Root-associated endophytic bacterial community composition and structure of three medicinal licorices and their changes with the growing year.

BACKGROUND: The dried roots and rhizomes of medicinal licorices are widely used worldwide as a traditional medicinal herb, which are mainly attributed to a variety of bioactive compounds that can be extracted from licorice root. Endophytes and plants form a symbiotic relationship, which is an important source of host secondary metabolites. RESULTS: In this study, we used high-throughput sequencing technology and high-performance liquid chromatography to explore the composition and structure of the endophytic bacterial community and the content of bioactive compounds (glycyrrhizic acid, liquiritin and total flavonoids) in different species of medicinal licorices (Glycyrrhiza uralensis, Glycyrrhiza glabra, and Glycyrrhiza inflata) and in different planting years (1-3 years). Our results showed that the contents of the bioactive compounds in the roots of medicinal licorices were not affected by the species, but were significantly affected by the main effect growing year (1-3) (P < 0.05), and with a trend of stable increase in the contents observed with each growing year. In 27 samples, a total of 1,979,531 effective sequences were obtained after quality control, and 2432 effective operational taxonomic units (OTUs) were obtained at 97% identity. The phylum Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, and the genera unified-Rhizobiaceae, Pseudomonas, Novosphingobium, and Pantoea were significantly dominant in the 27 samples. Distance-based redundancy analysis (db-RDA) showed that the content of total flavonoids explained the differences in composition and distribution of endophytic bacterial communities in roots of cultivated medicinal liquorices to the greatest extent. Total soil salt was the most important factor that significantly affected the endophytic bacterial community in soil factors, followed by ammonium nitrogen and nitrate nitrogen. Among the leaf nutrition factors, leaf water content had the most significant effect on the endophytic bacterial community, followed by total phosphorus and total potassium. CONCLUSIONS: This study not only provides information on the composition and distribution of endophytic bacteria in the roots of medicinal licorices, but also reveals the influence of abiotic factors on the community of endophytic bacteria and bioactive compounds, which provides a reference for improving the quality of licorice.

Preparative Separation and Purification of Liquiritin and Glycyrrhizic Acid from Glycyrrhiza uralensis Fisch by High-Speed Countercurrent Chromatography.

The technique of high-speed countercurrent chromatography (HSCCC) was applied to the preparative isolation and purification of liquiritin and glycyrrhizic acid from a crude extract of Glycyrrhiza uralensis Fisch for the first time. Using single factor and orthogonal design experiments, the best extraction conditions were 70% ethanol, 1:25 ratio of solid-to-liquid (w/v) and extracted 1.5 h at 80°C. The contents of liquiritin and glycyrrhizic acid in the crude extract were 1.3 and 5.3%, respectively. Using the two-phase solvent system of ethyl acetate-methanol-water (5:2:5, v/v), 6.0 mg liquiritin (the purity was 96.7%, and the recovery was 89.3%), and 20.5 mg glycyrrhizic acid (the purity was 98.9%, and the recovery was 77.1%) were obtained from 500 mg crude extraction by HSCCC, respectively. The retention rate of stationary phase was 51.0%. Their structures were identified by high-performance liquid chromatography, melting points, ultraviolet radiation, Fourier transform infrared (FTIR), Electrospray ionization mass spectrometry (ESI-MS), 1H Nuclear Magnetic Resonance (NMR) and 13C NMR spectra. The scavenging abilities of glycyrrhizic acid to 1,1-diphenyl-2-picrylhydrazyl and hydroxyl free radicals were stronger than those of liquiritin.

18ß-glycyrrhetyl-3-O-sulfate would be a causative agent of licorice-induced pseudoaldosteronism.

Licorice-induced pseudoaldosteronism is a common adverse effect in traditional Japanese Kampo medicine, and 3-monoglucuronyl glycyrrhetinic acid (3MGA) was considered as a causative agent of it. Previously, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate-30-glucuronide (1), one of the metabolites of glycyrrhizin (GL) in the urine of Eisai hyperbilirubinuria rats (EHBRs) treated with glycyrrhetinic acid (GA), and suggested that it is also a possible causative agent of pseudoaldosteronism. The discovery of 1 also suggested that there might be other metabolites of GA as causal candidates. In this study, we found 22α-hydroxy-18ß-glycyrrhetyl-3-O-sulfate (2) and 18ß-glycyrrhetyl-3-O-sulfate (3) in EHBRs' urine. 2 and 3 more strongly inhibited rat type 2 11ß-hydroxysteroid dehydrogenase than 1 did in vitro. When EHBRs were orally treated with GA, GA and 1-3 in plasma and 1-3 in urine were detected; the levels of 3MGA were quite low. 2 and 3 were shown to be the substrates of organic anion transporter (OAT) 1 and OAT3. In the plasma of a patient suffering from pseudoaldosteronism with rhabdomyolysis due to licorice, we found 8.6 µM of 3, 1.3 µM of GA, and 87 nM of 2, but 1, GL, and 3MGA were not detected. These findings suggest that 18ß-glycyrrhetyl-3-O-sulfate (3) is an alternative causative agent of pseudoaldosteronism, rather than 3MGA and 1.

Liquorice (Glycyrrhiza glabra): A phytochemical and pharmacological review.

In the last years, consumers are paying much more attention to natural medicines and principles, mainly due to the general sense that natural compounds are safe. On the other hand, there is a growing demand by industry for plants used in traditional medicine that could be incorporated in foods, nutraceuticals, cosmetics, or even pharmaceuticals. Glycyrrhiza glabra Linn. belongs to the Fabaceae family and has been recognized since ancient times for its ethnopharmacological values. This plant contains different phytocompounds, such as glycyrrhizin, 18ß-glycyrrhetinic acid, glabrin A and B, and isoflavones, that have demonstrated various pharmacological activities. Pharmacological experiments have demonstrated that different extracts and pure compounds from this species exhibit a broad range of biological properties, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antidiabetic activities. A few toxicological studies have reported some concerns. This review addresses all those issues and focuses on the pharmacological activities reported for G. glabra. Therefore, an updated, critical, and extensive overview on the current knowledge of G. glabra composition and biological activities is provided here in order to explore its therapeutic potential and future challenges to be utilized for the formulation of new products that will contribute to human well-being.

Inhibitory Effects of Baicalein Derived from Japanese Traditional Herbal Medicine on SN-38 Glucuronidation.

PURPOSE: The chemotherapeutic agent irinotecan is hydrolyzed to its active form SN-38 by human carboxyesterases, but SN-38 is converted into the inactive form SN-38G by hepatic UDP-glucuronosyltransferases (UGTs). The aim of the present study was to evaluate the inhibitory effects of two b-glucuronidase-treated Japanese traditional herbal medicines (kampo), Hange-Shashin-To (TJ-14) and Sairei-To (TJ-114) on SN-38 glucuronidation, and the deglycosylation of baicalin (BG) and glycyrrhizic acid (GL) derived from TJ-14 and TJ-114 to form their respective aglycones, baicalein (BA) and glycyrrhetinic acid (GA). METHODS: The inhibitory effects of b-glucuronidase-treated TJ-14 and TJ-114 on SN-38 glucuronidation by human liver microsomes were examined. BA and GA, which were enzymatically converted from BG and GL present in TJ-14 and TJ-114, were examined in the same manner. Furthermore, the enzymatic activities were measured by using recombinant UGT1A1 and UGT1A9 isoforms instead of human liver microsomes. BA, GA, SN-38, and their glycosides/glucuronides were analyzed with an LC-MS system. RESULTS: As regards the linear initial reaction rate, SN-38 glucuronidation by human liver microsomes was significantly inhibited by the addition of b-glucuronidase-untreated TJ-14 and TJ-114, but was more strongly inhibited by the addition of b-glucuronidase-treated TJ-14 and TJ-114. The results of LC-MS analysis and pharmacokinetic studies suggested that BA is the main inhibitor of SN-38 glucuronidation. In the Dixon plot, BA showed competitive inhibition of SN-38 glucuronidation, and the inhibition constant was 8.70 ± 3.24 mM. Previous reports, studies of recombinant UGT isoforms indicated that SN-38 glucuronidation was mainly catalyzed by UGT1A1. CONCLUSIONS: These findings strongly suggested that SN-38 glucuronidation is inhibited by BA. BA could act as a pharmacokinetic regulating factor associated with SN-38 glucuronidation. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

The association between consistent licorice ingestion, hypertension and hypokalaemia: a systematic review and meta-analysis.

There have been numerous case reports of severe adverse events including deaths following chronic licorice ingestion. The aim of the present study was to evaluate the effect of chronic ingestion of licorice on blood pressure, plasma potassium, plasma renin activity and plasma aldosterone. A search of MEDLINE, PubMed, EMBASE, CENTRAL, DARE, CINAHL and Current Contents Connect was performed from inception through to 26 April 2017. Trials that included a treatment group ingesting a product containing at least 100 mg of glycyrrhizic acid daily were selected. Pooled mean changes from baseline with 95% confidence intervals were calculated for diastolic blood pressure, systolic blood pressure, plasma potassium, plasma renin activity and plasma aldosterone using a random effects model. An assessment of dose-response was also undertaken. A total of 18 studies (n=337) were included in the meta-analysis. There was a statistically significant increase in mean systolic blood pressure (5.45 mm Hg, 95% CI 3.51-7.39) and diastolic blood pressure (3.19 mm Hg, 95% CI 0.10-6.29) after chronic ingestion of a product containing glycyrrhizic acid. Plasma potassium (-0.33 mmol l-1, 95% CI -0.42 to 0.23), plasma renin activity (-0.82 ngml-1 per hour, 95% CI -1.27 to -0.37) and plasma aldosterone (-173.24 pmol l-1, 95% CI -231.65 to -114.83) were all significantly decreased. A significant correlation was noted between daily dose of glycyrrhizic acid and systolic blood pressure (r2=0.55) and diastolic blood pressure (r2=0.65), but not for the other outcome measures. Hence, chronic licorice ingestion is associated with an increase in blood pressure and a drop in plasma potassium, even at modest doses. This is of particular relevance for individuals with existing cardiovascular disease.

Alternative to antibiotics against Pseudomonas aeruginosa: Effects of Glycyrrhiza glabra on membrane permeability and inhibition of efflux activity and biofilm formation in Pseudomonas aeruginosa and its in vitro time-kill activity.

The multi-drug resistance offered by Pseudomonas aeruginosa to antibiotics can be attributed towards its propensity to develop biofilm, modification in cell membrane and to efflux antibacterial drugs. The present study explored the activity of Glycyrrhiza glabra and one of its pure compounds, glycyrrhizic acid against P. aeruginosa and their mechanism of action in terms of the effect on membrane permeability, efflux activity, and biofilm formation were determined. Minimum inhibitory concentrations were determined by using broth dilution technique. The minimum bactericidal concentrations were assessed on agar plate. The MIC of the extract and glycyrrhizic acid was found to be 200 and 100 µg ml(-1), respectively. The MBC was found to be 800 and 400 µg ml(-1) in the case of extract and glycyrrhizic acid, respectively. Time -dependent killing efficacy was also estimated. Flowcytometric analysis with staining methods was used to determine the effect of extract and glycyrrhizic acid at 2 × MIC on different physiological parameters and compared it with the standard (antibiotic). The growth of P. aeruginosa was significantly inhibited by extract and the pure compound. The herbal extract and the glycyrrhic acid were also found to effective in targeting the physiological parameters of the bacteria that involve cell membrane permeabilization, efflux activity, and biofilm formation. This study reports the antipseudomonal action of Glycyrrhiza glabra and one of its compound and provides insight into their mode of action.

[STUDY OF COMPOSITION OF PLANT EXTRACTS, POSSESSING ANTIMICROBIAL EFFECT AGAINST VIBRIO CHOLERAE EL TOR, USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY].

AIM: Study the composition of plant extracts using high-performance liquid chromatography. (HPLC) and evaluation of their antimicrobial effect against Vibrio cholerae El Tor. MATERIALS AND METHODS: Qualitative and quantitative composition of plant extracts was studied using HPLC. Determination of sensitivity of microorganisms to plant extracts was carried out by diffusion into agar method and serial dilutions method. RESULTS: Antibacterial effect of water, water-alcohol and acetone extracts of roots of Limonium gmelinii L., Berberis vulgaris L. and Glycyrrhiza glabra L. was studied. The most effective methods of extraction of biologically active substances, possessing antimicrobial effect against various strains of V. cholerae El Tor, were determined. CONCLUSION: The use of HPLC allowed to establish the presence of catechines, alkaloids protoberberines and glycyrrhizic acid in xtracts, possessing antimicrobial effect against V. cholera El Tor strains.

[Optimization of ultrasonic extraction process for Xiaoqinglong granules by Box-Behnken in condition of medium scale].

This paper is to investigate the optimization conditions of ultrasonic technique for extraction process of Xiaoqinglong granules in medium scale. First of all, single factor experiment was used to determine the overall impact tendency and range of each factor； secondly, Box-Behnken method was used for optimization and detecting the content of paeoniflorin, ephedrine hydrochloride, glycyrrhizic acid of the liquid medicine. Their respective extraction rate was calculated and the comprehensive evaluation was carried out. The results were used as the evaluation basis for the efficacy of Xiaoqinglong granules ultrasonic extraction. The test results showed that the optimum extraction process of Xiaoqinglong granules by ultrasonic extraction was under the following conditions: ultrasonic power 600 W, liquid-solid ratio 10∶1, extraction for 31 min. Under this condition, the predicted value of extraction rate for Xiaoqinglong granules was 85.90%, and the test value was 85.87%. The mathematical model(P<0.01) established in this paper was significant, and can be used for the analysis and prediction of the ultrasonic extraction process of Xiaoqinglong granules.

HPLC-PDA Method for Simultaneous Determination of Nine Marker Components in Banhasasim-Tang.

A simple and accurate high-performance liquid chromatography-photodiode array (HPLC-PDA) detection method has been developed and validated for simultaneous determination of nine components-liquiritin, coptisine, baicalin, palmatine, berberine, wogonoside, baicalein, glycyrrhizin and wogonin-in the traditional Korean formula, Banhasasim-tang decoction. A Gemini C18 analytical column was used to separate the nine constituents and kept at 40°C by gradient elution with 0.1% (v/v) trifluoroacetic acid in distilled water (A) and acetonitrile (B) as mobile phases. The flow rate was 1.0 mL/min and the injection volume was 10 µL. The PDA detection wavelengths were set at 254, 275 and 350 nm. Calibration curves of all compounds showed good linearity with coefficients of determination ≥0.9998 within the test ranges. The limits of detection and quantification of all compounds were in the range 0.01-0.09 and 0.03-0.30 µg/mL, respectively. All recoveries of the nine marker compounds ranged from 98.65 to 103.22% with relative standard deviation (RSD) values <1.25%. The RSDs of intraday and interday precision were <1.13 and 1.83%, respectively. The concentrations of the nine marker constituents were 0.19-41.09 mg/g.

Anti-Inflammatory activities of licorice extract and its active compounds, glycyrrhizic acid, liquiritin and liquiritigenin, in BV2 cells and mice liver.

This study provides the scientific basis for the anti-inflammatory effects of licorice extract in a t-BHP (tert-butyl hydrogen peroxide)-induced liver damage model and the effects of its ingredients, glycyrrhizic acid (GA), liquiritin (LQ) and liquiritigenin (LG), in a lipopolysaccharide (LPS)-stimulated microglial cell model. The GA, LQ and LG inhibited the LPS-stimulated elevation of pro-inflammatory mediators, such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta and interleukin (IL)-6 in BV2 (mouse brain microglia) cells. Furthermore, licorice extract inhibited the expression levels of pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) in the livers of t-BHP-treated mice models. This result suggested that mechanistic-based evidence substantiating the traditional claims of licorice extract and its three bioactive components can be applied for the treatment of inflammation-related disorders, such as oxidative liver damage and inflammation diseases.

The influence of compatibility of processed radix Aconiti Kusnezoffii on the pharmacokinetic of four components in Glycyrrhiza uralensis Fisch.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhiza uralensis Fisch. and processed radix Aconiti kusnezoffii are the main components in many Chinese traditional patent medicines with the ratio of 1:1, which are used for treatment of rheumatoid arthritis, heart failure and so on. Glycyrrhizic acid, glycyrrhetic acid, liquiritigenin and isoliquiritigenin are the essential bioactive triterpenes and flavones in the extract of G. uralensis, which were analysis by a simple but accurate method. MATERIALS AND METHODS: In the present study, a specific HPLC method was developed and validated for simultaneous determination of pharmacokinetic parameters of glycyrrhizic acid, glycyrrhetic acid, liquiritigenin and isoliquiritigenin in G. uralensis after oral administration of single herb extract and a combination of two herbs extracts respectively. RESULTS: The calibration curves of the four components had good linearity higher than 0.9991 in the measured range. The intra-day and inter-day precisions (RSD) at different levels were both within 9.73%, and the accuracies (RE) were in the range of -7.9-8.0%. Compared with pharmacokinetic parameters of G. uralensis administered orally, values of AUC and Cmax of liquiritigenin and isoliquiritigenin decreased significantly (p<0.05), plasma concentrations of glycyrrhizic acid rose slightly and bimodal phenomenon of concentration-time of isoliquiritigenin and glycyrrhetinic acid disappeared after combined administration. DISCUSSION AND CONCLUSIONS: Some components in the extract of processed radix A. kusnezoffii showed different effects on the pharmacokinetics of the four ingredients in G. uralensis.

Screening compounds of Chinese medicinal herbs anti-Marek's disease virus.

CONTEXT: Marek's disease (MD) seriously threatens the world poultry industry and has resulted in great economic losses. Chinese medicinal herbs are a rich source for lead compounds and drug candidates for antiviral treatments. OBJECTIVE: To investigate the anti-MDV activity and mechanism of 20 compounds extracted from Chinese medicinal herbs. MATERIALS AND METHODS: Antiviral assay, time of addition experiments, and virucidal assay were performed on chicken embryo fibroblast cells. The 50% cytotoxic concentration and 50% effective concentration were determined and, accordingly, selectivity index and inhibition ratio were calculated. RESULTS: Antiviral assay showed dipotassium glycyrrhizinate (DG) and sodium tanshinone IIA sulfonate (STS) exhibited significantly inhibitory activity against MDV in a dose-dependent manner. EC50 of DG and STS were 893.5 ± 36.99 µg/mL and 54.82 ± 2.99 µg/mL, and selective index (SI) were >3.36 and >9.12, respectively. Time of addition experiment and virucidal assay demonstrated DG inhibited viral replication in the full replication cycle and inactivated MDV particles in non-time-dependent manner, but STS interfered with the early stage of MDV replication and inactivated MDV particles in a time-dependent manner. Moreover, both DG and STS promoted apoptosis of cells infected by MDV. DISCUSSION AND CONCLUSION: DG and STS have great potential for developing new anti-MDV drugs for clinic application.

Distribution patterns of the contents of five active components in taproot and stolon of Glycyrrhiza uralensis.

Wild or cultivated Glycyrrhiza uralensis FISCHER (G. uralensis) are the main source of licorice, and they contain the similar compounds, such as the triterpenoid saponins and flavonoids, but above two kinds of the components contents are low level in the cultivated licorice. To produce the high quality cultivated licorices, researchers studied the affecting factors about the compounds producing in the plant of licorice, and then found that the growth years, genetic differences and water deficit are all the important factors. In this paper, we found that there were different distribution patterns of the main five active components (FAC) including glycyrrhizin, liquiritin, isoliquiritin, liquiritigenin and isoliquiritigenin in the taproot and stolon of G. uralensis and maybe they are also important influence factors to the FAC contents of the licorices. In wild G. uralensis, the contents of FAC tended to be lower in the younger parts of the stolon, and in the cultivated G. uralensis taproot, the contents of glycyrrhizin, liquiritin and isoliquiritin tended to increase from top to end, contrary to the contents of liquiritigenin and isoliquiritigenin, which increased first and then decreased. Our results will contribute to the analyses of factors which influence the quality of licorice, and provide some reference for cultivating high quality licorices for herbal medicine.

Simultaneous quantification and inhibitory effect on LDL oxidation of the traditional Korean medicine, Leejung-tang.

BACKGROUND: Leejung-tang (LJT) is a traditional Korean herbal medicine for the treatment of gastrointestinal disorders. In this study, we performed quantification analysis of five marker components, liquiritin (1), ginsenoside Rg1 (2), ginsenoside Rb1 (3), glycyrrhizin (4), and 6-gingerol (5) in LJT using a high performance liquid chromatography-photodiode array (HPLC-PDA). In addition, we investigated the inhibitory effect on low-density lipoprotein (LDL) oxidation by the LJT sample. METHODS: Compounds 1-5 were separated within 35 min using a Gemini C18 column. The mobile phase used gradient elution with 1.0% (v/v) aqueous acetic acid (A) and 1.0% (v/v) acetic acid in acetonitrile (B). The flow rate was 1.0 mL/min and the detector was a photodiode array (PDA) set at 203 nm, 254 nm, and 280 nm. The inhibitory effect on LDL oxidation conduct an experiment on thiobarbituric acid reactive substance (TBARS) assay, relative electrophoretic mobility (REM) assay, and electrophoresis of ApoB fragmentation of LJT. RESULTS: Calibration curves of compounds 1-5 showed good linearity (r(2) ≥0.9995) in different concentration ranges. The recoveries of compounds 1-5 were in the range of 98.90-103.39%, with relative standard deviations (RSD) below 3.0%. The RSDs (%) of intra-day and inter-day precision were 0.10-1.08% and 0.29-1.87%, respectively. The inhibitory effect of LJT on Cu(2+)-induced LDL oxidation was defined by TBARS assay (IC50: 165.7 µg/mL) and REM of oxLDL (decrease of 50% at 127.7 µg/mL). Furthermore LJT reduced the fragmentation of ApoB of oxLDL in a dose-dependent manner. CONCLUSIONS: The established HPLC-PDA method will be helpful to improve quality control of LJT. In addition, LJT is a potential LDL oxidation inhibitor.

[Simultaneous separation and detection of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate by RP-HPLC and structure confirmation].

A simple, fast and sensitive analytical method for the simultaneous separation and detection of 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B by RP-HPLC and drug quality standard was established. The structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate have been confirmed. Reference European Pharmacopoeia EP7.0 version, British Pharmacopoeia 2012 version, National Drug Standards of China (WS 1-XG-2002), domestic and international interrelated literature were referred to select the composition of mobile phase. The experimental parameters including salt concentration, pH, addition quantities of organic solvent, column temperature and flow rate were optimized. Finally, the assay was conducted on a Durashell-C18 column (250 mm x 4.6 mm, 5 microm) with 0.01 mol x mL(-1) ammonium perchlorate (add ammonia to adjust the pH value to 8.2) -methanol (48 : 52) as mobile phase at the flow rate of 0.8 mL x min(-1), and the detection wavelength was set at 254 nm. The column temperature was 50 degrees C and the injection volume was 10 microL. The MS, NMR, UV and RP-HPLC were used to confirm the structures of principal component isomer and related substances of raw material drug of ammonium glycyrrhizinate. Under the optimized separation conditions, the calibration curves of 18 alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B showed good linearity within the concentration of 0.50-100 microg x mL(-1) (r = 0.999 9). The detection limits for 18alpha-glycyrrhizinic acid, 18beta-glycyrrhizinic acid, related substance A and related substance B were 0.15, 0.10, 0.10, 0.15 microg x mL(-1) respectively. The method is sensitive, reproducible and the results are accurate and reliable. It can be used for chiral resolution of 18alpha-glycyrrhizinic acid, 18Pbeta-glycyrrhizinic acid, and detection content of principal component and related substances of raw material drug of ammonium glycyrrhizinate. It is concluded that the separation of principal component isomer of raw material drug of ammonium glycyrrhizinate and the validity of the substance's structure assignments of retention time being 1.2 in the European pharmacopoeia EP7.0 version, British pharmacopoeia 2012 version remains open to question. It may be of practical value for the quality control of raw material drug, preparation, and Chinese herbal medicine of ammonium glycyrrhizinate.

Glycyrrhizic acid as the antiviral component of Glycyrrhiza uralensis Fisch. against coxsackievirus A16 and enterovirus 71 of hand foot and mouth disease.

ETHNOPHARMACOLOGICAL RELEVANCE: The radices of Glycyrrhiza uralensis Fisch. and herbal preparations containing Glycyrrhiza spp. have been used for thousands of years as an herbal medicine for the treatment of viral induced cough, viral hepatitis, and viral skin diseases like ulcers in China. Glycyrrhizic acid (GA) is considered the principal component in Glycyrrhiza spp. with a wide spectrum of antiviral activity. AIM: The present study attempt to validate the medicinal use of Glycyrrhiza uralensis for hand, foot and mouth disease (HFMD) and further to verify whether GA is an active antiviral component in the water extract of Glycyrrhiza uralensis. MATERIALS AND METHODS: Radices of Glycyrrhiza uralensis Fisch. were extracted with hot water. The chemical contents of the extract were profiled with HPLC analysis. The antiviral activity of the extract and the major components was evaluated against infection of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) on Vero cells. The cytopathic effect caused by the infection was measured with MTT assay. Infectious virion production was determined using secondary infection assays and viral protein expression by immunoblotting analysis. RESULTS: The extract at 1000 µg/ml suppressed EV71 replication by 1.0 log and CVA16 by 1.5 logs. The antiviral activity was associated with the content of GA in the extract since selective depletion of GA from the extract by acid precipitation resulted in loss of antiviral activity. In contrast, the acid precipitant retained antiviral activity. The precipitant at a concentration of 200 µg/ml inhibited EV71 and CVA16 replication by 1.7 and 2.2 logs, respectively. Furthermore, GA dose-dependently blocked viral replication of EV71 and CVA16. At 3 mM, GA reduced infectious CVA16 and EV71 production by 3.5 and 2.2 logs, respectively. At 5mM, CVA16 production was reduced by 6.0 logs and EV71 by 4.0 logs. Both EV71 and CVA16 are members of Enterovirus genus, time-of-drug addition studies however showed that GA directly inactivated CVA16, while GA anti-EV71 effect was associated with an event(s) post virus cell entry. CONCLUSIONS: This study validated the medicinal usefulness of radices Glycyrrhiza uralensis against the etiological agents of HFMD. In addition to the identification of GA as the antiviral component of Glycyrrhiza uralensis against EV71 and CVA16 infection, this study also reveals that GA inhibits EV71 and CVA16 with distinct mechanisms.

Simultaneous quantitative determination of nine active chemical compositions in traditional Chinese medicine Glycyrrhiza by RP-HPLC with full-time five-wavelength fusion method.

A new, simple, accurate and reliable full-time five-wavelength fusion method for the simultaneous separation and determination of nine active chemical compositions (liquiritin apioside, liquiritin, isoliquiritin apioside, ononin, isoliquiritin, liquiritigenin, calycosin, isoliquiritigenin, Glycyrrhizic acid monoammonium salt) in traditional Chinese medicine Glycyrrhiza was developed using reverse phase high-performance liquid chromatography (RP-HPLC) coupled with a diode-array detector (DAD). The chromatographic separation was performed on an Agilent TC-C18 column with gradient elution using 0.04% methanoic acid (A) and acetonitrile (B) at a flow rate of 1.0 mL min(-1) and UV detection at 248 nm, 250 nm, 276 nm, 362 nm, 370 nm. The standard curves were linear over the range of 2.1379-12.8272 µg for liquiritin apioside, 3.9299-23.5794 µg for liquiritin, 1.0432-6.2592 µg for isoliquiritin apioside, 0.8764-5.8584 µg for ononin, 1.0701-6.4205 µg for isoliquiritin, 1.3685-8.2111 µg for liquiritigenin, 0.3927-2.3563 µg for calycosin, 0.2498- 1.4986 µg for isoliquiritigenin, 2.0094-12.0564 µg for Glycyrrhizic acid monoammonium salt, respectively (r(2) > 0.9997). The recoveries and relative standard deviation (RSD) varied from 95.09% to 103.54% and 1.09% to 2.36%, respectively. The precision for all the analytes was less than 2.52%. The method indicated good performance in terms of precision, accuracy and linearity. The method enabled the simultaneous determination of nine active chemical compositions for quality control of Glycyrrhiza.

A green and efficient protocol for large-scale production of glycyrrhizic acid from licorice roots by combination of polyamide and macroporous resin adsorbent chromatography.

A green and efficient method for large-scale preparation of glycyrrhizic acid from licorice roots was developed by combination of polyamide and macroporous resin. The entire preparation procedure consisted of two simple separation steps. The first step is to use polyamide resin to remove licorice flavoniods from the licorice crude extract. Subsequently, various macroporous resins were tried to purify glycyrrhizic acid, and HPD-400 showed the most suitable adsorption and desorption properties. Under the optimized conditions, a large-scale preparation of glycyrrhizic acid from licorice roots was carried out. A 20 kg raw material produced 0.43 kg of glycyrrhizic acid using green aqueous ethanol as the solvent. The purity of glycyrrhizic acid was increased from 11.40 to 88.95% with a recovery of 76.53%. The proposed method may be also extended to produce large-scale other triterpenoid saponins from herbal materials.

[Rat intestinal absorption trait of peimine and peiminine in Thunberg fritillary bulb extract].

To study the in situ intestinal absorption kinetics and compatibility influence of peimine and peiminine in rats, the absorption of peimine and peiminine in small intestine (duodenum, jejunum and ileum) and colon of rats was investigated using in situ single-pass perfusion method and the drug content was measured by HPLC-ELSD. Perfusion rate, pH, concentration of drug, gender and bile duct ligation can significantly affect the absorption of peimine and peiminine, the Ka, and Papp values in the condition of pH 6.8 and pH 7.4 had significant difference (P<0.01), as drug concentration irlcreased, the absorption parameters of peimine and peiminine decreased, Ka and Papp between low concentrations and middle concentrations was significant difference (P<0.01). Verapamil can not affect Ka and Papp of peimine and peiminine which are in the extract (P> 0.05). Bitter almonds and licorice can significantly reduce the absorption of peimine and peiminine with the usual dose (P<0.01), extracted separately and together had no significant difference on Ka and Papp (P> 0.05). Experimental results show that the absorption features of peimine and peiminine are basically the same, both of them could be absorbed at all segments of the intestine in rats and had no special absorption window, and with significant differences between male and female individuals. The absorption of peimine and peiminine complies with the active transport and facilitated diffusion in the general intestinal segments. Bitter almond and licorice can reduce the intestinal absorption rate ofpeimine and peiminine.