A quality-comprehensive-evaluation-index-based model for evaluating traditional Chinese medicine quality.

BACKGROUND: Evaluating traditional Chinese medicine (TCM) quality is a powerful method to ensure TCM safety. TCM quality evaluation methods primarily include characterization evaluations and separate physical, chemical, and biological evaluations; however, these approaches have limitations. Nevertheless, researchers have recently integrated evaluation methods, advancing the emergence of frontier research tools, such as TCM quality markers (Q-markers). These studies are largely based on biological activity, with weak correlations between the quality indices and quality. However, these TCM quality indices focus on the individual efficacies of single bioactive components and, therefore, do not accurately represent the TCM quality. Conventionally, provenance, place of origin, preparation, and processing are the key attributes influencing TCM quality. In this study, we identified TCM-attribute-based quality indices and developed a comprehensive multiweighted multi-index-based TCM quality composite evaluation index (QCEI) for grading TCM quality. METHODS: The area of origin, number of growth years, and harvest season are considered key TCM quality attributes. In this study, licorice was the model TCM to investigate the quality indicators associated with key factors that are considered to influence TCM quality using multivariate statistical analysis, identify biological-evaluation-based pharmacological activity indicators by network pharmacology, establish real quality indicators, and develop a QCEI-based model for grading TCM quality using a machine learning model. Finally, to determine whether different licorice quality grades differently reduced the inflammatory response, TNF-α and IL-1ß levels were measured in RAW 264.7 cells using ELISA analysis. RESULTS: The 21 quality indices are suitable candidates for establishing a method for grading licorice quality. A computer model was established using SVM analysis to predict the TCM quality composite evaluation index (TCM QCEI). The tenfold cross validation accuracy was 90.26%. Licorice diameter; total flavonoid content; similarities of HPLC chromatogram fingerprints recorded at 250 and 330 nm; contents of liquiritin apioside, liquiritin, glycyrrhizic acid, and liquiritigenin; and pharmacological activity quality index were identified as the key indices for constructing the model for evaluating licorice quality and determining which model contribution rates were proportionally weighted in the model. The ELISA analysis results preliminarily suggest that the inflammatory responses were likely better reduced by premium-grade than by first-class licorice. CONCLUSIONS: In the present study, traditional sensory characterization and modern standardized processes based on production process and pharmacological efficacy evaluation were integrated for use in the assessment of TCM quality. Multidimensional quality evaluation indices were integrated with a machine learning model to identify key quality indices and their corresponding weight coefficients, to establish a multiweighted multi-index and comprehensive quality index, and to construct a QCEI-based model for grading TCM quality. Our results could facilitate and guide the development of TCM quality control research.

Identification of Pulsatilla chinensis (Bge.) Regel and look-alike species by UHPLC-TOF-MS using multivariate statistical analysis.

Pulsatillae Radix, the root of Pulsatilla chinensis (Bge.) Regel, is recorded in the Pharmacopoeia of the People's Republic of China and has been widely used for its pharmacological activities, such as anti-inflammatory, antioxidant, antibacterial, antitumor, and cardiovascular benefits. However, there are several look-alike species that can be marketed as Pulsatillae Radix. To distinguish P. chinensis (Bge.) Regel from its look-alikes, viz. Pulsatilla cernua (Thunb.) Bercht et Opiz., Pulsatilla dahurica (Fisch.) Spreng., Anemone tomeutosa (Maxim.) Pei., and Rhaponticum uniflorum (L.) DC, we used ultra high performance liquid chromatography with time-of-flight mass spectrometry combined with principal component analysis to compare their chemical compositions. Four ions, a (RT 8.98 min, m/z 1381.6671), b (RT 10.64 min, m/z 1219.6143), c (RT 11.52 min, m/z 1217.5978), and d (RT 13.6 min, m/z 749.4463), from P. chinensis (Bge.) Regel were identified as potential chemical markers to distinguish it from look-alike species using an unsupervised statistical model combined with orthogonal partial least-squares discriminant analysis. The results of this study provide an effective method for identifying and distinguishing P. chinensis (Bge.) Regel from similar plants.

A general procedure for establishing composite quality evaluation indices based on key quality attributes of traditional Chinese medicine.

Licorice, a medicinal herb and food flavor ingredient, has been widely used in traditional Chinese medicine (TCM) for the past 4000 years. In this study, we propose a new quality evaluation approach for licorice quality control based on the key quality attributes commonly used in TCM. The high quality of TCM formulations is ensured by verifying the genuine origin and implementing good agricultural and collection practices for each medicinal herb. In our study, the genuine production area, the harvest season, and the number of growth years were considered the key quality attributes of TCM. To ensure the representativeness of our analysis, we obtained a total of 158 licorice sample batches that differed in the number of growth years, the location of the production areas, and the season for harvesting. Initially, the 158 sample batches were subjected to ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). A preliminary screen identified 11 licorice compounds related to the three key quality attributes of TCM . An analysis by ultra-high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (UHPLC-TQ-MS/MS) verified the presence of 34 compounds in all licorice samples. These 34 compounds included the 11 compounds related to the three key quality attributes of the samples, along with other bioactive components identified in previous studies. After using UHPLC-TQ-MS/MS to assess the signal peak intensities of the 34 compounds, we selected 17 licorice compounds to establish sample content evaluation indices, which were determined by high-performance liquid chromatography at four different wavelengths in all 158 licorice sample batches. Finally, the screen identified nine compounds that were closely associated with the quality attributes of licorice based on principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Our results suggested that liquiritin and eight other compounds could be used as quality control indicators of licorice, which provided a foundation to establish the TCM quality composite evaluation index (TCM QCEI). In summary, this research concept can serve as a reference for research on quality markers and the evaluation of TCM.

UHPLC-QQQ-MS/MS assay for the quantification of dianthrones as potential toxic markers of Polygonum multiflorum Thunb: applications for the standardization of traditional Chinese medicines (TCMs) with endogenous toxicity.

BACKGROUND: The raw and processed roots of Polygonum multiflorum Thunb (PM) are commonly used in clinical practice to treat diverse diseases; however, reports of hepatotoxicity induced by Polygoni Multiflori Radix (PMR) and Polygoni Multiflori Radix Praeparata (PMRP) have emerged worldwide. Thus, it is necessary for researchers to explore methods to improve quality standards to ensure their quality and treatment effects. METHODS: In the present study, an ultra-high performance liquid chromatography triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) method was optimized and validated for the determination of dianthrones in PMR and PMRP using bianthronyl as the internal standard. Chromatographic separation with a gradient mobile phase [A: acetonitrile and B: water containing 0.1% formic acid (v/v)] at a flow rate of 0.25 mL/min was achieved on an Agilent ZORBAX SB-C18 column (2.1 mm × 50 mm, 1.8 µm). The triple quadrupole mass spectrometer (TQMS) was operated in negative ionization mode with multiple reaction monitoring for the quantitative analysis of six dianthrones. Moreover, compounds 5 and 6 were further evaluated for their cytotoxicity in HepaRG cells by CCK-8 assay. RESULTS: The UHPLC-QQQ-MS/MS method was first developed to simultaneously determine six dianthrones in PMR and PMRP, namely, polygonumnolides C1-C4 (1-4), trans-emodin dianthrones (5), and cis-emodin dianthrones (6). The contents of 1-6 in 90 batches of PMR were in the ranges of 0.027-19.04, 0.022-13.86, 0.073-15.53, 0.034-23.35, 0.38-83.67 and 0.29-67.00 µg/g, respectively. The contents of 1-6 in 86 batches of commercial PMRP were in the ranges of 0.020-13.03, 0.051-8.94, 0.022-7.23, 0.030-12.75, 0.098-28.54 and 0.14-27.79 µg/g, respectively. Compounds 1-4 were almost completely eliminated after reasonable processing for 24 h and the contents of compounds 5 and 6 significantly decreased. Additionally, compounds 5 and 6 showed inhibitory activity in HepaRG cells with IC50 values of 10.98 and 15.45 µM, respectively. Furthermore, a systematic five-step strategy to standardize TCMs with endogenous toxicity was proposed for the first time, which involved the establishment of determination methods, the identification of potentially toxic markers, the standardization of processing methods, the development of limit standards and a risk-benefit assessment. CONCLUSION: The results of the cytotoxicity evaluation of the dianthrones indicated that trans-emodin dianthrones (5) and cis-emodin dianthrones (6) could be selected as toxic markers of PMRP. Taking PMR and PMRP as examples, we hope this study provides insight into the standardization and internationalization of endogenous toxic TCMs, with the main purpose of improving public health by scientifically using TCMs to treat diverse complex diseases in the future.

Advances in Understanding the Metabolites and Metabolomics of Polygonum multiflorum Thunb: A Mini-review.

BACKGROUND: The roots of Polygonum multiflorum (PM) are a well-known traditional Chinese medicine, widely used to treat a variety of conditions in Southeast Asia, South Korea, Japan and other countries. It is known that Polygoni Multiflori Radix Praeparata (PMRP) may enhance the efficacy and reduce the toxicity of PM. However, reports of adverse reactions, such as hepatotoxicity, caused by PM or PMRP, have continuously appeared around the world, which increased the known risks of the medication and gradually gained the extensive attention of many researchers. The chemical constituents of PM that cause hepatotoxicity have not been distinctly elucidated using the traditional phytochemical screening. Recently, with the rapid development of metabolomics, there has been a growing need to explore the potential hepatotoxic components and mechanisms of PM. METHODS: The metabolites and metabolomics of PM were searched by the Web of Science, PubMed, Google scholar and some Chinese literature databases. RESULTS: A brief description of metabolites and metabolomics of PM is followed by a discussion on the metabolite- induced toxicity in this review. More than 100 metabolites were tentatively identified and this will contribute to further understanding of the potential hepatotoxic components of PM. Meanwhile, some toxic compounds were identified and could be used as potential toxic markers of PM. CONCLUSION: This review mainly outlines the metabolites and metabolomics of PM that have been identified in recent years. This study could help to clarify the potential hepatotoxic components and metabolic mechanisms of PM and provide a scientific reference for its safe clinical use in the future.

In vivo hepatotoxicity screening of different extracts, components, and constituents of Polygoni Multiflori Thunb. in zebrafish (Danio rerio) larvae.

Polygonum multiflorum Thunb. (PM) is a traditional Chinese medicine, commonly used to treat a variety of diseases. However, the hepatotoxicity associated with PM hampers its clinical application and development. In this study, we refined the zebrafish hepatotoxicity model with regard to the following endpoints: liver size, liver gray value, and the area of yolk sac. The levels of alanine aminotransferase, aspartate transaminase, albumin, and microRNAs-122 were evaluated to verify the model. Subsequently, this model was used to screen different extracts, components, and constituents of PM, including 70 % EtOH extracts of PM, four fractions from macroporous resin (components A, B, C, and D), and 19 compounds from component D. We found that emodin, chrysophanol, emodin-8-O-ß-D-glucopyranoside, (cis)-emodin-emodin dianthrones, and (trans)-emodin-emodin dianthrones showed higher hepatotoxicity compared to other components in PM, whereas polyphenols showed lower hepatotoxicity. To the best of our knowledge, this study is the first to identify that dianthrones may account for the hepatotoxicity of PM. We believe that these findings will be helpful in regulating the hepatotoxicity of PM.

Multiflorumisides HK, stilbene glucosides isolated from Polygonum multiflorum and their in vitro PTP1B inhibitory activities.

A phytochemical study on a 70% EtOH extract of dried roots of Polygonum multiflorum resulted in the isolation of four undescribed stilbene glucosides, namely multiflorumisides HK (1-4). The structures of the natural products were elucidated by 1D and 2D nuclear magnetic resonance (NMR) as well as mass spectroscopy analyses. Among them, multiflorumiside J (3) and multiflorumiside K (4) belong to rare tetramer stilbene glucosides. Moreover, the in vitro inhibitory activities against protein tyrosine phosphatase 1B (PTP1B) were evaluated and the putative biosynthetic pathway was proposed. Notably, compounds 1-4 showed the inhibitory activity against PTP1B with the IC50 values of 1.2, 1.7, 1.5 and 4.6 µm, respectively. Based on the obtained results, stilbene glucosides could be the potential PTP1B inhibitors of P. multiflorum.

Identification of Calculus Bovis and its mixed varieties by ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) combined with the principal component analysis (PCA) method.

The method of ultra-high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) was established and combined with principal component analysis (PCA) to identify natural Calculus Bovis, in vitro cultured Calculus Bovis and artificial Calculus Bovis. PCA, which was particularly powerful in dealing with multicollinearity and variables that outnumber the samples, was used to analyze the UHPLC-MS data of the processed samples, and potential markers were analyzed and described based on orthogonal partial least-squares discriminant analysis. According to the results in this study, the approach of combining UHPLC-QTOF-MS with PCA was proven to be credible and could be used to identify Calculus Bovis from in vitro cultured Calculus Bovis and artificial Calculus Bovis and to determine if there is Calculus Bovis in patented Chinese medicines that should contained Calculus Bovis medicinal materials.

Characterization and identification of the chemical constituents of Polygonum multiflorum Thunb. by high-performance liquid chromatography coupled with ultraviolet detection and linear ion trap FT-ICR hybrid mass spectrometry.

Dianthrone derivatives are minor constituents of Polygonum multiflorum Thunb. (PM). These derivatives are potential hepatotoxic components in PM. Fraction D6 contains many dianthrone derivatives and was successfully enriched using an efficient three-step approach. An effective and reliable high-performance liquid chromatography (HPLC) technique coupled with ultraviolet detection (UV) and a linear ion trap FT-ICR hybrid mass spectrometry (HPLC-UV/LTQ-FT-ICR-MS) method were successfully developed to separate and identify the dianthrones of the fraction D6. The characteristic diagnostic fragment ions and characteristic fragmentation pathway of the seven dianthrone standards, namely, Polygonumnolide B1 (S1), Polygonumnolide C3 (S2), Polygonumnolide C2 (S3), Polygonumnolide E (S4), Polygonumnolide A1 (S5), Polygonumnolide A2 (S6) and cis-emodin dianthrones (S7), were compared with unknown compounds in fraction D6, and 45 dianthrone derivatives were characterized or tentatively identified. Of these derivatives, 32 new dianthrone derivatives were tentatively characterized in PM. Therefore, LTQ-FT-ICR-MS combined with a selective enrichment method provided a powerful means for analyzing dianthrone derivatives. This study provides a meaningful basis for correcting some mistakes in previous studies, as well as further quality control and pharmacological and toxicological research.

Identification of velvet antler and its mixed varieties by UPLC-QTOF-MS combined with principal component analysis.

Many species of velvet antler have been used as traditional medicine for thousands of years; however, as medicinal materials, velvet antler derived from different animals have different clinical effects. To distinguish the differences and homologies, ultra-performance liquid chromatography-quadruple-time of flight mass spectrometry (UPLC-QTOF-MS) coupled with principal component analysis (PCA) was developed and applied to identify these antler samples derived from Cervus nippon Temminck, Cervus elaphus Linnaeus and Rangifer tarandus Linnaeus, which were first tested and compared at the molecular level of protein. The UPLC-MS data of the trypsin digested samples were subjected to PCA, and the potential markers based on peptide were depicted to illustrate their differences. With the integrated strategy combining UPLC-QTOF-MS with PCA, the results from this study indicated that the proposed methods could be successfully applied to distinguish reindeer antler from sika deer antler and red deer antler, which were prescribed in the Chinese Pharmacopoeia (2015 edition).

[Market quality surveillance and analysis of chinese medicinal materials and decoction pieces in 2017].

With the rapid development of traditional Chinese medicine (TCM) in China as well as the implementation of the four most strict requirements, the quality of Chinese medicinal materials and decoction pieces had beem improved in recent years, however new problems and challenges were occurred. All the data of Chinese medicinal materials and decoction piece in special inspection,supervision test and evaluation inspection of drug administration department to were summarized and analyzed evaluate and analyze of the quality of Chinese medicinal materials and decoction pieces in 2017. On this basis, the relevant quality control strategies and suggestions were put forward for the relevant departments of China Food and Drug Administration to formulate and implement regulatory measures, furthermore to improve drug standards, and ensure the safety of medication.

[Identification of Fel Serpentis and its adulterants with specific PCR method].

The study aims at developing a convenient and specific method for the identification of Fel Serpentis DNA. The methods of Fel Serpentis genomic DNA purification were tested and optimized, four pairs of specific primers for the amplification of COⅠ, Cyt b and 16S were designed. Then the best pair of primers were selected according to the specificity and efficiency. The DNA fragment about 400 bp was amplified from 20 kinds of Fel Serpentis, whereas no DNA fragment was amplified from other animal samples under the same condition. This method is specific,accurate and reproducible, which provides a useful tool for the quality control of Fel Serpentis.

Acute toxicity screening of different extractions, components and constituents of Polygonum multiflorum Thunb. on zebrafish (Danio rerio) embryos in vivo.

Polygonum multiflorum Thunb. has been used widely in East Asia in treatment of diseases associated with aging. However, there are many reports referred to the toxicity of P. multiflorum, especially for liver adverse reactions. The toxicity of it is caused by over dosage or by the herb itself remains unclear. The aim of this study was to study the toxicity of different extractions, components and constituents of P. multiflorum, which were assessed in zebrafish embryos. Firstly, the difference of extracting solvent to the toxicity of P. multiflorum was researched to probe the influence of usages to the safety of P. multiflorum. The toxicity of 70% EtOH extract is considerably higher than that of other extracts. Secondly, 70% EtOH extract was subjected to macroporous resin (DM-8) eluting with a gradient of water and EtOH (H2O, 25% EtOH, 40% EtOH and 95% EtOH) to give four components (A-D). The toxicity of the component (D) showed higher than the other components (A-C). Thus, the component (D) was taken more attentions to research. Lastly, study on the chemical constituents of the component (D), 27 compounds, including 7 anthraquinones (1-7), 8 stilbenes (8-15), 7 anthrones (16-22), 3 cinnamic acid amides (23-25), 2 naphthols (26-27) were isolated and assessed in zebrafish embryos. Compounds 1-3, 16-22 and 26-27 showed severe toxicity against the zebrafish embryos while other compounds, such as stilbenes, showed no obvious toxicity.

[The analysis of multivariate image and chemometrics in TLC fingerprinting of artificial cow-bezoar].

A method of thin-layer fingerprinting chromatogram of artificial cow-bezoar was established with the developing solvent consisting of cyclohexane, ethyl acetate, acetic acid and methanol (2∶7∶1∶2), and 10% sulfuric acid ethanol solution sprayed as colour-developing agent. After heated at 105 ℃, TLC was recorded as an image in ultraviolet light at 366 nm which was converted into grayscale. By the gray value extracted from the grayscale, the multivariate data obtained from TLC of samples could be analyzed by chemometric method. The results indicated that samples from different manufacturers could be distinguished by this method and some specific bands were found out. All in one, this simple and practical method was suitable for the evaluation of quality difference.

Distinguishing the Chinese materia medica Tiepishihu from similar Dendrobium species of the same genus using histological and microscopic method.

The Chinese Materia Medica, Tiepishihu, used as a tonic for over one thousand years, is a well-known precious medicine in China. According to the Chinese Pharmacopoeia, its source is the species Dendrobium officinale Kimura et Migo, which is distinguished from other species in Dendrobium genus. However, these species from the same genus are similar with Tiepishihu and caused confusion in the market. To find a quick and simple method to distinguish Tiepishihu from other similar species, histologic and microscopic methods were combined together to investigate the transverse section of stem of Tiepishihu and other similar species. Phloroglucinol test solution with hydrochloric acid was used to reveal the lignified tissue by staining the transverse section of Tiepishihu and similar species. Results revealed the unique identification characteristics to distinguish Tiepishihu from similar species, which were difficult to distinguish by other methods. The identification characteristics of Tiepishihu include the cells of vascular bundle sheath were stained red, parenchyma cells were not stained red. What's more, other species can be distinguished from each other with microscopic and histological characteristics. These characteristics proved stable and can be easily observed by normal light microscopic examination. This method is rapid, accurate, stable, and inexpensive.

Identification of four cornua by ultra-performance liquid chromatography with time-of-flight mass spectrometry coupled with principal component analysis.

An ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry method coupled with principal component analysis was developed and applied to the identification of Cornu Antelopis, Cornu Bubali, Cornu Naemorhedi, and Cornu Bovis. The data obtained from the trypsin-digested samples were subjected to principal component analysis to classify these four cornua. Additionally, marker peptides of the cornua were determined by orthogonal partial least-squares discriminant analysis, and fragmentation tandem mass spectra of these marker peptides were evaluated. The results from this study indicate that the proposed method is reliable, and it has been successfully applied to the identification of variants of cornua commonly used in traditional Chinese medicine.

Identification of Chinese Caterpillar Medicinal Mushroom, Ophiocordyceps sinensis (Ascomycetes) from Counterfeit Species.

Ophiocordyceps sinensis is a valuable traditional Chinese medicine with a high market price. In this study, a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) method based on 2 enzymes was developed to distinguish O. sinensis from 6 common counterfeit species. To verify the applicability of this method, we experimentally tested O. sinensis organisms, tablet preparations made from O. sinensis, and cultured mycelia isolated from O. sinensis. To validate the results from this PCR-RFLP method, all real samples were identified by internal transcribed spacer sequencing. This is, to our knowledge, the first time the PCR-RFLP method has been applied to identify O. sinensis. The selection of 2 restrictive enzymes for identification dramatically improved the accuracy and efficiency of this method. It is the great advantage of this method that sampling from either of 2 parts of O. sinensis-the fruiting body or the caterpillar body-would not cause any difference in the final experimental results. Therefore, this method is not only feasible for testing crude drugs of O. sinensis but it is also useful when the crude drugs are broken down into powder or made into tablets, demonstrating the promising prospect of application in quality control.

An integrated lipidomics and metabolomics reveal nephroprotective effect and biochemical mechanism of Rheum officinale in chronic renal failure.

Chronic renal failure (CRF) is a major public health problem worldwide. Earlier studies have revealed salutary effects of rhubarb extracts in CRF. In this study, we employed lipidomic and metabolomic approaches to identify the plasma biomarkers and to determine the effect of treatment with petroleum ether, ethyl acetate and n-butanol extracts of rhubarb in a rat model of CRF with adenine-induced chronic tubulointerstitial nephropathy. In addition, clinical biochemistry, histological evaluation and pro-fibrotic protein expression were analyzed. Significant changes were found between the CRF and control groups representing characteristic phenotypes of rats with CRF. Treatment with the three rhubarb extracts improved renal injury and dysfunction, either fully or partially reversed the plasma metabolites abnormalities and attenuated upregulation of pro-fibrotic proteins including TGF-ß1, α-SMA, PAI-1, CTGF, FN and collagen-1. The nephroprotective effect of ethyl acetate extract was better than other extracts. The differential metabolites were closely associated with glycerophospholipid, fatty acid and amino acid metabolisms. The results revealed a strong link between renal tubulointerstitial fibrosis and glycerophospholipid metabolism and L-carnitine metabolism in the development of CRF. Amelioration of CRF with the three rhubarb extracts was associated with the delayed development and/or reversal the disorders in key metabolites associated with adenine-induced CRF.

Detection of Gelatin Adulteration in Traditional Chinese Medicine: Analysis of Deer-Horn Glue by Rapid-Resolution Liquid Chromatography-Triple Quadrupole Mass Spectrometry.

Simultaneous identification of donkey-hide gelatin and bovine-hide gelatin in deer-horn glue was established by rapid-resolution liquid chromatography-triple quadrupole mass spectrometry. Water containing 1% NH4HCO3 was used for sample dissolution and trypsin was used for hydrolysis of the gelatins. After separation by a SB-C18 reversed-phase analytical column, collagen marker peptides were detected by mass spectrometry in positive electrospray ionization mode with multiple reaction monitoring. The method was specific, precise and reliable, and suitable for detection of adulterants derived from donkey-hide gelatin and bovine-hide gelatin in deer-horn glue.

Metabolomics insights into chronic kidney disease and modulatory effect of rhubarb against tubulointerstitial fibrosis.

Chronic kidney disease (CKD) is a major public health problem worldwide. Rhubarb has been shown to have nephroprotective and anti-fibrotic activities in patients with CKD. However, bioactive fractions and biochemical mechanism of anti-fibrotic properties of rhubarb remain unclear. Here we applied ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry together with univariate and multivariate statistical analyses to investigate the urinary metabolite profile in rats with adenine-induced CKD treated with the petroleum ether (PE)-, ethyl acetate (EA)- and n-butanol (BU)- extracts of rhubarb. Significant differences in renal function, kidney histopathology as well as metabolic profiles were observed between CKD and control rats. Changes in these parameters reflected characteristic phenotypes of CKD rats. We further identified a series of differential urinary metabolites for CKD rats, suggesting metabolic dysfunction in pathway of amino acid, purine, taurine, and choline metabolisms. Treatment with EA, BU and PE extracts of rhubarb improved renal function and histopathological abnormalities including interstitial fibrosis and inflammation, and either fully or partially reversed the abnormalities of the urinary metabolites. Among them, the nephroprotective effect of EA extract was stronger than BU and PE extracts. This work provides important mechanistic insights into the CKD and nephroprotective effects of different rhubarb extract against tubulo-interstitial fibrosis.