Pharmacokinetics-based comprehensive strategy to identify multiple effective components in Huangqi decoction against liver fibrosis

BackgroundHuangqi decoction (HQD) has been used to treat chronic liver diseases since the 11th century, but the effective components in HQD against liver fibrosis have not been definitively clarified.PurposeTo investigate and identify multiple effective components in HQD against liver fibrosis using a pharmacokinetics-based comprehensive strategy.MethodsThe absorbed representative components in HQD and their metabolites were detected in human plasma and urine using high-resolution mass spectrometry combined with a database-directed method, and then pharmacokinetics in multiple HQD components in human plasma was analyzed by ultra-performance liquid chromatography coupled with triple-quadruple mass spectrometry. Furthermore, the anti-fibrotic effect of potential effective HQD components was studied in LX-2 cells and that of a multi-component combination of HQD (MCHD) was verified in a mouse CCl₄-induced hepatic fibrosis model.ResultsTwenty-four prototype components in HQD and 17 metabolites were identified in humans, and the pharmacokinetic characteristics of 14 components were elucidated. Among these components, astragaloside IV, cycloastragenol, glycyrrhizic acid, glycyrrhetinic acid, liquiritigenin, and isoliquiritigenin downregulated the mRNA expression of α-SMA; cycloastragenol, calycosin-7-O-β-D-glucoside, formononetin, glycyrrhetinic acid, liquiritin, and isoliquiritin downregulated the mRNA expression of Col I; and calycosin, liquiritigenin, isoliquiritigenin, cycloastragenol, and glycyrrhetinic accelerated the apoptosis of LX-2 cells. MCHD reduced serum aminotransferase activity and hepatic collagen fibril deposition in mice with CCl₄-induced hepatic fibrosis.ConclusionUsing the pharmacokinetics-based comprehensive strategy, we revealed that multiple effective HQD components act together against liver fibrosis.     

Simultaneous Determination of Calycosin-7-O-β-d-Glucoside,Ononin, Calycosin,Formononetin, Astragaloside IV,and Astragaloside II in Rat Plasma After Oral Administration of Radix Astragali Extraction for Their Pharmacokinetic Studies by Ultra-Pressure Liquid Chromatography with Tandem Mass Spectrometry

A sensitive and reliable ultra-pressure liquid chromatography with tandem mass spectrometry (UPLC–MS) was developed and validated for simultaneous quantification of six main bioactive components, i.e., calycosin-7-O-β-d-glucoside, ononin, calycosin, formononetin, astragaloside IV, and astragaloside II in rat plasma after oral administration of the 95 % ethanol extraction from Radix Astragali. Plasma samples were extracted with Waters Oasis^TM HLB 1 cc (30 mg) Extraction Cartridges (SPE) separated on an UPLC? BEH C18 column and detected by MS with electro spray ionization interface in positive selective ion monitoring mode. Calibration curves offered linear ranges of two orders of magnitude with r ² > 0.99. The method had the lower limit quantification of 1.30, 0.73, 1.17, 2.33, 0.63, and 0.83 ng/mL for ononin, calycosin, calycosin-7-O-β-d-glucoside, formononetin, astragaloside IV, and astragaloside II, respectively, with precision less than 10 %. The RSD of intra- and inter-day variations ranged from 1.66 to 6.46 and 3.39 to 6.58 %. This developed method was applied subsequently to pharmacokinetic studies of the six compounds in rats successfully. The proposed method was for the first time to compare the pharmacokinetic difference between calycosin-7-O-β-d-glucoside and calycosin in rat plasma, so as between ononin and formononetin, and studied to the astragaloside II pharmacokinetics in rat plasma.     

HPLC-DAD-ELSD Combined Pharmacodynamics and Serum Medicinal Chemistry for Quality Assessment of Huangqi Granule

Objective

To more scientifically and reasonably control the quality of Huangqi Granules, preliminary studies on the pharmacodynamics and serum pharmacochemistry of this medicine were performed. DPPH and MTT experiments showed that water extracts of Huangqi Granules had good antioxidant activity and increased immunity. Timed blood samples collected 5 min, 15 min, and 30 min after oral administration of a set amount of Huangqi Granules were collected and tested using UPLC-ESI-MS/MS. As a result, calycosin-7-O-β-D-glucoside, ononin, calycosin, astragaloside IV, and formononetin were found to exist in rat blood after dosing, indicating that the five chemical compounds might have pharmacological activity, and based on this result, they were designated biomarkers for quality control of Huangqi Granules. Consequently, a simple, rapid and efficient method was developed in the present study for the simultaneous determination of the five characteristic compounds in Huangqi Granules using HPLC-DAD-ELSD.

Materials and Methods

The separation was performed using an Agilent Hypersil ODS column (4.6 × 250 mm, 5 μm) at 30 ℃. The mobile phase was composed of water (solvent A) and acetonitrile (solvent B) with a flow rate of 1 mL/min. The drift tube temperature of the ELSD system was set to 85 ℃, and the nitrogen pressure was 3.5 bar.

Results

All five characteristic compounds had good linear behavior with r2 values greater than 0.9972. The recoveries varied from 96.31% to 101.22%. Subsequently, the developed method was applied to evaluate the quality of Huangqi Granules from different batches, and hierarchical clustering analysis (HCA) was used to analyze the classification of the samples based on the values of the five compounds.

Conclusion

The established HPLC method combined with HCA proved to be effective to evaluate the quality of Huangqi Granules.     

Purification and Identification of Flavonoids from the Yellow Green Cocoon Shell (Sasamayu) of the Silkworm,Bombyx mori

Three quercetin glycosides, quercetin 5-O-β-D-glucoside, quercetin 7-O-β-D-glucoside, and quercetin 4′-O-β-D-glucoside, and two kaempferol glycosides, kaempferol 5-O-β-D-glucoside and kaempferol 7-O-β-D-glucoside, along with their aglycones, quercetin and kaempferol, were isolated from an ethanolic extract of Sasamayu cocoon shells. The chemical structures were characterized by chemical and spectroscopic methods including UV spectrometry and HPLC-ESI-MS. The five flavonol glycosides of the shell are different structurally from those of the leaves of mulberry (Morus alba). It was suggested that potent antioxidative activity in the cocoon is mainly due to flavonoid compounds since free radical scavenging activity was found in the cocoon flavonoids identified here.     

A multi-strategy platform for quality control and Q-markers screen of Chaiqin chengqi decoction

BackgroundAcute pancreatitis (AP) is an inflammatory disorder of the pancreas that is associated with substantial morbidity and mortality. Chaiqin chengqi decoction (CQCQD) has been proven clinically to be an effective treatment for AP for decades in West China Hospital. Quality control for CQCQD containing many hundreds of characteristic phytochemicals poses a challenge for developing robust quality assessment metrics.PurposeTo evaluate quality consistency of CQCQD with a multi-strategy based analytical method, identify potential quality-markers (Q-markers) based on drug properties and effect characteristics, and endeavor to establish CQCQD as a globally-accepted medicine.MethodsA typical analysis of constitutive medicinal plant materials was performed following the Chinese Pharmacopoeia. The extraction process was optimized through an orthogonal array (L₉(3⁴)) to evaluate three levels of liquid to solid ratio, soaking time, duration of extraction, and the number of extractions. An ultra-high-performance liquid chromatography (UHPLC) fingerprinting combined with absolute quantitation of multi chemical marker compounds, coupled with similarity, hierarchical clustering analysis (HCA), and principal component analyses (PCA) were performed to evaluate 10 batches of CQCQD. On the basis of systematic analysis of fundamental features of CQCQD in treating AP, the potential Q-marker screen was proposed through detection of quality transfer and efficacy for chemical markers. UHPLC coupled with quadrupole orbitrap mass spectrometry were used to determine compounds in medicinal materials, decoctions and plasma. Network pharmacology and taurolithocholic acid 3-sulfate induced pancreatic acinar cell death were used to evaluate the correlation between chemical markers and anti-pancreatitis activity. A cerulein induced AP murine model was used to validate quality assessed CQCQD batches at clinically-equivalent dose. The effective content of chemical markers was predicted using linear regression analysis on quantitative information between validated batches and the other batches.ResultsThe chemical markers and other physical and chemical indices in the original materials met Chinese Pharmacopoeia standards. A total of 22 co-existing fingerprint peaks were selected and the similarity varied between 0.946 and 0.990. Batch D10 possessed the highest similarity index. HCA classified the 10 batches into 2 main groups: 7 batches represented by D10 and 3 batches represented by D1. During the initial Q-marker screen stage, 22 compounds were detected in both plant materials and decoctions, while 13 compounds were identified in plasma. Network pharmacology predicted the potential targets and pathway of AP related to the 22 compounds. All 10 batches showed reduced necrosis below 60% with the best effect achieved by D10 (~40%). The spectrum-efficacy relationship analyzed by Pearson correlation analysis indicated that emodin, rhein, aloe emodin, geniposide, hesperridin, chrysin, syringin, synephrine, geniposidic acid, magnolol, physcion, sinensetin, and baicalein showed positive correlation with pancreatic acinar cell death protection. Similar to the in vitro evaluation, batch D10 significantly reduced total histopathological scores and biochemical severity indices at a clinically-equivalent dose but batch D1 did not. The content of naringin, narirutin and baicalin in batches D1, D5 and D9 consistently exceeds the upper limit of the predicted value. Eight markers whose lower limit is predicted to be close to 0 contributed less to the material basis for AP protection.ConclusionDespite qualified materials used for CQCQD preparation, the clinical effect depends on appropriate content range of Q-markers. Emodin, rhein, aloe emodin, magnolol, hesperidin, synephrine, baicalein, and geniposide are considered as vital Q-markers in the primary screen. This study proposed a feasible platform for producing highly consistent batches of CQCQD in future study.     

Core Bioactive Components Promoting Blood Circulation in the Traditional Chinese Medicine Compound Xueshuantong Capsule (CXC) Based on the Relevance Analysis between Chemical HPLC Fingerprint and In Vivo Biological Effects

Compound xueshuantong capsule (CXC) is an oral traditional Chinese herbal formula (CHF) comprised of Panax notoginseng (PN), Radix astragali (RA), Salvia miltiorrhizae (SM), and Radix scrophulariaceae (RS). The present investigation was designed to explore the core bioactive components promoting blood circulation in CXC using high-performance liquid chromatography (HPLC) and animal studies. CXC samples were prepared with different proportions of the 4 herbs according to a four-factor, nine-level uniform design. CXC samples were assessed with HPLC, which identified 21 components. For the animal experiments, rats were soaked in ice water during the time interval between two adrenaline hydrochloride injections to reduce blood circulation. We assessed whole-blood viscosity (WBV), erythrocyte aggregation and red corpuscle electrophoresis indices (EAI and RCEI, respectively), plasma viscosity (PV), maximum platelet aggregation rate (MPAR), activated partial thromboplastin time (APTT), and prothrombin time (PT). Based on the hypothesis that CXC sample effects varied with differences in components, we performed grey relational analysis (GRA), principal component analysis (PCA), ridge regression (RR), and radial basis function (RBF) to evaluate the contribution of each identified component. Our results indicate that panaxytriol, ginsenoside Rb₁, angoroside C, protocatechualdehyde, ginsenoside Rd, and calycosin-7-O-β-D-glucoside are the core bioactive components, and that they might play different roles in the alleviation of circulation dysfunction. Panaxytriol and ginsenoside Rb₁ had close relevance to red blood cell (RBC) aggregation, angoroside C was related to platelet aggregation, protocatechualdehyde was involved in intrinsic clotting activity, ginsenoside Rd affected RBC deformability and plasma proteins, and calycosin-7-O-β-D-glucoside influenced extrinsic clotting activity. This study indicates that angoroside C, calycosin-7-O-β-D-glucoside, panaxytriol, and protocatechualdehyde may have novel therapeutic uses.     

Thrombin-based discovery strategy of bioactive-chemical quality marker combination for pollen of Typha orientalis by metabolomics coupled with chemometrics

BackgroundIt is of utmost significance to choose the bioactive components as quality markers for ensuring the effectiveness of traditional Chinese medicine (TCM). Nonetheless, some markers are able to assess effectively the quality of TCM without considering the pharmacological mechanisms and intrinsic chemical complexities.ObjectiveThis underscores the need to discover new and efficient markers which can assess both quality and mechanism of action. Herein, a strategy of bioactive-chemical quality marker combination was proposed to improve the level of the quality control of TCM by metabolomics coupled with chemometrics.MethodsA four-step plan was followed. Firstly, acquisition of metabolic features and component characterization of different batches of pollen of Typha orientalis C.Presl were performed using UHPLC-Q-TOF/MS. Secondly, the direct inhibitory effects of pollen of T. orientalis on thrombin was assessed by using chromogenic substrate method together with HPLC. Thereafter, bioactive-chemical marker combination associated with anti-thrombin segregation was screened using supervised classifiers. Finally, quantitative assay and prediction-model of selected markers were established for guarantying the quality of pollen of T. orientalis.ResultsA total of 22 compounds were annotated based on comparison with previous work from pollen of T. orientalis by UHPLC-Q-TOF/MS. Citric acid and linolenic acid inhibited the thrombin activity with IC₅₀ values, 0.52 ± 0.02 and 0.51 ± 0.02 mg/mL, respectively. A bioactive-chemical marker combination including citric acid, linolenic acid, typhaneoside, and isorhamnetin-3-O-neohesperidoside were discovered and selected as quality markers for evaluation of pollen of T. orientalis according to their capacity for inhibiting thrombin.ConclusionThe thrombin-based discovery strategy of bioactive-chemical marker combination was a powerful tool for screening the quality markers for evaluation of pollen of T. orientalis.     

Polyphenol Components in Cultured Cells of Amacha (Hydrangea macrophylla Seringe var. Thunbergii Makino)

In the course of our studies on the polyphenol components of the cultured cells of amacha, seven polyphenol compounds were isolated as pure crystals. The chemical structures of these compounds were determined by analytical methods (IR, UV, NMR and MS spectra) and colour reactions, and the compounds were identified as phyllodulcin, hydrangenol, daphnetin-8-monomethylether, umbelliferone, phyllodulcin-8-β-D-glucoside, hydrangenol-8-β-D-glucoside and skiminin (umbelliferone-7-β-D-glucoside). This is the first report of the isolation of phyllodulcin-8-β-D-glucoside and daphnetin-8-monomethylether from a natural source and of the other compounds from the cells of higher plants in suspension culture.     

Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O-β-D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

Metabolomics-based optimal koji fermentation for tyrosinase inhibition supplemented with Astragalus radix

The present study was focused on improving the quality of rice koji by fermentation with a selected Aspergillus oryzae strain and a plant Astragalus radix. A. oryzae KCCM 60345 was used as main inoculant and the Astragalus radix was added as supplement in rice koji preparation. LC-MS based metabolite analysis and tyrosinase inhibitory activities were studied for different time periods. A. oryzae KCCM 60345 fermented rice koji supplemented with Astragalus showed higher tyrosinase inhibition activity at 4 d of fermentation and metabolite analysis with PCA and PLS-DA indicated differences in kojic acid, calycosin-7-O-β-D-glucoside, ononin, calycosin, and formononetin as compared with other forms of rice koji fermentation. By correlation analysis between metabolites and tyrosinase inhibitory activity, calycosin and kojic acid were identified as major tyrosinase inhibitors. Based on these results, we concluded that A. oryzae KCCM 60345 supplemented with Astragalus radix is useful for whitening effects, and we identified optimal conditions for rice koji preparation.     

Studies on the Chemical Constituents of Elsholtzia eriostachya Benth.

Sixteen constituents were isolated from the hot alcohoic extract of the whole plant of a Chinese medicinal herb Elsholtzia eriostachya Benth.. Twelve of them were elucidated by spectroscopic and chemical evidences as follows: dotriacontanoic acid （l）, β-sitosterol（2）, succinic acid （3）, aretigenin (5), 3-hydroxyarctiin （7）, luteolin-5-O-β-D-glucoside （8）, luteolin-7-O-β-D-glucoside (9), gentisic acid -5-O-β-D-glucopranoside (10), hyperoside (11), morin-7-O-β-D-glucopranoside （12）, isoskuranetin-7-O-β-D-neohesperidoside （14）, acacetin-7-O-β-D-rutinoside （15） 7 is a new compound, and eight compounds (3, 5, 8, 10,11,12,14 and 15) were isolated from Elsholtzia for the first time.     

Analysis of a Major Phenolic Glucoside and Biochemistry Compounds from Curculigo Orchioides Gaertn

Background:Orcinol-β-D-glucoside, which is also known as orcinol glucoside, is a major phenolic glucoside compound in the rhizome of the Curculigo orchioides Gaertn. This compound has many medicinal properties such as being antioxidant, immunomodulatory, antiosteoporosis, stress relief, antidepressant, etc.Methods:Determination of reducing sugar content by Bertrand’s method, determination of lipid content by Soxhlet method, determination of vitamin C content by iodine titration, determination of enzyme activity catalase by titration with KMnO4. Quantification of Orcinol-β-D-glucoside was conducted by HPLC analysis.Results:The Orcinol-β-D-glucoside of C. orchioides in Thuy Bang mountain was highest. Besides, the content of reducing sugars, vitamin C, enzyme catalase, and lipids of C. orchioides differed significantly among sites. In which, the reducing sugar and vitamin C of C. orchioides in Ngu Binh mountain was highest. Whereas, enzyme catalase was also highest in Thuy Bang mountain. However, the lipid content of C. orchioides was also highest in Huong Tho mountain.Conclusion:The result will contribute to providing the scientific basis for the selection of breeding, planting, development of C. orchioides in Thua Thien Hue province, as well as other provinces in Vietnam and opening new research directions for applications in the future.Key Words: Antioxidants, Hypoxidaceae, Medicinal Plants, Vietnam     

Distribution of Gastrointestinal Parasitic Infection in Domestic Pigs in the Republic of Korea: Nationwide Survey from 2020–2021

This study aimed to examine the distribution of gastrointestinal parasitic infections in domestic pigs in the Republic of Korea. From May 2020 to October 2021, 364 pig fecal samples were collected from 75 farms in 7 Provinces and microscopically examined. A total of 170 (46.7%) pigs were infected with at least one of the following parasites: Balantioides coli, strongyles, Ascaris suum, Trichuris suis, and coccidia. By parasite species, B. coli, strongyles, A. suum, T. suis, and coccidia oocysts or eggs were detected in 144 (39.6%), 24 (6.6%), 14 (3.8%), 4 (1.1%), and 1 (0.3%) samples, respectively. One hundred fifty-four, 15, and 1 cases showed single, double, and triple infections, respectively. Of the swine fecal samples from 75 farms, 69 specimens (92.0%) were infected with 1 or more parasites. All surveyed farms across the country exhibited a positive rate of over 30%, among which the highest positive rate was 65.0% in Chungcheongnam-do, and Jeollabuk-do was followed by 61.9%. Winter showed a statistically lower prevalence than other seasons. This study showed that gastrointestinal parasites are prevalent in pigs in Korea, although the diversity of parasites is low.     

Molecular-networking-guided discovery of species-specific markers for discriminating five medicinal Paris herbs

BackgroundParidis Rhizoma (PR) is a famous traditional herbal medicine. Apart from two officially recorded species, viz. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. (PPY) and P. polyphylla Smith var. chinensis (Franch.) Hara (PPC), there are still many other species used as folk medicine. It is necessary to understand the metabolic differences among Paris species.PurposeTo establish a strategy that can discover species-specific steroidal saponin markers to distinguish closely-related Paris herbs for quality and safety control.MethodsA new strategy of molecular-networking-guided discovery of species-specific markers was proposed. Firstly, the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was applied to obtain the MS and MS/MS data of all samples. Then, molecular networking (MN) was created using MS/MS data to prescreen the steroidal saponins for subsequent analysis. Next, the principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) models were established to discover potential markers. Finally, the verification, identification and distribution of chemical markers were performed.ResultsA total of 126 steroidal saponins were screened out from five species using MN. Five species were classified successfully by OPLS-DA model, and 18 species-specific markers were discovered combining the variable importance in the projection (VIP) value, P value (one-way ANOVA) and their relative abundance. These markers could predict the species of Paris herbs correctly.ConclusionThese results revealed that this new strategy could be an efficient way for chemical discrimination of medicinal herbs with close genetic relationship.     

Enzymatic Bioconversion of Cycloastragenol-6-O-β-D-glucoside into Cycloastragenol by a Novel Recombinant β-Glucosidase from Phycicoccus sp. Soil748

Cycloastragenol (CA), the genuine sapogenin of astragaloside from Astragalus membranaceus, exhibits diverse pharmaceutical activities. Recently, the efficient production of CA has received considerable attention due to rapidly increasing market demands. In this study, enzyme mining was conducted, based on skeleton and glycosyl similarity, to explore an efficient β-glucosidase for CA preparation. A novel β-glucosidase from Phycicoccus sp. Soil748 (Bgps) was discovered, possessing the efficient conversion rate for cycloastragenol-6-O-β-D-glucoside (CMG) into CA. The optimum temperature and pH value of Bgps were determined as 45 °C and 7.0. The results of kinetic analysis suggested that Bgps catalyzed deglycosylation of CMG more efficiently than other substrates. Furthermore, the optimal substrate concentration of Bgps was up to 80 mg/mL with the conversion rate as 99.2%, suggesting its potential application in CA industrial production by biotransformation.