Spatio-temporal selection of reference genes in the two congeneric species of Glycyrrhiza.

Glycyrrhiza, a genus of perennial medicinal herbs, has been traditionally used to treat human diseases, including respiratory disorders. Functional analysis of genes involved in the synthesis, accumulation, and degradation of bioactive compounds in these medicinal plants requires accurate measurement of their expression profiles. Reverse transcription quantitative real-time PCR (RT-qPCR) is a primary tool, which requires stably expressed reference genes to serve as the internal references to normalize the target gene expression. In this study, the stability of 14 candidate reference genes from the two congeneric species G. uralensis and G. inflata, including ACT, CAC, CYP, DNAJ, DREB, EF1, RAN, TIF1, TUB, UBC2, ABCC2, COPS3, CS, R3HDM2, were evaluated across different tissues and throughout various developmental stages. More importantly, we investigated the impact of interactions between tissue and developmental stage on the performance of candidate reference genes. Four algorithms, including geNorm, NormFinder, BestKeeper, and Delta Ct, were used to analyze the expression stability and RefFinder, a comprehensive software, provided the final recommendation. Based on previous research and our preliminary data, we hypothesized that internal references for spatio-temporal gene expression are different from the reference genes suited for individual factors. In G. uralensis, the top three most stable reference genes across different tissues were R3HDM2, CAC and TUB, while CAC, CYP and ABCC2 were most suited for different developmental stages. CAC is the only candidate recommended for both biotic factors, which is reflected in the stability ranking for the spatio (tissue)-temporal (developmental stage) interactions (CAC, R3HDM2 and DNAJ). Similarly, in G. inflata, COPS3, R3HDM2 and DREB were selected for tissues, while RAN, COPS3 and CS were recommended for developmental stages. For the tissue-developmental stage interactions, COPS3, DREB and ABCC2 were the most suited reference genes. In both species, only one of the top three candidates was shared between the individual factors and their interactions, specifically, CAC in G. uralensis and COPS3 in G. inflata, which supports our overarching hypothesis. In summary, spatio-temporal selection of reference genes not only lays the foundation for functional genomics research in Glycyrrhiza, but also facilitates these traditional medicinal herbs to reach/maximize their pharmaceutical potential.

Transcriptome and complete chloroplast genome of Glycyrrhiza inflata and comparative analyses with the other two licorice species.

In this study, we characterized the transcriptome and chloroplast genome of Glycyrrhiza inflata and performed comparative analyses with G. uralensis and G. glabra. 60,541unigenes were obtained from the transcriptome of G. inflata. The results of function annotation revealed a similar distribution of functional categories among three licorice species. By comparing chloroplast genomes of licorice species, it was demonstrated that the structure and the length of genome as well as gene content and gene order were highly similar. The phylogenetic tree, constructed with the mixed data of transcriptome and chloroplast genome, elucidated that G. inflata and G. glabra had a closer relationship than G. uralensis. Six regions were suggested as potential markers for the identification of three licorice species. In each licorice species, two unigenes were homologous to reference flavonol synthase. For G. inflata, 48 and 21 RNA editing sites were detected by PREP-Cp program and RNA-Seq data mapping, respectively.

Biological Effects of Licochalcones.

Medicinal plants and their natural bioactive molecules, are evaluated as the foundation for health preservation and care of humanity. The licorice root, known as "Radix Glycyrrhizae", is a perennial plant that comes from Mediterranean countries, central to southern Russia, Asia, Turkey, Iraq and Iran. The licorice root has been used in traditional Chinese medicines for centuries and has been defined as "the progenitor of herbs". The name 'Licorice' is derived from the ancient Greek word Glukurrhiza, meaning 'sweet root'. It consists of approximately 30 species, however, the most common ones consist of Glycyrrhiza glabra L., Glycyrrhiza uralensis Fisch and Glycyrrhiza Inflata. In addition, the licorice root contains chalcones, which are a part of an important class of natural products and are precursors of flavonoids. Chemically, chalcones are composed of two aromatic rings associated with α, ß-unsaturated α-carbon ketone, representing the prima nucleus of the structure. They have been classified, according to chemical structures, in Licochalcone A, B, C, D, E, F and G. This review aims to highlight all the in vitro and in vivo studies that have been conducted on the licochalcones, extracted from Glycyrrhiza species. The main effects are as follows: anti-inflammatory, antioxidant, anticancer, antimicrobial, antiviral, antiallergic, antidiabetic, hepatotoxic and osteogenic. It is important to implement the introduction of biologically active natural molecules from the bench (research) to the bedside (clinical practice). However, in the future, it is required to conduct additional studies to validate these biological effects.

Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model.

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used animal model for studying stroke is the middle cerebral artery occlusion (MCAO) model. Previous research studies have reported different infarct sizes even when the same experimental animal species was used under similar MCAO conditions. Therefore, we developed an improved experimental method to address this discrepancy. Mice were subjected to MCAO using a filament as the occlusion material to mimic human stroke conditions and filament thickness was optimized to establish more reproducible infarction volume. Mice treated with a methanol extract of Glycyrrhizae Radix et Rhizome (GRex) following stroke induction showed a significantly decreased total infarction volume and increased number of surviving cells relative to the untreated control group. This modified experimental protocol successfully and reproducibly demonstrated the beneficial effect of GRex on ischemic stroke.

[The molecular identification of licorice species and the quality evaluation of licorice slices].

Licorice is one of the most common herbs in traditional Chinese medicine, and classified as top grade in Shen Nong Ben Cao Jing. There are three different original plants of licorice stipulated in Chinese Pharmacopeia, Glycyrrhiza uralensis Fisch., Glycyrrhiza glabra L., and Glycyrrhiza inflata Bat. However, previous investigation showed that the pharmacodynamic effects of the three licorices were quite different. It is very difficult to identify them by the classical identification methods. In order to establish a fast and effective identification method, we collected 240 licorice plants from 21 populations of 7 provinces, and amplified their ITS and psbA-trnH sequences. ITS sequences with a full length of 616 bp and psbA-trnH sequences with a full length of 389 bp were obtained separately. Using DNAMAN to analyze these sequences, 4 variable sites were found in ITS sequences and 2 ITS haplotypes were determined, and 3 variable sites were found in psbA-trnH sequences and 4 psbA-trnH haplotypes were determined. With the combination analysis of ITS and psbA-trnH sequences, the molecular identification method of original licorice was established. Using this method, 40 samples of licorice slices collected from 4 main herbal material markets in China were identified successfully. Furthermore, the contents of 2 triterpenes, 18α-glycyrrhizic acid and 18ß-glycyrrhizic acid, and 4 flavonoids, liquiritin, isoliquiritin, liquiritigenin, and isoliquiritigenin in these licorice pieces were examined by HPLC and the results were analyzed using SPSS 21.0. This study provides a new method in identification of licorice, which may serve as a guideline for quality control of licorice slices.

Molecular and ecological investigations on the wild populations of Glycyrrhiza L. taxa distributed in the East Mediterranean Area of Turkey.

This paper covers studies on the molecular and ecological aspects of G. glabra var. glandulifera, G. flavescens ssp. flavescens and G. echinata collected from Hatay (Turkey); with the aim to better understand their genetic variation and ecological requirements for possible conservation programs. The material including total genomic DNA was extracted by the CTAB, and for PCR reaction, a total of 14 SSR primers developed for Medicago truncatula were used. PCR amplifications were performed in a Multigen® Thermal Cycler. Soil samples were analysed for their texture, pH, total soluble salts, calcium carbonate, total N content, total phosphorus and organic matter content. In order to see the association between genetic, ecological and geographical data, a similarity matrix was generated. Genetic similarity distances between genotypes were correlated with those of Eucledian distances obtained from ecological and geographical data. Analysis of molecular variance (AMOVA) was performed using GenAlEx 6.5 software to determine variation among and within genetic variations. The genetic analysis showed that the highest expected heterozygosity values were obtained from G. glabra while the lowest were obtained from G. echinata. In general heterozygosity values were low, especially for G. echinata. Therefore, variation appears to be lower within each species than among three species. The physical and chemical analysis of soil and plant samples indicates that mineral accumulation in plants is substantially affected by the soil characteristics. There is a need for identification of better strategies for the improvement of varieties, especially for small farmers managing marginal soils. More studies should be conducted in order to safeguard these taxa, especially G. glabra var. glandulifera which is collected intensively due to its economic value, the same is true for endemic taxon G. flavescens ssp. flavescens.

Rapid determination of flavonoids in licorice and comparison of three licorice species.

A simple, sensitive and fast method for the simultaneous quantitation of 15 flavonoids in licorice based on an ultra high performance liquid chromatography coupled with triple quadrupole electrospray tandem mass spectrometry had been established and validated in this study. The analysis was performed on an ACQUITY HSS T3 column with gradient elution using a mobile phase consisted of A (0.1% formic acid in water)/B (acetonitrile) at a flow rate of 0.4 mL/min. Satisfactory separation of these compounds was obtained in less than 9 min. All calibration curves showed good linearity (r(2) = 0.9940) during the test ranges. The precision, repeatability, accuracy, limit of detection and limit of quantification were also fully investigated. The validated method was successfully applied for the simultaneous quantitation of 15 flavonoids in 106 licorice samples which contained 83 batches of G. uralensis, 14 batches of G. glabra and 9 batches of G. inflata. Furthermore, multivariate statistical analysis (using principal components analysis) was performed to classify the samples based on the contents of the 15 analyzed compounds. The results showed that all of these licorice samples were rich in flavonoids, although their contents were obviously various, and the proposed method could serve as a prerequisite for quality control of licorice products.

[Determination of the Plant Origin of Licorice Oil Extract, a Natural Food Additive, by Principal Component Analysis Based on Chemical Components].

"Licorice oil extract" (LOE) (antioxidant agent) is described in the notice of Japanese food additive regulations as a material obtained from the roots and/or rhizomes of Glycyrrhiza uralensis, G. inflata or G. glabra. In this study, we aimed to identify the original Glycyrrhiza species of eight food additive products using LC/MS. Glabridin, a characteristic compound in G. glabra, was specifically detected in seven products, and licochalcone A, a characteristic compound in G. inflata, was detected in one product. In addition, Principal Component Analysis (PCA) (a kind of multivariate analysis) using the data of LC/MS or (1)H-NMR analysis was performed. The data of thirty-one samples, including LOE products used as food additives, ethanol extracts of various Glycyrrhiza species and commercially available Glycyrrhiza species-derived products were assessed. Based on the PCA results, the majority of LOE products was confirmed to be derived from G. glabra. This study suggests that PCA using (1)H-NMR analysis data is a simple and useful method to identify the plant species of origin of natural food additive products.

Metabolite Profiling and Classification of DNA-Authenticated Licorice Botanicals.

Raw licorice roots represent heterogeneous materials obtained from mainly three Glycyrrhiza species. G. glabra, G. uralensis, and G. inflata exhibit marked metabolite differences in terms of flavanones (Fs), chalcones (Cs), and other phenolic constituents. The principal objective of this work was to develop complementary chemometric models for the metabolite profiling, classification, and quality control of authenticated licorice. A total of 51 commercial and macroscopically verified samples were DNA authenticated. Principal component analysis and canonical discriminant analysis were performed on (1)H NMR spectra and area under the curve values obtained from UHPLC-UV chromatograms, respectively. The developed chemometric models enable the identification and classification of Glycyrrhiza species according to their composition in major Fs, Cs, and species specific phenolic compounds. Further key outcomes demonstrated that DNA authentication combined with chemometric analyses enabled the characterization of mixtures, hybrids, and species outliers. This study provides a new foundation for the botanical and chemical authentication, classification, and metabolomic characterization of crude licorice botanicals and derived materials. Collectively, the proposed methods offer a comprehensive approach for the quality control of licorice as one of the most widely used botanical dietary supplements.

Anti-hepatitis C virus compounds obtained from Glycyrrhiza uralensis and other Glycyrrhiza species.

Development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still much needed from clinical and economic points of view. Antiviral substances obtained from medicinal plants are potentially good targets to study. Glycyrrhiza uralensis and G. glabra have been commonly used in both traditional and modern medicine. In this study, extracts of G. uralensis roots and their components were examined for anti-HCV activity using an HCV cell culture system. It was found that a methanol extract of G. uralensis roots and its chloroform fraction possess anti-HCV activity with 50%-inhibitory concentrations (IC(50)) of 20.0 and 8.0 µg/mL, respectively. Through bioactivity-guided purification and structural analysis, glycycoumarin, glycyrin, glycyrol and liquiritigenin were isolated and identified as anti-HCV compounds, their IC(50) being 8.8, 7.2, 4.6 and 16.4 µg/mL, respectively. However, glycyrrhizin, the major constituent of G. uralensis, and its monoammonium salt, showed only marginal anti-HCV activity. It was also found that licochalcone A and glabridin, known to be exclusive constituents of G. inflata and G. glabra, respectively, did have anti-HCV activity, their IC(50) being 2.5 and 6.2 µg/mL, respectively. Another chalcone, isoliquiritigenin, also showed anti-HCV activity, with an IC(50) of 3.7 µg/mL. Time-of-addition analysis revealed that all Glycyrrhiza-derived anti-HCV compounds tested in this study act at the post-entry step. In conclusion, the present results suggest that glycycoumarin, glycyrin, glycyrol and liquiritigenin isolated from G. uralensis, as well as isoliquiritigenin, licochalcone A and glabridin, would be good candidates for seed compounds to develop antivirals against HCV.

Species-specific Standardisation of Licorice by Metabolomic Profiling of Flavanones and Chalcones.

INTRODUCTION: Major phenolics from licorice roots (Glycyrrhiza sp.) are glycosides of the flavanone liquiritigenin (F) and its 2'-hydroxychalcone isomer, isoliquiritigenin (C). As the F and C contents fluctuate between batches of licorice, both quality control and standardisation of its preparations become complex tasks. OBJECTIVE: To characterise the F and C metabolome in extracts from Glycyrrhiza glabra L. and Glycyrrhiza uralensis Fisch. ex DC. by addressing their composition in major F­C pairs and defining the total F:C proportion. MATERIAL AND METHODS: Three types of extracts from DNA-authenticated samples were analysed by a validated UHPLC/UV method to quantify major F and C glycosides. Each extract was characterised by the identity of major F­C pairs and the proportion of Fs among all quantified Fs:Cs. RESULTS: The F and C compositions and proportions were found to be constant for all extracts from a Glycyrrhiza species. All G. uralensis extracts contained up to 2.5 more Fs than G. glabra extracts. Major F­C pairs were B-ring glycosidated in G. uralensis, and A-/B-ring apiosyl-glucosidated in the G. glabra extracts. The F:C proportion was found to be linked to the glycosidation site: the more B-ring F-C glycosides were present, the higher was the final F:C proportion in the extract. These results enable the chemical differentiation of extracts from G. uralensis and G. glabra, which are characterised by total F:C proportions of 8.37:1.63 and 7.18:2.82, respectively. CONCLUSION: Extracts from G. glabra and G. uralensis can be differentiated by their respective F and C compositions and proportions, which are both useful for further standardisation of licorice botanicals.

Comparison of three officinal Chinese pharmacopoeia species of Glycyrrhiza based on separation and quantification of triterpene saponins and chemometrics analysis.

The dried roots and rhizomes of Glycyrrhiza species, named licorice, have been utilized as food as well as crude drugs in China for thousands of years. Glycyrrhiza species can be differentiated based on the morphologic features of their aerial part, i.e. leaf and fruit morphologies, but not on their root morphology, even though that is the medicinal part. In this paper, a fast and effective UPLC-TQ-MS/MS method was explored for better identification and quantitative investigation of triterpene saponins in licorice, which laid basis for chemical comparison of three officinal Chinese pharmacopoeia Glycyrrhiza species. The results showed that all of these licorice samples were rich in triterpene saponins but with significant difference corresponding to different Glycyrrhiza species. The proposed method could be useful in quality control and standardization of licorice raw materials and its products.

[Classification and distribution analysis of components in Glycyrrhiza using licorice compounds database].

In order to study the chemical components of licorice deeply and systematically, a licorice compounds database was established after the comprehensive summary of the compounds in Glycyrrhiza. The database was used to classify the licorice components anew in order to statistically analyze the distribution of each type of compounds and the compounds in the medical Glycyrrhiza plants. The results indicated that 422 compounds had been reported in Glycyrrhiza so far, and they could be categorized into 5 classes as flavoids, coumarins, triterpenoids, stilbenoids, and some others. Up to now, 170 compounds were isolated from G. uralensis, 134 compounds from G. glabra, 52 compunds from G. inflata, and 31 from G. yunnanensis. It is the first time to add categorization "stilbenoids", and "dibenzoylmethanes" classified as chalcones. In the meantime, the database is supposed to be convenient for further study.

Comparative metabolite profiling and fingerprinting of medicinal licorice roots using a multiplex approach of GC-MS, LC-MS and 1D NMR techniques.

Glycyrrhiza glabra, commonly known as licorice, is a popular herbal supplement used for the treatment of chronic inflammatory conditions and possesses anticancer and antiviral activities. This species contains a plethora of phytochemicals including terpenoids, saponins, flavonoids, polyamines and polysaccharides. The full complement of bioactive compounds has yet to be elucidated, a step necessary in order to explain its medicinal use. There are over 30 species in the Glycyrrhiza genus world-wide, most of which have been little characterized in terms of phytochemical or pharmacological properties. Here, large scale multi-targeted metabolic profiling and fingerprinting techniques were utilized to help gain a broader insight into Glycyrrhiza species chemical composition. UV, MS and NMR spectra of extracted components were connected with NMR, MS, and multivariate analyses data from Glycyrrhiza glabra, Glycyrrhiza uralensis, Glycyrrhiza inflata and Glycyrrhiza echinata. Major peaks in (1)H NMR and MS spectra contributing to the discrimination among species were assigned as those of glycyrrhizin, 4-hydroxyphenyl acetic acid, and glycosidic conjugates of liquiritigenin/isoliquiritigenin. Primary metabolites profiling using GC-MS revealed the presence of cadaverine, an amino acid, exclusively found in G. inflata roots. Both LC-MS and NMR were found effective techniques in sample classification based on genetic and or geographical origin as revealed from derived PCA analysis.

[HPLC fingerprint spectrum of honey-fried Radix Glycyrrhizae].

OBJECTIVE: To establish the HPLC fingerprint of the pieces of honey-fried Radix Glycyrrhizae. METHOD: Using the reverse-performance liquid chromatography, method was performed on a Hyperclone ODS C18 column (4.6 mm x 250 mm, 5 microm) and acetonitrile-0.1% phosphoric acid was selected as mobile phase gradient elution were adopted. RESULT: Established HPLC fingerprint of Radix et Rhizoma glycyrrhizae pieces were established, and the results of methodological study met the technical requirements for fingerprinting. CONCLUSION: The HPLC method is stable, accurate, and reliable to provide a scientific basis of quality control standard for the honey-fried Radix et Rhizoma glycyrrhizae.

[Classification of licorice based on inorganic elements characteristics].

The present study focused on the ecological characteristics of medicinal plants Glycyrrhiza uralensis from two typical ecological environments with two different growth patterns respectively. The authors detected the contents of 16 kinds of inorganic elements in 24 licorice samples with two different producing areas (i. e. Gansu and Inner-Mongolia) and growth patterns (i. e., wild species and the cultivated), using the methods of ICP-MS and ICP-AES after microwave-assisted digestion. With the systematic analytic methods, including the analysis of total element distribution, Q-type cluster analysis of the characteristic elements, and the comparison of element and the ratio of two elements among the samples one by one, the authors constructed the inorganic element fingerprint chromatogram of G. uralensis based on the contents of the 16 inorganic elements (K, Ca, Na, Mg, P, Ca, Cr, Mn, Fe, S, Ni, Cu, Zn, Sr, Mo and Sn). Based on the characteristic elements selected by principal component analysis, the result of Q-type cluster analysis showed consistency with the growth pattern of licorice. By comparing the differences of the inorganic elements in different samples, the authors discovered that the combination of elements Mo and Sr not only provides the bases for the growth pattern of licorice, but also can be used as a diagnostic criteria for the division of its producing area. This study also indicated that the content ratios of Na : P and K : Ca can also provide reliable references for the assessment of different production patterns. It gives insight into the differences in the inorganic element of licorice with different producing area and production pattern treatments. In conclusion, the method we founded here turned out to be intuitive, informative, and highly accurate, and can be used to reveal the characteristic of inoganic elements in medicinal plant.

Identification of Glycyrrhiza species by direct analysis in real time mass spectrometry.

DART (Direct Analysis in Real Time)-MS is a novel mass spectrometric ion source, and allows the analysis of most compounds at ambient pressure and ground potential by producing [M+H]+ molecular ion species. Using this method, we examined the compounds characteristic of several kinds of licorices. For the analysis of Glycyrrhiza inflata Batalin, the peak at m/z 339 originates mainly from [M+H]+ of licochalcone A (LA), a species-specific compound. This peak was hardly detected in G. glabra Linné and G. uralensis Fischer. These results indicate that G. inflata can be differentiated from the other two species by detection of LA peaks using DART-MS analysis.

Anti-proliferative effect of licochalcone A on vascular smooth muscle cells.

Licochalcone A, a flavonoid found in licorice root (Glycyrrhiza glabra), is known for its anti-microbial activity and its reported ability to inhibit cancer cell proliferation. In the present study, we investigated whether licochalcone A inhibits rat vascular smooth muscle cell (rVSMC) proliferation. Our data indicate that 5 microM licochalcone A inhibited platelet-derived growth factor (PDGF)-induced rVSMC proliferation, possibly through its ability to block the progression of the cell cycle from G1 to S phase. In addition, 5 microM licochalcone A significantly inhibited the PDGF-induced expression of cyclin A, cyclin D1, CDK2, and CDK4, and the phosphorylation of Rb. Licochalcone A also reversed the decrease in p27(kip1) expression reduced by PDGF. Finally, licochalcone A inhibited the PDGF-induced activation of extracellular signal-regulated kinase (ERK)1/2. Together, these data provide the first evidence that licochalcone A can regulate rVSMC proliferation and suggest that licochalcone A inhibits the proliferation of rVSMCs by suppressing the PDGF-induced activation of the ERK1/2 pathway and Rb phosphorylation, resulting in cell cycle arrest.

[Study on the identification of standard and false Gancao by Fourier transform infrared spectroscopy].

Standard Gancao (Glycyrrhiza uralensis Fisch) and false Ciguogancao (Glycyrrhiza pallidiflata Batal) were identified fast, nondestructively by Fourier transform infrared spectroscopy (FTIR) combined with derivative spectra and two-dimensional correlation spectroscopy (2D) in the present article. The result shows that although the two kinds of Gancao belong to one genus, there are some certain differences in their chemical components that are reflected in the IR spectra, but with some similarity and dissimilarity in the IR spectra. The two kinds of Gancao are quite different from each other in second derivative spectra and 2D spectra. Based on the differences reflected in the IR and 2D IR, the standard gancao can be identified from the false easily and clearly. The result also proved that there is a relationship between the IR spectra and the chemical components of the herbs. This method is fast, accurate and nondestructive, and the wastage of sample is less. The fast, accurate property of 2D spectroscopy makes it a powerful and new approach to evaluating medicinal herbs impersonally.

[Dual-index sequence analytical method for IR fingerprint spectra of the chloroform extract of Radix Glycyrrhizae].

OBJECTIVE: To investigate a new method for the analysis of IR fingerprint spectra of Radix Glycyrrhizae. METHOD: Two indexes, common peak ratio and variation peak ratio, are used to compare the IR spectra of various Radix Glycyrrhizae samples, and the values are calculated by means of sequent analysis. RESULT AND CONCLUSION: The dual-index sequence method provides a good approach to discriminate Radix Glycyrrhizae samples of different species and geographical origins.