Cistanche Polysaccharides Enhance Echinacoside Absorption in Vivo And Affect The Gut Microbiota

Contact: emily.li@wecistanche.com

Zhifei Fu, et al

Abstract

The polysaccharides and phenylethanoid glycosides from Cistanche deserticola have been demonstrated to have various health benefits, however, the interactive effect between these two kinds of compounds in vivo is not in detail known. The objective of this study was to investigate the synergistic actions of cistanche polysaccharides with phenylethanoid glycoside and the effects of polysaccharides on gut microbiota. Sprague-Dawley rats were fed with different kinds of cistanche polysaccharides for 20 days, on the last day, all rats were administered the echinacoside at 100 mg/kg. The results were compared mainly on the difference in pharmacokinetic parameters, gut microbiota composition, and short-chain fatty acids contents. The results indicated that all the cistanche polysaccharides, including crude polysaccharides, high molecular weight polysaccharides, and low molecular weight polysaccharides, could regulate the gut microbiota diversity, increase beneficial bacteria and particularly enhance the growth of Prevotella spp. as well as improve the production of short-chain fatty acids and the absorption of echinacoside. By exploring the synergistic actions of polysaccharides with small molecules, these fifindings suggest that cistanche polysaccharides, particularly low molecular weight polysaccharides, could be used as a gut microbiota manipulator for health promotion.

Keywords: Cistanche deserticola polysaccharide Echinacoside Gut microbiota

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1. Introduction

In recent years, it has become clear that polysaccharides modulate the composition of the gut microbiota and that changes can mediate multiple nutritional absorptions [1], producing physiological function [2,3]. Polysaccharides from traditional Chinese medicine (TCM) have been demonstrated to be ideal candidates for gut microbiota regulation and showed prebiotic effects. In the digestive, the polysaccharides and some small molecular compound contact inevitably, show some effects on biological activities [4]. As one kind of the major active ingredient in Panax ginseng, which has been widely used for thousands of years, the polysaccharides showed good improvement in the intestinal metabolism and absorption of ginsenosides in vivo [5]. Codonopsis pilosula polysaccharides showed the promotion effect of the absorption of saponins in dextran sulfate sodium-induced colitis mice [6]. Ganoderma lucidum polysaccharides, especially the high molecular weight fraction, were revealed to have prebiotic functions in high fat diet-induced gut dysbiosis mice models [7]. Longan polysaccharides regulated the intestinal microbiota and the gut metabolites (increasing the contents of pyruvate and butanoate) to produce immunomodulatory activities [8].

Cistanche deserticola, as a Traditional Chinese Medicine, has been widely used as a dietary supplement in China. Pharmacological studies demonstrated that it exhibited neuroprotective, immunomodulatory, hormonal balancing, anti-fatigue, anti-inflammatory, hepatoprotection, anti-oxidative, anti-bacterial, anti-viral, and anti-tumor effects [9]. Chemical analysis showed that phenylethanoid glycosides and polysaccharides were the main active constituents. Echinacoside, the main phenylethanoid glycoside in C. deserticola, has shown wide pharmacological actions, however, its druggability is limited due to the poor bioavailability, fast and extensive metabolism in vivo. Studies have verified echinacoside could be metabolized by the gut microbiota in vitro [10,11]. However, the evidence in vivo has not been investigated. As we know, the intestinal microbiota played an important role in mediating the metabolizing bioactivity of herbal medicines. Therefore, the main aim of this work was to investigate the influence of cistanche polysaccharides on the gut microbiota and the effects on the metabolism of echinacoside.

2. Materials and methods

2.1. Materials and chemicals

Cistanche deserticola Y. C. Ma was obtained from Alashan, Inner Mongolia Mandela sand industry development Co., LTD, China. They were authenticated by professor Lijuan Zhang (Tianjin University of Traditional Chinese Medicine). A voucher specimen was deposited in our laboratory. The standard substance such as echinacoside, acteoside, and chlorogenic acid was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). The purity of each standard was ≥98%. Acetonitrile and methanol of chromatography grade were purchased from Fisher Scientific (Pittsburgh, PA). Formic acid was purchased from Anaqua Chemical Supply Inc. (Wilmington, USA). The other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

Male Sprague-Dawley rats (200–220 g) were supplied by Beijing HFK Bioscience CO., LTD (Beijing, China, license No. SCXK (Jing) 2014- 0004). All animals were bred and housed under SPF conditions in the animal facility. The experiment was performed according to the guidelines established by the animal care and uses a committee of TJUTCM and conformed to the guide for the care and use of laboratory animals published by the U.S. National Institutes of Health (NIH Publication number 85–23, revised 1996). After 1 week acclimation period, rats were randomly divided into four groups: control group, total polysaccharide group, high molecular polysaccharide group, and low molecular polysaccharide group. Each group comprised 6 rats, which were bred in the same cage.

2.2. Cistanche polysaccharides preparation

3.0 kg of Cistanche deserticola Y. C. Ma was cleaned and dried at 40 °C in the air-drying oven, then was crushed into a coarse powder. The dry coarse powder was extracted with 85% ethanol aqueous (v/v) at 60 °C two times, and 2 h for each time. The solid-liquid ratios were 1:12 and 1:10, respectively. The herbal residues were extracted with water at 100 °C another two times, the extract time and solid-liquid ratios were the same with 85% ethanol. The water solution was combined and concentrated under reduced pressure and then precipitated in 80% ethanol, standing under 4 °C for 24 h. The precipitate was isolated through centrifugation at 8000 rpm for 10 min at 4 °C and then lyophilized to get the total polysaccharides (ZT). ZT was then dissolved in deionized water and filtrated on a 100 kDa hollow fiber ultrafiltration membrane, the retentive infiltrate high molecular polysaccharides (HM) were gathered. The permeated filtrate was filtrated with a 6 kDa ultrafiltration membrane. The retentive filtrate middle molecular polysaccharides (MM) and the permeated filtrate low molecular polysaccharides (LM) were obtained.

2.3. Primary characterization of cistanche polysaccharides

The polysaccharide's content was determined by the sulfuric acidphenol method using glucose as a standard [12]. Total protein content was determined by the BCA method [13]. The monosaccharide composition analysis was conducted by HPLC (UltiMate 3000 system) equipped with a SinoPak C18 column (5 μm, 150 mm × 4.6 mm) and diode array detector (VWD-3000). The chromatographic separation of PMP (1-phenyl-3- methyl-5-pyrazolone) pre-column derivatives was carried out using 100 mM phosphate buffer and acetonitrile at a ratio of 82:18 (v/v, %) as a mobile phase at a flow rate of 1.0 mL/min. A 10 μL of each sample was injected and a run time of analysis of 30 min with detection at 245 nm was employed. Data acquisition and processing were performed using Chromeleon Software 7.1 (Dionex, Sunnyvale, CA, USA).

The molecular weight (Mw) was determined by high-performance size exclusion chromatography (HPSEC, UltiMate 3000 system), equipped with a TSK gel G3000 PWXL (7.8 × 300.0 mm, Tosoh, Japan) and TSK gel GMPWXL (7.8 × 300.0 mm, Tosoh, Japan) column and a RefractoMax521 refractive index detector (RID). The samples were eluted with 0.1 moL/L Na2SO4 at a flow rate of 0.5 mL/min. The temperature of the column was maintained at 35 °C. Dextran as the standards and the standard curve was plotted according to the elution time plotted against the logarithm of molecular weight.

2.4. Pharmacokinetic study

Blood samples were collected from the inner canthus at 0 h, 0.083 h, 0.167 h, 0.25 h, 0.5 h, 0.75 h, 1.0 h, 2.0 h, 3.0 h, 4.0 h, 6.0 h, 8.0 h, 10 h, 12 h and 24 h after administration of echinacoside (100 mg/kg). Plasma samples were obtained by centrifuging for 10 min at 7500 pm. To 100 μL of the plasma sample, 10 μL of IS (chlorogenic acid) solution, and 500 μL of methanol were added. The mixture was then vortexed for 3 min and centrifugation at 14,000 rpm for 10 min. The supernatant was collected and dried in nitrogen at room temperature. Subsequently, the residue was dissolved in 100 μL 50% aqueous methanol, centrifugation at 14,000 rpm for 10 min again. Finally, 3 μL of the supernatant was injected into the LC-MS/MS system for analysis. The samples were analyzed using the method according to a previously reported. The mobile phase consisted of CH3CN (solvent A) and H2O (containing 0.1% HCOOH; solvent B) using the following gradient: 0–1 min, 10% A; 1–8 min, 10–90% A; 8–9 min, 90–10% A; 9–11 min: 10% A. The sample was separated on the ACQUITY HSS T3 column (1.8 μm, 100 mm × 2.1 mm i.d.). The flow rate was 0.3 mL/min and the column temperature was set at 35 °C. In the MS analysis, negative ionization mode was used, with the following optimized parameters: capillary voltage, 2.5 kV; desolvation temperature, 450 °C. Nitrogen was used as desolvation and cone gas, at a flow rate of 900 L/h and 150 L/h, respectively. Quantification was thus performed using MRM of the transitions of m/z 785.29 → 623.26 for echinacoside, m/z 353.06 → 191.07 for chlorogenic acid (IS), m/z 623.24 → 161.09 for acteoside. The cone voltage for echinacoside, IS and acteoside were 100 V, 32 V, and 74 V; collision energy, 32 V, 14 V, and 30 V.

2.5. Short-chain fatty acids (SCFAs) study

Male Sprague-Dawley rats were orally administered ZT, HM, and LM for 3 weeks. Determination of SCFAs (acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, valeric acid, and hexanoic acid) by gas chromatography (GC) was done as described previously with a few modifications [14]. Briefly, 0.5 g fecal sample was dispersed in 2.5 mL of Milli-Q water, vortexed for 10 min, and 7.5 mL 1% HCl/ethanol solution containing internal standards (butyric acid 24.0 μg/mL and 2- ethyl hexanoic acid 1.32 μg/mL as internal standards) were added. The mixtures were homogenized for 5 min and centrifuged at 14,000 rpm for 10 min at 4 °C. Five microliters of supernatant were injected in split mode at a ratio of 30: 1. The carrier gas was N2 (99.999%) at 1 mL/ min. The make-up gas (N2) was at 25 mL/min. The flow rate of H2 and air were 30 mL/min and 300 mL/min, respectively. Fecal samples were collected on the 20th day of this study and immediately stored at −80 °C.

2.6. Stool collection, DNA extraction, and 16S rRNA gene sequencing

Approximately 200 mg of a fresh fecal sample was collected in a 1.5 mL sterile Eppendorf tube from per rat and stored at −80 °C for later DNA preparation. DNA was extracted from the stool using the QIAamp DNA Stool Mini Kit (QIAGEN). The amount of DNA was determined using a NanoDrop spectrophotometer (Thermo Electron Corporation); the integrity and size were checked by 1.0% agarose gel electrophoresis. According to the concentration, DNA was diluted to 1 ng/μL using sterile water.

16S rRNA was amplified using a specific primer (V4: 515F-806R) with the barcode. All PCR reactions were carried out with Phusion® HighFidelity PCR Master Mix (New England Biolabs). Mix equal volume of 1× loading buffer (contained SYB green) with PCR products and operate electrophoresis on 2% agarose gel for detection. The bright main strips between 400 and 450 bp were chosen for further experiments. PCR products were mixed in EU density ratios. Then, mixture of PCR products were purified with Qiagen Gel Extraction Kit (Qiagen, Germany).

Sequencing libraries were constructed using TruSeq® DNA PCRFree Sample Preparation Kit (Illumina, USA) following the manufacturer's recommendations and index codes were added. The library quality was assessed on the Qubit® 2.0 Fluorometer (Thermo Scientific) and Agilent Bioanalyzer 2100 system. The library was sequenced on an Illumina HiSeq 2500 platform and 250 bp paired-end reads were generated.

2.7. Bioinformatics analysis

The barcode and primer sequence cutting off, sequence assembly, and data filtration according to the QIIME (V 1.7.0). Sequences with ≥97% similarity were assigned to the same OTUs. The OTUs are annotated as SILVA labels. The alpha-diversity indices evaluating gut microbial community richness (the ACE and Chao1 metrics) and community diversity (the Shannon and Simpson metrics) were calculated using Qiime (Version 1.9.1). Beta diversity analysis was used to investigate the structural variations of the microbial communities using UniFrac distance. Differences among all groups were tested by the Tukey test and Wilcox test. The principal coordinate analysis (PCoA) was applied to explain the compositional differences among the four groups using R packages. The linear discriminant analysis effect size (LEfSe) method is specifically designed for biomarker discovery and explanation sequencing data. And linear discriminant analysis (LDA) was used to calculate the effect size of each differentially gut microbes and the characteristics associated with each group of rats. The correlation analysis was implemented by using R packages gcorrplot (Version 1.1.463).

SCFAs data were analyzed using the ANOVA of SPSS 22.0.

3. Results and discussion

3.1. Preparation and chemical analysis of cistanche polysaccharides

Cistanches Herba is used as both food and medicine in China. Polysaccharides isolated from it have attracted great attention due to their signifificant activities. In this study, the four parts of cistanche polysaccharides (ZT, HM, MM, and LM) were obtained by the method of water extraction, alcohol precipitation, and ultrafiltration. The chemical compositions of four parts of polysaccharides are shown in Table 1.

Total sugar contents of the samples by using a standard curve of glucose. The result showed that the yields of HM and LM were higher than MM. Polysaccharides showed diverse pharmacological properties, especially immune activity. Studies have shown that the monosaccharide content and type, molecular weight, type of substituent group play an important role in the activities of polysaccharides [15]. The different parts of the monosaccharide composition of cistanche polysaccharides were similar, and mannose (Man), galacturonic acid (GalUA), glucose (Glc), galactose (Gal), and arabinose (Ara) were the main monosaccharides.

However, the contents were different among the four parts. Glucose and arabinose were the main components of ZT and HM, while glucose was the main sugar unit of LM, besides, little rhamnose and glucuronic acid (GlcUA) were also detected in these fractions.

The relative molecular weights of the samples were investigated by high-performance size exclusion chromatography (HPSEC). According to the standard curve of the dextrans and retention time in the columns, the main peak molecular weights in ZT were observed at 1.68 × 106 Da and 1.70 × 104 Da, in HM were determined to be 1.54 × 106, 2.06 × 105, and 4.81 × 104, in MM and LM were determined to be 1.09 × 104 and 2.05 × 104, respectively. The yields of them were listed in Table 1 and the yield of MM was low and not enough for the animal experiment. Previous studies have indicated the structure-dependent effects of cistanche polysaccharides on diverse bioactivities [16]. Further experiments are still needed, especially purification, Fifine structural analysis should be applied for the structure-activity study

3.2. Pharmacokinetics study

The pharmacokinetics of the echinacoside was investigated by ultra-high-performance liquid chromatography triple quadrupole mass spectrometry (UPLC and Xevo TQ-S) after oral administration of echinacoside. The mean plasma concentration-time profiles of echinacoside were shown in Fig. 1. According to the drug and statistics software (DAS, version 2.0), the pharmacokinetics of echinacoside fifit a one-compartment model, and the other parameters in ZT, HM, and LM groups were summarized in Table 2, including the time to reach the maximum concentration (Tmax), maximum concentration (Cmax), area under the concentration-time curve (AUC), and mean residence time (MTR). A pharmacokinetic study showed that the average Cmax of echinacoside in ZT, HM, and LM groups were found to be 646.4 ng/mL, 732.0 ng/ml, and 898.4 ng/mL which were 1.6, 1.8 and 2.2 times as compared to the control group (406.8 ng/ml), respectively. By pretreating with polysaccharides, AUC(0-24 h) of echinacoside were respectively increased by 6.6%, 36.7%, and 63.6% in ZT, HM, and LM groups, compared with the control group, which proved that the polysaccharides showed the activity of promoting the absorption of a small molecule. These results were quite similar to that the chitooligosaccharides improved the bioavailability of phenylethanoid glycosides in Fructus Forsythia extract [17] and the polysaccharides of Polygonum multiflorum improved the biopharmaceutical properties of 2, 3, 5, 4′-tetrahydroxy-stilbene- 2-O- β-D-glucoside in normal Sprague-Dawley rats [18]. Gut microbiota contains a variety of drug-metabolizing related enzymes and the mechanisms of the microbiome shapes drug pharmacokinetics and pharmacodynamics are still unknown [19]. Polysaccharides may nourish the gut microbiota by increasing the immune system, absorbing nutrients, and reducing the growth of pathogens [20,21]. Although literature reported that cistanche polysaccharides could increase echinacoside metabolites (such as acteoside) in vitro, we didn't find the coexisting polysaccharides have benefited for echinacoside converts into acteoside in vivo [10,11].

3.3. SCFAs content in rat feces

Gut microbiota fermented polysaccharides, producing various bioactive polymers and metabolites, such as SCFAs [3]. As the main metabolites of polysaccharides, SCFAs were analyzed in the feces. The SCFAs, including acetic acid, propionic acid, and butyrate have been reported to have beneficial effects on glucose, cholesterol, and lipids metabolism. Acetate is the main SCFAs in the colon and is an important substrate for cholesterol synthesis, propionate is a substrate for gluconeogenesis in the liver and butyrate is the preferred energy substrate of colonocytes and has anti-inflflammatory in the gut [22,23].

Our results showed that acetate was the major SCFAs formed in the feces for all groups, followed by butyrate and propionate, while the concentration of valerate and branched SCFAs (isobutyric acid and isovaleric acid) were lower. The acetate has been shown to increase enzyme activity in the liver tissue, resulting in better blood sugar control [24]. On the 20th day (Fig. 2), the LM group had a signifificantly higher level of acetate (p b 0.05), the proportion of propionate tended to be decreased, and the proportion of butyrate tended to be increased although the change was insignificant in feces compared to the control group.

However, the HM group had a signifificantly lower proportion of butyrate than the control group (p b 0.05). The contents of total SCFAs between LM and HM were opposite trend compared to the control group and only the LM group had a signifificantly difference.

Many factors affect the level of SCFAs in the feces, including the composition and the amount of gut microbiota, the dietary source, and the time it takes for food to pass through the digestive tract [22]. Accumulating studies show that polysaccharides' structure, including composition of monosaccharide residues, degree of polymerization, both can affect fermentation properties and SCFAs production [25]. From our results, we can conclude that cistanche polysaccharides modulate SCFAs production and different structure polysaccharides have a different effects.

3.4. Cistanche polysaccharides induced core changes in composition and diversity of gut microbiota

The bacterial composition of the fecal samples (n = 4) was examined using an Illumina high-throughput sequencing technique. The library was sequenced on an Illumina HiSeq2500 platform and 250 bp paired-end reads were produced. They were processed for taxonomic identification and diversity analysis. Rarefaction curves (Fig. 3a) showed that, for each sample, bacterial species richness leveled off as sampling depth increased, indicating that the sequencing effort was sufficient and that the total diversity within the sample was captured. The Shannon curve (Fig. 3b) showed that the number of unique bacterial species increased as the number of identifified sequences increased. Saturation of the Shannon curves means the sequencing data was enough to cover all the bacteria species.

Alpha diversity analysis is used to measure the diversity within a sample. Chao1, ACE, Shannon, and Simpson diversity indices were calculated by Tukey's test to describe the alpha diversity. The diversity analysis based on ACE and Chao 1 indexes revealed the community richness of the intestinal microbiota at the OTU level. ACE and Chao 1 indices were signifificantly lower in the fecal samples of the LM group compared with the control group (p b 0.05) (Fig. 4a, b), implying that the species richness of the gut microbiota was decreased by LM feeding. The diversity analysis based on Shannon and Simpson indexes revealed the community diversity of the intestinal microbiota. There was no obvious change in the Shannon and Simpson diversity indexes when treated with cistanche polysaccharides compared with the control group.

Cistanche polysaccharides induced major changes in taxa. Moreover, in four groups N98% of the OTUs were related to the phyla Bacteroidetes, Firmicutes, and Proteobacteria. The most predominant phylum was Bacteroidetes contributing 55.09%, 57.89%, 52.72%, and 69.04% of the fecal microbiota in control, ZT, HM, and LM groups respectively, followed by Firmicutes, contributing 41.39%, 38.03%, 42.15%, and 28.15% respectively (Fig. 5a, b). Compared with the control group, the ZT group was shown to result in weak effects on the relative abundance of the major phyla, except the low abundance of Actinobacteria was signifificantly increased (MetaStat, p b 0.05). In the HM group, Proteobacteria was significantly increased (p b 0.01). This indicated that HM Cistanche polysaccharide could promote the enrichment of Proteobacteria to affect the metabolism of substances in rats. In the LM group, the abundance of the Bacteroidetes, whose main contributors were Prevotellaceae and Prevotellaceae_UCG-001 increased signifificantly (p b 0.05) and the abundance of Firmicutes, including Peptococcaceae, decreased signifificantly (p b 0.01). Members of the Prevotella and Bacteroides were previously described as dietary fiber degraders [26], producing metabolic endproducts such as succinate and acetate [27]. Such fifindings confirmed previous studies showing that some Prevotella spp. can degrade complex plant polysaccharides. A previous reports also showed that the two dominant bacterial divisions, the increased ratio of the phyla Firmicutes/Bacteroidetes are associated with obesity [28]. The lower abundance of the former and the higher abundance of the latter in the LM group suggested LM could help shed pounds.

Out of 72 detected families, the taxa top10 were isolated and analyzed (Fig. 5c, d). The Lachnospiraceae and Ruminococcaceae are two of the most abundant families found in the mammalian gut environment and have been associated with the maintenance of gut health [29]. In present study, families Bacteroidales_S24-7_group (39.11 ± 6.04%, 36.88 ± 4.44%, 28.37 ± 4.01%, 35.81 ± 7.91%), Prevotellaceae (10.66 ± 3.84%, 15.14 ± 5.50%, 19.65 ± 8.97%, 21.05 ± 8.39%), Lachnospiraceae (18.21 ± 5.20%, 15.09 ± 3.29%, 12.13 ± 7.14%, 13.12 ± 6.82%), and Ruminococcaceae (9.64 ± 0.88%, 10.44 ± 2.45%, 11.27 ± 4.70%, 8.19 ± 1.72%) were dominating in the each group. S24-7 family has been reported that contained abundances of carbohydrate degrading enzymes [30]. Prevotellaceae presented a rising and Lachnospiraceae presented a declining tendency in the polysaccharides group compared with the control group [31]. It was assumed that there were no corresponding cistanche polysaccharides active enzymes in Bacteroidales_S24-7_group and Lachnospiraceae or carbohydrate-binding modules of bacteria at polysaccharides. Prevotella genera have been reported in association with polysaccharide utilization loci and carbohydrate-active enzymes [32]. In order to find the relationship between bacteria and SCFAs, Spearman correlation analysis was applied. Some genera such as Bacteroides, Prevotella, and Lactobacillus in gut microbiota play an important role in the hydrolyzation of polysaccharides into SCFAs [33]. Our results also showed that the Bacteroidaceae correlated positively and dominated microbiota produced more butyrate [34], while the Prevotellaceae correlated positively and dominated microbiota produced more acetate. Lactobacillaceae and Ruminococcaceae dominated microbiota produced more propionate (Fig. 6).

Beta diversity analysis was used to investigate the structural variations of the gut microbiota across the samples using UniFrac distance metrics. Differences in the Unifrac distances were analyzed by the Tukey and Wilcox test (Fig. 7). Using uniFrac unweighted analysis, we found that LM feed mice cluster away from other groups. An apparent clustering pattern was identifified for control, ZT, HM, and LM groups.

The abundance of these microbial was differentially modulated with polysaccharide treatments. To identify the specific bacterial taxa in each group, the composition of the microbiota from the control, ZT, HM, and LM groups were compared by the linear discriminant analysis effect size (LEfSe) method. Linear Discriminant Analysis (LDA) is used to calculate the effect size of each differentially abundant feature. The LDA scores threshold is higher than the 4 were list, indicating a higher relative community abundance in the corresponding group than in other groups. Using this method, we found that 19 OTUs were signifificantly different in all groups, namely, 6 OTUs in the control group were higher, only 1 OTU in the ZT group was higher, 10 OTUs in the HM group were higher and 2 OTUS in LM group were higher than other groups (Fig. 8). The abundance of g_Prevotellaceae_ UCG_001 in LM was increased signifificantly (8.47 ± 1.9% in LM, 0.40 ± 0.09% in the Control group, p b 0.01). The result was consistent with the report that Porphyra haitanensis and Ulva prolifera polysaccharides could obviously promote the proliferation of Prevotellaceae UGC-001 (p b 0.05) and Rikenellaceae RC9 (p b 0.01) [35].

4. Conclusions

It is clear that there is a relationship between TCM chemical constituents, gut microbiota, and certain metabolites resulting from gut microbial. Our current fifindings provided a method for studying echinacoside metabolism synergies with different kinds of polysaccharides based on gut microbiota. Gut microbiota can metabolize TCM and TCM can affect the growth of gut microbiota. On one hand, after polysaccharides' oral administration into the intestine, a variety of gut microbiota encode metabolic enzymes, playing an important role in polysaccharides decomposition, and to produce bioactive metabolites. On the other hand, the contributions of polysaccharides have some impact on the balance of gut microenvironmental in the process. Through comparison of with or without polysaccharides, we found that polysaccharides although indigested by the host directly, potentially acted as prebiotics, leading to favorable changes in the gut microbiota by promoting the growth of probiotics. Cistanche polysaccharides regulated the gut microbiota diversity, increase beneficial bacteria and especially low molecular polysaccharides enhanced the growth of Prevotella spp. Furthermore, the improved gut microbiota then enhanced the absorption of echinacoside in the Cistanche deserticola. But the reason is not entirely clear, may be a result of the combined defects of the gut microbiota, SCFAs, and immunity, and further research is necessary for a disease model. It is hard to prove the effects of prebiotics in a healthy animal models whose microbiota community is stable and resilient. Cistanche deserticola polysaccharides may be developed as probiotic products to regulate the gut microbiota and can be applied to the synergistic metabolism of small molecules in the future. These data will help us to design interventions to prevent vulnerable groups of people to improve their health.

Declaration of competing interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments

Authors gratefully acknowledge the National Natural Science Foundation of China (Grant No. 81830112 and 81904058)

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