Gentianae Radix Research Articles

Extraction efficiency of loganic acid and gentiopicroside from Gentianae radix et rhizoma by deep eutectic solvent-based ultrasound-assisted extraction

This study describes the extraction efficiency of loganic acid and gentiopicroside from Gentianae radix et rhizoma using the deep eutectic solvent-based ultrasound-assisted extraction (DES-UAE) method. A comparison was made against 12 deep eutectic solvents (DESs) as alternative extraction solutions. The extraction efficiency of DES-4 (prepared from choline chloride and lactic acid) exceeded that of other DESs. A single factor experiment and response surface methodology (RSM) were adopted, and the optimal processing parameters of DES-UAE were: the water content of DES-4 was 19.9 %, solid/liquid was 29.3 mg/ml, extraction temperature was 50.8 °C and extraction time was 26.8 min. Under these conditions, the extraction efficiency of loganic acid and gentiopicroside was 7.6986 mg/100 g. The results of scanning electron microscopy (SEM) indicated that the unextracted powder exhibited a granular shape with rough surface and complete structure. Moreover, the ultrasonic extraction of Gentianae radix et rhizoma powder with DES-4 as extraction solution caused significant damage to the surface structure of the powder, creating numerous fine pores on the surface. This suggested that DES-4 had higher permeability and erosive force, which effectively destroyed the cell wall.

Integrating deep learning and near-infrared spectroscopy for quality control of traditional Chinese medicine extracts

Extraction is a key fundamental step in producing most traditional Chinese medicine (TCM) products. Influenced by factors such as herbs and production techniques, the quality of TCM extracts may vary significantly, leading to high variability between batches and unstable quality of the products. Currently, high-performance liquid chromatography (HPLC) and other analytical methods have been employed for the quality control of TCM extracts, but these methods are time-consuming, environmentally unfriendly, and complicated in sample pre-treatment, which cannot meet the needs of actual production. Therefore, a rapid and effective method for the quality analysis and control of TCM extracts is urgently needed. In this study, the traditional Chinese medicine Radix Gentianae Macrophyllae (RGM) was taken as the research object, and a quantitative analysis model for the active ingredients loganic acid (LA) and gentiopicroside (GPS) was constructed based on near-infrared (NIR) spectroscopy, combining with convolutional neural network (CNN) and gated recurrent unit (GRU). Moreover, the multi-task mean square error (MTMSE) loss function proposed in this paper was used to train the model to ensure the prediction accuracy for different tasks. Meanwhile, this model integrated Bayesian optimization to automatically find the optimal hyperparameters without manual involvement. The results showed that the CNN-GRU model had higher prediction accuracy compared with CNN, BP, and PLS models. In addition, the feasibility of the CNN-GRU model was comprehensively analyzed. Finally, the feature extraction process of the CNN-GRU model was visualized to enhance the interpretability. This research creatively integrated deep learning with near-infrared spectroscopy to achieve rapid and accurate analysis of the active ingredient content of TCM extracts, providing new ideas and methods for quality control of TCM preparations.

Huangqin-Huanglian Decoction Protects Liver against Non-alcoholic Fatty Liver Disease in High Fat-diet Mice.

Traditional Chinese medicine (TCM) has the advantage of low toxicity of natural ingredients, multiple targets and effects, and low medication costs. It has unique advantages for metabolic and chronic diseases. Huangqin-Huanglian decoction (HQHLD) is composed of Scutellariae Radix, Coptidis Rhizoma, Rehmanniae Radix, and Gentianae Radix Et Rhozima; it has great potential for the treatment of NAFLD with the modern pharmacological research and TCM theory, but there is still a relative lack of research on the potential targets and pharmacological effects of HQHLD. In this work, we have used network pharmacology to predict the targets and signaling pathways of HQHLD, and validated NAFLD-related targets using the HFD model in order to explore more therapeutic drugs and methods for NAFLD. We collected the HQHLD ingredients and NAFLD targets through TCMSP, ETCM, DisGeNET, HGMD, MalaCards, OMIM, and TTD, built ingredients-target networks by Cytoscape, and screened key ingredients in HQHLD. DAVID and Metascape databases were used for GO functional enrichment analysis and KEGG pathway enrichment analysis, respectively. Molecular docking of the key ingredients and key targets was performed by AutoDock. We verified the effect of HQHLD on high-fat diet (HFD) mice by measuring the weight, liver weight index, and the level of TG, TC, LDL-C, and HDLC. HE staining and oil-red staining were performed to detect the damage and fat accumulation in the liver. The changes in INSR, PPAR-α, PPAR-γ, TNF-α, and caspase3 were experimented with WB. With the network pharmacology analysis, we found quercetin, baicalein, sitosterol, wogonin, oroxylin-A, glycyrrhizin, hydroberberine, berberine, sesamin, and carotene to be the main ingredients in HQHLD. According to KEGG pathway analysis, INSR, AKT, JNK1, PPAR-α, PPAR-γ, and the other 16 targets are the main targets of HQHLD in the treatment of NAFLD. We took HFD mice as the in vivo model of NAFLD. Our results showed that HQHLD could reduce liver weight, and TG and LDL-C levels, and increase HDL-C level in serum. By HE and oil red staining, we found that HQHLD could protect the morphology of hepatocytes and reduce fat in the liver. We also found HQHLD to protect the liver by increasing the expression of INSR and PPAR-α, and reducing the expression of PPAR-γ, TNF-α, and caspase3 in the liver. In conclusion, our study has firstly studied the main ingredients and key targets of HQHDL in treating NAFLD by network pharmacology analysis, and preliminarily confirmed that HQHLD could alleviate NAFLD in a multi-target way by lowering fatty acids, and decreasing insulin resistance, inflammation, and apoptosis in the liver.

Comparative analysis of four iridoids between Gentiana rigescens and G. cephalantha from the sympatric and allopatric distributions

Gentiana rigescens Franch. Ex Hemsl. is listed as an original species of Gentianae radix et rhizoma which is also an endangered plant in Gentianaceae. G. cephalantha Franch. Ex Hemsl., a sister species to G. rigescens, is usually used as a substitute of this endangered species in folk because of its similar morphologic features and abundant resources. To investigate reasonability of the replacement, four iridoids, namely gentiopicroside, swertiamarin, sweroside, and amarogentin were determined based on HPLC method and compared between G. rigescens and G. cephalantha from the sympatric and allopatric distributions. Total contents of the four iridoids were 9.05%–9.77% with an average value of 9.45% for G. rigescens, and 9.01%–12.73% with an average value of 11.20% for G. cephalantha, respectively. The results showed that contents of the iridoids in G. cephalantha was significantly higher than those in G. rigescens (P < 0.001), including the samples from the sympatric distribution. Meanwhile, samples from the middle altitude possessed higher content than those from the low and high altitudes. Moreover, hierarchical cluster analysis revealed well regularity of the iridoids content between G. rigescens and G. cephalantha, except the sample Gr3 which indicated that there were obviously genetic differences between the two species. This study could provide scientific evidence on substituting for G. rigescens with G. cephalantha based on contents of the active components which would further expand medicinal resources and be beneficial to conservation of G. rigescens.

Characterization of the Chemical Constituents in Radix gentianae by Ultra-High Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry.

Radix gentianae (RG) is a traditional Chinese medicine used for the treatment of acute and chronic hepatitis in clinic. However, the chemical profile of RG is still unconfirmed, which hindered the progress of pharmacological study and clinical application. In this study, ultra-high performance liquid chromatography together with quadrupole time-of-flight mass spectrometry techniques were employed to separate and characterize the chemical constituents in RG. Under the optimized conditions, a total of 60 compounds were rapidly identified or tentatively characterized. Results indicated that iridoid glucosides, flavonoids, organic acids, amino acids, saccharides and nucleosides were major constituents in RG. It is concluded the established method can help to clarify the substance basis and provide useful information for ascertaining the bioactive constituents and action mechanism of RG.

Gentiopicroside Ameliorated Ductular Reaction and Inflammatory Response in DDC-induced Murine Cholangiopathies Model.

Cholangiopathies comprise a spectrum of diseases without curative treatments. Pharmacological treatments based on bile acid (BA) metabolism regulation represent promising therapeutic strategies for the treatment of cholangiopathies. Gentiopicroside (GPS), derived from the Chinese medicinal herb Gentianae Radix, exerts pharmacological effects on bile acid metabolism regulation and oxidative stress. The present study aims to investigate the effect of GPS on 3,5-diethoxycarbonyl-1,4dihydrocollidine (DDC)-induced cholangiopathy. Two independent animal experiments were designed to evaluate the comprehensive effect of GPS on chronic DDC diet-induced cholangiopathy, including bile duct obliteration, ductular reaction, BA metabolism reprogramming, liver fibrosis, oxidative stress and inflammatory responses. In the first pharmacological experiment, three doses of GPS (5, 25 and 125 mg/kg) were injected intraperitoneally into mice fed a DDC diet for 14 days. DDC induced a typical ductular reaction, increased periductal fibrosis and mixed inflammatory cell infiltration in the portal areas. GPS treatment showed dose-dependent improvements in the ductular reaction, BA metabolism, fibrosis, oxidative stress and inflammatory response. In the second experiment, a high dose of GPS was injected intraperitoneally into control mice for 28 days, resulting in no obvious histologic changes and significant serologic abnormalities in liver function. However, GPS inhibited DDC-induced oxidative stress, serum and hepatic BA accumulation, proinflammatory cytokine production, and immunocyte infiltration. Specifically, the GPS-treated groups showed decreased infiltration of monocyte-derived macrophages and CD4+ and CD8+ T lymphocytes, as well as preserved Kupffer cells. GPS alleviated chronic DDC diet-induced cholangiopathy disorder by improving the ductular reaction, periductal fibrosis, oxidative stress and inflammatory response. Its dosage-dependent pharmacological effects indicated that GPS warrants its further evaluation in clinical trials for cholangiopathy.

Exploring the mechanism of Gentiana rigescens Franch. in the treatment of pulmonary fibrosis based on network pharmacology

Pulmonary fibrosis (PF) is a severe chronic interstitial lung disease with high mortality, and there is currently a lack of definite and effective treatment methods. Studies have found that gentiopicroside, a secoiridoid glycoside compound derived from plants of the Gentiana genus, can significantly improve pulmonary inflammation and fibrosis lesions in mice with pulmonary fibrosis. However, the mechanism of its anti-fibrotic effect is unclear. Therefore, in this study, we employed network pharmacology's virtual computer technology to investigate the potential mechanism underlying the anti-pulmonary fibrosis effect of Gentianae Radix et Rhizome (Longdan in Chinese, GRR). Our findings offer valuable theoretical insights and serve as a guiding reference for future experimental investigations. A total of 10 active compounds and 920 drug-target proteins were identified from the TCMSP database. The compound-target-pathway-disease network showed that GRR could potentially treat PF by regulating the MDM2, ERBB2 and VEGFA, signaling pathways through its key targets, including AKT1, TNF, and MAPK1. The protein‒protein interaction network revealed that these targets had strong interactions with each other, indicating a potential synergistic effect of GRR in treating PF. The GO and KEGG enrichment analyses further supported the potential anti-pulmonary fibrosis mechanisms of GRR, including regulating the inflammatory response, ECM-receptor interaction, and TGF-beta signaling pathways. Our study provides a systematic analysis of the potential anti-pulmonary fibrosis mechanisms of GRR based on network pharmacology. These findings could contribute to the development of novel treatments for PF and provide a basis for further experimental studies.

A Study of Gentianae Radix et Rhizoma Class Differences Based on Chemical Composition and Core Efficacy.

(1) Background: Establishment of a method for evaluating Gentianae Radix et Rhizoma (GRR) classes based on chemical composition and core efficacy; (2) Methods: Liquid chromatography-mass spectrometry (LC-MS) was used to determine the chemical constituents of GRR-first class (GF) and GRR-second class (GS). The cell viability, liver function, oxidative stress enzyme activity, and inflammatory factor levels of GF and GS on H2O2-induced HepG2 cells were determined with CCK-8, ELISA, and biochemical methods, and the antioxidant activity of the two was evaluated using bioefficacy; ELISA, biochemical methods, real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) method, and Western blot (WB) were used to determine the liver function, oxidative stress enzyme activity, inflammatory factor levels, and expression of related genes and proteins in mice with acute liver injury (ALI) model induced with 0.3% CCl4 olive oil solution after gavage administration; (3) Results: GF and GS had the same types of components, but the cyclic enol ether terpenes such as morinlon goside c, loganin, gentiopicroside, and swertiamarin differed significantly between the two; the effect of GF on CCl4-induced acute hepatic injury in C57BL/6 mice was stronger compared to GS. It helped alleviate weight loss, increase hepatic and splenic indices, improve hepatic lobular structure and hepatocyte status, inhibit collagen deposition, enhance oxidative stress and anti-inflammatory-related genes and protein expression, and decrease apoptotic genes and proteins more significantly than GS; (4) Conclusions: In this study, we established a GRR class evaluation method combining chemical composition and core medicinal effects, which can rapidly determine the differential composition of GF and GS, detect the quality of GRR through antioxidant bioefficacy, and validate it with in vivo experiments, which provides references for the evaluation of the class of GRR and the rational use of medication in the clinic.

Effect of postharvest processing on quality traits of Radix Gentianae Macrophyllae: A integrative analysis of metabolomics and proteomics

The dried roots and rhizomes of Radix Gentianae Macrophyllae are widely used as food material or medicinal crops. “Sweating” is a traditional postharvest processing method, the basic processing procedure consists of softening, stacking and drying. The aim of this paper is to unveil the scientific connotation responsible for the “Sweating” processing in Radix Gentianae Macrophyllae during postharvest. Thus, the effect of different postharvest processing methods on the metabolic pathways of Radix Gentiasnae Macrophyllae was studied by the non-targeted metabolomic technique in combination with the label-free proteomics approach. The results showed that the differentially accumulated metabolites and abundant proteins were mainly enriched in the pathways of phenylalanine, tyrosine and tryptophan biosynthesis, polyphenols and terpenoids biosynthesis. “Sweating” has a greater up-regulation effect on these pathways than “Non-sweating”, and can induce protein expression and metabolite accumulation associated with the quality traits of Radix Gentianae Macrophyllae. The results provide a detailed explanation of the scientific connotation of crucial steps of “Sweating” processing wherein opportunities existed for taking appropriate measures to enhance the accumulation of bioactive ingredients. These findings will serve as significant references for enhancing the postharvest processing technology of Radix Gentianae Macrophyllae and similar plants, resulting in higher product quality for food or plant materials production.

Conservation planning for the endemic and endangered medicinal plants under the climate change and human disturbance: a case study of Gentiana manshurica in China.

Human activities and climate change have significantly impacted the quantity and sustainable utilization of medicinal plants. Gentiana manshurica Kitagawa, a high-quality original species of Gentianae Radix et Rhizoma, has significant medicinal value. However, wild resources have experienced a sharp decline due to human excavation, habitat destruction, and other factors. Consequently, it has been classified as an Endangered (EN) species on the IUCN Red List and is considered a third-level national key-protected medicinal material in China. The effects of climate change on G. manshurica are not yet known in the context of the severe negative impacts of climate change on most species. In this study, an optimized MaxEnt model was used to predict the current and future potential distribution of G. manshurica. In addition, land use data in 1980, 2000, and 2020 were used to calculate habitat quality by InVEST model and landscape fragmentation by the Fragstats model. Finally, using the above-calculated results, the priority protection areas and wild tending areas of G. manshurica were planned in ZONATION software. The results show that the suitable area is mainly distributed in the central part of the Songnen Plain. Bio15, bio03, bio01, and clay content are the environmental variables affecting the distribution. In general, the future potential distribution is expected to show an increasing trend. However, the species is expected to become threatened as carbon emission scenarios and years increase gradually. At worst, the high suitability area is expected to disappear completely under SSP585-2090s. Combined with the t-test, this could be due to pressure from bio01. The migration trends of climate niche centroid are inconsistent and do not all move to higher latitudes under different carbon emission scenarios. Over the past 40 years, habitat quality in the current potential distribution has declined yearly, and natural habitat has gradually fragmented. Existing reserves protect only 9.52% of G. manshurica's priority conservation area. To avoid extinction risk and increase the practicality of the results, we clarified the hotspot counties of priority protection area gaps and wild tending areas. These results can provide an essential reference and decision basis for effectively protecting G. manshurica under climate change.

The importance of herbal medications in the treatment of rhinosinusitis in adults and children: an example of a five-ingredient herbal medicine

This review paper presents current recommendations for the treatment of acute and chronic rhinosinusitis according to theEuropean Position Paper on Rhinosinusitis and Nasal Polyps 2020, EPOS 2020. A particularly important and new element ofthese recommendations is to emphasize the role of phytotherapy as a rational treatment method, documented by a randomized,double-blind placebo-controlled trial (DBPC). An example of rational phytotherapy of rhinosinusitis is the BNO 1016 formulationrecommended in EPOS 2020, known under the trade name of Sinupret. Each of the pharmaceutical forms of BNO 1016 coatedtablets (powdered drug; dry extract) and oral drops (ethanol extract), is produced on the basis of the same combination of5 herbal substances such as: gentian root (Gentianae radix), primrose flower (Primulae flos), sorrel herb (Rumicis herba),elderflower (Sambuci flos) and verbena herb (Verbenae herba). BNO 1016 has the status of a drug with well-establishedtherapeutic use, with efficacy and safety confirmed in numerous clinical and preclinical studies. Used in the treatment of acuteuncomplicated rhinosinusitis BNO 1016 has antiviral, secretolytic, anti-inflammatory and antibacterial effects confirmed inpharmacological studies. Importantly, the drug Sinupret can be used in different age groups and therapeutic regimens (monoand polytherapy), at any stage of sinusitis from cold disease to bacterial rhinosinusitis (in combination with other drugs).

Assessment of the Quality of Herbal Teas from Šabac, Serbia in Terms of the Content of Heavy Metals.

Chemical components of teas have received great interest because they are related to health. In this work, data on the determination of foreign matter, loss on drying/water content, total ash and ash insoluble in hydrochloric acid are presented. The content of seven heavy metals including Cu, Fe, Mn, Zn, Ni, Cd and Pb were determined by atomic absorption spectrometry in samples of several herbal teas: Matricariae flos, Thymi herba, Menthae piperitae folium, Betulae folium, Quercus cortex, Gentianae radix, Frangulae cortex, Althaeae radix, Uvae ursi folium and Glycyrrhizae radix collected from Šabac’s market, Serbia. The sample preparation procedure involved dry digestion and dissolution of the ash in 6M HCl and then in 0.1 M HNO3. Herbal teas showed the concentration of heavy metals Cu, Fe, Mn, Zn and Ni in the range: 2.9 ± 0.1 – 22.2 ± 0.9 mg/kg, 118.5 ± 1.1 – 755.5 ± 2.5 mg/kg, 19.0 ± 5.8 – 561.0 ± 1.9 mg/kg, 6.5 ± 0.4 – 242.5 ± 1.4 mg/kg and 2.5 ± 0.1 – 10 ± 1.1 mg/kg, respectively. The level of copper in all samples was uniform. The highest content of Fe was in Thymi herba, while Mn and Zn were at maximum in Betulae folium. The levels of toxic heavy metals Cd and Pb were below the detection limit. The obtained values were compared with data available from literature. The herbal tea samples analyzed contained essential heavy metals (Cu, Fe, Mn, Zn) and probably essential in trace (Ni), and could obey the daily dietary requirements. Noncancer health risk assessment detected that the herbal teas of Betulae folium and Frangulae cortex can manifest some health risk to consumers.

Simultaneous Quantitative Analysis of the Major Bioactive Compounds in Gentianae Radix and its Beverages by UHPSFC-DAD.

This study presents the first ultra-high performance supercritical fluid chromatography–diode array detector (UHPSFC–DAD) assay for simultaneous quantitation of secoiridoids, iridoids, xanthones, and xanthone glycosides in Gentiana lutea L. Separation was reached within 12 min on an Acquity UPC2 BEH 2-EP column using CO2 and methanol with 5.5% water as mobile phases. Method validation for nine selected marker compounds (gentisin, isogentisin, swertiamarin, sweroside, gentiopicroside, loganic acid, amarogentin, gentioside, and its isomer) confirmed the assay’s sensitivity, linearity, precision, and accuracy. The practical applicability was proven by the analysis of 13 root specimens and 10 commercial liquid preparations (seven liqueurs and three clear spirits). In all root batches, the secoiridoid gentiopicroside dominated (2.1–5.6%) clearly over all other metabolites. In the liqueurs, the metabolite content and distribution were extremely variable: while gentiopicroside was the main compound in four liqueurs, sweroside dominated in one preparation and loganic acid in two others. In contrast, measurable amounts of the metabolites were not detected in any of the examined clear spirits.

Roburic acid attenuates osteoclastogenesis and bone resorption by targeting RANKL-induced intracellular signaling pathways.

Excessive activity of osteoclasts contributes to skeletal diseases such as osteoporosis and osteolysis. However, current drugs targeting osteoclast have various deficiencies, placing natural compounds as substitutions of great potential. Roburic acid (RA) is a triterpenoid exacted from Radix Gentianae Macrophyllae, which exhibits inhibitory effects on inflammation and oxidation. By employing an in vitro osteoclastogenesis model, this study investigates the effects and mechanisms of RA on intracellular signaling induced by receptor activator of nuclear factor-κB ligand (RANKL). As expected, RA at a concentration scope from 1 to 10 μM dampened the osteoclast differentiation of bone marrow macrophages (BMMs) but without cell toxicity. Interestingly, RA showed no effect on osteoblastogenesis in vitro. Furthermore, RA mitigated F-actin ring formation, hydroxyapatite resorption, and gene expression in osteoclasts. Mechanistically, RA suppressed TNF receptor-associated factor 6 (TRAF6), the crucial adaptor protein following RANKL-RANK binding. On the one hand, RA downregulated the nuclear factor-κB (NF-κB) activity, extracellular regulated protein kinases (ERK) phosphorylation, and calcium oscillations. On the other hand, RA upregulated the antioxidative response element (ARE) response and the protein expression of heme oxygenase (HO)-1. These upstream alterations eventually led to the suppression of the nuclear factor of activated T cells 1 (NFATc1) activity and the expression of proteins involved in osteoclastogenesis and bone resorption. Furthermore, by using an ovariectomized (OVX) mice model, RA was found to have therapeutic effects against bone loss. On account of these findings, RA could be used to restrain osteoclasts for treating osteoporosis and other osteolytic diseases.

Rhinitis, rhinosinusutus and phytotherapy

Acute rhinitis and acute rhinosinusitis (ARS) have viral etiology in the most of cases, and their therapy is aimed at alleviating clinical symptoms and preventing new episodes. The indications for antibiotic therapy are severe bacterial ARS, ARS with complications, and recurrent bacterial ARS. Such a categorical approach is due to the increase of antibiotic resistance, which leads to the popularization of efficient phytotherapy. Modern herbal medical products comply with general recommendation and standards of efficacy, safety and quality, and their effectiveness has been proven in clinical trials along with synthetic drugs. In acute upper respiratory tract infections, herbal medical drugs stimulate the immune system, relieve clinical symptoms, and improve the patient’s quality of life. A significant emphasis in phytotherapy is given to complex drugs synthesized on the basis of phytoneering. One of such remedies is the herbal medicinal product containing the different parts of 5 medicinal herbs: Gentian root (Gentianae radix), Primrose flowers (with calyx) (Primulae flos (cum calycibus)), Common sorrel herb (Rumicis herba), Elder flowers (Sambuci flos) and Vervain herb (Verbenae herba). This herbal medicinal product shows secretolytic, secretomotor, antiinflammatory, antiviral and mild antibacterial farmacological effects. The article provides an analysis of studies in vitro and in vivo proving the anti-inflammatory effect of Sinupret®, its potentiating effect on nasal mucosa mucociliary transport as well as efficacy and safety in patients with rhinosinusitis.

A new strategy for the preparation of total iridoids from Radix Gentianae Macrophyllae and anti-inflammatory profile digesting by UPLC-Q-TOF-MS characterization coupled with PLS analysis

Radix Gentianae Macrophyllae (RGM) is a well-known traditional Chinese medicine with anti-inflammatory, analgesia and gastric motility strengthening effects. The iridoids are believed to be the RGM bioactive ingredients, however, an effective strategy for its total iridoids extraction and purification is lacking. In addition, a reliable method for the bioactive iridoid constituents digesting needs to be developed. In the present work, followed by process optimization via response surface modeling and Box-Behnken analysis, the maximum yield of total RGM iridoids was predicted to be 7.75 % from rhizomes of Gentiana macrophylla Pall. via three cycles of extraction in 69.16 % ethanol at liquid-solid ratio 12.27 (mL/g) for 110.91 min. The crude iridoid extract was further purified using HPD-300 macroporous resin to give the enriched product with the iridoid content up to 73.27 %. The anti-inflammatory effects of refined iridoid extracts were quantitatively measured to validate the purification procedure. The refined extract was detected to significantly inhibit the expression of NO through expression inhibition of COX-2, iNOS in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells with the IC50 of 10.49 μg/mL, which was stronger than that of crude extract with the IC50 of 51.11 μg/mL, indicating the iridoid compounds were enriched from crude extract through the purification process. Subsequently, an UPLC-QTOF-MS method was established for qualitative and quantitative analysis of the iridoid constituents in the crude and refined extracts. In addition, based on the UPLC-QTOF-MS data, 6 principle iridoids that give the most important support for the anti-inflammatory activities were digested by means of PCA and PLS analysis. The current study developed a new and effective strategy for optimal extraction, purification and identification of natural bioactive iridoids from Radix Gentianae Macrophyllae, which facilitates its further applications in pharmaceuticals and/or foods industry.

Anti-inflammatory target prediction of Gentianae Radix et Rhizoma based on network pharmacology and construction of a bioassay method for its quality control

Based on the heat-clearing and detoxifying effects of Gentianae Radix et Rhizoma, the network pharmacology is mainly used to predict the potential targets of Gentianae Radix et Rhizoma for anti-inflammatory activity and to perform the experimental verification. A method for detecting the biological potency of Gentianae Radix et Rhizoma based on verifiable targets has been established to provide a reference for improving the quality evaluation and control standards of Gentianae Radix et Rhizoma. High performance liquid chromatography can be used to construct chemical fingerprints of different batches of Gentianae Radix et Rhizoma. Constructing a component-target-disease network of Gentianae Radix et Rhizoma for its anti-inflammatory activity was applied to screen potential anti-inflammatory components and related targets of Gentianae Radix et Rhizoma, and to verify the target of Gentianae Radix et Rhizoma by using biological evaluation methods. Detecting the biological potency of different batches of Gentianae Radix et Rhizoma extracts was used to inhibit COX-2 enzyme activity based the verifiable target cyclooxygenase-2(COX-2). The results showed that different batches of Gentianae Radix et Rhizoma accorded with the pharmacopoeia testing regulations, and the chemical fingerprints have a high similarity(similarity&gt;0.93), suggesting that there is no significant difference in the characteristics of the chemical components. Based on network pharmacology predictions, 18 candidate targets were found to have potential direct interactions with the ingredients in Gentianae Radix et Rhizoma. Among them, the most important target is COX-2. Based on the experimental verification of recombinant human COX-2 protease activity inhibition, Gentianae Radix et Rhizoma can inhibit the COX-2 enzyme activity in a dose-dependent manner. It can function with a low concentration(0.75 mg·mL~(-1)), which preliminarily confirmed the accuracy of network pharmacology prediction. The biological potency detection method of Gentianae Radix et Rhizoma based on COX-2 inhibitory activity was optimized and established. The qualitative response parallel line method was used to calculate the biological potency of anti-inflammatory activity, which ranged from 23.04 to 46.60 U·mg~(-1). For network pharmacology prediction, it can screen and clarify the possible targets of traditional Chinese medicine rapidly, which can guide the establishment of a biological evaluation method for the quality of medicinal materials with related activities. Compared with chemical fingerprints, the biological potency testing can better detect quality fluctuations of traditional Chinese medicine.

Network pharmacology integrated with molecular docking reveals the common experiment-validated antipyretic mechanism of bitter-cold herbs

Ethnopharmacological relevanceBitter-cold herbs have been used to clearing heat and expelling damp in clinical practice in China for thousands of years.Aim of the study: This study aimed to investigate the common molecular mechanism of bitter-cold herbs through network pharmacology analysis, molecular docking and experimental validation in vivo. Materials and methodsNetwork pharmacological analysis integrated with molecular docking was employed to identify the active compounds and core action targets of the bitter-cold herbs. Then, the yeast-induced pathological model was established, and the antipyretic effect of the herbs was evaluated by checking rectal temperatures of the mice hourly. Lastly, the protein expression of core targets was examined to reveal the antipyretic mechanism. ResultsA total of 52 lead compounds from the four bitter-cold herbs, Phellodendri Chinensis Cortex (PCC), Sophorae Flavescentis Radix (SFR), Gentianae Radix Et Rhozima (GRER) and Coptidis Rhizoma (CR), and 248 compounds-related targets were screened out with PTGS2 ranking the first. The results from molecular docking showed that 22 compounds adopted the same orientation as aspirin and had an excellent stability in the active site pocket of PTGS2. Furthermore, these herbs exerted potential therapeutic effects through 38 related pathways. On the other hand, the outcome of animal experiments showed that they could significantly attenuate the yeast-induced mice fever with dose-dependent relationship. Further experimental results demonstrated that administration of yeast suspension raised protein expression of PTGS2 significantly, which was evidently inhibited in the high or low-dose groups of GRER as well as in the low-dose group of SFR (P < 0.01) though a higher expression of PTGS2 was shown in the low-dose group of CR compared with FM group (P < 0.01). ConclusionsThe bitter-cold herbs can alleviate fever response and their antipyretic effect may mainly be attributed to regulating the expression of PTGS2 after the formation of ligand-receptor/PTGS2 complexes, and their active compounds might be nominated as antipyretic lead-ligand candidates.

Hepatoprotective effect and metabonomics studies of radix gentianae in rats with acute liver injury

Context As a well-known traditional Chinese medicine for protecting the liver, the mechanism of Radix Gentianae (RG) remains unclear. Objective The hepatoprotective effect and metabonomics of RG were studied to explore the molecular and metabolic mechanisms of RG protecting the liver. Materials and methods Sprague-Dawley rats were divided into control and model group (n = 10, orally given distilled water), intervention group (4 subgroups, n = 10, prophylactically and orally given 0.63, 2.5 and 5.6 g/kg RG and 0.2 g/kg bifendatatum for 7 d). On day 7 of the intervention, all rats except the control were injected intraperitoneally with 2.5% carbon tetrachloride vegetable oil solution (1.5 mL/kg) to induce liver injury. After 24 h of carbon tetrachloride injection, rat serum and liver tissue were collected for determining AST, ALT, TNF-α, MCP-1, IL-6, SOD, MDA, GSH, and GSH-PX. Rat serum was used for analysing endogenous metabolism by UPLC-Q-TOF-MS. Results Different doses of RG can significantly decrease the levels of AST, ALT, TNF-α, MCP-1, IL-6 and MDA, and increase the levels of SOD, GSH, and GSH-PX in rats with liver injury (p < 0.05; TNF-α, and IL-6, p < 0.05 only at 5.6 g/kg dose). Eight biomarkers of liver injury were obtained in serum metabonomics, involving five significant metabolic pathways. RG can improve steroid biosynthesis, linoleic acid metabolism, porphyrin and chlorophyll metabolism, and fatty acid biosynthesis. Conclusion RG demonstrated a good ability to protect the liver and improving endogenous metabolism in rats with liver injury. This can help us understand the mechanism of RG and more clinical verifications were inspired.

A new and effective evaluation method for Radix Gentianae Macrophyllae herbs based on 2-phenylethyl β-d-glucopyranoside, 2-methoxyanofinic acid and gentioxepine by UPLC-QTOF-MS exploring and HPLC-DAD quantification.

Radix Gentianae Macrophyllae (RGM) is a traditional Chinese medicine belonging to the Gentiana genus and including four species of herbs. Owing to the lack of characteristic constituents, it is difficult to discriminate RGMs of different origins or even differentiate between herbs in the same genus. The current research aimed to explore the characteristic aromatic compounds, verify their significance in distinguishing different origins of RGM herbs and provide a simple and effective quality evaluation method. A selective extraction method was developed for noniridoid compounds and the extract was then subjected to UPLC-QTOF-MS analysis to obtain the RGM-MS ion pair database for noniridoid components. An HPLC-DAD quantitative analysis method was further developed based on characteristic aromatic compounds (2-phenylethyl β-d-glucopyranoside, 2-methoxyanofinic acid and gentioxepine) after the ion screening in the MS database. By means of principal component analysis and hierarchical clustering analysis analysis, the significant relationship between aromatic compounds contents and different species of RGM was revealed. As a result, the significance of 2-phenylethyl β-d-glucopyranoside, 2-methoxyanofinic acid and gentioxepine in distinguishing four species of RGM herbs was verified and a sensitive and reproducible HPLC-DAD method was established using these markers, which can be used for the classification and quantitative analysis of RGMs.