Jinhua Tao

Simultaneous determination of loganin, morroniside, catalpol and acteoside in normal and chronic kidney disease rat plasma by UPLC-MS for investigating the pharmacokinetics of Rehmannia glutinosa and Cornus officinalis Sieb drug pair extract.

A sensitive and rapid method for determination of loganin, morroniside, catalpol and acteoside in rat plasma after oral administration of Rehmannia glutinosa Libosch and Cornus officinalis Sieb drug pair based on ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS). Chromatographic separation was achieved using an Acquity UPLC BEH C18 column (100mm×2.1mm, 1.7μm) at a flow rate of 0.4mL/min, using gradient mode containing 0.1% formic acid in water and acetonitrile were used as the mobile phase A and B. Loganin, morroniside, catalpol, acteoside and the internal standard (chloramphenicol) were detected by selected reaction monitoring in the negative ion mode with the mass transition of m/z 451.0→179.0 (morroniside), m/z 435.0→227.0 (loganin), m/z 407.1→199.1 (catalpol), m/z 623.2→161.0 (acteoside) and m/z 320.8→151.9 (chloramphenicol), respectively. All calibration curves showed good linearity (r>0.991). The precision was evaluated by intra-day and inter-day assays and the RSD% were all within 9.58%. The recovery ranged from 67.62 to 80.14%. The method was successfully applied to pharmacokinetic study of the analytes in normal and doxorubicin-induced chronic kidney disease rat plasma.

Comparative pharmacokinetics of the main compounds of Shanzhuyu extract after oral administration in normal and chronic kidney disease rats.

ETHNOPHARMACOLOGICAL RELEVANCEnPharmacokinetic studies on traditional Chinese medicine are useful to evaluate and predict the drug efficacy and safety. The renal impairment may affect drug clearance and other pharmacokinetic processes which can increase toxicity and drug to drug interactions or cause ineffective therapy. Pharmacokinetic studies in pathological status rats might be meaningful for revealing the action mechanism and improving clinical medication of the herb medicine.nnnMATERIALS AND METHODSnA highly sensitive and rapid ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method with multiple-reaction monitoring (MRM) mode was developed and validated for simultaneous quantitation of morroniside and loganin in normal and doxorubicin-induced chronic kidney disease (CKD) rat plasma after oral administration of Shanzhuyu (fruit of Cornus officinalis) extract.nnnRESULTSnBoth calibration curves gave satisfactory linearity (r>0.99) at linear range of 1.96-1962.5ngmL(-1) for morroniside, 1.53-1531.25ngmL(-1) for loganin. The precision and accuracy of the in vivo study were assessed by intra-day and inter-day assays. The percentages of relative standard deviation (RSD) were all within 9.58% and the accuracy (RE) was in the -6.02% to 8.11% range. The extraction recoveries of morroniside, loganin and internal standard (IS) were all >67.62% and the matrix effects ranged from 95.07% to 102.75%.nnnCONCLUSIONSnThe pharmacokinetic behavior of morroniside and loganin in normal and CKD rat plasma was determined in this paper. The significant different pharmacokinetic parameters might partly result from the changes of P-glycoprotein and metabolic enzymes in the pathological state. The pharmacokinetic research in the pathological state might provide more useful information to guide the clinical usage of the herb medicine.

Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for rapid analysis of the metabolites of morroniside produced by human intestinal bacteria

Morroniside, the most abundant iridoid glycoside in the valuable traditional Chinese medicine Fructus Corni, exhibits various pharmacological activities and biological effects. Intestinal flora plays an important role in the metabolism of drug compounds, which might lead to the variation of ethnopharmacological profile of the medicine. However, little is known of the interactions of the morroniside with human intestinal bacteria. In this study, different pure bacteria were isolated from human feces and their capability to convert morroniside were investigated. The metabolites of morroniside were analyzed by ultra high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) technique using Metabolynx™ software. Parent compound and three metabolites were detected and tentatively identified based on the characteristics of their protonated ions. The parent is proposed to be metabolized by three main metabolic pathways including deglycosylation, dehydroxylation and methylation. Morroniside was firstly metabolized to its aglycone (M1), and then was further converted to dehydroxylated aglycone (M2) and methylated aglycone (M3). This is the first report of the metabolism of morroniside by human intestinal bacteria. These metabolites might influence the biological activities of morroniside in vivo, which could affect the clinical effects of medicines. Thus, the study on the metabolism of morroniside by human intestinal bacteria is very helpful to unravel how traditional medicines work.

Comparative pharmacokinetics of catalpol and acteoside in normal and chronic kidney disease rats after oral administration of Rehmannia glutinosa extract

In this study, a sensitive and robust ultra-performance liquid chromatography-mass spectrometry method with multiple-reaction monitoring mode was developed, validated, and applied to determine pharmacokinetics of catalpol and acteoside in normal and doxorubicin-induced chronic kidney disease rats after oral administration of Rehmannia glutinosa extract. The lower limits of quantification for catalpol and acteoside in rat plasma were 2.62 and 0.61 ng/mL, with a signal-to-noise ratio of ≥10. Precision and accuracy studies showed that catalpol and acteoside plasma concentrations were within the 10% range in all studies. The extraction recoveries of catalpol and acteoside were both >68.24% and the matrix effects ranged from 96.59 to 101.62%. The method was successfully applied to the pharmacokinetic study of catalpol and acteoside after oral administration of RG extract to normal and model rats, respectively. This study might further support the traditional use of RG to treat kidney diseases clinically.

Biodiversity and Antimicrobial Activity of Endophytic Fungi in Angelica sinensis

Abstract Objective To systematically investigate the biodiversity, ecological distribution, and antimicrobial activities of the endophytic fungi in Angelica sinensis growing in several natural habitats in China. Methods The isolation, culture, and identification of microorganism and mycelium growth inhibition test were adopted, and the relative data were analyzed by the statistical methods. Results A total of 206 isolates of endophytic fungi representing 22 species were collected at different time periods from A. sinensis in three locations. Melanconiaceae (45.1%) was the most prevalent followed by Dematiaceae (34.0%), mycelia sterilia (17.0%), Tuberculariaceae (2.9%), Moniliaceae (0.5%), and Leptostromataceae (0.5%). The main genera were Gongromella Ribaldi, Coniosporieae Lk. ex Fr., Fusella Sacc., Myxormia B. ex Br., Ozonium Lk. ex Fr., Pestalotia de Not., Phacodium Pers. ex Wallr., and Sphaceloma de Barry. Endophytes from the samples of Min county showed more diversity, percentage colonization, and species richness compared to other two locations. Endophytic colonization frequency was also greater in Min county (56.5%) than those in Heqing county (29.5%) and Baoxing county (17.0%). As to endophytic community in different plant tissues, the maximum endophytes species richness and diversity appeared in root tissues rather than in stem and leaf tissues from each location. Leaf samples were colonized by greater numbers of endophytes relative to the stem and root samples of the same location. Antimicrobial assay of the 206 endophytes showed that 20% was capable of inhibiting plant pathogenic fungi and eight strains displayed strong inhibition. Conclusion Endophytic fungi isolated from A. sinensis are specific in location, tissue, and season, and diverse in species. They are potential sources of antimicrobial agents which might improve the stress resistance and growth of this medicinal plant and play the important biological functions in ecosystem. Therefore, endophytic fungi will explore a new way to control plant diseases and develop healthful Chinese crude drugs.

Dynamic changes of flavonoids in Abelmoschus manihot different organs at different growth periods by UPLC–MS/MS

Abelmoschus manihot (Linn.) Medicus has been clinically used to treat chronic kidney disease, oral ulcers, burns, and dysmenorrhea in China for many centuries. The major pharmacologically-active components of A. manihot are flavonoids. In this study, a rapid and highly sensitive UPLC-MS/MS analysis method was established and successfully applied to the simultaneous determination of five major flavonoids (rutin, hyperoside, isoquercitrin, quercetin, and myricetin) in different parts of A. manihot harvested at ten growth periods. Under the optimized chromatographic conditions, good separation for five target components was obtained on an Acquity UPLC BEH C18 column within 18min. The total contents of the five investigated flavonoids in A. manihot roots, stems, leaves and flowers ranged from 2.86 to 123.7μg/g, 46.39 to 141.0μg/g, 929.4 to 3096μg/g, and 10,150 to 19,390μg/g, respectively, indicating that the total flavonoids in the four parts could be mainly arranged in a decreasing order as flower>leaf>stem>root. The peak of total flavonoids in flowers and leaves appeared at G8 and G9, respectively. These results will be helpful for the determination of the suitable harvest time of A. manihot and the improvement of the utility value of the disused parts.

Xiexin Tang improves the symptom of type 2 diabetic rats by modulation of the gut microbiota

Type 2 diabetes mellitus (T2DM), a chronic metabolic disease which severely impairs peoples’ quality of life, currently attracted worldwide concerns. There are growing evidences that gut microbiota can exert a great impact on the development of T2DM. Xiexin Tang (XXT), a traditional Chinese medicine prescription, has been clinically used to treat diabetes for thousands of years. However, few researches are investigated on the modulation of gut microbiota community by XXT which will be very helpful to unravel how it works. In this study, bacterial communities were analyzed based on high-throughput 16S rRNA gene sequencing. Results indicated that XXT could notably shape the gut microbiota. T2DM rats treated with XXT exhibited obvious changes in the composition of the gut microbiota, especially for some short chain fatty acids producing and anti-inflammatory bacteria such as Adlercreutzia, Alloprevotella, Barnesiella, [Eubacterium] Ventriosum group, Blautia, Lachnospiraceae UCG-001, Papillibacter and Prevotellaceae NK3B31 group. Additionally, XXT could also significantly ameliorate hyperglycemia, lipid metabolism dysfunction and inflammation in T2DM rats. Moreover, the correlation analysis illustrated that the key microbiota had a close relationship with the T2DM related indexes. The results probably provided useful information for further investigation on its active mechanism and clinical application.

Characterization and immunomodulatory activity of polysaccharides from the stems and leaves of Abelmoschus manihot and a sulfated derivative

BACKGROUNDnAbelmoschus manihot (Linn.) Medicus is a traditional herbal medicine whose flowers, stems and leaves exhibit widely pharmacological activities. However, only the flowers have long been used as medicine while the stems and leaves were mainly discarded and burned, which undoubtedly caused enormous waste of these resources and serious environment pollution. Many researches have indicated that bioactivities of polysaccharides were significantly improved after sulfation. The aim of this study was to investigate the characterization and immunomodulatory activity of polysaccharides from stems and leaves of A. manihot and a sulfated derivative.nnnRESULTSnA mixed neutral polysaccharide (SLAMP-a) and two acidic polysaccharides (SLAMP-c and SLAMP-d) were obtained from stems and leaves of A. manihot by DEAE-cellulose chromatography. SLAMP-a was a water-insoluble mixture while its sulfated derivative (S-SLAMP-a3), prepared with aminosulfonic acid, was a homogeneous polysaccharide with excellent solubility. The average molecular weights of S-SLAMP-a3, SLAMP-c and SLAMP-d were 1044.2kDa, 477.8kDa and 264.2kDa respectively. SLAMP-a and its sulfate mainly contained glucose, and SLAMP-c and SLAMP-d were both composed of mannose, rhamnose, glucuronic acid, glucose, galactose, and arabinose. In vitro study indicated that S-SLAMP-a3, SLAMP-c and SLAMP-d exhibited significant immunomodulatory activity, while SLAMP-a showed little effects.nnnCONCLUSIONnSLAMP-c and SLAMP-d from A. manihot stems and leaves could be explored as immunomodulatory agents, which would provide a way to utilize these enormously discarded resources and avoid massive waste. Additionally, the neutral polysaccharide, SLAMP-a, could also be developed after sulfation, suggesting that these disused resources would be further used effectively.

Metabolic profiles of the Flos Abelmoschus manihot extract by intestinal bacteria from the normal and CKD model rats based on UPLC‐Q‐TOF/MS

Flos Abelmoschus manihot is a traditional herbal medicine widely used in clinical practice to tackle chronic kidney disease (CKD) for thousands of years. Nowadays, many studies indicate that gut bacteria are closely related to the progression of CKD and CKD-related complications. In this study, a UPLC-Q-TOF/MS method coupled with the MetaboLynx™ software was established and successfully applied to investigate the metabolites and metabolic profile of Flos A. manihot extract by intestinal bacteria from normal and CKD rats. Eight parent components and eight metabolites were characterized by their protonated ions. Among these compounds, 15 were detected in the two group samples while M16 was only determined in the CKD model samples. Compared with the quercetin-type glycosides, fewer myricetin-type and gossypetin-type metabolites were obtained in the two group samples. These metabolites suggested that deglycosylation and methylation are the major metabolic pathways of Flos A. manihot extract. Few differences of metabolite classes were observed in the two group samples. However, the concentrations of aglycones such as quercetin, myricetin and gossypetin in the normal samples were notably higher than those in the CKD model samples. The results are important in unravelling the pharmacological effects of A. manihot and clarifying its mechanism of action in vivo.

Metabolites of Rehmannia glutinosa Libosch extract by intestinal bacteria from normal and chronic kidney disease rats in vitro

Rehmannia glutinosa Libosch is a traditional Chinese medicine clinically applied to treat kidney disease for thousands of years. However, the interaction between the herb medicine and the gut microbiome remains unknown. In this study, a rapid automated analysis method, ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q/TOF MS) technique combined with Metabolynx™ software, was first established and successfully applied for screening and identification of the metabolites of the main bioactive components in Rehmannia glutinosa extract by intestinal bacteria from normal and chronic kidney disease (CKD) rat feces. When compared with blank samples, the two parent compounds (catalpol and acteoside) plus six metabolites were detected in the two groups of bacterial samples. Catalpol was first deglycosylated to its aglycone and subsequently to hydrogenated catalpol aglycone. Acteoside was converted to the De-caffeic acid moieties acteoside, hydroxytyrosol, and considerable amounts of caffeic acid. The latter was methylated to the corresponding caffeic acid methyl ester. Caffeic acid and its methylated product were the main metabolites. However, compared with the model group, intestinal bacteria from normal rats showed stronger metabolism capability and larger amounts of the metabolites were detected. These results will be helpful for the further investigation of the pharmacokinetic study of Rehmannia glutinosa Libosch in vivo.