethanol to obtain the P. notoginseng extract. The detailed information about P. notoginseng and PNS extraction were described in our former publication.
Gut microbiota stock was activated with GAM broth and then centrifuged to collect the gut microbiota precipitation. Leibovitz’s L-15 medium was added to re-suspend the precipitate as the gut microbiota work solution. Gut microbiota work solution, P. notoginseng extract stock solution in dimethyl sulphoxide (DMSO) and Leibovitz’s L-15 medium were mixed as incubation system incubated at 37 ℃ for 48 h. The reaction mixtures were successively extracted by ethyl acetate and n-butanol, and evaporated under nitrogen. At last, the samples were reconstituted with methanol before subjected to HPLC analysis. The specific plan was described in our previous study7.
Bifidobacterium adolescentis and Lactobacillus rhamnosus were cultured in Reinforced Clostridium Medium （RCM）and Brain Heart Infusion Medium （BHI）broth containing 10% FBS for 24 h before incubation 48 h with PNS at 37 ℃. The reaction mixtures were extracted as the same with above-mentioned methods.
Relative quantification of metabolites by HPLC- MS
The PNS metabolites biotransformed by intestinal microbiota were quantified by comparing the five main metabolites between LF-PF and HF-HP diet group on an HPLC-ESI-MS/MS system, which consisted of a SHIMADZU Nexera X2 HPLC system (SHIMADZU, Tokyo, Japan) and AB SCIEX Triple Quad TM 6500 mass spectrometers equipped with electrospray ionization (AB SCIEX, CA, USA). Take our previous method and adjust it accordingly, the chromatographic separation was performed on an Phenomenex LUNA C18 (2) Reversed Phase (150 mm × 2.0 mm, 5 μm) with a gradient elution of 0.1% formic acid in water (A) and ACN (B) at a flow rate of 0.3 mL/min. The gradient profile was optimized below, 0–2 min: 35%–65% B, 2–6 min: 65%–70% B, 6–7 min: 70%–72% B, 7–7.5 min: 72%–85% B, 7.5–8 min: 85%–85% B, 8–11 min: 85%–95% B,11–12 min: 95%–100% B. The injection volume was 2 μL and the temperature of column was set at 40 ℃. The following mass spectrometer parameters were selected in positive ion mode,spray voltage:4500 V; temperature:350 ℃; collision gas: 10 psi; curtain gas: 35 psi; ion source gas 1: 55 psi; ion source gas 2: 50 psi.
Five metabolites including GF1, Rh2, GC-K, PPD, PPT and digoxin (IS) were mixed and dissolved in methanol to validate this method. For intra-day precision, the dissolved standards were analyzed three times within one day, while they were determined in triplicate for three continuous days for inter-day precision. Relative standard deviations (RSDs) were calculated to assess the variations. Selectivity was investigated by comparing the spectra of blank human gut microbiota with IS or with analytes and IS to exclude the peaks of endogenous components in incubating system.
DNA extraction and PCR amplification
Microbial genomic DNA was extracted from each sample and stored in -20 ℃ using the Qiagen QIAamp DNA Stool Mini Kit (Qiagen). DNA concentration and molecular weight were estimated using a nanodrop instrument (Thermo Scientific), and the purity of DNA were monitored on 1% agarose gels. Subsequently, DNA was diluted to 1 ng/μL using sterile water. The variable region V3-V4 of the bacteria 16S rRNA gene from each sample were amplified using the bacterial universal primer 338F 5'- ACTCCTACGGGAGGCAGCAG-3' and 806R 5’-barcode GGACTACHVGGGTWTCTAAT-3’, while barcode is a six-base unique sequence to each sample. All PCR reactions were carried out in 20 μL of reactions with 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase, and 10 ng of template DNA. PCR thermal cycling conditions were initial denaturation at 98 ℃ for 1 min, followed by 30 cycles of denaturation at 98 ℃ for 10 s, annealing at 50 ℃ for 30 s and elongation at 72 ℃ for 30 s, with a final extension at 72 ℃ for 5 min.
MiSeq sequencing of 16S rRNA gene amplicons
The PCR products of the same sample were mixed firstly, and then extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s instructions. Referring to the preliminary quantitative results of electrophoresis, QuantiFluor™ -ST blue fluorescence quantitative system was used to detect and quantify the PCR products. Purified amplicons were pooled in equimolar and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard protocols.
Paired-end reads were assigned to samples based on their unique barcode and truncated by cutting off the barcode and primer sequence, and then merged using FLASH (V1.2.7, http://ccb.jhu.edu/software/FLASH/). Quality filtering on the raw tags were performed to obtain the clean tags according to the QIIME2 software (Version 2018.11，https://qiime2.org) quality control process. The Dada2 plugin was used for quality filtering , and then the Vsearch plugin was used to remove the chimera of the data to obtain Clean Data. Sequences analysis was performed using Vsearch plugin, and sequence with ≥ 97% similarity was assigned to the same OTUs. Representative sequence for each OTU was screened and annotated for taxonomic information based on the GreenGene Database (http://greengenes.lbl.gov/cgi-bin/nph-index.cgi). QIIME2 software package in-house Perl scripts were used to analyze alpha- (within samples) and beta- (among samples) diversity with normalized data to evaluate differences of samples in species richness and complexity.
In order to analyze alpha-diversity, Shannon index, Faith’s index and Pielou’s index were used to indicate the richness and diversity of the community, phylogenetic diversity and evenness, respectively. Observed OTUs were used to indicate abundance. The results were visualized using R software (Version 2.15.3). In order to analyze beta- diversity，Bray-Curtis distance and Jaccard distance were calculated according to phylogenetic measures, while Unweighted Unifrac distance and Weighted Unifrac distance were employed for the sequence measures
A heat map was constructed with a cluster tree using the Microeco bioinformatics cloud (https://www.bioincloud.tech). LEfSe analysis was performed using LEfSe software to assess the effective size (LED> 2) of each differentially abundant taxon, while the cladogram was displayed according to effective size.
Spearman’s correlation analysis and Student’s t-test were performed using SPSS software (Version 23). Significant differences were set as * p < 0.05 and ** p < 0.01 or q < 0.01.