Chemical composition analysis of P. oleracea extract

Using UHPLC-QE Orbitrap platform to annotate P. oleracea water and ethanol extracts, a total of 2831 metabolites were annotated. The components of the P. oleracea ethanol extraction group (PE) extract are Nicotinurate, Pantothenic Acid, and Genistein, L-Homocysteine, Furylacetone, Biotin, 3,4-Dihydroxyphthalate, etc. Genistein, Biotin, Engeletin, and Ephedrine are common active ingredients in plants, purslane alcohol extracts are mainly plant active ingredients and some growth promoting substances. The main components of purslane water extract (PW) are Thymine, Sorbic acid, L-Homocysteine, Nicotinurate, Biotin, Methyloscitric acid, Rotenone, etc. Sorbic acid, Rotenone, Engeletin are active ingredients in plants. The detailed results of ingredient content are shown in Table 1

Table 1 Content of water and ethanol extracts from P. oleraceaEffect of P. oleracea on excretion of coccidian oocysts

As shown in Table 2, the OPG values of the PL, PH, PW, PE, and DIC groups on day 7 were 11200, 7883, 5367, 5783, and 733, respectively. The OPG values were significantly lower in the PW and DIC groups than the CON group (p ≤ 0.05 and 0.01, respectively). On day 14, the OPG values were significantly lower in the PW and DIC groups than the CON group (p ≤ 0.01). On the final day of treatment (day 28), the OPG values were significantly lower in the PH, PW, and PE groups than the CON group (p ≤ 0.05), while there was no significant difference between the DIC and CON groups (p > 0.05). The OPG values of each P. oleracea treatment group at specific time points are shown in the line graph presented in Fig. 2a. The OPG values of the different P. oleracea treatment groups had decreased as compared to the initial values, with the most obvious decrease in the DIC group. After 14 days, the OPG value of the DIC group suddenly increased, while the OPG values of the other drug groups were relatively stable. DIC had the strongest anti-coccidian effect, but efficacy was only maintained for 14 days, followed by the PW group. This finding suggests that DIC has a relatively short-term anti-coccidian effect. A heat map of the OPG values over time is presented in Fig. 2b. After medication, the color of the lambs gradually turned white, with the highest proportion and degree observed in the DIC group. Some of the sheep appeared blue on day 14. However, the heat map of the different treatment groups indicates a trend of turning white. Changes to the OPG values of individual sheep collectively reflect the overall change to the group.

Table 2 Coccidia oocyst excretion of different treatment groups of P.oleracea (a, b, c values with different superscripts differ significantly (P ≤ 0.05)Fig. 2

Effects of different treatments on coccidia in lambs. a line chart of mean OPG values of the different treatment groups. b Heat map of changes to OPG values, where higher OPG values are indicated by brighter colors. c Line graph of changes to OPG values. Analysis of variance was used to compare the OPG values. Asterisks indicate significant differences (*0.01 < p ≤ 0.05; **0.001 < p ≤ 0.01; ***p ≤ 0.001)

Treatment with P. oleracea and the extracts tended to decrease the OPG values. However, anti-coccidian effect was weaker in the PL group than the PH, PW, and PE groups. There were significant differences in OPG values between the PW and CON group on days 7, 14, and 21, with the lowest OPG value on the 42th day of the experiment (p ≤ 0.05). There were also significant differences in OPG values between the PE and CON groups on days 7 and 14, and between the PH and CON groups on the 28th day of the experiment (p ≤ 0.05). These results show the P. oleracea extracts had better anti-coccidian effects, which may be due to greater release of effective components of P. oleracea after extraction. Although DIC initially exhibited strong anti-coccidian effects on the 7th and 14th day of the experiment (p ≤ 0.01), the OPG value immediately increased with no difference as compared to the CON group. Notably, the coccidia were not completely eradicated in the PH, PW, and PE groups, but infection was ameliorated for prolonged periods.

Effect of P. oleracea on GP

As shown in Fig. 3a, the mean BW of lambs on days 14 and 42 was higher in each P. oleracea treatment group as compared to the CON group. On day 14, the mean BW was significantly higher in the PE group than the DIC group (p ≤ 0.05). As shown in Fig. 3b, the mean DWG during the 14 day treatment period was significantly higher in the PH and PW groups than the PL group (p ≤ 0.05), and significantly higher in the PE group than the PL and DIC groups (p ≤ 0.01). As shown in Fig. 3c, the mean DWG was higher in each treatment group than the CON group throughout the entire 42 day experimental period. The mean DWG was highest in the PH, PW, and PE groups, but there was no significant difference as compared to the CON group (p > 0.05). Collectively, these results demonstrate that supplementation with P. oleracea can improve the GP of sheep and a single dose of DIC has relatively limited effects.

Fig. 3

Effect of different treatments on GP of sheep. a Mean weight of sheep in each group at different times. b Mean DWG in each group during the dosing period (14 days). c Mean DWG of each group throughout the trial period (42 days). (*0.01 < p ≤ 0.05; **0.001 < p ≤ 0.01; ***p ≤ 0.001)

P. oleracea is beneficial to maintain the composition of the GM

The roles of the GM in health and disease are well established. In the present study, 16S rRNA analysis was conducted to clarify the effects of dietary additives on the composition of the GM. The term “clean reads” refers to high-quality reads obtained after quality control of the original sequence (Fig. 4a). As shown in Fig. 4b, the richness of the GM (ACE index) was significantly higher in the PH, PW, and PE groups than the CON group (p ≤ 0.05), while there was no difference in the PL and DIC groups as compared to the CON group. The diversity of the GM (Shannon index) was significantly higher in the PH and PW groups than the CON group (p ≤ 0.05; Fig. 4b; Fig. 4c), and even more pronounced in the PE group than the CON group (p ≤ 0.01), while there was no significant difference between the PL and CON groups. These results indicate that the high-dose of P. oleracea as well as the water and ethanol extracts increased the diversity of the GM, while the low-dose of P. oleracea also improved diversity, but to a lesser extent. A dilution curve is generally used to reflect the reliability of the diversity results of sequencing data (Amato KR, et al. 2013). As shown in Fig. 4d, the dilution curve gradually flattened, indicating that the diversity results are reliable.

Fig. 4

Effect of P. oleracea on the overall structure of the GM. a Sample coverage of the six treatment groups. b Chao1 indices of the six treatment groups. c Shannon indices of the six treatment groups. d Dilution curves to compare the richness of species among samples and to assess the size of the cohort. e Venn diagrams of the OTUs of each group. f PCoA plots based on Spearman distances are colored by time point. The significance of differences among the groups was determined by analysis of similarity. g NMDS among groups was conducted by one-way ANOVA (*p < 0.05; **p < 0.01)

Clustering OTUs at 97% similarity reflects differences in sequencing data. As shown in Fig. 4e, the number of OTUs was lower in the CON group than the PH and PW groups, demonstrating that P. oleracea influenced the composition of the GM. In order to further study the similarities and differences in the composition of the GM among the groups, non-metric multidimensional scaling (NMDS) and principal co-ordinate analysis (PCoA) were performed. Cluster analysis uses a tree structure to describe and compare similarities among multiple samples. As demonstrated by the results of NMDS (Fig. 4f) and PCoA (Fig. 4g), the CON group is similar in distance to the PH and PE groups demonstrate that the colony structure of the lambs, when treated with P. oleracea, is similar to the colony composition of normal lambs. The large distance between the DIC group and other groups indicates that the use of DIC has changed the colony composition of GM. The effects of different properties of chemical drugs and traditional Chinese medicine on GM may vary.

Differences in dominant microbial communities among the treatment groups

At the phylum level (Fig. 5a), 90% of the GM of the six groups were mainly composed of Firmicutes and Bacteroides. The abundances of Firmicutes in the PL, PH, PW, PE, DIC, and CON groups were 65.07, 81.13, 72.99, 76.32, 68.30, and 64.00%, respectively, while the relative abundances of Bacteroides were 15.13, 10.94, 15.91, 14.64, 21.05, and 18.07%. As shown in Fig. 5b, the relative abundance of Firmicutes was significantly higher in the PH group than the CON and PL groups (p ≤ 0.01). Meanwhile, the abundance of Firmicutes was significantly lower in the DIC group than the PH group (p ≤ 0.05). The relative abundance of Bacteroides was lower in the TCM groups than the CON group, but this difference was not significant (p > 0.05). Firmicutes is believed to help the absorb food calories and leads to weight gain, suggesting that weight gain associated with P. oleracea might be related to an increase in the abundance of Firmicutes. Also, the relative abundance of Bacteroides was higher in the DIC group than the CON group and gradually decreased with an increase in the dose of P. oleracea.

Fig. 5

The dominant members of the GM at the phylum and genus levels. a Composition of the GM (top 8 phyla) of each group. b Relative abundance of Firmicutes and Bacteroides of the six groups. c Relative abundances of the top 30 genera of each group. d Relative abundances of Lachnospiraceae and Rikenellacae of the six groups/ e F/B ratio. f and g Taxonomic cladogram obtained by LEfSe of the six groups. Biomarker taxa are highlighted with colored circles and shaded areas. The diameter of each circle reflects the abundance of the taxa. b–d, Relative abundance data were analyzed by one-way ANOVA (*p < 0.05; **p < 0.01)

At the genus level, the GM of the six groups were mainly composed of Lachnospiraceae, Rikenellaceae, and Oscillospiraceae. As shown in Fig. 5c, the abundances of Lachnospiraceae in the PL, PH, PW, PE, DIC, and CON groups were 18.31, 15.49, 16.90, 17.50, 12.62, and 15.99%, respectively, while the abundances of Rikenellaceae were 7.26, 6.00, 8.62, 7.11, 13.17, and 11.47%. Although there was no significant difference among the six groups, DIC was found to reduce the relative abundance of Lachnospiraceae (p ≤ 0.01; Fig. 5d). However, the abundance of Rikenellaceae was increased in the DIC group and decreased in the other TCM treatment groups (Fig. 5d).

The F/B ratio is as an index to evaluate imbalances in the GM caused by various illnesses. As compared to the CON group, the F/B ratio was significantly increased in all of the TCM groups (p ≤ 0.05), but decreased in the DIC group (Fig. 5e). These results indicate that P. oleracea can improve the structure and maintain the stability of the GM.

LEfSe (linear discriminant analysis effect size) analysis identified significant differences in GM structure at the phylum level among the treatment groups (Fig. 5f). The results of linear discriminant analysis showed that o__Monglobales, c__clostridia, and p__Fimicutes were the dominant bacteria in the PH group, whereas o__Oscillospiraceae, g__Monglobales, and g __unclassified__Veillonellales__Selenomonadales were the dominant bacteria in the PW group. The main differentiated microbial species between the DIC and other groups were o__Bacteroidales, f__Planococcaceac, and g __unclassified__Peptostreptococcaceae (Fig. 5g). These results suggest that P. oleracea enhanced the proportions of beneficial bacteria, increased the F/B ratio, and restored the natural balance of the GM.

P. oleracea increased the gut metabolite content

The main purpose of metabolomics analysis is to identify metabolites with significant biological activities in order to elucidate the related metabolic processes and underlying mechanisms. The results of OPLS-DA identified various differential metabolites among the treatment groups. As shown in Fig. 6a, there were significant differences in the differential metabolites between the PH and CON groups. The permutation test (R2Y = 0.996 and Q2 = 0.866) confirmed that the OPLS-DA results were reliable. There were also significant differences in metabolites between the PW and CON groups (R2Y = 0.993 and Q2 = 0.528) (Fig. 6b).

Fig. 6

Effects on different fecal metabolites in sheep. a OPLS-DA score chart and permutation test (PH vs. CON). b OPLS-DA score chart and permutation test (PW vs. CON). The OPLS-DA score chart is often used to directly show the classification effect of the model. c PLS-DA of the microbial metabolites. d Heat map of tissue metabolites. e Log converted values of the differential metabolites among the groups. f Map of differential metabolite abundance, where the name of the differential pathway is shown on the vertical axis and the DA score is shown on the horizontal axis. The DA score reflects the overall change to all metabolites of the metabolic pathway, where a score of 1 indicates an upward trend and a score of − 1 indicates a downward trend in the expression of all annotated metabolites in the pathway

In addition, the PLS-DA results showed significant separation among the PH, PW, DIC, and CON groups (Fig. 6c), indicating the reliability of the alternative experimental model. In total, 1675 metabolites were identified and quantified in this study, which included lipids, amino acids, carbohydrates, organic acids, and esters. Cluster heat map analysis identified differences in metabolites and relative changes to metabolite concentrations among the groups. The metabolite concentrations were similar between the PW and PH groups, while there were significant differences between the DIC and CON groups (Fig. 6d).

After conducting qualitative and quantitative analyses of the detected metabolites, Fold Change values and abundance scores of the groups were compared (Fig. 6e, f). As shown in Fig. 6e, the metabolites kedaricidin chromophora, levanbiose, prunasin, and 3-hydroxycosanoyl-CoA were 2.17, 1.98, 1.72, and 1.55 times higher respectively in the PH group than the CON group. Levanbiose is reported to prevent dental caries and weight gain, and correct GM imbalance. Prunasin also has the ability to nourish the stomach and diuresis. High doses of P. oleracea may increase the abundance of metabolites that promote digestion, growth, and improve GM. Meanwhile, the metabolites biotin sodium, prunasin, and acadesine (drug) were 1.03, 0.67, and 0.66 times higher, respectively, in the PW group than the CON group, dihydroxyl aminic acid, gamma aminobutyric acid were 2.15, 1.02 times lower, The PW group is similar to the PH group, the content of bioactive components levambios, prunasin, and biotin sulfone are relatively increased compared to the CON group. And central nervous system inhibitory neurotransmitters that play an important role in metabolites γ- The decrease in the abundance of aminobutyric acid and acute toxic dihydroxy amino acid suggests that purslane may upregulate the abundance of substances that promote cell growth, fatty acid production, and fat and amino acid metabolism, thereby promoting body growth.

As shown by the map of metabolite abundance scores presented in Fig. 6f, the dots are distributed on the right side of the axis and the longer line segments indicate greater overall expression of the pathway. The differences in metabolite abundance scores between the PH and CON groups showed that the Arginine synthesis pathway, Biosynthesis of type ll polyketide products, Tetracycline biosynthesis, and Biosynthesis of vancomycin group antibiotics pathways were upregulated, while the thiamine metabolism pathway was downregulated (Fig. 6f). The metabolic pathways of Pantothenate and CoA biosynthesis and Naphthalene degradation were upregulated in the PW group, while Atrazine degradation, Clavulanic acid biosynthesis, and Sulfur relay system were down-regulated. Similar to the results of Fold Change values, the use of P. oleracea can up-regulate metabolic pathways such as arginine, Pantothenate and CoA that provide energy and active substances. The DIC group showed down-regulation of phenylalanine, tyrosine, and tryptophan biosynthesis; biosynthesis of enedyne antibiotics, Pseudomonas aeruginosa, Fluor benzoate degradation. In summary, P. oleracea has increased the abundance of certain beneficial bacteria, which promote the up-regulation of metabolic pathways of nutrients and active substances, maintain intestinal health, promote growth and development, and resist pathogens.