Characterization of the active ingredients in PT and DHT extracts

The total phenolic contents estimated by the Folin-Ciocalteu method were 11.6 g GAE (gallic acid equivalent)/100 g and 9.12 g GAE/100 g in DHT and PT, respectively. Furthermore, UHPLC analysis identified the significant ingredients in the two tea extracts. Higher concentrations of EGC (epigallocatechin), C (catechin), EC (epicatechin) and EGCG (epigallocatechin gallate) were present in DHT than in PT. In contrast, higher levels of GA (gallic acid), ECG (epicatechin-3-gallate), TF (theaflavin), and TB (theabrownin) were found in PT (Table 1).

Table 1 Comparison of compound contents of DBT and PT (mg/g) Sequencing metadata and microbial diversity in the gastrointestinal tracts of the mice

After removing low-quality reads and chimeras, 19,23,284 high-quality 16 S rRNA gene sequences (V4 region: 533–786 bp) with no chimeras were obtained. The average was 80136.83 ± 86.63 sequences per sample, ranging from 80,034 to 80,344. The average length of these sequences was 411 bp, and they belonged to 1703 operational taxonomic units (OTUs) based on 97% similarity. Each sample had an average of 567.67 ± 7.09 OTUs. After calculation, the effective data accounted for 98%. The average Q20 was 80.67, and the average GC ratio was 52.79% (Additional file 1: Table S2).

The microbial community richness (alpha diversity) was measured using the Chao1 index, and species were identified. The indices did not differ significantly between the P and R groups. However, the Chao1 and Observed species were notably different in the bacterial community structure of P vs. H and R vs. H. Microbial community diversity was significantly higher in mice treated with PTP and DHTP than in the control group. (Fig. 1A, B, C and D).

Fig. 1

Microbial diversity in the gastrointestinal tracts of the mice species diversity curve, A Chao1 index, B Observed species; Statistical map of species significant differences between groups, C Chao1 index, D Observed species; E Based on unweighted_unifrac level of PCOA analysis result graph, Each point in the figure represents a sample, and samples from the same group are represented by the same color

The beta diversity representing the differences among the H, P, and R mice was examined using weighted and unweighted UniFrac distances. PCoA (Principal coordinates analysis) visualized the distances. The mice treated with ddH2O, Dian Hong tea polyphenols, and Pu-erh polyphenols harbored distinct microbial taxa. The microbial communities in the mice belonging to the H, P, and R groups clustered together and separated along the principal coordinate axis based on membership (Fig. 1E). ANOSIM (Analysis of similarities) (P < 0.01) identified significant differences in the community memberships between the different groups. Thus, distinct microbial community structures existed among the mice treated with different tea polyphenols.

Differences in the microbial communities of mice treated with ddH2O2, Dian Hong tea polyphenols, and Pu-erh tea polyphenols

The specific taxonomic groups of species (e.g., kingdom, phylum, class, order, family, genus, and species) were identified. Two groups of bacteria, the Firmicutes and Bacteroidetes, dominated the intestinal microbiota of these mice, accounting for more than 97% of reads. The relative abundance of these two predominant microbes (phylum level) differed between the mice groups. The mice in the H, P, and R groups contained 67.48%, 70.11%, and 77.65% of Firmicutes, and 28.41%, 26.65%, and 19.44% of Bacteroidetes (Fig. 2A). Lactobacillus, Lachnospiraceae, Ruminococcaceae, Alistipes, Alloprevotella, Turicibacter, Bacteroides, Desulfovibrio, Faecalibaculum, and Parasutterella were dominant at the genus level (Fig. 2B).

Fig. 2

Differences in the microbial communities of mice treated with ddH2O, Dianhong black tea, and Pu-erh tea polyphenols. A Histogram of relative abundance of species at the gate level; B Column Chart of Relative Species Abundance at Genus Level; C Venn Graph; D LDA value distribution histogram

The Venn diagrams consisted of three groups and 620 OTUs via clustering. The cluster numbers of OTUs in the R, H, and P groups were 569, 560, and 574, respectively. Of these, the R and H groups shared 528 OTUs in the overlapping portion, the P and H groups shared 531 OTUs, and the P and R groups shared 532 OTUs. Moreover, 19 species were unique to the P group, nine to the H group, and 17 to the R group (Fig. 2C).

The LEfSe (Linear discriminant analysis Effect Size) tool focuses on the significant differences and biological relevance. It identified specific genera that were differentially distributed among the H, P, and R groups. Furthermore, 22 genera were differentially represented among the three groups, 12 genera were more abundant in the P group (e.g., Lactobacillaceae, Lactobacillus spp. Bacilli, Lactobacillales, Ruminococcaceae, Clostridium papyrosolvens, Ruminococcaceae spp. Erysipelotrichales, Erysipelotrichia, Alloprevotella spp. and Prevotellaceae). Further, six genera were more abundant in the R group (e.g., Lachnospiraceae, unidentified Lachnospiraceae spp. Clostridiales, Clostridia, Firmicutes, and Clostridiales bacterium CIEAF 020) (Fig. 2D).

Effect of tea polyphenols on the body weight of mice

There was no significant difference in the average body weight of mice in each group at the beginning. During the experimental period, no significant difference was seen in the food and water intake among the mice in different groups. However, treatment with DHTP and PTP for 14 days caused a much slower weight gain in the R and P groups than in the H group. The rates of weight gain in the R and P groups were 5.27% and 10.29%, respectively, while it was 12.74% in the H group (Fig. 3A).

Fig. 3

Effects of Dian Hong tea and Pu-erh tea polyphenols on the body parameters of mice. A Body weights (g). B T-AOC activity in serum and liver. Serum: U/mL, Liver: U/g. C SOD activity in serum and liver. Serum: U/mL, Liver: U/g. *p < 0.05, **p < 0.01, ***p < 0.005 vs. the H group

Analysis of antioxidant capacity of serum and liver tissue in mice

To assess the antioxidant capacity of the two tea polyphenols, we measured the activities of T-AOC and SOD in mouse serum and liver. T-AOC and SOD activities in serum and liver tissue of groups R and P were significantly higher than those in group H. (Fig. 3B, C). T-AOC activity in serum samples was 1.70 ± 0.09 U/mL, 1.87 ± 0.09 U/mL, and 0.92 ± 0.04 U/mL, respectively, while SOD activity was 16.48 ± 1.10 U/mL, 16.72 ± 0.99 U/mL, and 10.12 ± 0.63 U/mL, respectively. T-AOC activity in the liver tissue was 1.46 ± 0.08 U/g, 1.67 ± 0.10 U/g, and 1.19 ± 0.07 U/g, respectively, and SOD activity was 13.38 ± 0.85 U/g, 15.52 ± 1.76 U/g, and 11.07 ± 0.84 U/g.

Transcriptome data analysis

The liver transcriptomic analysis was performed by sequencing the RNA samples from three liver tissues in each group. A total of 1,268,484,156 raw sequencing reads were generated, and 1,217,618,868 clean reads remained after filtering (Additional file 1: Table S3).

Principal Component analysis (PCA) was performed on the gene expression values (FPKM) of all samples to evaluate the difference between groups and the duplication of samples within groups. The samples between LH, LR, and LP groups were dispersed, but the samples within groups were clustered together (Fig. 4B).

Fig. 4

Transcriptome analysis of mice liver. A Venn diagram of coexpression. B Results of the Principal component analysis. The abscissa is the first principal component, and the ordinate is the second principal component. C Statistical histogram of the number of differentially compared genes in combination. Grey and blue represent upregulated and downregulated differential genes, respectively. The numbers on the bars represent the number of differential genes. LH Liver tissue samples from the H group of mice, LP Liver tissue samples from the P group of mice, LR Liver tissue samples from the R group of mice

Identification of differentially expressed genes

The gene expression levels of LH, LR, and LP were quantified and compared. The differentially expressed genes (DEGs) were obtained using the criteria: log2 (Fold Change) > 0 and padj < 0.05. A total of 988, 1416, and 564 DEGs were identified in the LR vs. LH, LP vs. LH, and LR vs. LP groups, respectively. Among these DEGs, 369, 697, and 204 DEGs were uniquely expressed in LR vs. LH, LP vs. LH, and LR vs. LP groups, respectively. Moreover, 31 DEGs were commonly expressed in all groups (Fig. 4A).

Significantly upregulated or downregulated genes were identified by DEGseq. Compared with the LH group, the LP group exhibited 1417 DEGs (730 upregulated and 687 downregulated genes), and the LR group showed 988 DEGs (514 upregulated and 474 downregulated genes). Further, the LR group showed 564 DEGs (277 upregulated and 287 downregulated genes) when compared with the LP group (Fig. 4C).

GO analysis for DEGs

The top 30 significant GO terms for DEGs involved between LR vs. LH and LP vs. LH groups are depicted. The DEGs were mainly enriched in the sterol biosynthetic process, fatty acid metabolic process, monocarboxylic acid metabolic process, secondary alcohol biosynthetic process, proteasome complex, endopeptidase complex, oxidoreductase activity, cofactor binding, vitamin B6 binding, etc. Most genes were involved in the fatty acid metabolic process and antioxidant activity (Fig. 5A, B).

Fig. 5

GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analyses. A LP vs. LH. B LR vs. LH. The abscissa is the ratio of the number of differential genes on the GO Term to the total number of differential genes, and the ordinate is the GO Term. LH Liver tissue samples from the H group of mice, LP Liver tissue samples from the P group of mice LR Liver tissue samples from the R group of mice. The abscissa is the KEGG pathway, and the ordinate is the significance level of pathway enrichment. LH Liver tissue samples from the H group of mice, LP Liver tissue samples from the P group of mice, LR Liver tissue samples from the R group of mice. C LP vs. LH. D LR vs. LH.

Pathways responsive to Dianhong and Pu-erh tea polyphenols treatment

The DEGs identified between the LR vs. LH group were associated with the biosynthesis of unsaturated fatty acids, PPAR signaling pathway, p53 signaling pathway, peroxisomes, fatty acid metabolism, cholesterol metabolism, etc. (Fig. 5D). The DEGs between the LP vs. LH groups were related to proteasomes, steroid biosynthesis, protein processing in the endoplasmic reticulum, glycerophospholipid metabolism, p53 signaling pathway, etc. (Fig. 5C). The peroxisome and p53 signaling pathways were noteworthily associated with the LP and LR regulatory pathways. Most pathways regulated by PTP and DHTP were related to lipid metabolism and antioxidant action.

Expression of genes involved in lipid metabolism and the antioxidation pathway

The relative mRNA expression of six genes involved in the antioxidation signaling pathway (COX-2, iNOS, IκB-α, Cu/Zn-SOD, Mn-SOD, GSH-Px) and six genes associated with the lipid metabolism signaling pathway (PPAR-α, LDLR, CPT-1a, C/EBP-α, FAS, SREBP-1c) in the liver tissues was measured (Fig. 6).

Fig. 6

The relative mRNA expression levels of genes related to lipid metabolism and antioxidants in the liver. *p < 0.05 vs. the H group

After treatment with Dian Hong tea polyphenols, the expression of the COX-2, iNOS, and IκB-α genes in the liver was significantly reduced to 0.70-, 0.60-, 0.83-fold, while the expression of the Cu/Zn-SOD, Mn-SOD, GSH-Px genes was significantly increased to 2.64-, 1.97-, 2.43-fold compared to the mice in the LH group (P < 0.01). In the Pu-erh tea polyphenols treated mice, the mRNA expression of the COX-2, iNOS, and IκB-α genes was significantly downregulated to 0.30-, 0.29-, 0.48-fold. And the expression of Cu/Zn-SOD, Mn-SOD, and GSH-Px genes was upregulated to 4.32-, 2.71-, and 8.56-fold. In addition, higher expression of PPAR-α (2.43- and 8.56-fold), LDLR (2.96- and 5.84-fold), and CPT-1a (2.49- and 4.06-fold) was observed in these two groups. Further, lower expression of C/EBP-α (0.32- and 0.16-fold), FAS (0.78- and 0.58-fold), and SREBP-1c (0.56- and 0.38-fold) was recorded.