Quality evaluation of natural monomer flavors for Chinese tobacco industry based on dual mode combined Fourier transform ion cyclotron resonance mass spectrometry and isotopic fine structures.

RATIONALE: Natural monomer flavors can modify the taste of cigarettes. However, no report was published to establish the quality control method for their chemical compositions. METHODS: In this study, licorice, a traditional natural monomer flavor used in tobacco aroma processing, was selected, and the fingerprint was developed by high-performance liquid chromatography (HPLC). Next, the chemical markers of samples from different places of origin were discovered by multivariate statistical analysis. Then, its chemical constituents were identified by combination of HPLC-Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), direct infusion FT-ICR-MS (DI-FT-ICR-MS), and the technology of isotopic fine structures (IFSs). Moreover, its characteristic constituents were quantitatively analyzed using HPLC. RESULTS: The 14 common peaks were assigned in the fingerprint, and 8 of them were considered as qualitative markers by multivariate statistical analysis. A total of 42 chemical constituents were detected using HPLC-FT-ICR-MS, and 13 of them were unambiguously identified by references. Meanwhile, the elemental compositions of other eight unknown chemical components were decisively determined using IFSs. Subsequently, the contents of five characteristic constituents in 11 batches of samples were determined. CONCLUSIONS: The integration strategy established here can discover and quantify the chemical markers for improving the quality control standard of natural monomer flavor of licorice. It is expected that the strategy will be valuable for further quality control of other natural monomer flavors in Chinese tobacco industry.

Exploring the dietary and therapeutic potential of licorice (Glycyrrhiza uralensis Fisch.) sprouts.

ETHNOPHARMACOLOGICAL RELEVANCE: This research substantiates the traditional use of Glycyrrhiza uralensis Fisch. for liver health, with scientific evidence of the non-toxic and lipid-lowering properties of licorice sprout extracts. The sprouts' rich mineral and amino acid content, along with their strong antioxidant activity, reinforce their value in traditional medicine. These findings bridge ancient herbal practices with modern science, highlighting licorice's potential in contemporary therapeutic applications. AIM OF THE STUDY: The study aimed to investigate the dietary and medicinal potential of G. uralensis sprouts by assessing their safety, nutritional content, and antioxidant properties using both plant and animal models. Specifically, the study sought to determine the effects of different sizes of licorice sprouts on lipid metabolism in human liver cancer cells and their overall impact on rat health indicators. MATERIALS AND METHODS: The study examined the effects of aqueous and organic extracts from G. uralensis sprouts of varying lengths on the cytotoxicity, lipid metabolism, and antioxidant activity in HepG2 cells, alongside in vivo impacts on Sprague-Dawley rats, using MTT, ICP, and HPLC. It aimed to assess the potential health benefits of licorice sprouts by analyzing their protective effects against oxidative stress and their nutritional content. RESULTS: Licorice sprout extracts from G. uralensis demonstrated no cytotoxicity in HepG2 cells, significantly reduced lipid levels, and enhanced antioxidant activities, with the longest sprouts (7 cm) showing higher mineral, sugar, and arginine content as well as increased glycyrrhizin and liquiritigenin. In vivo studies with Sprague-Dawley rats revealed weight gain and improved antioxidant enzyme activities in blood plasma and liver tissues after consuming the extracts, highlighting the sprouts' dietary and therapeutic potential. CONCLUSIONS: This study is the first to demonstrate that G. uralensis sprouts, particularly those 7 cm in length, have no cytotoxic effects, reduce lipids, and have high mineral and antioxidant contents, offering promising dietary and therapeutic benefits.

Improving the geographical origin classification of Radix glycyrrhizae (licorice) through hyperspectral imaging assisted by U-Net fine structure recognition.

Radix glycyrrhizae (licorice) is extensively employed in traditional Chinese medicine, and serves as a crucial raw material in industries such as food and cosmetics. The quality of licorice from different origins varies greatly, so classification of its geographical origin is particularly important. This study proposes a technique for fine structure recognition and segmentation of hyperspectral images of licorice using deep learning U-Net neural networks to segment the tissue structure patterns (phloem, xylem, and pith). Firstly, the three partitions were separately labeled using the Labelme tool, which was utilized to train the U-Net model. Secondly, the obtained optimal U-Net model was applied to predict three partitions of all samples. Lastly, various machine learning models (LDA, SVM, and PLS-DA) were trained based on segmented hyperspectral data. In addition, a threshold method and a circumcircle method were applied to segment licorice hyperspectral images for comparison. The results revealed that compared with the threshold segmentation method (which yielded SVM classifier accuracies of 99.17%, 91.15%, and 92.50% on the training set, validation set, and test set, respectively), the U-Net segmentation method significantly enhanced the accuracy of origin classification (99.06%, 94.72% and 96.07%). Conversely, the circumcircle segmentation method did not effectively improve the accuracy of origin classification (99.65%, 91.16% and 92.13%). By integrating Raman imaging of licorice, it can be inferred that the U-Net model, designed for region segmentation based on the inherent tissue structure of licorice, can effectively improve the accuracy origin classification, which has positive significance in the development of intelligence and information technology of Chinese medicine quality control.

Effect of subcritical water temperature on the structure, antioxidant activity and immune activity of polysaccharides from Glycyrrhiza inflata Batalin.

In this study, the polysaccharide from Glycyrrhiza inflata Batalin extracted by hot water (HW-GP) was further physically modified with subcritical water to obtain novel polysaccharides (SW-GP). Comparative analysis was conducted to examine the disparities in conformation and bioactivity between HW-GP and SW-GP, aiming to precisely regulate the structure of the polysaccharides and enhance their bioactivity by controlling subcritical water temperature. The results showed that, compared with HW-GP, subcritical water modification (100-160 °C) not only significantly reduced the molecular weight of polysaccharides (from 5.586 × 105 g/mol to 1.484 × 105 g/mol), but also modulated the intermolecular interaction forces, which maintain the conformation of the polysaccharides, including electrostatic and hydrophobic interactions, thereby dynamically transforming the polysaccharide chain conformation from triple helix to random coil, and the strength of the chain conformation shifted from rigid to flexible. In addition, the modification of the SW-GP structure by subcritical water also enhanced its biological activity. SW-GP (140 °C) with low molecular weight and semi-rigid triple helix conformation showed the best scavenging effect on the DPPH, ABTS, and hydroxyl radicals, and exhibited excellent antioxidant activity. SW-GP (130 °C) with medium molecular weight and semi-rigid triple helix conformation significantly promoted the proliferation and phagocytosis of RAW264.7 cells, as well as increased the release levels of NO, TNF-α, IL-6, and IL-1ß, and the immunomodulatory activity was much higher than that of other polysaccharides. These findings confirmed the feasibility of using subcritical water temperature as a regulatory feature for the structure and bioactivity of glycyrrhiza polysaccharides, which may have reference significance for the modification of polysaccharides with heightened bioactivity.

In silico network pharmacology study on Glycyrrhiza glabra: Analyzing the immune-boosting phytochemical properties of Siddha medicinal plant against COVID-19.

Immunosenescence is a pertinent factor in the mortality rate caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The changes in the immune system are strongly associated with age and provoke the deterioration of the individual's health. Traditional medical practices in ancient India effectively deal with COVID-19 by boosting natural immunity through medicinal plants. The anti-inflammatory and antiviral properties of Glycyrrhiza glabra are potent in fighting against COVID-19 and promote immunity boost against the severity of the infection. Athimadhura Chooranam, a polyherbal formulation containing Glycyrrhiza glabra as the main ingredient, is recommended as an antiviral Siddha herb by the Ministry of AYUSH. This paper is intended to identify the phytoconstituents of Glycyrrhiza glabra that are actively involved in preventing individuals from COVID-19 transmission. The modulated pathways, enrichment study, and drug-likeness are calculated from the target proteins of the phytoconstituents at the pharmacological activity (Pa) of more than 0.7. Liquiritigenin and Isoliquiritin, the natural compounds in Glycyrrhiza glabra, belong to the flavonoid class and exhibit ameliorative effects against COVID-19. The latter compound displays a higher protein interaction to a maximum of six, out of which HMOX1, PLAU, and PGR are top-hub genes. ADMET screening further confirms the significance of the abovementioned components containing better drug-likeness. The molecular docking and molecular dynamics method identified liquiritigenin as a possible lead molecule capable of inhibiting the activity of the major protease protein of SARS-CoV-2. The findings emphasize the importance of in silico network pharmacological assessments in delivering cost-effective, time-bound clinical drugs.

Network pharmacology and in vitro experimental verification unveil glycyrrhizin from glycyrrhiza glabra alleviates acute pancreatitis via modulation of MAPK and STAT3 signaling pathways.

Acute pancreatitis (AP) is a severe gastrointestinal inflammatory disease with increasing mortality and morbidity. Glycyrrhiza glabra, commonly known as Liquorice, is a widely used plant containing bioactive compounds like Glycyrrhizin, which possesses diverse medicinal properties such as anti-inflammatory, antioxidant, antiviral, and anticancer activities. The objective of this study is to investigate the active components, relevant targets, and underlying mechanisms of the traditional Chinese medicine Glycyrrhiza glabra in the treatment of AP. Utilizing various computational biology methods, we explored the potential targets and molecular mechanisms through Glycyrrhizin supplementation. Computational results indicated that Glycyrrhizin shows promising pharmacological potential, particularly with mitogen-activated protein kinase 3 (MAPK3) protein (degree: 70), forming stable complexes with Glycyrrhizin through ionic and hydrogen bonding interactions, with a binding free energy (ΔGbind) of -33.01 ± 0.08 kcal/mol. Through in vitro experiments, we validated that Glycyrrhizin improves primary pancreatic acinar cell injury by inhibiting the MAPK/STAT3/AKT signaling pathway. Overall, MAPK3 emerges as a reliable target for Glycyrrhizin's therapeutic effects in AP treatment. This study provides novel insights into the active components and potential targets and molecular mechanisms of natural products.

A bibliometric review of Glycyrrhizae Radix et Rhizoma (licorice) research: Insights and future directions.

ETHNOPHARMACOLOGICAL RELEVANCE: Glycyrrhizae Radix et Rhizoma, a Chinese herb known as licorice, is frequently incorporated in traditional Chinese medicine (TCM) formulations, due to its significant medicinal value and sweet taste. Despite licorice's merits, no systematic scientometric study has yet been conducted to analyze licorice research trends over the past 25 years. AIM OF THE STUDY: We conducted this study with the aim to provide researchers with a comprehensive overview of research advances in the application of licorice as a TCM ingredient and to offer valuable insights to guide future endeavors in this research field. METHODS: We selected licorice-related research papers published between 1997 and 2021 from the Web of Science Core Collection then conducted a scientometric analysis using VOSviewer and CiteSpace software tools. RESULTS: A total of 4883 licorice-related publications, including 4511 research papers, 372 review papers, and their cited references, were included in the analysis. Most of these articles were authored by researchers in China (36.8%), including major contributors Wang Ying, Ye Min, and Zhang Yu. The Journal of Ethnopharmacology (impact factor = 5.4) hosted the greatest number of papers (145 articles). Keyword cluster analysis revealed three keyword categories indicating that current licorice research is focused on licorice quality control and identification of licorice active ingredients and associated pharmacological mechanisms. CONCLUSION: This study provides a comprehensive overview of licorice-related research trends over the past 25 years as based on quantitative and qualitative analyses of published licorice-related articles. The results of this multi-level analysis of licorice research related to TCM formulations, chemical compositions, and pharmacological effects should provide valuable reference data and insights to guide future research endeavors in this field.

A cytosine analogue 5-azacitidine improves the accumulation of licochalcone A in licorice Glycyrrhiza inflata.

Licochalcone A (LCA) is a characteristic compound of Glycyrrhiza inflata with anti-inflammatory, antioxidant and antitumor activities. However, G. inflata produces LCA in low quantities that does not meet the market demand. In this study, we found that DNA methylation inhibitor 5-azacitidine (5-azaC) successfully improved the LCA contents in G. inflata seedlings. Transcriptome analysis revealed a series of differentially expressed genes (DEGs), including transcription factors such as MYB, ERF, WRKY, and some structural genes related to flavonoid biosynthesis. However, whole genome bisulfite sequencing (BS-seq) results showed little effect of the 5-azaC treatment on the alteration of DNA methylation on these genes, indicating the possibility that 5-azaC acts as a stimulus, but not an epigenetic modulation factor to improve the LCA content in G. inflata. Additionally, we applied the 5-azaC treatment to field plants and hairy roots and successfully increased the LCA contents in both cases. This research demonstrates the feasibility of 5-azaC treatments in future applications to improve plant production of LCA.

Investigation of the principle of concoction by using the processing excipient Glycyrrhiza uralensis Fisch. juice to reduce the main toxicity of Dioscorea bulbifera L. and enhance its main efficacy as expectorant and cough suppressant.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea bulbifera L. (Rhizoma Dioscoreae Bulbiferae; RDB) is commonly used as an expectorant and cough suppressant herb but is accompanied by severe hepatotoxicity. Using the juice of auxiliary herbs (such as Glycyrrhiza uralensis Fisch. (Glycyrrhizae Radix et Rhizoma; GRR) juice) in concocting poisonous Chinese medicine is a conventional method to reduce toxicity or increase effects. Our previous study found that concoction with GRR juice provided a detoxifying effect against the major toxic hepatotoxicity induced by RDB, but the principle for the detoxification of the concoction is unknown to date. AIM OF THE STUDY: The principle of concoction was investigated by using the processing excipient GRR juice to reduce the major toxic hepatotoxicity of RDB, and the efficacy of RDB as an expectorant and cough suppressant was enhanced. MATERIALS AND METHODS: In this study, common factors (RDB:GRR ratio, concocted temperature, and concocted time) in the concoction process were used for the preparation of each RDB concocted with GRR juice by using an orthogonal experimental design. We measured the content of the main toxic compound diosbulbin B (DB) and serum biochemical indicators and performed pathological analysis in liver tissues of mice to determine the best detoxification process of RDB concocted with GRR juice. On this basis, the biological mechanisms of target organs were detected by Western blot and enzyme-linked immunosorbent assay at the inflammation and apoptosis levels. Further, the effects of RDB on expectorant and cough suppressant with GRR juice were evaluated by the conventional tests of phenol red expectorant and concentrated ammonia-induced cough. Lastly, the major compounds in the GRR juice introduced to RDB concoction were determined. RESULTS: RDB concocted with GRR juice significantly alleviated DB content, serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase levels, and improved liver pathological damages. The best detoxification process was achieved by using an RDB:GRR ratio of 100:20 at 120 °C for 20 min. Further, RDB concocted with GRR juice down-regulated the protein levels of nuclear factor kappa B (NF-κB), cyclooxygenase 2 (COX-2), and Bcl-2 related X protein (Bax) in the liver and enhanced the expectorant and cough suppressant effects of RDB. Finally, liquiritin (LQ) and glycyrrhizic acid (GA) in the GRR juice were introduced to the RDB concoction. CONCLUSION: Concoction with GRR juice not only effectively reduced the major toxic hepatotoxicity of RDB but also enhanced its main efficacy as an expectorant and cough suppressant, and that the rationale for the detoxification and/or potentiation of RDB was related to the reduction in the content of the main hepatotoxic compound, DB, the introduction of the hepatoprotective active compounds, LQ and GA, in the auxiliary GRR juice, as well as the inhibition of NF-κB/COX-2/Bax signaling-mediated inflammation and apoptosis.

Disruption of a licorice cellulose synthase-derived glycosyltransferase gene demonstrates its in planta role in soyasaponin biosynthesis.

KEY MESSAGE: CRISPR-Cas9-mediated disruption of a licorice cellulose synthase-derived glycosyltransferase gene, GuCSyGT, demonstrated the in planta role of GuCSyGT as the enzyme catalyzing 3-O-glucuronosylation of triterpenoid aglycones in soyasaponin biosynthesis. Triterpenoid glycosides (saponins) are a large, structurally diverse group of specialized metabolites in plants, including the sweet saponin glycyrrhizin produced by licorice (Glycyrrhiza uralensis) and soyasaponins that occur widely in legumes, with various bioactivities. The triterpenoid saponin biosynthetic pathway involves the glycosylation of triterpenoid sapogenins (the non-sugar part of triterpenoid saponins) by glycosyltransferases (GTs), leading to diverse saponin structures. Previously, we identified a cellulose synthase-derived GT (CSyGT), as a newly discovered class of triterpenoid GT from G. uralensis. GuCSyGT expressed in yeast, which could transfer the sugar glucuronic acid to the C3 position of glycyrrhetinic acid and soyasapogenol B, which are the sapogenins of glycyrrhizin and soyasaponin I, respectively. This suggested that GuCSyGT is involved in the biosynthesis of glycyrrhizin and soyasaponin I. However, the in planta role of GuCSyGT in saponin biosynthesis remains unclear. In this study, we generated GuCSyGT-disrupted licorice hairy roots using CRISPR-Cas9-mediated genome editing and analyzed the saponin content. This revealed that soyasaponin I was completely absent in GuCSyGT-disrupted lines, demonstrating the in planta role of GuCSyGT in saponin biosynthesis.

[Screening of quality markers and activity verification of Glycyrrhizae Radix et Rhizoma based on small molecule compound-protein interaction].

In order to solve the problem of weak correlation between quality control components and efficacy of Glycyrrhizae Radix et Rhizoma, this study detected the interaction between small molecular chemical components of Glycyrrhizae Radix et Rhizoma and total proteins of various organs of mice by fluorescence quenching method to screen potential active components. The 27 chemical components in Glycyrrhizae Radix et Rhizoma were detected by HPLC and their deletion rates in 34 batches of Glycyrrhizae Radix et Rhizoma were calculated. Combined with the principle of component effectiveness and measurability, the potential quality markers(Q-markers) of Glycyrrhizae Radix et Rhizoma were screened. RAW264.7 macrophage injury model was induced by microplastics. The cell viability and nitric oxide content were detected by CCK-8 and Griess methods. The levels of inflammatory factors(TNF-α, IL-1ß, IL-6, CRP) and oxidative stress markers(SOD, MDA, GSH) were detected by the ELISA method to verify the activity of Q-markers. It was found that the interaction strength between different chemical components and organ proteins in Glycyrrhizae Radix et Rhizoma was different, reflecting different organ selectivity and 18 active components were screened out. Combined with the signal-to-noise ratio of the HPLC chromatographic peaks and between-run stability of the components, seven chemical components such as liquiritin apioside, liquiritin, isoliquiritin apioside, isoliquiritin, liquiritigenin, isoliquiritigenin and ammonium glycyrrhizinate were finally screened as potential Q-markers of Glycyrrhizae Radix et Rhizoma. In vitro experiments showed that Q-markers of Glycyrrhizae Radix et Rhizoma could dose-dependently alleviate RAW264.7 cell damage induced by microplastics, inhibit the secretion of inflammatory factors, and reduce oxidative stress. Under the same total dose, the combination of various chemical components could synergistically enhance anti-inflammatory and antioxidant effects compared with the single use. This study identified Q-markers related to the anti-inflammatory and antioxidant effects of Glycyrrhizae Radix et Rhizoma, which can provide a reference for improving the quality control standards of Glycyrrhizae Radix et Rhizoma.

PPARα/γ-Targeting Amorfrutin Phytocannabinoids from Aerial Parts of Glycyrrhiza foetida.

An LC-MS/MS-guided analysis of the aerial parts of Glycyrrhiza foetida afforded new phenethyl (amorfrutin)- and alkyl (cannabis)-type phytocannabinoids (six and four compounds, respectively). The structural diversity of the new amorfrutins was complemented by the isolation of six known members and the synthesis of analogues modified on the aralkyl moiety. All of the compounds so obtained were assayed for agonist activity on PPARα and PPARγ nuclear receptors. Amorfrutin A (1) showed the highest agonist activity on PPARγ, amorfrutin H (7) selectively targeted PPARα, and amorfrutin E (4) behaved as a dual agonist, with the pentyl analogue of amorfrutin A (11) being inactive. Decarboxyamorfrutin A (2) was cytotoxic, and modifying its phenethyl moiety to a styryl or a phenylethynyl group retained this trait, suggesting an alternative biological scenario for these compounds. The putative binding modes of amorfrutins toward PPARα and PPARγ were obtained by a combined approach of molecular docking and molecular dynamics simulations, which provided insights on the structure-activity relationships of this class of compounds.

Tracking the effect of roasting and fermentation on the metabolites of licorice root (Glycyrrhiza glabra L.) using UPLC-MS analysis combined with multivariate statistical analysis.

BACKGROUND: Roasting, honey-roasting and fermentation are the most common pre-processing procedures of licorice roots. They were shown to noticeably change the composition of extracts. In this work, the common alterations in licorice secondary metabolites by processing were interpreted. Comprehensive metabolic profiling of different studied samples was undergone. METHODS: UPLC-QqQ-MS/MS analysis coupled to various chemometric analysis models was implemented to unravel the effect of different pre-processing procedures on the chemical profile of licorice samples. RESULTS: UPLC-QqQ-MS/MS analysis designated 133 chromatographic peaks with saponins, flavonoids, chalcones and pterocarpans being the most abundant groups. Triterpene saponins dominated the secondary metabolites in the aqueous extracts, with fermented samples showing the highest relative amounts. Meanwhile the ethanol extracts showed significant amounts of chalcones. Melanoidins were only detected in roasted and honey roasted samples. Multivariate models indicated that roasting of samples induced a greater effect on the polar metabolites rather than nonpolar ones. Variable of importance (VIP) plot indicated that glycyrrhizin and its hydrolysis product glycyrrhetinic acid, trihdroxychalcone diglycoside, glabrone and glabridin are the main chemical features responsible for the discrimination of samples. CONCLUSION: Coupling UPLC-MS/MS to multivariate analysis was a successful tool that unveiled the significant effect of different pre-processing methods on the chemical profile of processed and unprocessed licorice samples. Moreover, such coupling unraveled the discriminatory chemical compounds among tested samples that can be employed as markers for the processing procedure of licorice.

Quality consistency evaluation of chemical composition and pharmacology of Shaoyao-Gancao decoction dispensing granules and traditional decoction.

Dispensing granules of Chinese medicine (DGCM) have emerged as a more convenient alternative to traditional decoction (TD) of Chinese medicine, gaining popularity in recent years. However, the debate surrounding the consistency of DGCM compared to TD remains unresolved. In this study, three batches of Baishao and Gancao DGCM were obtained from manufacturers A, B, and C, and 15 batches of crude drugs were procured from hospital pharmacies for the preparation of dispensing granule decoction (DGD) and TD of Shaoyao-Gancao decoction (SGD). The HPLC-UV method was employed to determine the levels of gallic acid, paeoniflorin, albiflorin, liquiritin, liquiritin apioside, isoliquiritin apioside, isoliquiritin, glycyrrhizic acid, and isoliquiritigenin. The analgesic and antispasmodic effects were assessed using the hot plate and acetic acid writhing test in mice. To evaluate the consistency of chemical constituents and pharmacological effects between the two decoctions, the Criteria Importance Though Intercriteria Correlation (CRITIC) method combined with chemometrics was employed. Grey relation analysis (GRA) was used to assess the comprehensive quality consistency of the two decoctions. The CRITIC results revealed certain differences in chemical constituents and pharmacological effects between the selected DGCM and TD. Notably, DGD-A/C exhibited a significant difference from TD (p > 0.05), whereas DGD-B demonstrated no significant difference from TD (p > 0.05). The GRA analysis demonstrated that the overall quality consistency between DGD-B and TD was the highest among the three manufacturers. This study presents a method for evaluating the quality consistency of DGCM and TD of SGD, offering novel insights into the evaluation of consistency between DGCM and TD.

Research Progress on Structural Modification of Effective Antitumor Active Ingredients in Licorice.

Licorice, a widely used traditional Chinese medicine, contains more than 300 flavonoids and more than 20 triterpenoids, which have potential medicinal value and can prevent the growth of tumor cells by blocking the cell cycle, affecting the regulation of the apoptosis gene of tumor cells, and inhibiting tumor cell angiogenesis. However, many of the compounds in licorice still have the drawbacks of poor solubility, significant toxic side effects, and low antitumor activity. This article reviews the structural modification of effective antitumor active ingredients in licorice, thus providing a theoretical basis for further investigation of licorice and the development of new antitumor drugs.

Exploring the effect and mechanism of Haizao Yuhu decoction containing three variants of glycyrrhiza on goiter using an integrated strategy of network pharmacology and RNA sequencing.

ETHNOPHARMACOLOGICAL RELEVANCE: Haizao Yuhu decoction (HYD) is a classic Chinese herbal formula described in the surgical monographs of the Ming Dynasty "Waikezhengzong." It has been widely used to treat goiter for approximately 500 years and found to be particularly effective. HYD contains glycyrrhiza and sargassum. This pair of herbs belongs to "18 incompatible medicaments" of traditional Chinese medicine theory. Although these two herbs are opposite, our preliminary study proved that they have superior effect when added into HYD at 2 times the dose of Chinese Pharmacopoeia. However, the species of glycyrrhiza in HYD that are the most effective have not been recorded in ancient Chinese medical texts. According to the Chinese Pharmacopoeia, glycyrrhiza is divided into the following three species: Glycyrrhiza uralensis Fish., G. glabra L., and G. inflata Bat. The effect of HYD containing different species of glycyrrhiza and their mechanisms remain to be further explored. AIM OF THE STUDY: To investigate the effect of HYD containing three species of glycyrrhiza on goiter, and to elucidate the molecular mechanism using network pharmacology combined with RNA sequencing (RNA-seq). MATERIALS AND METHODS: A rat model of goiter was established by 14 days of intragastric gavage of propylthiouracil (PTU), and the rats were treated for 4 weeks with HYD containing three different species of glycyrrhiza. The body weight and rectal temperature of rats were tested weekly. At the end of the experiment, the serum and thyroid tissues of rats were collected. The effect of the three HYDs was assessed based on general observations (including body weight, rectal temperature, and living status of rats), absolute/relative thyroid weight, thyroid function (including triiodothyronine, thyroxine, free triiodothyronine, free thyroxine, and thyroid-stimulating hormone levels), and thyroid tissue pathology. Next, we explored their pharmacological mechanisms using network pharmacology combined with RNA-seq and validated key targets using real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR), western blotting (WB), and immunofluorescence (IF) assays. RESULTS: The three HYDs reduced the absolute/relative weights of thyroid tissues and improved the pathological structure, thyroid function, and general findings of rats with goiter. Overall, the effect of HYD-G. uralensis Fish. (HYD-U) was better. Results from network pharmacology and RNA-seq jointly suggested that both the pathogenesis of goiter and the mechanism of action of HYD for goiter were related to the phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt) pathway. We validated the key targets in the pathway, namely, vascular endothelial growth factor (VEGF) A, VEGF receptor 2, phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) and its encoded protein PI3K (p85), AKT serine/threonine kinase 1 (AKT1), phospho-AKT and cyclin D1 using RT-qPCR, WB, and IF assays. The PI3K-Akt pathway was hyperactivated in rats with PTU-induced goiter, whereas the three HYDs could inhibit the pathway. CONCLUSION: This study confirmed the definite effect of the three HYDs in the treatment of goiter, and HYD-U was found to be more effective. The three HYDs inhibited angiogenesis and cell proliferation in goiter tissue by inhibiting the PI3K-Akt signaling pathway.

Holistically assessing the quality consistency of compound liquorice tablets from similarities of both all chemical fingerprints and the integrated dissolution curves by systematically quantified fingerprint method.

In recent years, traditional analytical methods have failed to meet the widespread use of multi-component Chinese pharmaceutical formulations. To solve this problem, this study proposed a comprehensive analytical strategy using compound liquorice tablets (CLTs) as an example, both in terms of chemical quality and dissolution curve consistency. Firstly, the peak purity of the two wavelengths was checked using dual-wavelength absorbance coefficient ratio spectra (DARS) to avoid the fingerprint bias caused by peak purity. Secondly, liquid-phase dual-wavelength tandem fingerprint (DWTF) of 38 batches of CLTs was established for the first time. The two analytical methods were also evaluated using the systematically quantified fingerprint method (SQFM), and the 38 batches of samples were classified into two grades with good quality consistency. Quantitative analysis of the five markers of CLTs was performed simultaneously using the standard curve method (SCM) and the quantitative analysis of multiple components by single marker (QAMS). The results showed no significant differences between the two analytical methods (p > 0.5). In addition, the in vitro dissolution of CLTs in two media (pure water and pH = 4.5 medium) was determined by the total UV fingerprint dissolution assay. The similarity of the dissolution curves was also analyzed by combining the f2 factor and the dissolution-systematically quantified fingerprint method (DSQFM). The result showed that most of the samples had f2 > 50 and Pm satisfied the range of 70-130%. Finally, a principal component analysis (PCA) model was developed to combine the evaluation parameters of chemical fingerprint and dissolution curves for comprehensive analysis of the samples. In this study, a chromatographic and dissolution-based quality analysis method was proposed, which effectively overcomes the shortcomings of previous analytical methods and provides a scientific analytical method for the quality control of natural drugs.

Breast cancer prevention with liquiritigenin from licorice through the inhibition of aromatase and protein biosynthesis in high-risk women's breast tissue.

Breast cancer risk continues to increase post menopause. Anti-estrogen therapies are available to prevent postmenopausal breast cancer in high-risk women. However, their adverse effects have reduced acceptability and overall success in cancer prevention. Natural products such as hops (Humulus lupulus) and three pharmacopeial licorice (Glycyrrhiza) species have demonstrated estrogenic and chemopreventive properties, but little is known regarding their effects on aromatase expression and activity as well as pro-proliferation pathways in human breast tissue. We show that Gycyrrhiza inflata (GI) has the highest aromatase inhibition potency among these plant extracts. Moreover, phytoestrogens such as liquiritigenin which is common in all licorice species have potent aromatase inhibitory activity, which is further supported by computational docking of their structures in the binding pocket of aromatase. In addition, GI extract and liquiritigenin suppress aromatase expression in the breast tissue of high-risk postmenopausal women. Although liquiritigenin has estrogenic effects in vitro, with preferential activity through estrogen receptor (ER)-ß, it reduces estradiol-induced uterine growth in vivo. It downregulates RNA translation, protein biosynthesis, and metabolism in high-risk women's breast tissue. Finally, it reduces the rate of MCF-7 cell proliferation, with repeated dosing. Collectively, these data suggest that liquiritigenin has breast cancer prevention potential for high-risk postmenopausal women.

Chalcone derivatives from licorice inhibit human and rat gonadal 3ß-hydroxysteroid dehydrogenases as therapeutic uses.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine, licorice (the roots of Glycyrrhiza glabra and G. inflata) has been used to treat inflammation and sexual debility for over 1000 years. Pharmacological studies have identified many biologically active chalcone derivatives from licorice. AIM OF THE STUDY: Human 3ß-Hydroxysteroid dehydrogenase 2 (h3ß-HSD2) catalyzes the formation of precursors for sex hormones and corticosteroids, which play critical roles in reproduction and metabolism. We explored inhibition and mode action of chalcones of inhibiting h3ß-HSD2 and compared it with rat 3ß-HSD1. MATERIALS AND METHODS: We investigated the inhibition of 5 chalcones on h3ß-HSD2 and compared species-dependent difference with 3ß-HSD1. RESULTS: The inhibitory strength on h3ß-HSD2 was isoliquiritigenin (IC50, 0.391 µM) > licochalcone A (0.494 µM) > licochalcone B (1.485 µM) > echinatin (1.746 µM) >chalcone (100.3 µM). The inhibitory strength on r3ß-HSD1 was isoliquiritigenin (IC50, 0.829 µM) > licochalcone A (1.165 µM) > licochalcone B (1.866 µM) > echinatin (2.593 µM) > chalcone (101.2 µM). Docking showed that all chemicals bind steroid and/or NAD+-binding site with the mixed mode. Structure-activity relationship analysis showed that strength was correlated with chemical's hydrogen bond acceptor. CONCLUSION: Some chalcones are potent h3ß-HSD2 and r3ß-HSD1 inhibitors, possibly being potential drugs to treat Cushing's syndrome or polycystic ovarian syndrome.

Metagenomics insights into responses of rhizobacteria and their alleviation role in licorice allelopathy.

BACKGROUND: Allelopathy is closely associated with rhizosphere biological processes, and rhizosphere microbial communities are essential for plant development. However, our understanding of rhizobacterial communities under influence of allelochemicals in licorice remains limited. In the present study, the responses and effects of rhizobacterial communities on licorice allelopathy were investigated using a combination of multi-omics sequencing and pot experiments, under allelochemical addition and rhizobacterial inoculation treatments. RESULTS: Here, we demonstrated that exogenous glycyrrhizin inhibits licorice development, and reshapes and enriches specific rhizobacteria and corresponding functions related to glycyrrhizin degradation. Moreover, the Novosphingobium genus accounted for a relatively high proportion of the enriched taxa and appeared in metagenomic assembly genomes. We further characterized the different capacities of single and synthetic inoculants to degrade glycyrrhizin and elucidated their distinct potency for alleviating licorice allelopathy. Notably, the single replenished N (Novosphingobium resinovorum) inoculant had the greatest allelopathy alleviation effects in licorice seedlings. CONCLUSIONS: Altogether, the findings highlight that exogenous glycyrrhizin simulates the allelopathic autotoxicity effects of licorice, and indigenous single rhizobacteria had greater effects than synthetic inoculants in protecting licorice growth from allelopathy. The results of the present study enhance our understanding of rhizobacterial community dynamics during licorice allelopathy, with potential implications for resolving continuous cropping obstacle in medicinal plant agriculture using rhizobacterial biofertilizers. Video Abstract.