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History and the immunostimulatory effects of heat-processed licorice root products with or without honey
Abstract
Ethnopharmacological relevance
In traditional Chinese medicine, the dried root of Glycyrrhiza uralensis Fisch. (licorice root) is usually used after stir-baked with honey. However, in Japanese traditional Kampo medicine, processed licorice root is prepared by roasting without honey.
Aim of the study: We summarized our previous studies on the processed licorice root products to review the effectiveness of the processing for licorice root.
Materials and methods
We summarized our previous studies about processed licorice root. The first report was about investigating the successive literatures of traditional medicine in China and Japan about the processing of licorice root. Next was the report about chemically analyzing for prepared various kinds of processed licorice root samples. The last reports were evaluating in vitro effects of the extracts of these samples and heated honey on granulocyte colony-stimulating factor (G-CSF) secretion in cultured intestinal epithelial cells.
Results
Before Song dynasty in mainland China, the processing of licorice root for the internal usage had been roasted without any drug adjuvants. Then, clinicians had also used honey-roasted licorice to treat throat pain since Song dynasty, and honey-roasted licorice has been used as the substitute to roasted licorice since the end of Qing dynasty. While the descriptions using honey have been disappeared in 18th century in Japan. We found that the conversion between liquiritigenin and isoliquiritigenin or between liquiritin and isoliquiritin in licorice root by heating was accelerated by using honey as drug adjuvant. The inducible effect of G-CSF of licorice root was not augmented by roasting, but significantly augmented by stir-baked with honey. Heated honey also had this activity, and isomaltose contributed the appearance of this activity among the constituents in honey. The best activity was appeared when isomaltose was heated at 180 °C for 60 min or at 200 °C for 15–30 min, and the average molecular weight of the active product was 790 kDa.
Conclusions
By our previous studies, we believe that the processing method in China is better than that in Japan for licorice root, since the immunostimulatory effects are appeared in honey used as drug adjuvant when honey is heated. Among the ingredients of honey, isomaltose can be used as the marker compound to choose a conforming honey product for the processing of licorice root.
... The "using Chinese botanical drugs to process other Chinese botanical drugs" method is the principle of processing the primary medicine by using a certain TCM as processing adjuvant in the processing technology, which could attenuate drug toxic reaction (Huang et al., 2011;Su et al., 2016;Shan et al., 2018;Jiang T. et al., 2021;Zhang K. et al., 2021;Ota and Makino, 2022) or (and) increase the synergistic effect (Huang et al., 2011;Lu et al., 2018;Wang J. et al., 2019;Liao et al., 2021;Kong et al., 2022;Wang T. et al., 2022). Angelicae Sinensis Radix (ASR) is sweet in taste and warm in nature (Chen Q. et al., 2021), and has been proved to have a variety of pharmacological effects such as hepatoprotective , anti-oxidation (Nai et al., 2021), and its main pharmacological components have been proved to significantly reduce liver damage induced by carbon tetrachloride , ethanol (Wang et al., 1997), and concanavalin A . ...
Rhizoma Dioscoreae Bulbiferae (RDB) was effective on relieving cough and expectorant but accompanied by severe toxicity, especially in hepatotoxicity. A previous study found that processing with Angelicae Sinensis Radix (ASR) reduced RDB-induced hepatotoxicity. However, up to now, the optimized processing process of ASR-processed RDB has not been explored or optimized, and the detoxification mechanism is still unknown. This study evaluated the detoxification technology and possible mechanism of processing with ASR on RDB-induced hepatotoxicity. The optimized processing process of ASR-processed RDB was optimized by the content of diosbulbin B (DB), the levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and histopathological analysis. The processing detoxification mechanism was evaluated by detecting the antioxidant levels of nuclear factor E2 related factor 2 (Nrf2) and its downstream heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1), glutamylcysteine ligase catalytic subunit (GCLM), and the levels of downstream antioxidant factors of Nrf2. Besides, the antitussive and expectorant efficacy of RDB was also investigated. This work found that processing with ASR attenuated RDB-induced hepatotoxicity, which can be verified by reducing the levels of ALT, AST, and ALP, and reversing the pathological changes of liver histomorphology. And the optimized processing process of ASR-processed RDB is “processing at a mass ratio of 100:20 (RDB:ASR) and a temperature of 140°C for 10 min.” Further results corroborated that the intervention of processed products of ASR-processed RDB remarkably upregulated the Nrf2/HO-1/NQO1/GCLM protein expression levels in liver, and conserved antitussive and expectorant efficacy of RDB. The above findings comprehensively indicated that the optimized processing process of ASR-processed RDB was “processing at a mass ratio of 100:20 (RDB:ASR) and a temperature of 140°C for 10 min,” and the processing detoxification mechanism involved enhancing the level of Nrf2-mediated antioxidant defense in liver as a key target organ.
... The chemical composition of licorice changed after processing with honey. Honey used as an adjuvant can accelerate the mutual transformation of liquiritin and isoliquirtin in ZGC (Ota and Makino, 2022). In addition, the content of liquiritin, liquiritin apioside, and glycyrrhizin in ZGC has significantly reduced as well . ...
Licorice is well known for its ability to reduce the toxicity of the whole prescription in traditional Chinese medicine theory. However, honey-fired licorice (ZGC for short), which is made of licorice after being stir-fried with honey water, is more commonly used for clinical practice. The metabolism in vivo and detoxification-related compounds of ZGC have not been fully elucidated. In this work, the chemical constituents in ZGC and its metabolic profile in rats were both identified by high ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The network pharmacology was applied to predict the potential detoxifying ingredients of ZGC. As a result, a total of 115 chemical compounds were identified or tentatively characterized in ZGC aqueous extract, and 232 xenobiotics (70 prototypes and 162 metabolites) were identified in serum, heart, liver, kidneys, feces, and urine. Furthermore, 41 compounds absorbed in serum, heart, liver, and kidneys were employed for exploring the detoxification of ZGC by network pharmacology. Ultimately, 13 compounds (five prototypes including P5, P24, P30, P41 and P44, and 8 phase Ⅰ metabolites including M23, M47, M53, M93, M100, M106, M118, and M134) and nine targets were anticipated to be potential mediums regulating detoxification actions. The network pharmacology analysis had shown that the ZGC could detoxify mainly through regulating the related targets of cytochrome P450 and glutathione. In summary, this study would help reveal potential active ingredients in vivo for detoxification of ZGC and provided practical evidence for explaining the theory of traditional Chinese medicine with modern technology.
In this study, an immunocompromised rat model was established by long-term diet control and excessive swimming and treated with extracts of raw licorice (RL) and honey-roasted licorice (HL) to explore the immunomodulatory efficacy and gut microbiota composition with their metabolites. Pharmacological experiment confirmed that honey-roasting enhanced the ability of RL to regulate inflammatory cytokines and gut mucosal-layer homeostasis. The analysis of 16S rRNA sequences showed that both RL and HL regulated the balance of gut microbiota, but HL exhibited better effects than RL in regulating microbiota closely related to inflammatory cytokines (Coriobacteriaceae_UCG-002) as well as short-chain fatty acids-producing bacteria (Dubosiella and Ruminococcus). Moreover, HL improved the contents of butyrate and isobutyrate in feces more significantly than RL. In conclusion, the balance of gut microbiota and short-chain fatty acids might be one of the pathways that honey-roasting improve the immunomodulatory efficacy of licorice.
Abbreviations: ANOVA: analysis of variance; BCA: bicinchoninic acid; ELISA: enzyme-linked immunosorbent assay; HRP: horseradish peroxidase; LDA: the linear discriminant analysis; LEfSe: the linear discriminant analysis effect size; MS: mass spectrometry; OTU: operational taxonomic units; PBS: phosphate buffered saline; PCoA: principal coordinate analysis; PVDF: polyvinylidene fluoride; RIPA: radio immunoprecipitation assay; SCFAs: short-chain fatty acids;
A component of licorice polysaccharide (GPS-1) was extracted from licorice, its primary structure was identified and characterized for the first time, and its immunomodulatory activity was studied. Crude licorice polysaccharide was isolated and purified by DEAE sepharose FF ion-exchange column chromatography and Chromdex 200 PG gel filtration column chromatography to obtain a purified Glycyrrhiza polysaccharide named GPS-1. NMR and methylation analysis revealed that GPS-1 is composed of homogalacturonan (HG)-type pectin with 4)-D-GalpA-(1 as the backbone. This study of GPS-1 also examined its significant role in regulating immune activity in vitro and in vivo . As a result, GPS-1 promoted the secretion of IFN-γ and IL-4 in mice and increased the proportion of CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD8 ⁺ T lymphocytes in their spleens. Dendritic cells (DCs) treated with GPS-1 showed promotion of DC maturation, antigen presentation, and phagocytic capacity. The results suggest that GPS-1 is a potential immunomodulator that stimulates the immune system by regulating multiple signaling pathways. Combined with our characterization of the primary structure of GPS-1, the present investigation provides the basis for future study of the form-function relationship of polysaccharides.
We have previously discovered that heated honey but not unheated honey could induce the secretion of granulocyte-colony stimulating factor (G-CSF) in the MCE301 intestinal epithelial cells. The objective of this study was to identify compounds in honey that could contribute to this activity. We bought several kinds of commercial honey samples derived from different flowers, as well as corn syrup samples, in the markets of China and Japan, and heated them at 180 °C for 30 min. MCE301 cells were treated with the medium containing the samples, and G-CSF levels in the medium were measured by ELISA. By comparing their activities and sugar contents, we discovered that isomaltose was primarily implicated. The optimum heating conditions for isomaltose were at 180 °C for 60 min or at 200 °C for 15-30 min, and these time- and temperature-dependencies were similar to those of honey in our previous study. When heated isomaltose was partitioned by dialysis, the active ingredients were transferred into a high-molecular-weight fraction. By size-exclusion HPLC analysis, the average molecular weight of heated isomaltose was 790 kDa. When heated isomaltose was hydrolyzed by acids, glucose was subsequently produced. Maltose, sucrose, turanose, and trehalose did not exhibited any activity when heated at 180 °C for 60 min, indicating that the glucose groups with α(1 → 6)-binding in the isomaltose molecule play important roles in its activity when oxidatively polymerized by heat. The stimulating activity of heated isomaltose was inhibited by toll-like receptor 4 (TLR4) inhibitor, suggesting that heated isomaltose activates TLR4 to induce G-CSF. Since G-CSF is clinically used for cancer patients to accelerate their recovery from neutropenia following chemotherapy or accompanied with aplastic anemia, these findings indicate that honey which contains high level of isomaltose could improve immunosuppressive conditions when honey is heated, and that heated isomaltose might be of potential therapeutic use in patients with compromised immunity caused by chemotherapeutic agents.
Phenolics and flavonoids in honey are considered as the main phytonutrients which not only act as natural antioxidants, but can also be used as floral markers for honey identification. In this study, the chemical profiles of phenolics and flavonoids, antioxidant competences including total phenolic content, DPPH and ABTS assays and discrimination using chemometric analysis of various Chinese monofloral honeys from six botanical origins (acacia, Vitex, linden, rapeseed, Astragalus and Codonopsis) were examined. A reproducible and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was optimized and validated for the simultaneous determination of 38 phenolics, flavonoids and abscisic acid in honey. Formononetin, ononin, calycosin and calycosin-7-O-β-d-glucoside were identified and quantified in honeys for the first time. Principal component analysis (PCA) showed obvious differences among the honey samples in three-dimensional space accounting for 72.63% of the total variance. Hierarchical cluster analysis (HCA) also revealed that the botanical origins of honey samples correlated with their phenolic and flavonoid contents. Partial least squares-discriminant analysis (PLS-DA) classification was performed to derive a model with high prediction ability. Orthogonal partial least squares-discriminant analysis (OPLS-DA) model was employed to identify markers specific to a particular honey type. The results indicated that Chinese honeys contained various and discriminative phenolics and flavonoids, as well as antioxidant competence from different botanical origins, which was an alternative approach to honey identification and nutritional evaluation.
Licorice (root and rhizome of Glycyrrhiza uralensis Fisch.) is sometimes used as crude drug after processing. In this report, we prepared roasted licorice with or without honey using 3 lots of crude drug samples derived from wild G. uralensis, and analyzed the constituents in unprocessed, roasted, and honey-roasted licorice samples by high performance liquid chromatography–electrospray ionization-ion trap-time of flight mass spectrometry (HPLC–ESI-IT-TOF-MSⁿ) with principal component analysis. We found that the areas of 41 peaks were noticeably changed by processing. Among them, the areas of 12 peaks, viz. isoliquiritin, isoliquiritigenin, glucoisoliquiritin, 6″-O-acetylisoliquiritin, 6″-O-acetylisoliquiritin apioside, glycyrrhetinic acid 3-O-glucuronide, 5 kinds of sugar-derivatives and one compound whose molecular weight was 386 Da were increased by roasting in all 3 lots, and those peak areas were increased by higher heating temperatures. Among the increased peaks, 3 kinds of sugar-derivatives had larger areas, and 6″-O-acetylisoliquiritin had lower areas than those in honey-roasted licorice. Those sugar-derivatives were the only characteristics differing between honey-roasted licorice and roasted licorice. Meanwhile, the areas of 9 peaks, four of them identified as 6″-O-acetylliquiritin, 6″-O-acetylliquiritin apioside, formononetin and gancaonin l, were decreased by roasting in all 3 lots, but there were no differences between roasted licorice with or without honey. Those compounds whose amounts were changed by processing could be used as markers for the quality control of roasted and honey-roasted licorice.
Factors in honey that improve wound healing are poorly understood, but are thought to include lipopolysaccharide (LPS), apalbumin-1 and -2, and a 5.8 kDa component that stimulate cytokine release from macrophages.
To characterize the ability of New Zealand honeys to elicit the release of tumor necrosis factor-α (TNF-α) from monocytic cell lines as a model for early events within a wound site.
The ability of kanuka (Kunzea ericoides), manuka (Leptospermum scoparium), and clover (Trifolium spp.) honeys to stimulate the release of TNF-α from monocytic cell lines THP-1 and U937 was assayed by ELISA.
All three honeys stimulated TNF-α release from THP-1 cells, with kanuka honey being the most active. The activity of kanuka honey was associated with a high molecular weight (>30 kDa) component that was partially heat labile and inhibitable with polymyxin B. LPS concentrations in the honeys were too low to adequately explain the level of immunostimulation. The contribution of type II arabinogalactan proteins (AGPs) we recently identified in kanuka honey was tested, as AGPs are known immunostimulators. AGPs purified from kanuka honey stimulated the release of TNF-α from THP-1 and U937 cells.
Here we demonstrated that AGPs we recently identified in kanuka honey have immunostimulatory activity. We propose that the immunostimulatory properties of individual honeys relate to their particular content of LPS, apalbumins, the 5.8 kDa component and AGPs.
The immunostimulatory activity of kanuka honey may be particularly dependent on AGPs derived from the nectar of kanuka flowers.
Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.
Jungle honey (JH) is collected from timber and blossom by wild honey bees that live in the tropical forest of Nigeria. JH is used as a traditional medicine for colds, skin inflammation and burn wounds as well as general health care. However, the effects of JH on immune functions are not clearly known. Therefore, we investigated the effects of JH on immune functions and antitumor activity in mice. Female C57BL/6 mice were injected with JH (1 mg/mouse/day, seven times intra-peritoneal). After seven injections, peritoneal cells (PC) were obtained. Antitumor activity was assessed by growth of Lewis Lung Carcinoma/2 (LL/2) cells. PC numbers were increased in JH-injected mice compared to control mice. In Dot Plot analysis by FACS, a new cell population appeared in JH-injected mice. The percent of Gr-1 surface antigen and the intensity of Gr-1 antigen expression of PC were increased in JH-injected mice. The new cell population was neutrophils. JH possessed chemotactic activity for neutrophils. Tumor incidence and weight were decreased in JH-injected mice. The ratio of reactive oxygen species (ROS) producing cells was increased in JH-injected mice. The effective component in JH was fractionized by gel filtration using HPLC and had an approximate molecular weight (MW) of 261. These results suggest that neutrophils induced by JH possess potent antitumor activity mediated by ROS and the effective immune component of JH is substrate of MW 261.
Kampo (traditional Japanese herbal) medicines are taken orally due to which the gastric mucosal immune system may act as one of the major targets for the expression of pharmacological activity. The inner surface of the intestinal tract possesses a large area of mucosal membranes, and the intestinal epithelial cells sit at the interface between a lumen and a lymphocyte-rich lamina propria. The cross talk that occurs between these compartments serves to maintain intestinal homeostasis, and the cytokine network plays an important role in the cross talk. In this study, the effect of Hochuekkito (HET), one of Kampo medicines, on cytokine secretion of intestinal epithelial cells was investigated. When murine normal colonic epithelial cell-line MCE301 cells were stimulated with HET, the contents of granulocyte colony-stimulating factor (G-CSF) in the conditioned medium were significantly increased in dose- and time-dependent manners. The enhanced G-CSF gene transcription in MCE301 cells by the stimulation of HET was observed by RT-PCR. The enhanced G-CSF secretion by HET was also observed in C3H/HeJ mice-derived primary cultured colonic epithelial cells. When the HET was fractionated, only the polysaccharide fraction (F-5) enhanced the G-CSF secretion of MCE301 cells, and the activity of F-5 lost after the treatment of periodate that can degrade the carbohydrate moiety. These results suggest that HET enhances secretion of G-CSF from colonic epithelial cells and the polysaccharide is one of the active ingredients of HET. The enhanced G-CSF secretion by HET may partly contribute to the clinically observed various pharmacological activities of HET including immunomodulating activity.
A cDNA sequence coding for murine granulocyte colony-stimulating factor (G-CSF) has been isolated from a cDNA library prepared with mRNA derived from murine fibrosarcoma NFSA cells, which produce G-CSF constitutively. Identification of murine G-CSF cDNA was based on the cross-hybridization with human G-CSF cDNA under a low-stringency condition. The cDNA can encode a polypeptide consisting of a 30-amino acid signal sequence, followed by a mature G-CSF sequence of 178 amino acids with a calculated Mr of 19,061. The nucleotide sequence and the deduced amino acid sequence of murine G-CSF cDNA were 69.3% and 72.6% homologous, respectively, to the corresponding sequences of human G-CSF cDNA. The murine G-CSF cDNA, when introduced into monkey COS cells under the simian virus 40 promoter, could direct the synthesis of a protein that can stimulate the granulocyte colony formation from mouse bone marrow cells and support the proliferation of murine NFS-60 myeloid leukemia cells.
We examined the in vivo effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) in primates (cynomolgus monkeys) treated with subcutaneous doses of rhG-CSF for 14-28 d. A dose-dependent increase in the peripheral white blood cells (WBC) was seen, reaching a plateau after 1 wk of rhG-CSF treatment. The elevation of WBC was due to an increase in the absolute neutrophil count. These results demonstrate that rhG-CSF is a potent granulopoietic growth and differentiation factor in vivo. In cyclophosphamide (CY)-induced myelosuppression, rhG-CSF was able to shorten the time period of WBC recovery in two treated monkeys to 1 wk, as compared to more than 4 wk for the control monkey. Its ability to significantly shorten the period of chemotherapy-induced bone marrow hypoplasia may allow clinicians to increase the frequency or dosage of chemotherapeutic agents. In addition, the increase in absolute numbers of functionally active neutrophils may have a profound effect in the rate and severity of neutropenia-related sepsis. Furthermore, the activities reported here indicate a potential role for rhG-CSF in the treatment of patients with myelodysplastic syndrome, congenital agranulocytosis, radiation-induced myelosuppression, and bone marrow transplantation.
Honey is used as a therapy to aid wound healing. Previous data indicate that honey can stimulate cytokine production from human monocytes. The present study further examines this phenomenon in manuka honey. As inflammatory cytokine production in innate immune cells is classically mediated by pattern recognition receptors in response to microorganisms, bacterial contamination of honey and the effect of blocking TLR2 and -4 on stimulatory activity were assessed. No vegetative bacteria were isolated from honey; however, bacterial spores were cultured from one-third of samples, and low levels of LPS were detected. Blocking TLR4 but not TLR2 inhibited honey-stimulated cytokine production significantly. Cytokine production did not correlate with LPS levels in honey and was not inhibited by polymyxin B. Further, the activity was reduced significantly following heat treatment, indicating that component(s) other than LPS are responsible for the stimulatory activity of manuka honey. To identify the component responsible for inducing cytokine production, honey was separated by molecular weight using microcon centrifugal filtration and fractions assessed for stimulatory activity. The active fraction was analyzed by MALDI-TOF mass spectroscopy, which demonstrated the presence of a number of components of varying molecular weights. Additional fractionation using miniaturized, reverse-phase solid-phase extraction resulted in the isolation of a 5.8-kDa component, which stimulated production of TNF-alpha via TLR4. These findings reveal mechanisms and components involved in honey stimulation of cytokine induction and could potentially lead to the development of novel therapeutics to improve wound healing for patients with acute and chronic wounds.
There is abundant processing theory in the system of traditional Chinese medicine. The deep excavation and research of that is conducive to promoting the development of science of Chinese materia medica (CMM), ensuring the quality of CMM prepared in ready-to-use forms and promoting the clinical rational use of drugs. Based on the current research status of CMM processing theory, using the representative drugs as the object, modern pharmacological research as a guide, the contents of the relevant studies of “raw and cooked different use” theory, “carbonic herbs for hemostasis” theory and theory of auxiliary material action were elaborated. The review provided reference for the further research on traditional processing theory and also provided a scientific basis for the clinical rational use of processed products of CMM. © 2018, Editorial Office of Chinese Traditional and Herbal Drugs. All right reserved.
Ethnopharmacological relevance:
In traditional Chinese medicine (TCM), honey has been used as an additive in the heat-processing of herbal medicines to enhance their immunostimulatory activities.
Aim of the study:
We investigated the immunostimulatory activity of heated honey in vitro and in vivo.
Materials and methods:
For the in vitro study, we compared the differences among the inducible effects of honey subjected to various heating conditions on granulocyte colony-stimulating factor (G-CSF) secretion from the cultured enterocytes and investigated the active ingredient. For the in vivo study, we conducted a survival test of mice infected by Streptococcus pyogenes with and without oral administration of heated honey.
Results:
We found that heating the honey induced the appearance of G-CSF secretions from the cultured enterocytes, and that this appearance depended on the heating temperature and time. No G-CSF secretions appeared when honey was not heated. Mice infected with Streptococcus pyogenes that were fed heated honey revealed prolonged survival. The active ingredient in heated honey was a high-molecular compound with about 730kDa. When this compound was hydrolyzed, galactose, glucose, rhamnose, α-ribofuranose β-ribofuranose 1,5':1',5-dianhydride, and 5-hydroxymethylfurfural were generated.
Conclusions:
Heated honey reveals immunostimulatory activity both in vitro and in vivo. These results support the scientific evidences of the TCM theory.
Ethnopharmacological relevance:
Licorice (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 study:
We 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 methods:
We 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 24 h, and the concentrations of G-CSF in the media were measured using enzyme-linked immunosorbent assay (ELISA).
Results:
Licorice 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.
Conclusions:
The 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.
In China, the crude drug licorice ("kanzo" in Japanese, "gancao" in Chinese) has been used both dried and roasted as the situation demands from ancient times. The meaning of "roasted licorice" is simply roasted and honey-roasted in ancient and modern times, respectively. However, it is not clear medicinal purposes of processed licorice or why licorice processed with honey began to be used. We researched ancient literature and found that the main objective of roasting was to change the property of licorice from cool to warm (i.e., dried licorice had the effect of draining fire), while roasted licorice was used as an energy supplement, having a digestive effect and thus warming the body. Meanwhile, doctors began using honey-roasted licorice to treat throat pain from the Song dynasty, and then at the end of the Qing dynasty, honey-roasted licorice was expected to have the same effects of roasted licorice (i.e., supplementing energy and having a digestive effect).
This study aimed to assess 5-hydroximethylfurfural and carbohydrates (fructose, glucose, and sucrose) in 13 stingless bee honey samples before and after thermal treatment using a capillary electrophoresis method. The methods were validated for the parameters of linearity, matrix effects, precision, and accuracy. A factorial design was implemented to determine optimal thermal treatment conditions and then verify the postprocedural 5-HMF formation, but once 5-HMF were <LOQ was not possible to build a response surface. The methods applied to samples obtained from Brazil, expressed good linearity, precision, and accuracy. None of the thirteen in natura samples presented 5-HMF, and carbohydrate levels ranged from 48.59% to 69.36%. In the same conditions of thermal treatment, Apis mellifera honey presented higher 5-HMF content than stingless bee honey. Results suggest that a high temperature related to briefer thermal treatment could be an efficient way to extend shelf life without affecting 5-HMF content in stingless bee honey.
There is a wealth of information about the nutritional and medicinal properties of honey. However, honey may contain compounds that may lead to toxicity. A compound not naturally present in honey, named 5-hydroxymethylfurfural (HMF), may be formed during the heating or preservation processes of honey. HMF has gained much interest, as it is commonly detected in honey samples, especially samples that have been stored for a long time. HMF is a compound thatmay be mutagenic, carcinogenic and cytotoxic. It has also been reported that honey can be contaminated with heavy metals such as lead, arsenic, mercury and cadmium. Honey produced from the nectar of Rhododendron ponticum contains alkaloids that can be poisonous to humans,while honey collected from Andromeda flowers contains grayanotoxins,which can cause paralysis of limbs in humans
and eventually leads to death. In addition, Melicope ternata and Coriaria arborea from New Zealand produce toxic honey that can be fatal. There are reports that honey is not safe to be consumed when it is collected from Datura plants (from Mexico and Hungary), belladonna flowers and Hyoscamus niger plants (fromHungary), Serjania lethalis (fromBrazil), Gelsemiumsempervirens (from the American Southwest), Kalmia latifolia, Tripetalia paniculata and Ledum palustre. Although the symptoms of poisoning due to honey consumption may differ depending on the source of toxins, most common symptoms generally include dizziness, nausea, vomiting, convulsions, headache, palpitations or even death. It has been suggested that honey should not be considered a completely safe food.
In traditional Chinese medicine (TCM), licorice is usually processed with honey and traditionally used in decoction form. However, the influence of honey-roasting on the main pharmacological activities and the water-soluble active constituents of licorice has not been reported. The aim of the present study is to determine whether honey-roasting can modify the main pharmacological activities and the active constituents of licorice. According to licorice clinical application and processing method, the mainly related pharmacological activities of crude licorice, processed licorice and refined honey, such as enhancing immune function, relieving cough, eliminating phlegm and detoxication, were compared. The results showed that honey-roasting obviously reinforced the licorice activity of enhancing Pi-deficiency mice's immune function, and significantly weaken the licorice activity of relieving cough, removing phlegm and detoxication. However, honey didn't show the significant activity of relieving cough, removing phlegm and detoxication. The influence of honey-roasting on the chemical compositions in licorice slice and licorice decoction was investigated by using HPLC. The results showed that the content and the decocting quantity of mainly 5 active glycosides in licorice, i.e. liquiritin apioside, liquiritin, licuraside, isoliquiritin and glycyrrhizin, obviously changed after processing; glycyrrhizin and liquiritin obviously decomposed during honey-roasting. In conclusion, honey-roasting obviously modified the main pharmacological activities and the water-soluble compositions of licorice. The modification was not cause by honey only. This finding may shed some light on understanding the differences in the therapeutic values of crude and processed licorice.
Neo-formed contaminants (NFCs) are compounds forming during heating or preservation processes and exhibiting possible harmful effects to humans. Among the several NFCs described in literature, Acrylamide and 5-hydroxymethylfurfural (HMF) have attracted the attention of the scientific community in recent years. Both acrylamide and HMF are considered as probably or potentially carcinogenic to humans or might be metabolized by humans to potentially carcinogenic compounds. Acrylamide and HMF are mainly formed through Maillard Reaction and can be regarded as the most important heat-induced contaminants occurring in bread and bakery products. Acrylamide is carcinogen in rodent and some recent epidemiological studies have highlighted the association between dietary acrylamide and an increased risk of some types of cancer. HMF has been recently shown to be converted in vivo to 5-sulfoxymethylfurfural (SMF) which is a genotoxic compound. Dietary intake of HMF is in the order of mg/kg far above that of other food toxicants. In this paper, the latest available data on acrylamide and HMF have been reviewed focusing on available mitigation strategies, metabolism, dietary exposure, and toxicity. The results from the epidemiological studies about acrylamide and cancer risk and their relevance have been discussed, the major gaps of knowledge have been identified and the perspective of ongoing and future research was established.
a b s t r a c t Size-exclusion chromatography (SEC) and activity-guided fractionation of honeys allowed the isolation of high molecular weight brown compounds, ranging in size from 66 to 235 kDa that exhibited peroxyl rad-ical-scavenging activity. Their concentrations, antioxidant activity and degree of browning increased after heat-treatment of honeys, suggesting that they represent melanoidins. Chemical analysis of mela-noidins demonstrated the presence of proteins, polyphenols and oligosaccharides. Heat-treatment caused an increased incorporation of phenolics into high molecular weight melanoidins and drastically decreased the protein content in these fractions with a concomitant appearance of high molecular weight protein–polyphenol complexes of reduced solubility. LC–ESI–MS demonstrated the presence of oligosac-charide moieties, supporting the postulated origin of melanoidins. The changes in the phenolic content of melanoidins from heated honeys were strongly correlated with their oxygen radical absorbance capacity (ORAC) values (R = 0.75, p < 0.0001), indicating that polyphenols contribute to the antioxidant activity of melanoidins. In summary, honey melanoidins are multi-component polymers consisting of protein– polyphenol–oligosaccharide complexes. A direct interaction between polyphenols and melanoidins resulted in a loss or gain of function for melanoidin antioxidant activity.
To establish the HPLC fingerprint of the pieces of honey-fried Radix Glycyrrhizae.
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.
Established HPLC fingerprint of Radix et Rhizoma glycyrrhizae pieces were established, and the results of methodological study met the technical requirements for fingerprinting.
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.
Several glycoproteins that control blood-cell production and function have been purified and sequenced. The four colony-stimulating factors interact in a complex way to regulate the differentiation and maturation of the granulocyte and macrophage lineages and have potential applications for the clinical manipulation of blood-cell production.
We have recently purified murine granulocyte colony-stimulating factor (G-CSF), a regulatory glycoprotein which stimulates granulocyte colony formation from committed murine precursor cells in semi-solid agar cultures. G-CSF is one of a family of colony-stimulating factors that regulate the growth and differentiation of granulocytes and macrophages. While the other murine CSFs (granulocyte-macrophage (GM)-CSF, macrophage (M)-CSF and multi-CSF) show little or no differentiation-inducing activity on murine myelomonocytic leukaemia cell lines, G-CSF (or MGI-2(6)) is able to induce the production of terminally differentiated cells from WEHI-3B and other myeloid leukaemia cell lines. More importantly, G-CSF-containing materials suppress the self-renewal of myeloid leukaemia stem cells in vitro and the leukaemogenicity of treated myeloid leukaemic cells in vivo. It is important to identify the human analogue of murine G-CSF so that its effectiveness on human myeloid leukaemia cells can be assessed. Here we show that an analogue of G-CSF does exist among the CSFs produced by human cells and that the murine and human molecules show almost complete biological and receptor-binding cross-reactivities to normal and leukaemic murine or human cells. The human G-CSF analogue is identified as a species of CSF that we have previously described as CSF-beta.
As a continuing study of chemical characterization of crude drug processing, we have been analyzing the chemical constituents in licorice roots before and after processing. At first, we analyzed chemical constituents in licorice roots of various origins. Next, we have developed the HPLC analytical method, by which saponins and flavonoids, major constituents in various licorice roots, were determined simultaneously in a quantitative manner. In this paper, by means of the HPLC analytical method, chemical constituents in licorice roots, processed and unprocessed, were determined. It was found that nonglycosidic flavonoid constituents were mostly lost while root bark removing, whereas, in roasted licorice roots, sugar chains in the saponin and glycosidic flavonoid constituents were hydrolyzed stepwise during roasting through hydrothermolysis.
We produced an immortalized colonic epithelial cell line, MCE301, using fetal mice transgenic for the temperature-sensitive simian virus 40 large T-antigen gene. MCE301 cells showed epithelial-like morphology and maintained tight connections with neighboring cells. The cells grew at a permissive temperature (33 degrees C), but the growth of the cells was significantly prevented at the nonpermissive temperature (39 degrees C). The cells expressed large T-antigen at 33 degrees C but not at 39 degrees C. MCE301 cells were not transformed, as judged by the absence of anchorage-independent growth in soft agar gel and lack of tumor formation in nude mice. Electron microscopic studies showed that the cells formed microvilli-like structures on the cell surface and junctional complexes such as tight junctions and desmosomes between the cells. The cells expressed cytosketal (acidic cytokeratins and actin), basement membrane (laminin and collagen type IV) and junctional complex proteins (ZO-1 and desmoplakin I + II), as judged by specific antibodies. Fetal bovine serum, epidermal growth factor, insulin-like growth factor and insulin significantly increased the cell growth at 33 degrees C. Moreover, MCE301 cells expressed colonic mucin Muc2 mRNA as demonstrated by reverse transcriptase-polymerase chain reaction, indicating that the cells originate from mucus-secreting cells. Alkaline phosphatase, a brush border-associated enzyme, was detected in the cells. Sodium butyrate (2 mM), an inducer of cellular differentiation, markedly elevated alkaline phosphatase activity. Thus, the present mouse colonic epithelial cell line MCE301 possessing these unique characteristics should provide a useful in vitro model of colonic epithelium.
In herbal medicine, licorice is usually processed using a roasting procedure which might modify the chemical compositions in licorice. To test this hypothesis, licorice root samples were roasted under various conditions (with or without honey) and subsequently extracted by refluxing with 95% ethanol. The analysis of chemical compositions of licorice root extracts was achieved by capillary electrophoresis. The running buffer has been optimized to be 50 mM sodium tetraborate (pH 9.01) containing 5 mM beta-cyclodextrin. Thermal decomposition of glycyrrhizin, which was a major ingredient in licorice, was first studied in detail, indicating the conversion of glycyrrhizin to glycyrrhetinic acid. The licorice extracts were then analyzed to indicate the above thermal conversion did occur in the licorice samples. This finding may shed some light on understanding the differences in the therapeutic values of raw versus roasted licorice in herbal medicine.
High-dose clindamycin (CLDM) and benzylpenicillin (PCG) are the recommended chemotherapeutic remedies for toxic shock-like syndrome caused by group A streptococci. One reason for this is that it has been shown that CLDM suppresses the expression of some exoproteins, e.g., SpeB, SpeA, and streptolysin O (Slo). We analyzed the effects of antibiotics on the production of whole exoproteins by two-dimensional gel electrophoresis. Unexpectedly, we found that the levels of several exoproteins, Slo, NAD(+)-glycohydrolase (Nga), M protein, and Sic, were increased by CLDM treatment, although we also confirmed previous findings that the levels of various exoproteins, including SpeB, were decreased. The increases in exoprotein levels were also detected by using other protein synthesis inhibitor antibiotics: erythromycin, kanamycin, tetracycline, chloramphenicol, and linezolid. Peptidoglycan synthesis inhibitors (such as PCG, cefazolin, and imipenem), DNA replication inhibitors (such as gatifloxacin), and an RNA polymerase inhibitor (rifampin) did not have significant effects on exoprotein production. The combination of CLDM and PCG had no advantageous effects with regard to exoprotein production compared to the effect achieved with CLDM alone. We also analyzed the transcriptional levels of slo and nga by reverse transcription-PCR and found that this change was also detected at the transcriptional level. Furthermore, the phenomenon was seen not only in strains of the M1 serotype but also in strains of the other M serotypes. Our study suggests that the clinical effectiveness of CLDM might be due to the inhibition of the production of a limited number of exoproteins.
The objective of this study was to evaluate the immunomodulatory effects of the purified glycyrrhiza polysaccharides (GP) on the activity of macrophages. A purified fraction of water-soluble polysaccharides, with estimated molecular weight of 10 kDa, was isolated from Glycyrrhiza uralensis Fish using ion exchange and size exclusion chromatography. The results indicate that GP increased the pinocytic activity, the production of nitric oxide (NO), interleukin-1 (IL-1), IL-6 and IL-12 in a dose-dependent manner. The production of IL-1 was induced by GP at a dose of 10 microg/mL; but, NO, IL-6 and IL-12 was significantly induced at 100 microg/mL. A time-dependent enhancement showed that the production of IL-1, NO and IL-12 were significantly increased within 6 h. Superoxide anion (O(2)(-)) production by macrophages from GP-treated mice was higher than that of cells from untreated mice. Moreover, cells from both untreated and treated mice responded to phorbol 12-myristate 13-acetate (PMA) treatment; however, the O(2)(-) production was higher in the cells from treated mice than that of cells from untreated mice. Our data suggest that the beneficial therapeutic effects of GP may be attributed partly to its ability to modulate macrophage immune functions.
Rpt. Songs including detailed annotation of materia medica (本草歌括詳注)
- Liang
Rpt. The invention of materia medica (本草発明)
- Huang
Rpt. Amplified clauses of materia medica (本草衍句)
- Huang
Rpt. Amplification on materia medica (本草衍義)
- Kou
Rpt. Dongyuan's trials of effective prescriptions (東垣試效方)
- Li
History of the titles of materia medica for decoctions (湯液本草) and cishinanzhi (此事難知)
- Mayanagi
Rpt. Great compedium of Broad Benefit in materia medica (広益本草大成)
- Okamoto
Rpt. Records of Japanese name and its synonyms in drug products (和名集并異名製剤記)
- Bai
Rpt. Handbook of studying drugs (研薬指南)
- He
Rpt. Master Lei’s Discourse on medicinal processing (雷公炮炙論)
- Lei
Rpt. Compendium of materia medica jinling book (本草綱目金陵本)
- Li
Comparison of the names and origins of crude drugs used in ethical Kampo extract formulation and listed in the European and the United States Pharmacopoeias with the Pharmacopoeias of East Asian countries
- Makino
Rpt. Records of different names in materia medica (本草異名記)
- Manase
Rpt: supplement to prescriptions Worth a Thousand Gold pieces (千金翼方)
- Sun
Rpt. Collecting Envelope in medicine (医学彙函)
- Nie
Rpt: collective Commentaries on Classics of materia medica (本草経集注)
- Tao
Rpt. Materia medica for decoctions (湯液本草)
- Wang
Rpt. Sacred treasures of drug properties, Efficacies, and toxicities, new edition (新編霊宝薬性能毒)
- Unknown
HPLC with switching wavelength simultaneous determination of seven constituents in licorice and its processed products
- Zhou
Rpt. Promise story of materia medica (本草約言)
- Xue
Effects on immunologic active of Glycyrrizae Radix et Rhizome pieces before and after processed with honey
- Zhang
Fingerprint of ethyl acetate extract of Glycyrrhizae Radix et Rhizoma Praeparta
- Su