Intellectual Property Protection and High Quality Development of Radix Glycyrrhizae for Treatment of COVID-19

Radix Glycyrrhizae is the dominant native medicinal material variety in the north and northwestern medicinal materials producing areas.It is a main Chinese medicine of effective TCM drugs and formulas for the treatment of new coronavirus disease(COVID-19).This paper introduces the medicinal value of Radix Glycyrrhizae,involving the labeling,cultural heritage,and creative intellectual property rights of the Chinese medicinal materials,analyzes the poverty-stricken areas that are ecologically suitable for Radix Glycyrrhizae cultivation,and the superior counties and production bases of the Chinese medicinal materials.Besides,mainly from the aspects of perfect intellectual property rights,the establishment of authentic medicinal material production bases,and the construction of quality control systems,etc.,it discusses the rural revitalization strategy and the development strategy of traditional Chinese medicine(TCM),agricultural intellectual property protection and high-quality development strategy of Radix Glycyrrhizae.

A systematic summary of natural compounds in Radix Glycyrrhizae

Objective： To accumulate data from studies on the compounds identified from Gancao （Radix Glycyrrhizae, GC）, andthen systematically summarize and classify these compounds according to their structural characteristics. Methods： Fivedatabases （CNKI, VIP, Wanfang data, CBM, and Pubmed） were used to search for studies on the chemical structure ofcompounds from GC. The retrieval time of the respective databases was from their inception to March 2016. Accordingto the inclusion and exclusion criteria, the papers were carefully screened, and the data were extracted. Results： A totalof 653 compounds from GC were collated from 252 articles, including flavonols （n = 201） belonging to 10 subgroups,terpenes and saponins （n = 167）, coumarins （n = 30） belonging to 3 subgroups, aliphatics （n = 206）, aromatics （n = 35）,and others （n = 14）. The flavones, represented by liquiritin and liquiritigenin, were the most reported compounds isolatedfrom GC, followed by terpenes and saponins such as glycyrrhizic acid and glycyrrhetinic acid. Conclusion： The morethan 600 natural compounds in GC may be responsible for GC’s anti-inflammatory, phlegm-expelling, cough- andpain-relieving activities.

Simultaneous Determination of Saponins in Radix Glycyrrhizae, Notoginseng and Ginseng by High Performance Liquid Chromatography

To establish a method for determining five saponins(notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, ginsenoside Rb1 and ammonium glycyrrhizinate) in Glycyrrhizae, Notoginseng and Ginseng, the high performance liquid chromatography with diode array detector(HPLC-DAD) method was applied to an Inertsil ODS-SP column(4.6 mm×250 mm, 5 μm) with a mobile phase consisting of acetonitrile-0.05% phosphoric acid in a gradient elution manner. The flow rate was 1.0 mL/min. The column temperature was 30 ℃ and the detection wavelengths were 203 nm and 237 nm, respectively. The linear ranges were 0.700,0—7.000,0 μg for R1(r=1.000,0), 0.751,1— 7.511,4 μg for Rg1(r=1.000,0), 0.677,2—6.771,6 μg for Re(r=1.000,0), 0.733,9—7.339,1 μg for Rb1(r= 1.000,0), and 0.540,0—5.399,8 μg for ammonium glycyrrhizinate(r=0.999,9), respectively. In addition, their average recoveries were 100.28%, 105.83%, 104.09%, 99.36% and 98.54%, respectively. The relative standard deviations(RSDs) of precision, reproducibility and recovery were all less than 1.5%. The results indicate that the method is simple, accurate and reproducible so that it can be used for the simultaneous determination of the five saponins in Chinese patent medicines containing the three kinds of herbs.

Screening for cyclooxygenase 2 inhibitors from natural compounds of Radix Glycyrrhizae using computer simulation

Objective： To explore the molecular basis of the effects of Gancao （Radix Glycyrrhizae, GC） on inflammation throughthe inhibition of cyclooxygenase 2 （COX-2）. Methods： The Discovery Studio 4.5 System was used to predict thephysicochemical properties of GC molecular compounds. The Ligand Profiler was used to screen for natural GCcomponents that could combine with the COX-2 pharmacophores. The AutoDock Vina 1.1.2 software was used for themolecular docking of the natural GC components with the COX-2 protein. Results： The aromatics were the closest to thenon-steroidal anti-inflammatory drugs in terms of the three properties, namely molecular weight, molecular surface area,and molecular solubility, followed by the flavonoids; whereas the terpenoids/saponins differed most from thenon-steroidal anti-inflammatory drugs in terms of the three properties; and the aliphatics were inconsistent. One hundredand eighteen small molecules were obtained through the pharmacophore screening using GC. The molecular bindingenergy （MBE） results demonstrated that the MBE value of the flavonoids/aromatics, obtained from their binding with theCOX-2 protein, was lower than that obtained from their binding with the substrate, metabolism of arachidonic acid,whereas the MBE value of the aliphatics/terpenoids, obtained from their binding with the COX-2 protein, was higherthan that obtained from their binding with the substrate, arachidonic acid. Finally, further filtration, based on thephysicochemical properties and the molecular binding energies of the small molecules, was carried out. Forty-twonatural GC components, including 35 flavonoid and 7 aromatic constituents, with low binding energies and potentialinhibitory effects on COX-2, were screened. Conclusion： Using the three-step program, pharmacophore screening,molecular docking, and physicochemical properties analysis, we screened out 35 flavonoid molecules and 7 aromaticmolecules, which may be potential COX-2 inhibitors, from GC. Two of the 35 flavonoid molecules （licochalcone A andglabridin） have been confirmed in the laboratory to have inhibitory effects on COX-2. Our findings provide a materialbasis for the development of non-steroidal GC drugs.

Variations in the Composition of Fructus Evodiae after Processing with Radix Glycyrrhizae Extract

Objective： To evaluate the changes in the concentrations of five components in Fructus Evodiae used in Chinese medicine, including evodiamine and glycyrrhizic acid, during processing of Fructus Evodiae with Radix Glycyrrhizae extract by using high performance liquid chromatography （HPLC） and to provide a scientific basis for different clinical uses of processed and unprocessed Fructus Evodiae. Methods： The concentrations of the Fructus Evodiae components in processed or unprocessed Fructus Evodiae were evaluated by HPLC using a YMC J＇sphere ODS-H80 column （4.6 mm × 250 ram, 5 μm） with acetonitrile-water-tetrehydrofuran-acetic acid （41：59：1：0.2, v/v/v/v） as the mobile phase. The detection wavelength was 225 nm, the column temperature was 35 %, the flow rate was 1.0 mL/min, and the injection volume was 10 μL. The concentrations of the Radix Glycyrrhizae components were determined by HPLC with a KromasiI-C18 column （4.6 mm × 250 mm, 4 μm） and a gradient elution of acetonitrile （A） and 0.05% aqueous phosphoric acid （B） as the mobile phase. The detection wavelength was 237 nm, the column temperature was 35 ℃, the flow rate was 1.0 mlEmin, and the injection volume was 10μL. Results： The calibration curves of evodia lactone, evodiamine, rutaecarpine, liquiritin, and glycyrrhizin showed good linear relationships （r〉0.99）. The recoveries of evodia lactone, evodiamine, rutaecarpine, liquiritin, and glycyrrhizin were 96.59%, 104.18%, 101.91%, 97.75%, and 97.95%, respectively. The concentrations of the components in processed Fructus Evodiae were obviously different to those in unprocessed Fructus Evodiae. Conclusions： The developed method is rapid and accurate. The results provide a reference for processed Fructus Evodiae and the changes that could be expected in its effects compared to unprocessed Fructus Evodiae.

Identification of potential anti-pneumonia pharmacological components of Glycyrrhizae Radix et Rhizoma after the treatment with Gan An He Ji oral liquid

Glycyrrhizae Radix et Rhizoma,a traditional Chinese medicine also known as Gan Cao(GC),is frequently included in clinical prescriptions for the treatment of pneumonia.However,the pharmacological components of GC for pneumonia treatment are rarely explored.Gan An He Ji oral liquid(GAHJ)has a simple composition and contains GC liquid extracts and paregoric,and has been used clinically for many years.Therefore,GAHJ was selected as a compound preparation for the study of GC in the treatment of pneumonia.We conducted an in vivo study of patients with pneumonia undergoing GAHJ treatments for three days.Using the intelligent mass spectrometry data-processing technologies to analyze the metabolism of GC in vivo,we obtained 168 related components of GC in humans,consisting of 24 prototype components and 144 metabolites,with 135 compounds screened in plasma and 82 in urine.After analysis of the metabolic transformation relationship and relative exposure,six components(liquiritin,liquiritigenin,glycyrrhizin,glycyrrhetinic acid,daidzin,and formononetin)were selected as potential effective components.The experimental results based on two animal pneumonia models and the inflammatory cell model showed that the mixture of these six components was effective in the treatment of pneumonia and lung injury and could effectively downregulate the level of inducible nitric oxide synthase(iNOS).Interestingly,glycyrrhetinic acid exhibited the strongest inhibition on iNOS and the highest exposure in vivo.The following molecular dynamic simulations indicated a strong bond between glycyrrhetinic acid and iNOS.Thus,the current study provides a pharmaceutical basis for GC and reveals the possible corresponding mechanisms in pneumonia treatment.

Simultaneous determination of five major components of Glycyrrhizae Radix et Rhizoma in Xiaoer Zhike Tangjiang with standardized reference extract

In this study, we developed a novel and simple HPLC-DAD method for simultaneous qualitative and quantitative determination of five major components of Glycyrrhizae Radix et Rhizoma （GRR） in Xiaoer Zhike Tangjiang （XEZKTJ） with standardized reference extract （SRE）. The five analytes （liquiritin apioside, liquiritin, isoliquiritin apioside, liquirigenin and glycyrrhizic acid） were well separated with good linearity, precision, stability and repeatability. The recovery rates ranged from 95.69% to 100.80%. The content of the five compounds in 34 batches of commercial XEZKTJ products was determined using standardized GRR extract （SRE method） and individual chemical reference standards （CRS method）. Highly similar results were obtained from the two methods, demonstrating the feasibility of the proposed SRE method. Taken together, we proposed an efficient and low-cost way to perform multi-component quality control of XEZKTJ in this study.

Metabolomics of Fuzi-Gancao in CCl4 induced acute liver injury and its regulatory effect on bile acid profile in rats

BACKGROUND Fuzi(Radix aconiti lateralis)-Gancao(Radix glycyrrhizae)is one of the most classical drug pairs of traditional Chinese medicine.In clinical practice,decoctions containing Fuzi-Gancao(F-G)are often used in the treatment of liver diseases such as hepatitis and liver failure.AIM To investigate the metabolomics of F-G in CCl4 induced acute liver injury in rats and its regulatory effect on the bile acid profile.METHODS The pharmacodynamic effect of F-G on CCl4 induced acute liver injury in rats was evaluated,and an ultra performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the simultaneous determination of 92 metabolites from multiple pathways was established to explore the protective metabolic mechanism of F-G in serum on the liver.RESULTS Twenty-four differential metabolites were identified in serum samples.The primary bile acid biosynthetic metabolic pathway was the major common pathway in the model group and F-G group.Subsequently,a UPLC-MS/MS method for simultaneous determination of 11 bile acids,including cholic acid,ursodeoxycholic acid,glycochenodeoxycholic acid,glycochenodeoxycholic acid,taurocholic acid,glycocholic acid,chenodeoxycholic acid,deoxycholic acid,taurochenodeoxycholic acid,taurocholic acid,and glycinic acid,was established to analyze the regulatory mechanism of F-G in serum.F-G decreased the contents of these 11 bile acids in serum in a dose-dependent manner compared with those in the model control group.CONCLUSION F-G could protect hepatocytes by promoting the binding of free bile acids to glycine and taurine,and reducing the accumulation of free bile acids in the liver.F-G could also regulate the compensatory degree of taurine,decreasing the content of taurine-conjugated bile acids to protect hepatocytes.

An Herbal Formula (GPC) Suppresses the Releases of Pro-Inflammatory Effectors in Lipopolysaccharide/Peptidoglycan-Activated RAW264.7 Macrophages and Reduces the Extent of Chemical-Induced Acute/Chronic Inflammation in Rodents

Pharyngitis (an inflammation in the pharynx) is a commonly occurring symptom of upper respiratory tract infection in patients suffering from common cold. The high prevalence of upper respiratory tract infection necessitates a safe and effective antiinflammatory agent for pharyngitis. Chinese herbal medicine, which has been clinically prescribed for thousands of years, may offer a basis for the treatment of common cold as well as the amelioration of pharyngitis. In the present study, we investigated the in vitro and in vivo anti-inflammatory activities of a Chinese herbal formula, namely GPC, which is comprised of Glycyrrhiza Radix, Platycodonis Radix, Citri Reticulatae Pericarpium, Phyllanthi Fructus and Taraxaci Herba. Incubation with GPC (30, 100 and 300 μg/mL) suppressed the releases of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and nitric oxide in lipopolysaccharide/peptidoglycanactivated RAW264.7 macrophages. In addition to the cell-based study, long-term treatment with GPC (0.35, 1.05 and 2.10 mL/kg/day × 30 doses) was found to reduce the extent of inflammation in animal models of carrageenan-induced paw edema (acute inflammation) as well as cotton pellet-induced granuloma formation (chronic inflammation) in mice. The ability of GPC to enhance the tracheobronchial expectorant action suggested its immunomodulatory activity in the respiratory tract. This postulation was supported by the observation that GPC reduced the degree of pharyngitis and reversed the changes in plasma TNF-α and IL-6 levels in capsaicininduced pharyngitis in rats. The ensemble of results suggests that GPC may offer a promising prospect for alleviating the extent of pharyngitis by virtue of antiinflammatory activities.

Identification and comparative analysis of the major chemical constituents in the extracts of single Fuzi herb and FuziGancao herb-pair by UFLC-IT-TOF/MS

AIM: The aim of this work was to establish a specific and sensitive method to comprehensively investigate and compare chemical constituents of Fuzi-Gancao herb pair(FG), consisting of Aconitum carmichaelii Debeaux(Fuzi, Chinese) and Roast Radix Glycyrrhizae(Glycyrrhiza glabra L., Gancao, in Chinese) and Fuzi alone to explore the underlying interaction mechanism of FG. METHOD: An ultra-fast liquid chromatography-ion trap/time-of-flight mass spectrometry(UFLC/MS-IT-TOF) method using diazepam as internal standard was developed for the identification and semi-quantitative analysis of the phytochemical constituents of Fuzi and FG. Chromatographic separation was achieved on a UFLC column using a gradient program with 40 mmol?L-1 ammonium acetate and acetonitrile as the mobile phase. RESULTS: Fifty-one of the sixty compounds, including forty-five C19-diterpenoid alkaloids and six C20-diterpenoid alkaloids were tentatively identified in the extracts of Fuzi and FG through accurate mass measurements and fragmentation patterns. Comparing the contents of these alkaloids in these two extracts, it was found that the diester-diterpenoid alkaloids(DDAs) and the alkylolamine-diterpenoid alkaloids(ADAs) were increased, while the monoester-diterpenoid alkaloids(MDAs) were decreased in the extracts of FG. CONCLUSION: This work provided comprehensive information for the quality control of Fuzi preparations, and the further investigation on the compatibility mechanisms of FG.

Comprehensive Metabolic Profiling of Modified Gegen Qinlian Decoction by Ultra-high-Performance Liquid Chromatography-Diode Array Detection-Q-Exactive-Orbitrap-Electrospray Ionization-Mass Spectrometry/Mass Spectrometry and Application of High-Performance

Objective:Gegen Qinlian decoction(GQD)is a classical traditional Chinese medicine formulation which has been used for almost 2000 years.At Guang'anmen Hospital,Beijing,a modified GQD version(m GQD)with seven instead of four herbal ingredients has been applied to treat Type 2 diabetes.Quality control is a crucial prerequisite for the therapeutic application of herbal medicines.For the identification of products derived from classical GQD,the Chinese Pharmacopeia requires the analysis of only three marker compounds.Because m GQD is a more complex mixture containing seven herbs and hundreds of constituents,the pharmacopoeia method for GQD is inadequate.Materials and Methods:A more comprehensive characterization of the formula's constituents has been developed using ultra-high-performance liquid chromatography-diode array detection(UHPLC-DAD)-Q-Exactive-mass spectrometry(MS)in electrospray ionization positive and negative mode.Moreover,a new method for the fingerprint analysis of m GQD via high-performance thin-layer chromatography(HPTLC)has been established.Results:Altogether,91 compounds have been assigned to their originating plants and 84 substances were identified either by comparison with authentic references or with data from the literature.The HPTLC method is based on the application of two different mobile phases and is able to detect both lipophilic and hydrophilic constituents of m GQD.Conclusions:The modified GQD was extensively characterized by UHPLC combined with DAD and Q-Exactive Orbitrap high-resolution MS detection,leading to the assignment and identification of compounds present in the decoction.In addition,a new method for the fingerprint analysis of the m GQD using HPTLC was established,which allows fast and simple identification of the herbal ingredients in the mixture.