Comparing steamed and wine-stewed Rehmanniae Radix in terms of Yin-nourishing effects via metabolomics and microbiome analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Rehmanniae Radix Praeparata (RRP), the processed root of Rehmannia glutinosa, has been widely used to treat Yin deficiency syndrome in traditional Chinese medicine. RRP is available in two forms: processed by steaming with water (SRR) or processed by stewing with yellow rice wine (WRR). Previous work has documented chemical differences in the secondary metabolomes and glycomes of SRR and WRR. AIM OF THE STUDY: This study aimed to compare SRR and WRR in terms of Yin-nourishing effects via metabolomics and microbiome analysis. MATERIALS AND METHODS: ICR mice were orally administered with thyroxine for 14 d to induce Yin deficiency. Changes in biochemical indices and histopathology were detected. Serum metabolomics analysis and microbial 16S rRNA sequencing were performed to compare the therapeutic effects and mechanisms between SRR and WRR in treating thyroxine-induced Yin deficiency. RESULTS: Both SRR and WRR decreased serum T3, T4 and MDA levels, and increased SOD activity. SRR more effectively decreased serum Cr, and ameliorated kidney injury, while WRR showed better regulation on ratio of cAMP/cGMP and serum TSH, and relieved thyroid injury. Both SRR and WRR regulated tyrosine, glycerophospholipid, and linoleic acid metabolism and the citric acid cycle. Additionally, SRR regulated fatty acid metabolism, while WRR influenced alanine, aspartate and glutamate metabolism, and bile acid biosynthesis. SRR significantly enriched the genera Staphylococcus and Bifidobacterium in the gut microbiome, while WRR significantly enriched the genera Akkermansia, Bacteroides and Parabacteroides, and decreased the abundance of Lactobacillus. CONCLUSIONS: SRR displayed better protective effects on kidney, while WRR showed stronger effects on thyroid in thyroxine-induced Yin deficient mice. These differences might be due to different regulating effects of SRR and WRR on the metabolome and gut microbiota.

Gut Microbiota Mediates the Protective Effects of Traditional Chinese Medicine Formula Qiong-Yu-Gao against Cisplatin-Induced Acute Kidney Injury.

Our previous study found that Qiong-Yu-Gao (QYG), a traditional Chinese medicine formula derived from Rehmanniae Radix, Poria, and Ginseng Radix, has protective effects against cisplatin-induced acute kidney injury (AKI), but the underlying mechanisms remain unknown. In the present study, the potential role of gut microbiota in the nephroprotective effects of QYG was investigated. We found that QYG treatment significantly attenuated cisplatin-induced AKI and gut dysbiosis, altered the levels of bacterial metabolites, with short-chain fatty acids (SCFAs) such as acetic acid and butyric acid increasing and uremic toxins such as indoxyl sulfate and p-cresyl sulfate reducing, and suppressed histone deacetylase expression and activity. Spearman's correlation analysis found that QYG-enriched fecal bacterial genera Akkermansia, Faecalibaculum, Bifidobacterium, and Lachnospiraceae_NK4A136_group were correlated with the altered metabolites, and these metabolites were also correlated with the biomarkers of AKI, as well as the indicators of fibrosis and inflammation. The essential role of gut microbiota was further verified by both the diminished protective effects with antibiotics-induced gut microbiota depletion and the transferable renal protection with fecal microbiota transplantation. All these results suggested that gut microbiota mediates the nephroprotective effects of QYG against cisplatin-induced AKI, potentially via increasing the production of SCFAs, thus suppressing histone deacetylase expression and activity, and reducing the accumulation of uremic toxins, thereby alleviating fibrosis, inflammation, and apoptosis in renal tissue. IMPORTANCE Cisplatin-induced acute kidney injury is the main limiting factor restricting cisplatin's clinical application. Accumulating evidence indicated the important role of gut microbiota in pathogenesis of acute kidney injury. In the present study, we have demonstrated that gut microbiota mediates the protective effects of traditional Chinese medicine formula Qiong-Yu-Gao against cisplatin-induced acute kidney injury. The outputs of this study would provide scientific basis for future clinical applications of QYG as prebiotics to treat cisplatin-induced acute kidney injury, and gut microbiota may be a promising therapeutic target for chemotherapy-induced nephrotoxicity.

Gut microbiota influenced the xenograft MC38 tumor growth potentially through interfering host lipid and amino acid metabolisms, basing on the integrated analysis of microbiome and metabolomics.

Gut microbiota is associated with tumor progress and host metabolic disorder, but whether gut microbiota regulation can affect cancer growth through interfering host metabolism maintains unknown yet. Here, we used combined antibiotics (ABX) to build an extremely altered gut microbiota ecosystem and study its influence on the xenograft MC38 tumor as well as the associations of the effects with host metabolisms. The MC38 tumor bearing mouse was treated with ABX (vancomycin, neomycin and imipenem-cilastatin) to build the extremely altered microbiota ecosystem, the gut microbiota diversity alteration was determined by 16S rRNA based gene sequencing. The effects of the altered microbiota on tumor were assessed by cell apoptosis and growth rate of the tumor. The potential metabolic biomarkers and involved metabolism pathways were screened out by UPLC-QTOF-MS/MS based untargeted metabolomics and KEGG analysis respectively. The correlations between key metabolites and microbiota were analyzed by Spearman correlation analysis. Compared with the un-treated mice, the tumor growth of ABX-treated mice was significantly suppressed, and the cell apoptosis was obviously promoted. The gut microbiota diversity was decreased significantly with the dominant bacteria phylum Bacteroidetes and Firmicutes replaced by Proteobacteria, which involved 14 significantly altered bacteria genera. Four potential targeted metabolism pathways, including sphingolipid, glycerophospholipid, arginine-proline and primary bile acid metabolism, were screened out, and the involved key metabolites such as ceramide, phosphatidylethanolamine, phosphatidylcholine, taurocholic acid and L-proline were correlated significantly with the altered bacteria genera. Through the integrated analysis of microbiome and metabolomics, it was revealed that gut microbiota regulation may inhibit the xenograft MC38 tumor growth potentially by interfering host lipid and amino acid metabolisms, such as sphingolipid, glycerophospholipid, primary bile acid and arginine-proline metabolisms in this case.

Protective effects of Poria cocos and its components against cisplatin-induced intestinal injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Poria cocos (Schw.) Wolf (Poria) is a well-known traditional medicinal fungus. It has been considered to possess spleen-invigorating (Jianpi) effects in traditional Chinese medicine, and is used clinically to treat spleen deficiency (Pixu) with symptoms of intestinal disorders such as diarrhea, indigestion, mucositis and weight loss. THE AIM OF THIS STUDY: To investigate the protective effects of Poria and its three component fractions (Water-soluble polysaccharides, WP; alkali-soluble polysaccharides, AP; triterpene acids, TA) on cisplatin-induced intestinal injury and explore the underlying mechanisms. MATERIALS AND METHODS: C57BL/6 mice were treated with Poria powder (PP), WP, AP and TA by oral gavage respectively for 13 days, and intraperitoneally injected with 10 mg/kg of cisplatin on day 10 to conduct a cisplatin-induced intestinal injury model. Pathological changes of ileum and colon were examined using H&E staining. The composition of gut microbiota and the alteration of host metabolites were characterized by 16S rDNA amplicon sequencing and UPLC-QTOF-MS/MS based untargeted metabolomics analysis. RESULTS: PP and WP attenuated the cisplatin-induced ileum and colon injury, and WP alleviated the weight loss and reversed the elevation of IL-2, IL-6 in serum. Both PP and WP could mitigate cisplatin-induced dysbiosis of gut microbiota, in particular PP and WP decreased the abundance of pathogenic bacteria including Proteobacteria, Cyanobacteria, Ruminococcaceae and Helicobacteraceae, while WP promoted the abundance of probiotics, such as Erysipelotrichaceae and Prevotellaceae. Moreover, WP attenuated the cisplatin-induced alteration of metabolic profiles. The levels of potential biomarkers, including xanthine, L-tyrosine, uridine, hypoxanthine, butyrylcarnitine, lysoPC (18:0), linoleic acid, (R)-3-hydroxybutyric acid, D-ribose, thiamine monophosphate, indolelactic acid and plamitic acid, showed significant correlations with intestinal flora. CONCLUSIONS: PP and WP possess protective effects against cisplatin-induced intestinal injury via potentially regulating the gut microbiota and metabolic profiles.

Independent or integrative processing approach of metabolite datasets from different biospecimens potentially affects metabolic pathway recognition in metabolomics.

In metabolomics studies, metabolic pathway recognition (MPR) is performed by software tools to screen out the significant pathways disturbed by diseases or reinstated by drugs. To achieve MPR, the significantly changed metabolites determined in different biospecimens (e.g. plasma and urine) are analyzed either independently (metabolites from each biospecimen as a dataset) or integratively (metabolites from all biospecimens as a dataset). However, whether the choice of these two processing approaches affects the results of MPR remains unknown. In this study, this issue was addressed by selecting evaluation of the effects of the herbal medicine Rehmanniae Radix (RR) on anemia and adrenal fatigue by UPLC-QTOF-MS/MS-based metabolomics as an example. The significant pathways disturbed by the modeling of anemia and adrenal fatigue and those reinstated by treatments with raw and processed RR were recognized using MetPA software tool (MetaboAnalyst 3.0), and compared by independent and integrative processing of the significantly changed metabolites determined in plasma and urine. The results showed that the two processing approaches could yield different impact values of pathways and thereby recognize different significant pathways. The differences appear to happen more easily when metabolites from different biospecimens shared the same metabolic pathway. Such pathway could be recognized as a significant pathway by integrative processing but could be excluded by independent processing due to the converged and dispersed importance contributions of the involved metabolites to MPR in the two processing approaches. This issue should concern researchers because MPR is crucial not only to understanding metabolomics data but also to guiding subsequent mechanistic research.

A dereplication strategy for identifying triterpene acid analogues in Poria cocos by comparing predicted and acquired UPLC-ESI-QTOF-MS/MS data.

INTRODUCTION: Triterpene acids from the dried sclerotia of Poria cocos (Schw.) Wolf (poria) were recently found to possess anti-cancer activities. Identification of more triterpene acid analogues in poria is worthwhile for high throughput screening in anti-cancer drug discovery. OBJECTIVE: To establish an efficient dereplication strategy for identifying triterpene acid analogues in poria based on ultra-performance liquid chromatography with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS). METHODOLOGY: The structural characteristics and mass spectrometric data profiles of known triterpene acids previously reported in poria were used to establish a predicted-analogue database. Then, the quasi-molecular ions of components in a poria extract were automatically compared with those in the predicted-analogue database to highlight compounds of potential interest. Tentative structural identification of the compounds of potential interest and discrimination of isomers were achieved by assessing ion fragmentation patterns and chromatographic behaviour prediction based on structure-retention relationship. RESULTS: A total of 62 triterpene acids were unequivocally or tentatively characterised from poria, among which 17 triterpene acids were tentatively identified for the first time in poria. CONCLUSION: This study provided more structure information of triterpene acids in poria for future high throughput screening of anti-cancer candidates. It is suggested that this semi-automated approach in which MS data are automatically compared to a predictive database may also be applicable for efficient screening of other herbal medicines for structural analogues of proven bioactives.

Time segment scanning-based quasi-multiple reaction monitoring mode by ultra-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry for quantitative determination of herbal medicines: Moutan Cortex, a case study.

In this study, a time segment scanning-based quasi-multiple reaction monitoring (quasi-MRM) mode was proposed to improve the quantitative performance of UPLC-QTOF-MS/MS. To achieve the quasi-MRM mode, a strategy to select the ion pair (precursor and product ions) of each analyte was adopted as follows. First, a stable and abundant ion by quadrupole was set as precursor ion in MS scan mode. Second, the fragment ions of the precursor ion formed via collision-induced dissociation were measured by time-of-flight (TOF) in MS/MS scan mode; a characteristic, stable and abundant fragment ion (or precursor ion in case of fragment ion unavailable) was designated as the product ion. Third, the detection specificity and sensitivity of the product ion by TOF were strengthened through time segment scanning over a narrowed mass scan range. The proposed quasi-MRM mode achieved simultaneous quantification of fifteen major components in Moutan Cortex, a widely used medicinal herb, as well as its sulfur-fumigated samples. The quasi-MRM mode was methodologically compared with the other two quantitative modes commonly used in the UPLC-PDA-QTOF-MS/MS apparatus, namely UPLC-PDA and extracted ion analysis. The results demonstrated that the quasi-MRM mode performed better in specificity, sensitivity and linearity. The quasi-MRM mode was further validated with regard to precision, accuracy and stability. The research deliverables indicate that the proposed mode improved the quantitative capability of UPLC-QTOF-MS/MS, and therefore could serve as a potential mode for QTOF-MS/MS-based quantification of herbal medicines.

UPLC-QTOF-MS/MS-guided isolation and purification of sulfur-containing derivatives from sulfur-fumigated edible herbs, a case study on ginseng.

In this study, a novel ultra-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UPLC-QTOF-MS/MS)-guidance strategy was proposed for preparation of sulfur-containing derivatives in sulfur-fumigated edible herbs. Being versatile in both chromatographic separation and mass spectrometric detection, UPLC-QTOF-MS/MS was inducted into each experimental step for multifaceted purposes including finding, tracking, purity determination and structural elucidation of targeted compounds as well as UPLC-HPLC chromatographic conditions transplantation, whereby the isolation and purification procedures were greatly facilitated. Using this strategy, a new sulfur-containing ginsenoside Rg1 derivative (named compound I) was obtained from sulfur-fumigated ginseng. The chemical structure of compound I was elucidated to be (3ß, 6α, 12ß)-3, 12-dihydroxydammar-25-ene-6, 20-diylbis-ß-d-glucopyranoside, 24-sulfonic acid by QTOF-MS/MS, 1H-NMR and 13C-NMR analysis, and its generation mechanisms by sulfur-fumigation were accordingly discussed. The research deliverable suggests that the UPLC-QTOF-MS/MS-guidance strategy is promising for targeted preparation of sulfur-containing derivatives from sulfur-fumigated edible herbs.

Chemomics-based marker compounds mining and mimetic processing for exploring chemical mechanisms in traditional processing of herbal medicines, a continuous study on Rehmanniae Radix.

Exploring processing chemistry, in particular the chemical transformation mechanisms involved, is a key step to elucidate the scientific basis in traditional processing of herbal medicines. Previously, taking Rehmanniae Radix (RR) as a case study, the holistic chemome (secondary metabolome and glycome) difference between raw and processed RR was revealed by integrating hyphenated chromatographic techniques-based targeted glycomics and untargeted metabolomics. Nevertheless, the complex chemical transformation mechanisms underpinning the holistic chemome variation in RR processing remain to be extensively clarified. As a continuous study, here a novel strategy by combining chemomics-based marker compounds mining and mimetic processing is proposed for further exploring the chemical mechanisms involved in herbal processing. First, the differential marker compounds between raw and processed herbs were rapidly discovered by untargeted chemomics-based mining approach through multivariate statistical analysis of the chemome data obtained by integrated metabolomics and glycomics analysis. Second, the marker compounds were mimetically processed under the simulated physicochemical conditions as in the herb processing, and the final reaction products were chemically characterized by targeted chemomics-based mining approach. Third, the main chemical transformation mechanisms involved were clarified by linking up the original marker compounds and their mimetic processing products. Using this strategy, a set of differential marker compounds including saccharides, glycosides and furfurals in raw and processed RR was rapidly found, and the major chemical mechanisms involved in RR processing were elucidated as stepwise transformations of saccharides (polysaccharides, oligosaccharides and monosaccharides) and glycosides (iridoid glycosides and phenethylalcohol glycosides) into furfurals (glycosylated/non-glycosylated hydroxymethylfurfurals) by deglycosylation and/or dehydration. The research deliverables indicated that the proposed strategy could advance the understanding of RR processing chemistry, and therefore may be considered a promising approach for delving into the scientific basis in traditional processing of herbal medicines.