Licorice Products Research Articles

Effects of different post-harvest processing methods on changes in the active ingredients of licorice based on LC-MS and plant metabolomics.

Licorice, the dried roots and rhizomes of the Glycyrrhiza uralensis Fisch., holds a prominent status in various formulations within the realm of Chinese medicinal practices. The traditional processing methods of licorice hinder quality assurance, thus prompting Chinese medicine researchers to focus on the fresh processing methods to enhancing processing efficiency and quality. This study aimed to identify the differential compounds of licorice between traditional and fresh processing methods and provide a scientific basis for the fresh processing of licorice and for further research on the processing mechanism. A methodology integrating ultra-performance liquid chromatography with quadrupole-time-of-flight tandem mass spectrometry combined with multivariate statistical analysis was employed to characterize the differential compounds present in licorice between traditional processing and fresh processing. The results derived from principal component analysis and heat map analyses underscored significant differences in the content of bioactive compounds between the two processing methods. By applying conditions of VIP > 1.5 and p < 0.05, a total of 38 differential compounds were identified through t tests, and the transformation mechanisms of select compounds were illustrated. The adoption of fresh processing techniques not only improved processing efficiency but also significantly enhanced the preservation of bioactive compounds within licorice. This research has established a rapid and efficient analytical method for the identification of differential compounds present in differently processed licorice products.

Matrine and Oxymatrine: evaluating the gene mutation potential using in silico tools and the bacterial reverse mutation assay (Ames test).

The quinolizidine alkaloids matrine and its N-oxide oxymatrine occur in plants of the genus Sophora. Recently, matrine was sporadically detected in liquorice products. Morphological similarity of the liquorice plant Glycyrrhiza glabra with Sophora species and resulting confusion during harvesting may explain this contamination, but use of matrine as pesticide has also been reported. The detection of matrine in liquorice products raised concern as some studies suggested a genotoxic activity of matrine and oxymatrine. However, these studies are fraught with uncertainties, putting the reliability and robustness into question. Another issue was that Sophora root extracts were usually tested instead of pure matrine and oxymatrine. The aim of this work was therefore to determine whether matrine and oxymatrine have potential for causing gene mutations. In a first step and to support a weight-of-evidence analysis, in silico predictions were performed to improve the database using expert and statistical systems by VEGA, Leadscope (Instem®), and Nexus (Lhasa Limited). Unfortunately, the confidence levels of the predictions were insufficient to either identify or exclude a mutagenic potential. Thus, in order to obtain reliable results, the bacterial reverse mutation assay (Ames test) was carried out in accordance with OECD Test Guideline 471. The test set included the plate incorporation and the preincubation assay. It was performed with five different bacterial strains in the presence or absence of metabolic activation. Neither matrine nor oxymatrine induced a significant increase in the number of revertants under any of the selected experimental conditions. Overall, it can be concluded that matrine and oxymatrine are unlikely to have a gene mutation potential. Any positive findings with Sophora extracts in the Ames test may be related to other components. Notably, the results also indicated a need to extend the application domain of respective (Q)SAR tools to secondary plant metabolites.

Glycyrrhiza glabra L. Extracts and Other Therapeutics against SARS-CoV-2 in Central Eurasia: Available but Overlooked.

In Central Eurasia, the availability of drugs that are inhibitors of the SARS-CoV-2 virus and have proven clinical efficacy is still limited. The aim of this study was to evaluate the activity of drugs that were available in Kazakhstan during the acute phase of the epidemic against SARS-CoV-2. Antiviral activity is reported for Favipiravir, Tilorone, and Cridanimod, which are registered drugs used for the treatment of respiratory viral infections in Kazakhstan. A licorice (Glycyrrhiza glabra) extract was also incorporated into this study because it offered an opportunity to develop plant-derived antivirals. The Favipiravir drug, which had been advertised in local markets as an anti-COVID cure, showed no activity against SARS-CoV-2 in cell cultures. On the contrary, Cridanimod showed impressive high activity (median inhibitory concentration 66 μg/mL) against SARS-CoV-2, justifying further studies of Cridanimod in clinical trials. Tilorone, despite being in the same pharmacological group as Cridanimod, stimulated SARS-CoV-2 replication in cultures. The licorice extract inhibited SARS-CoV-2 replication in cultures, with a high median effective concentration of 16.86 mg/mL. Conclusions: The synthetic, low-molecular-weight compound Cridanimod suppresses SARS-CoV-2 replication at notably low concentrations, and this drug is not toxic to cells at therapeutic concentrations. In contrast to its role as an inducer of interferons, Cridanimod is active in cells that have a genetic defect in interferon production, suggesting a different mechanism of action. Cridanimod is an attractive drug for inclusion in clinical trials against SARS-CoV-2 and, presumably, other coronaviruses. The extract from licorice shows low activity against SARS-CoV-2. At the same time, high doses of 2 g/kg of this plant extract show little or no acute toxicity in animal studies; for this reason, licorice products can still be considered for further development as a safe, orally administered adjunctive therapy.

Species identification and quality evaluation of licorice in the herbal trade using DNA barcoding, HPLC and colorimetry

ABSTRACT Licorice is a product with the same origin as medicine and food and is widely used in traditional Chinese medicine and the food, cosmetics and tobacco industries. With the increasing demand for licorice in the herbal market, there is also increasing concern about complex varieties and quality deterioration. Considering the importance of the quality and safety of commercial licorice products, we used DNA barcoding, HPLC and colorimetry to distinguish and evaluate commercial licorice products. Here, 52 samples could be accurately identified into three kinds of licorice, Glycyrrhiza uralensis Fisch. (Gu), Glycyrrhiza inflata Bat. (Gi) and Glycyrrhiza glabra L. (Gg), by internal transcribed spacer (ITS) and trnH-psbA sequences. Among them, the S27 sample was identified as Gi but had been incorrectly labeled as Gg. Based on HPLC combined with stoichiometric analysis, the contents of liquiritin, glycyrrhizic acid and isoliquiritin contents were significantly different between Gu and Gg, and Gi and Gg showed significant differences only in isoliquiritigenin content. The chemical components and color difference parameters showed that isoliquiritigenin had a significant correlation with the color parameter b*. The above results show that DNA barcoding can quickly and accurately distinguish species, but it cannot provide data on metabolites or other quality information beyond the plant raw materials. Techniques that combine of DNA barcoding, HPLC and colorimetry can more easily distinguish and evaluate the types and quality of commercial licorice products. This study provides good data support for the safety and quality of licorice in the Chinese market.

Development of Highly Sensitive Chemiluminescence Enzyme Immunostaining Assay to Determine Glycyrrhizin Content Using Anti-glycyrrhizin Monoclonal Antibody

Licorice, the root of Glycyrrhiza spp., is used in a large number of herbal medicines, such as traditional Chinese medicines, Japanese Kampo medicines, and therapeutic drugs. Since glycyrrhizin (GL) is among the main components in licorice and exhibits numerous beneficial pharmacological activities, the content of GL directly affects biological activity. The quality control based on GL content is an important factor in ensuring biological activity; however, the content of GL in licorice varies depending on plant cultivation environment, genetic factors, and species type. Previously, we prepared an anti-GL monoclonal antibody (anti-GL mAb) and employed it in various immunochemical assays for quality control of licorice and licorice-based products. In this study, we employed the anti-GL mAb in chemiluminescence enzyme immunostaining (CLEIS) to develop a very simple, rapid, specific, and sensitive quality control assay for licorice products, with a limit of detection of 3.9 ng. Furthermore, the CLEIS assay enabled semiquantitative analysis of GL in Kampo medicines. Our results showed that multiple samples can be simultaneously analyzed using CLEIS, and it is a useful tool for determining GL content, as well as ensuring chemical quality control of licorice-containing products and herbal medicines.

Glycyrrhizinic acid in licorice products on the Danish market

Glycyrrhizinic acid (GA) is a natural compound found in licorice root and its extract, and is used in a large number of food products. It has been documented that excess consumption of licorice can cause adverse health effects, due to their content of GA. Mandatory labelling rules are therefore in place in the EU to inform consumers of the possible adverse health effects. In this study, 219 samples of confectionery, ice cream, and tea with licorice were sampled in Denmark and analyzed for their GA content. The content of GA was measured with an UPLC-UV method. The results showed that 10% of the samples were inadequately labelled. The samples in each category reaching the highest GA content were confectionery made of pure licorice extract at 23,154 mg/kg, ice cream at 1690 mg/kg, and brewed tea at 1203 mg/L. An intake of 4.3 g of confectionery, 59 g of ice cream, or 83 ml of tea, would surpass the provisionary limit of 100 mg GA/day for regular ingestion. In addition to this health risk scenario from high content products, a larger consumption of licorice products with a lower GA content could also pose a health risk. We suggest campaigns to increase public awareness of the potential adverse health effects of excessive licorice consumption. In addition, it was investigated if 18β-glycyrrhetinic acid (GE) was liberated during the processing steps of licorice production. GE is the pharmacologically active aglucone of GA. 18β-Glycyrrhetinic acid was not identified in any product.

Analysis of international competitiveness of the China's licorice industry from the perspective of global trade

Ethnopharmacological relevanceLicorice is a multi-purpose plant raw material, which is widely used in the pharmaceutical industry and food industry, cosmetic industry, etc. It has a wide application in various countries and regions around the world. Aim of studyThis paper studied the trade situation of licorice-related products among major countries and regions in the world, providing a practical reference for the sustainable development of the global licorice industry. Materials and methodsThe licorice trade data of licorice-related products came from the United Nations Commodity Trade Database and China Customs data. We analyzed the world's major trading countries by using international market share (IMS), trade competitiveness index (TC), average export price (AEP) and average import price (AIP), and analyzed global trade flows with chord diameter. ResultsUzbekistan, Kazakhstan and Iran mainly export licorice raw materials and low value-added products. China is both a producer and a consumer of licorice raw materials and licorice products. The processing trade of the licorice industry in China has advantages, and the structure of import and export trade has been continuously improved. The United States, France, Germany and other developed countries are still important consumers who rely on the intellectual property rights and brand advantages of licorice products, which have stronger global trade radiation capacity. ConclusionsChina's trade structure has been optimized and its industrial competitiveness has been enhanced. China's experience can be used for reference by other countries, especially those with rich licorice resources among the SCO members.

Chemometric analysis and chemical characterization for the botanical identification of Glycyrrhiza species (G. glabra, G. uralensis, G. inflata, G. echinata and G. lepidota) using liquid chromatography-quadrupole time of flight mass spectrometry (LC-QToF)

Licorice is obtained from the roots of several species of Glycyrrhiza (Fabaceae), a genus containing about 20 species. The Chinese Pharmacopeia specifies three Glycyrrhiza species (G. glabra, G. uralensis, G. inflata). In an effort to unambiguously differentiate roots of five Glycyrrhiza species (G. glabra, G. uralensis, G. inflata, G. echinata, and G. lepidota), a LC-QToF method integrated with chemometric tools has been applied, based on chemical profiling of two major classes of saponins and phenolics. Multivariate statistical analysis, together with mass fragmentation of protonated/deprotonated molecular species, resulted in diagnostic fragment ions which were helpful in identification of core structural skeletons as well as functional groups appended to core structures. Roots named in the Chinese Pharmacopeia showed higher content of the triterpene saponinglycyrrhizinalong with phenolics including species-specific components. Most species-specific components were confirmed by comparison with reference standards and were utilized in statistical models to differentiate aerial from root parts. This study investigated 105 licorice samples and 11 licorice dietary supplements using LC-QToF data combined with PCA and PLS-DA models. Species-specific markers including glabridin, glycybridins, hispaglabridins, glabrol from G. glabra, glycycoumarin, licoflavonol, licoisoflavone A/B from G. uralensis, and licochalcones A-E from G. inflata were identified. Additionally, macedonosides and yunganosides were identified from G. lepidota and G. echinata. The investigated licorice dietary supplements were found to contain G. glabra. Furthermore, this study demonstrated the usefulness of these tools for quality assessment of licorice products

Analysis and occurrence of matrine in liquorice raw materials - Exclusion of its application as pesticide

ABSTRACT A quick and selective analytical method was developed employing LC-MS/MS for the quantitation of matrine. This is known to be a natural ingredient of Sophora ssp. and is suggested to be a potential contaminant, e.g. in herbal raw materials from liquorice or confectionery products based on liquorice. To prove that the finding of matrine in liquorice roots does not originate from an active use of pesticides, wild collection areas, as well as geographical, legal and economic aspects have been studied with the help of experienced traders and suppliers in cooperation with local liquorice producers. An LC-MS/MS method was successfully developed and applied for monitoring of raw material and semi-finished products (N = 104) and afterwards a model test was performed to show that findings of matrine in liquorice products originates from a co-harvesting of nearby growing Sophora roots during the manual collection of liquorice roots.

DEVELOPMENT AND VALIDATION OF ULTRAVIOLET–VISIBLE SPECTROPHOTOMETRIC METHOD FOR ESTIMATION OF GLYCYRRHIZIN IN METHANOLIC EXTRACT OF GLYCYRRHIZA GLABRA L

Objective: The pure form of glycyrrhizin was bought from Yucca Enterprises, Mumbai-37, India. The pure form of drug used in treatment of acne vulgaris disease. Pharmacognostical studies of Glycyrrhiza glabra L. indicated surprising antibacterial action against Propionibacterium acnes. &#x0D; Methods: A simple, rapid, accurate, precise, and economic spectrophotometric technique for estimation of glycyrrhizin in methanolic extract of G. glabra L. have been developed. Glycyrrhizin exhibit absorbance most at 254 nm when phosphate buffer (pH-6.8) methanol is used as solvent in 70:30 proportion, so absorbance was once measured at the identical wave lengths for the determination of glycyrrhizin. Glycyrrhizin obeys the Beer Lambert’s law in the concentration range of 4–24 μg/ml.&#x0D; Results: This method was validated according to International Council for Harmonization guidelines and can be adopted for the general analysis of glycyrrhizin in hydroalcoholic extract of G. glabra. The approach is simple, rapid, safe, accurate, affordable, and beneficial for standardization of the licorice products.&#x0D; Conclusion: The results applied in the routine analysis and quality control of pharmaceutical dosage forms containing glycyrrhizin.

Fingerprint analysis and Q-marker prediction of processed liquorice products

Licorice has long been regarded as one of the most popular herbs, with a very wide clinical application range. Whether being used alone or as an ingredient in prescription, it has an important role which cannot be ignored. However, the efficacy and chemical constituents of licorice will change after honey-processing. Therefore, it is necessary to find quality markers before and after honey-processing to lay the foundation for a comprehensive evaluation of the differences between raw and processed licorice pieces. HPLC-DAD was employed to establish fingerprints of raw and processed licorice. Multivariate statistical analysis methods including principal component analysis(PCA) and orthogonal partial least squares discrimination analysis(OPLS-DA) were applied to screen out the differential components before and after processing of licorice. Based on network pharmacology, the targets and pathways corresponding to the differential components were analyzed with databases such as Swiss Target Prediction and Metascape, and the &quot;component-target-pathway&quot; diagram was constructed with Cytoscape 3.6.0 software to predict the potential quality markers. A total of 17 common peaks were successfully identified in the established fingerprint, and seven differential components were selected as potential quality markers(licoricesaponin G2, glycyrrhizic acid, liquiritigenin, liquiritin, isoliquiritin, liquiritin apioside and isoliquiritigenin). The HPLC fingerprint method proposed in this study was efficient and feasible. The above seven differential chemical components screened out as potential quality markers of licorice can help to improve and promote the overall quality. These researches offer more sufficient theoretical basis for scientific application of licorice and its corresponding products.

Comparison of Chinese licorice (Glycyrrhiza uralensis) granules and water extracts and investigation of their antibacterial activities for veterinary application

IntroductionAlthough Chinese licorice (Glycyrrhiza uralensis) granules are widely used in the treatment of human diseases, there are few reports on its veterinary applications. Chinese licorice granules may have multiple advantages compared to its water extract obtained by decoction (tang). The procedure for making granules avoids the extraction steps, significantly reducing the medicine preparation time and making dosage measurements more accurate, thus providing the potential for broader applications. This study compared the glycyrrhizin content and antimicrobial efficacy between Chinese licorice granules and its water extract to determine whether Chinese licorice granules are a reliable alternative to water extract. MethodsThe glycyrrhizin content of Chinese licorice granules was compared to that of water extractions from Chinese licorice using qualitative thin-layer chromatography (TLC) and quantitative high-performance liquid chromatography (HPLC). The efficacy of these two Chinese licorice products was determined by in vitro antibacterial experiments. ResultsTLC analysis showed that Chinese granules had fluorescent spots in the same corresponding positions as Chinese licorice standards and the same yellow fluorescent spot as the glycyrrhizin standards. The content of glycyrrhizin in granules and water extracts was 3.288 ± 0.0376 and 2.721 ± 0.0199 mg/mL respectively as detected by HPLC. HPLC chromatographic fingerprints showed that granules had a 94.9 % similarity with the licorice standard solution, compared to a 91.5 % similarity of the water extraction. In vitro antibacterial assays proved that the granules had higher efficacy than the water extracts, which was comparable to the efficacy of standards licorice solution against Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli and Salmonella enteric. ConclusionsThe data shows that the licorice granules exhibit higher antibacterial efficacy than its water extractions. Our results suggest that Chinese licorice granules may be an alternative to its water extract for treating animals for bacterial infections.

Evaluation of Glycyrrhizic Acid Content in Licorice Roots and Products of its Processing by HPLC-UV Method

Introduction. The actual form of packaging licorice roots for water extraction are filter paper sachets. New forms of licorice root processing provide the proper technological properties of raw materials for filter paper sachets.Aim. A comparative assessment of the of glycyrrhizic acid (GA) content in licorice roots (LR), produced in the Russian Federation and in licorice raw materials of various processing methods by HPLC-UV.Materials and methods. The objects of research were crushed LR, packaged in packs of industrial production and laboratory-industrial samples of coarse powder (CP), cut-pressed granules (CPG) and compositions proposed for packaging in filter paper sachets. The determination of GA content was carried out on an Agilent 1200 high performance liquid chromatography system equipped with a photodiode array detector (Agilent Technologies, USA). The stationary phase is column Phenomenex Luna® C18 (2) 250×4.6 mm. The composition of the mobile phase – 5% orthophosphoric acid water solution: acetonitrile (60:40). A flow rate – 1.0 ml/min, isocratic elution mode. The temperature of the column – 30 °C. The sample injected volume – 10 µl. Detection was performed at a wavelength of 254 nm; run time – 15 minutes.Results and discussion. The GA content in CP, CPG and the composition of CP and CPG –was determined in the ratio of 80:20 (composition). It was established that the GA content varies in the LR in the range – 7.08–9.17%, CP – 3.87–3.90%, CPG – 6.88–7.08%, and the composition – 4.44–4.88%.Conclusion. The use of HPLC-UV method for the standardization of licorice roots of domestic production and the roots of various forms of processing is very promising. The technique can be recommended for inclusion in the State Pharmacopoeia of the Russian Federation of a subsequent edition, which will allow harmonizing the quality requirements of domestic licorice raw materials with the requirements of foreign pharmacopoeias.

Comparison of the inducible effects of licorice products with or without heat-processing and pre-treatment with honey on granulocyte colony-stimulating factor secretion in cultured enterocytes

Ethnopharmacological relevanceLicorice (the roots and rhizomes of Glycyrrhiza uralensis Fisch.) is occasionally used as crude drug following processing including roasting or honey-roasting (soaking with honey before roasting) in traditional Japanese Kampo medicine and traditional Chinese medicine (TCM). Aim of the studyWe investigated the differences in the inducible effect of processed licorice products on granulocyte colony-stimulating factor (G-CSF) secretion in cultured intestinal epithelial cells and elucidated the active ingredients in both unprocessed and processed licorice products. Materials and methodsWe prepared heat-processed licorice with or without pretreatment with honey, and fractionated the extracts by Sephadex G-100. Enterocyte-like differentiated MCE301 cells were incubated in media comprising a hot water extract of licorice products for 24h, and the concentrations of G-CSF in the media were measured using enzyme-linked immunosorbent assay (ELISA). ResultsLicorice extract induced G-CSF secretion in MCE301 cells, and the active ingredients of licorice were high molecular compounds. Although the roasted licorice extract exhibited the activity similar to that of the unprocessed licorice extract, honey-roasted licorice extracts exhibited a significantly higher inducible effect on G-CSF secretion in the cells than that of unprocessed or roasted licorice extracts without pretreatment with honey. This enhanced activity was dependent on the temperature and heating time. ConclusionsThe enhanced inducible effect of honey-roasted licorice on G-CSF secretion might be attributed to the combined effect of licorice-derived high molecular compounds and heated-honey-derived compounds. The results of this study can scientifically explain the objective of processing via honey-roasting in TCM theory.

Ultra high performance liquid chromatography coupled with triple quadrupole mass spectrometry and chemometric analysis of licorice based on the simultaneous determination of saponins and flavonoids.

Licorice is among the most popular herbal medicines and frequently used in traditional medicine, food products, and cosmetics. In China, only Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L. are officially used and are usually processed with honey prior to use. To maintain the quality of commercially available herbal products, a simple, rapid, and reliable ultra high performance liquid chromatography with triple quadrupole mass spectrometry was developed to investigate the major active constituents of commercially available licorice products. Nineteen components were accurately determined, including eight triterpenoid saponins, one triterpene, and ten flavonoids. Subsequently, multivariate statistical analysis methods were employed to further explore and interpret the experimental data. The results indicated that liquiritin apioside may be considered as a candidate index for the quality control of licorice as well as 18β-glycyrrhizic acid and liquiritin. In addition, both 18β-glycyrrhizic acid and licorice-saponin G2 can be used for discrimination between crude and honey-processed licorice. Furthermore, using 18β-glycyrrhizic acid and liquiritin as markers, this work revealed that the quality of licorice products may have declined in recent years. This highlights the need for additional effort focused on good agricultural practice during the processing of licorice. In summary, this study provides a valuable reference for the quality assessment of licorice.

To quit or not: Vulnerability of women to smoking tobacco

ABSTRACTTobacco smoking is currently on the rise among women, and can pose a greater health risk. In order to understand the nature of the increase in smoking prevalence among women, we focused on the vulnerability of women to smoking behaviors—smoking cessation or tobacco addiction—and performed a systematic review of the socioeconomic and intrinsic factors as well as tobacco ingredients that affect women's susceptibility to smoking tobacco. We observed that nicotine and other tobacco components including cocoa-relatives, licorice products, and menthol aggravate tobacco addiction in women rather than in men. Various genetic and epigenetic alterations in dopamine pathway and the pharmaco-kinetics and -dynamic factors of nicotine also showed potential evidences for high susceptibility to tobacco addiction in women. Therefore, we suggest systemic approaches to prevent tobacco smoking–related health risks, considering gene–environment–gender interaction.

Determination of 2-Methylimidazole, 4-Methylimidazole, and 2-Acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole in Licorice Using High-Performance Liquid Chromatography–Tandem Mass Spectrometry Stable-Isotope Dilution Analysis

A quick and selective analytical method was developed for the simultaneous quantitation of 2-methylimidazole, 4-methylimidazole, and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole, which are known to be formed by Maillard reactions. The methodology reported here employs stable-isotope dilution analysis (SIDA) using 4-methylimidazole-d6 and [(13)C6]-2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole as internal standards. It was successfully applied in a model assay to show that the addition of ammonium chloride during the manufacture of licorice promotes imidazole formation depending on the added amount of ammonium chloride without the well-known impact of present caramel food colorings. Furthermore, a monitoring assay of 29 caramel coloring-free licorice products showed that both 4-methylimidazole and 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole are endogenously generated in detectable quantities. None of the samples showed 2-methylimidazole levels above the limit of detection, 50 μg/kg.

Development of a Monoclonal Antibody-Based Immunochemical Assay for Liquiritin and Its Application to the Quality Control of Licorice Products

Liquiritin was reacted with a keyhole limpet hemocyanin (KLH) to synthesize a liquiritin-KLH conjugate as an immunogen for mice. A hybridoma cell line named 2F8 secreted a monoclonal antibody (mAb) against liquiritin, which was applied to an enzyme-linked immunosorbent assay (ELISA) for liquiritin. ELISA showed a good linear range from 0.39 to 25 μg/mL of liquiritin. The maximum relative standard deviation (RSD) values for the intra-assay and interassay were approximately 5%. The recovery rates of liquiritin were in the range of 100.9-103.7%, and the concentrations of liquiritin in various licorice roots, as determined by ELISA, showed a good correlation with those analyzed by high-performance liquid chromatography (HPLC; R² = 0.948). These results suggested that ELISA with anti-liquiritin mAb could be a simple, rapid, convenient, and accurate method for the high-throughput analysis of liquiritin in various licorice products including liqueurs, sweets, and food supplements.

Ochratoxin A and Aflatoxins in Liquorice Products

The occurrence of ochratoxin A (OTA) and aflatoxins (AFs) in liquorice products made in Italy was surveyed. Twenty-eight samples of dried liquorice extract and fifty-four of liquorice confectionery (liquorice content between 2 and 10%) were collected from retail outlets located in northern Italy. After extraction and purification through an immunoaffinity column, OTA and AFs were analysed using both HPLC-FLD and HPLC-MS/MS. OTA occurred in all samples of dried liquorice extract and in 61% of samples of liquorice confectionery, showing very high values for the former (mean 89.6 µg kg-1, maximum value 990.1 µg kg-1), and relatively low levels for the latter (mean 0.96 µg kg-1, maximum value 8.3 µg kg-1). The contribution of dried liquorice extract to OTA intake appears to be non-negligible for children, who are potentially high consumers. AF contamination resulted very low: AFB1 was detected only in 15.8% of samples (maximum value 7.7 µg kg-1, mean 0.38 and 0.41 µg kg-1 for dried liquorice extract and liquorice confectionery, respectively); the other AFs were not detected. To our knowledge, it is the first time that AFB1 has been detected in liquorice extract samples.

Determination of ochratoxin A in liquorice products using HPLC-based analytical methods. Part II: harmonised method and method validation study

On the basis of the outcome of an European proficiency test series conducted on behalf of the CAOBISCO (Association of the Chocolate, Biscuit and Confectionery Industries of the EU) expert group on ochratoxin A, a new harmonised method was developed for the analysis of ochratoxin A in liquorice extracts. This method works without the use of halogenated solvents because, as the proficiency test showed, an aqueous extraction solution can be used instead of, for example, chloroform, whose use is restricted in the EU. The main objective of this method validation study was to check the performance of this harmonised method. To carry out the method validation study, a set of three different test samples (one liquorice powder and two liquorice pastes) and a liquorice powder sample with an indicated range of ochratoxin A (a so-called sunshine sample) was distributed to 21 laboratories in ten countries throughout Europe and to one laboratory in the USA. The study was evaluated according to internationally recognised guidelines. In terms of its repeatability and reproducibility for determining ochratoxin A in liquorice extracts with a relative standard deviation for repeatability (RSDr) of between 6.68 and 19.95 and a relative standard deviation for reproducibility (RSDR) of between 17.39 and 29.08 the performance of the harmonised method was found to be in the accepted range of the EU directive for the analysis of mycotoxins in several foodstuffs.