Multiomics profiling of the therapeutic effect of Dan-deng-tong-nao capsule on cerebral ischemia-reperfusion injury.

BACKGROUND: Stroke is a complex physiological process associated with intestinal flora dysbiosis and metabolic disorders. Dan-deng-tong-nao capsule (DDTN) is a traditional Chinese medicine used clinically to treat cerebral ischemia-reperfusion injury (CIRI) for many years. However, little is known about the effects of DDTN in the treatment of CIRI from the perspective of gut microbiota and metabolites. PURPOSE: This study aimed to investigate the regulatory roles of DDTN in endogenous metabolism and gut microbiota in CIRI rats, thus providing a basis for clinical rational drug use and discovering natural products with potential physiological activities in DDTN for the treatment of CIRI. METHODS: The chemical composition of DDTN in vitro and in vivo was investigated using ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLCHRMS), followed by target prediction using reverse molecular docking. Secondly, a biological evaluation of DDTN ameliorating neural damage in CIRI was performed at the whole animal level. Then, an integrated omics approach based on UHPLCHRMS and 16S rRNA sequencing was proposed to reveal the anti-CIRI effect and possible mechanism of DDTN. Finally, exploring the intrinsic link between changes in metabolite profiles, changes in the intestinal flora, and targets of components to reveal DDTN for the treatment of CIRI. RESULTS: A total of 112 chemical components of DDTN were identified in vitro and 10 absorbed constituents in vivo. The efficacy of DDTN in the treatment of CIRI was confirmed by alleviating cerebral infarction and neurological deficits. After the DDTN intervention, 21 and 26 metabolites were significantly altered in plasma and fecal, respectively. Based on the fecal microbiome, a total of 36 genera were enriched among the different groups. Finally, the results of the network integration analysis showed that the 10 potential active ingredients of DDTN could mediate the differential expression of 24 metabolites and 6 gut microbes by targeting 25 target proteins. CONCLUSION: This study was the first to outline the landscapes of metabolites as well as gut microbiota regulated by DDTN in CIRI rats using multi-omics data, and comprehensively revealed the systematic relationships among ingredients, targets, metabolites, and gut microbiota, thus providing new perspectives on the mechanism of DDTN in the treatment of CIRI.

Unraveling spatial metabolome of the aerial and underground parts of Scutellaria baicalensis by matrix-assisted laser desorption/ionization mass spectrometry imaging.

BACKGROUND: Scutellaria baicalensis Georgi, a traditional Chinese medicine, is clinically applied mainly as the dried root of Scutellaria baicalensis, and the aerial parts of Scutellaria baicalensis, its stems and leaves, are often consumed as "Scutellaria baicalensis tea" to clear heat, dry dampness, reduce fire and detoxify, while few comparative analyses of the spatial metabolome of the aerial and underground parts of Scutellaria baicalensis have been carried out in current research. METHODS: In this work, Matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI) was used to visualize the spatial imaging of the root, stem, and leaf of Scutellaria baicalensis at a high resolution of 10 µm, respectively, investigating the spatial distribution of the different secondary metabolites in the aerial and underground parts of Scutellaria baicalensis. RESULTS: In the present results, various metabolites, such as flavonoid glycosides, flavonoid metabolites, and phenolic acids, were systematically characterized in Scutellaria baicalensis root, stem, and leaf. Nine glycosides, 18 flavonoids, one organic acid, and four other metabolites in Scutellaria baicalensis root; nine glycosides, nine flavonoids, one organic acid in Scutellaria baicalensis stem; and seven flavonoids and seven glycosides in Scutellaria baicalensis leaf were visualized by MALDI-MSI. In the underground part of Scutellaria baicalensis, baicalein, wogonin, baicalin, wogonoside, and chrysin were widely distributed, while there was less spatial location in the aerial parts. Moreover, scutellarein, carthamidin/isocarthamidin, scutellarin, carthamidin/isocarthamidin-7-O-glucuronide had a high distribution in the aerial parts of Scutellaria baicalensis. In addition, the biosynthetic pathways involved in the biosynthesis of significant flavonoid metabolites in aerial and underground parts of Scutellaria baicalensis were successfully localized and visualized. CONCLUSIONS: MALDI-MSI offers a favorable approach for investigating the spatial distribution and effective utilization of metabolites of Scutellaria baicalensis. The detailed spatial chemical information can not only improve our understanding of the biosynthesis pathways of flavonoid metabolites, but more importantly, suggest that we need to fully exert the overall medicinal value of Scutellaria baicalensis, strengthening the reuse and development of the resources of Scutellaria baicalensis aboveground parts.

Pharmacokinetic-pharmacodynamic modeling of the active components of Shenkang injection in rats with chronic renal failure and its protective effect on damaged renal cells.

The study aimed to explore the pharmacokinetic and pharmacodynamic alterations of the active components of Shenkang injection (i.e. hydroxy saffron yellow pigment A [HSYA], tanshinol, rheum emodin, and astragaloside IV) in rats with chronic renal failure (CRF), and establish a pharmacokinetic-pharmacodynamic model (PK-PD model) in order to provide a scientific and theoretical basis for the rational clinical use of Shenkang injection. Sprague-Dawley (SD) rats were randomly divided into a normal group, model group, and Shenkang injection group. A rat model of CRF was induced by adenine gavage and then followed by drug administration via tail vein injection. Orbital blood was collected at different timepoints and the blood concentrations of the four active components were measured by UHPLC-Q-Orbitrap HRMS. Serum levels of creatinine (Scr), urea nitrogen (BUN), and uric acid (UA) were determined using an automatic biochemical analyzer. A PK-PD model was established, and DAS 3.2.6 software was used for model fitting as well as statistical analysis. TGF-ß1 was utilized to induce normal rat kidney cells to construct a renal fibrosis model to investigate the protective effect of the pharmacological components on renal fibrosis. The pharmacokinetic analysis of hydroxy saffron yellow pigment A, tanshinol, rheum emodin, and astragaloside IV based on UHPLC-Q-Orbitrap HRMS was stable. The linear regression equations for the four active components were as follows: Y = 0.031X + 0.0091 (R2  = 0.9986) for hydroxy saffron yellow pigment A, Y = 0.0389X + 0.164 (R2  = 0.9979) for tanshinol, Y = 0.0257X + 0.0146 (R2  = 0.9973) for rheum emodin, and Y = 0.0763X + 0.0139 (R2  = 0.9993) for astragaloside IV, which indicated good linear relationships. The methodological investigation was stable, with the interday and intraday precision RSD <10%. Meanwhile, the recoveries ranged between 90% and 120%, in accordance with the requirements for in vivo analysis of drugs. Compared with the model group, the levels of Scr, BUN, and UA were significantly decreased after 20 min in the Shenkang injection group (p < 0.01). The PK-PD model showed that the four active components in the Shenkang injection group could fit well with the three effect measures (i.e. Scr, BUN, and UA), with the measured values similar to the predicted values. The cell model of renal fibrosis showed that the connective tissue growth factor and FN1 protein expression levels were significantly lower in the Shenkang injection group than those in the model group, and the cell fibrosis was improved. The established method for in vivo analysis of Shenkang injection was highly specific, with good separation of the components and simple operation. The total statistical moment could well integrate the pharmacokinetic parameters of the four active components. After treatment with Shenkang injection, all indexes in the administered group improved and showed significant inhibition of renal cell fibrosis in vitro. This study could provide scientific reference ideas for the clinical rational use of traditional Chinese medicine.

Rapid qualitative profiling and quantitative analysis of Juglandis Mandshuricae Cortex and seven flavonoids by ultra-high performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry.

Juglandis Mandshuricae Cortex is the bark of Juglans mandshurica Maxim., which has been used as a folk medicine plant in China and India. In this study, an ultra-high performance liquid chromatography-quadrupole/orbitrap high-resolution mass spectrometry method was developed to clarify and quantify the chemical profiling of Juglandis Mandshuricae Cortex rapidly. A total of 113 compounds were characterized. Among them, seven flavonoids were simultaneously quantified in 15 min, including myricetin, myricetrin, taxifolin, kaempferol, quercetin, quercitrin, and naringenin. The method was validated for accuracy, precision, and the limits of detection and quantification. All calibration curves showed a good linear relationship (r > 0.9990) within test ranges. The intra- and inter-day relative standard deviations were less than 2.16%. Accuracy validation showed that the recovery was between 95.6 and 101.3% with relative standard deviation values below 2.85%. The validated method was successfully applied to determine the contents of seven flavones in Juglandis Mandshuricae Cortex from seven sources and the contents of these places were calculated respectively. This method provides a theoretical basis for further developing the medicinal value of Juglandis Mandshuricae Cortex.

Integrated UPLC-MS/MS and UHPLC-Q-orbitrap HRMS Analysis to Reveal Pharmacokinetics and Metabolism of Five Terpenoids from Alpiniae oxyphyllae Fructus in Rats.

BACKGROUND: Alpiniae oxyphyllae Fructus (AOF), a traditional Chinese medicine (TCM), is widely used in the treatment of urinary, gastrointestinal and neurologic diseases in China. Although terpenoids are the main active ingredients of AOF, there are few researches on their pharmacokinetics and metabolism. METHODS: In this study, a sensitive, rapid, accurate and novel ultra high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established to evaluate the pharmacokinetic behavior of five terpenoids (oxyphyllenodiol B, (4S*,5E,10R*)-7-oxo-tri-nor-eudesm-5-en-4ß-ol, 7-epi-teucrenone, (+)- (4R,5S,7R)-13-hydroxynootkatone, (E)-labda-12,14-dien-15(16)-olide-17-oic acid) in rats after oral administration of AOF extracts. 27 metabolic metabolites of the five terpenoids were identified by ultra high performance liquid chromatography -Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap HRMS) based on precise mass and fragment ions. RESULTS: The established pharmacokinetic analysis method showed good linearity over a wide concentration range, and the lower quantitative limit (LLOQ) ranged from 0.97 to 4.25 ng/mL. Other validation parameters were within the acceptable range. In addition, 27 metabolites were identified in plasma, urine and feces samples, and the metabolic pathways of five terpenoids were mainly focused on glucoside conjugation, dehydration, desaturation and glycine conjugation. CONCLUSION: This is the first study on the pharmacokinetics and metabolism of five terpenoids in AOF, illuminating the disposal process of terpenoids in vivo. It was expected that the results of this study would provide some references for the apprehension of the action mechanism and the further pharmacological study of five terpenoids in AOF.

[Quality control of Dandeng Tongnao Ruanjiaonang based on UPLC fingerprint combined with chemical pattern recognition].

To establish the ultra performance liquid chromatography (UPLC) fingerprint of Dandeng Tongnao Ruanjiaonang and conduct a systemic, comprehensive quality evaluation of the drug by combining with a chemical pattern recognition method. In this study, Waters UPLC ultra-high performance liquid chromatography instrument and ACQUITY UPLCHSS T3 chromatographic colum n were employed to perform the separation with acetonitrile-0.1% formic acid aqueous solution as the mobile phase for gradient elution; and the detection wavelength was set at 256 nm to establish the UPLC fingerprint of 10 batches of Dandeng Tongnao Ruanjiaonang. Then, the further quality assessment of the drug was carried out by similarity evaluation, Cluster Analysis(CA), Principal Component Analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Finally, 77 peaks were recognised as common peaks in the fingerprint, and 15 peaks of them were identified using standard references. The similarity value of these 10 batches of drugs was all above 0.960, indicating a relatively stable quality. But minor differences were still discovered between the batches of the drug by CA and PCA. Finally, 6 common peaks were recognised as the quality makers using OPLS-DA method. The analysis method established in this study was scientific, accurate, reliable and simple; fingerprint combined with chemical pattern recognition technique can be used to systematically and comprehensively evaluate the drug quality of Dandeng Tongnao Ruanjiaonang; what's more, it could also provide a reference for the quality control of traditional Chinese medicine and its preparations at the same time.

Metabolomics approach in lung tissue of septic rats and the interventional effects of Xuebijing injection using UHPLC-Q-Orbitrap-HRMS.

Sepsis is the dysregulated host response to an infection which leads to life-threatening organ dysfunction. Metabolomic profiling in bio-fluid or tissue is vital for elucidating the pathogenesis of sepsis and evaluating therapeutic effects of medication. In this study, an untargeted metabolomics approach was applied to study the metabolic changes in lung tissue of septic rats induced by cecal ligation and puncture (CLP) and investigate the treatment effects of Xubijing injection (XBJ). Metabolomics analyses were performed on ultra-high performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high-resolution accurate mass spectrometry (UHPLC-Q-Orbitrap-HRMS) together with multivariate statistical analysis. A total of 26 differential metabolites between CLP and sham-operated group were identified. The altered metabolic pathways included energy metabolism, amino metabolism, lipid metabolism, fatty acid metabolism and hormone metabolism. Among the 26-varied metabolites, 15 were significantly regulated after XBJ treatment. The metabolic pathway network of sepsis was drawn to interpret the pathological feature of lung damage caused by sepsis and the underlying regulating mechanism of XBJ on the molecular levels. Our findings display that LC-MS-based metabolomics is a useful tool for uncovering the underlying molecular mechanism of sepsis, and XBJ may exert therapeutic effect by regulating multiple metabolic pathways.

A homologues prediction strategy for comprehensive screening and characterization of C21 steroids from Xiao-ai-ping injection by using ultra high performance liquid chromatography coupled with high resolution hybrid quadrupole-orbitrap mass spectrometry.

Because of the complicated chemical composition of Traditional Chinese Medicines, their chemical profile study has been a great challenge. In the present work, a homologues prediction strategy for rapid screening and identification of C21 steroids in Xiao-ai-ping injection was developed by using an ultra high performance liquid chromatography coupled with high resolution hybrid quadrupole-orbitrap mass spectrometry. This strategy was characterized by the design of C21 steroidal skeleton, substituent group and glycan chain in an orderly way, which could quickly and efficiently screen the interested precursor ions. As a result, a total of 95C21 steroids including 47 potential new ones were identified or tentatively identified, which greatly expanded our knowledge of C21 steroids in Xiao-ai-ping injection. The results indicated that the homologues prediction strategy not only provided an efficient technique to screen and identify target constituents, but also offered a new perspective for discovery new components in Traditional Chinese Medicines.

Chemical profiling and quantification of Dan-Deng-Tong-Nao-capsule using ultra high performance liquid chromatography coupled with high resolution hybrid quadruple-orbitrap mass spectrometry.

Dan-Deng-Tong-Nao capsule (DDTN) was a traditional Chinese medicine (TCM) formula, and has been widely used for the treatment of stroke clinically which caused by blood stasis. However, the bioactive substances and mechanism are unclear because of the complex compositions in DDTN. In this research, An ultra high-performance liquid chromatography (UHPLC) coupled with hybrid quadruple-orbitrap mass spectrometry (Q-Orbitrap MS) method was utilized to identify the chemical constituents of DDTN. In total, 102 compounds including diterpenes, lactones, flavonoids, and phenolic acids were identified by the accurate masses and fragmentation pathways, and 18 of them were unambiguously determined by comparison of reference standards. Besides, 12 representative compounds were simultaneously quantification analyzed and successfully applified for detecting in 9 batches of DDTN samples by UHPLC-Q-Orbitrap MS in parallel reaction monitoring (PRM) mode. The proposed approach was validated to be satisfied in terms of linearity (0.9954-0.9999), LOD (0.771ng/mL), LOQ (2.568ng/mL), intra-day precision ( <2.68%), inter-day precision ( <4.52%), repeatability ( <2.96%), stability ( <3.21%), and recovery (94.6-105.5%). The results indicate that the method of combining UHPLC with Q-Orbitrap MS is practical and efficient for the chemical clarification in DDTN, and has great potential for the integrating quality control of other traditional Chinese medicines.

Chemical profiling and quantification of ShenKang injection, a systematic quality control strategy using ultra high performance liquid chromatography with Q Exactive hybrid quadrupole orbitrap high-resolution accurate mass spectrometry.

ShenKang injection is traditional Chinese medicine used to treat chronic renal failure in China. It is a compound preparation that consists of four herbs: Rhubarb, Salvia miltiorrhiza, Safflower and Radix Astragali. We developed an ultra high pressure liquid chromatography coupled with Q Exactive hybrid quadrupole-orbitrap high resolution accurate mass spectrometry tandem mass spectrometry method to analyze its chemical compositions, and a total of 90 compounds were identified from ShenKang injection. Among them, 19 major compounds were unambiguously detected by comparing with reference standards. Meanwhile, 13 representative compounds selected as quality control markers were simultaneously quantified in ShenKang injection samples. Chromatographic separation was accomplished on a Waters ACQUITY HPLC® HSS C18 column using gradient elution. The method was validated with respect to linearity, sensitivity, accuracy and precision, reproducibility and stability. And the validated method was successfully applied for simultaneous determination of 13 bioactive compounds in ShenKang injection from ten batches of samples by liquid chromatography with mass spectrometry. The results were analyzed by principal components analysis method, and three compounds had a significant relationship with the quality control of ShenKang injection. This research established a rapid and reliable method for the integrating quality control, including qualitation and quantification of ShenKang injection.