Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis / 生物工程学报

Genistein and its monoglucoside derivatives play important roles in food and pharmaceuticals fields, whereas their applications are limited by the low water solubility. Glycosylation is regarded as one of the effective approaches to improve water solubility. In this paper, the glycosylation of sophoricoside (genistein monoglucoside) was investigated using a cyclodextrin glucosyltransferase from Penibacillus macerans (PmCGTase). Saturation mutagenesis of D182 from PmCGTase was carried out. Compared with the wild-type (WT), the variant D182C showed a 13.42% higher conversion ratio. Moreover, the main products sophoricoside monoglucoside, sophoricoside diglucoside, and sophoricoside triglucoside of the variant D182C increased by 39.35%, 56.05% and 64.81% compared with that of the WT, respectively. Enzymatic characterization showed that the enzyme activities (cyclization, hydrolysis, disproportionation) of the variant D182C were higher than that of the WT, and the optimal pH and temperature of the variant D182C were 6 and 40℃, respectively. Kinetics analysis showed the variant D182C has a lower Km value and a higher kcat/Km value than that of the WT, indicating the variant D182C has enhanced affinity to substrate. Structure modeling and docking analysis demonstrated that the improved glycosylation efficiency of the variant D182C may be attributed to the increased interactions between residues and substrate.

Quality Analysis and Suggestion for Ginkgo Folium Tablets and Ginkgo Folium Capsules / 中国实验方剂学杂志

Objective:To conduct quality evaluation of Ginkgo Folium preparations by analyzing the national evaluation sampling test results， analyze the quality differences， and put forward suggestions for the improvement of quality standards and market supervision. Method:The contents of total flavonol glycosides and terpene lactones in Ginkgo Folium tablets and Ginkgo Folium capsules were determined according to the methods of determination in the 2015 edition of Chinese Pharmacopoeia （the first volume）， and the contents of free flavonoids （quercetin， kaempferide and isorhamnetin） and sophoricoside in Ginkgo Folium preparations were determined according to related supplementary testing method of Ginkgo Folium tablets and Ginkgo Folium capsules issued by National Medical Products Administration. The quality differences of Ginkgo Folium preparations from different batches and different manufacturers were compared according to the contents of total flavonol glycosides， terpene lactones， free flavonoids and sophoricoside in 328 batches of Ginkgo Folium tablets and Ginkgo Folium capsules manufactured by 48 enterprises. Result:Quality of 328 batches of Ginkgo Folium tablets and Ginkgo Folium capsules was in accordance with the standard， but the contents of terpene lactones and total flavonol glycosides were all distributed in a wide range， and the quality of samples varied greatly among different enterprises. Conclusion:It is recommended that each enterprise should optimize the production process and strictly control the raw materials to ensure the consistency between different batches of samples.

Identification of all herb medicines in Fuhuang Tablet / 中草药

Objective To establish the identification method of Typha angustifolia (stir-bake to black), Sophora japonica, Sanguisorba officinalis (stir-bake to black), and Rheum palmatum in Fuhuang tablets. Methods HPLC method were studied to identify typhaneoside and isorhamnetin-3-O-neoheptanoside in T. angustifolia, and sophoricoside in S. japonica. HPLC conditions were as follows: Agilent TC-C18 (2) column (250 mm × 4.6 mm, 5 μm) with a gradient elution at a temperature of 30 ℃; Phosphate buffer and acetonitrile were used as the mobile phase with a flow rate of 1.0 mL/min; The detection wavelength was 254 nm. The acid hydrolysis-HPLC method was explored to identify S. officinalis (stir-bake to black). TLC identification method was performed to identify R. palmatum. Results HPLC method can identify typhaneoside, isorhamnetin-3-O-neoheptanoside, and sophoricoside synchronously. Acid hydrolysis method can identify S. officinalis (stir-bake to black) by HPLC and TLC can identify R. palmatum. Conclusion The three methods are simple and accurate with high sensitivity and good specificity, which can be used to identify all herbal medicines in Fuhuang tablets.

Effects of different processing methods on content of flavonoids from Sophorae Fructus / 中草药

Objective: To compare the content of flavonoids from Sophorae Fructus by different processing methods. Methods: HPLC method with Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) was used in the experiment; Methanol-0.4% acetic acid was used as mobile phase, with gradient elution; Column temperature was set as 30℃; The flow rate was 1.0 mL/min and detection wavelength was 256 nm. Results: Genistin: crude (0.86%) > stir-frying with honey (0.67%) > carbonizing by stir-frying (0.48%); Rutin: crude (3.0%) > stir-frying with honey (2.2%) > carbonizing by stir-frying (0.88%); Sophoricoside: crude (8.08%) > stir-frying with honey (5.73%) > carbonizing by stir-frying (3.58%); Quercetin: crude (0.04%) < stir-frying with honey (0.05%) < carbonizing by stir-frying (0.12%); Genistein: crude (0.06%) < stir-frying with honey (0.08%) < carbonizing by stir-frying (0.21%); Kaempferide: crude (0.01%) < stir-frying with honey (0.02%) < carbonizing by stir-frying (0.54%). Conclusion: Among the flavonoids from Sophorae Fructus after processing, the content of flavonoid glycosides is reduced and the content of flavonoid aglycone is increased simultaneously, which may be related to the different functions of crude Sophorae Fructus, Sophorae Fructus stir-fried with honey, and Sophorae Fructus carbonized by stir-frying pieces.

Antagonism of Sophoricoside from Sophorica japonica on IL-5 / 천식및알레르기

BACKGROUND: Allergic disease is an inflammatory disease, whose main inflammatory cells are eosinophils, mast cells, and T lymphocytes. From that point, new therapeutic targets on allergic inflammation focusing on them are under investigation. We extracted four isoflavonoids from Sophorica japonica such as sophi, orobol, genistin, and genistein which has been known as PTK antagonist. We documented those three iso-flavonoids except genistein had an anti-inflammatory effect as potent as dexamethasone on carageen-induced ear model. Also they had antagonism on the Y-16 cell line, whose growth is dependent on IL-5. OBJECTIVES: From above results, in this experiment, we tried to find antagonistic effects of those compounds on IL-5 using the inhibition of eosinophil activation and survival in vitro and possibility of anti-allergic medicine. METHODS: LTC4 by RIA and ECP for degranulation by UniCAP(TM), which had been used previously were activation markers. RESULTS: Among those compounds, sophi was the most potent antagonist on IL-5 induced LTC4 release, degranulation, and survival. Orobol and genistin also had antagonism on them, but genistein, an antagonist of PTK didn't show any antagonistic effects. CONCLUSION: From these results, we concluded those three iso-flavonoids were IL-5 antagonist, and the mechanism of it might not be through PTK signaling.

O-glucogenistein inhibits eosinophil recruitment and nasal allergic symptoms in a murine model of nasal allergy / 천식및알레르기

BACKGROUND: Infiltration of eosinophils in the nasal mucosa is a consistent feature of nasal allergic inflammation. Various cytokines, especially interleukin-5(IL-5), were identified to play important roles in the infiltration and activation of eosinophils in nasal mucosa. Our previous study found that among 4 kinds of sophoricosides extracted from Sophora japonica, named sophi, orobol, genistin, and genistein, 3 compounds except genistein known as protein tyrosine kinase(PTK) inhibitor had anti-inflammatory and anti-IL-5 effects, and sophi was the most potent. OBJECTIVE: The goal of this study was to investigate the antagonism of sophi on the nasal eosinophilia in ovalbumin(OA)-sensitized murine nasal allergy model. METHODS: Male BALB/c mice sensitized intraperitoneally and then topically with OA were treated with sophi(10 or 30mg/kg) or anti-mouse IL-5 monoclonal antibody(anti-IL-5 mAb, 1mg/Kg) intravenously 1 hour before challenge. The effect of sophi on the infiltration of eosinophils into the nasal mucosa, peripheral blood eosinophilia, nasal symptom, and OA-specific IgE antibody production were evaluated. Results: Administration of sophi(10, 30mg/kg) significantly inhibited the nasal eosinophil infiltration and nasal symptom compared to that of anti-IL-5 mAb. But eosinophil count inthe peripheral blood and the titer of OA-specific IgE were not affected by sophi. CONCLUSION: Sophi inhibited not only the tissue eosinophilia but also the acute nasal allergic symptom. These findings suggest that sophi has anti-eosinophilic cytokine activity and also plays blockade of early allergic reaction. Taken together, sophi may be a candidate for new anti-allergic medicine.

Antagonism of sophoricoside from sophorica japonica on GM-CSF-induced cosinophil activation / 천식및알레르기

BACKGROUND: allergic disease is an inflammatory disease, whose main inflammatory cells are eosinophils, mast cells, and T lymphocytes. From that point, new therapeutic targets on allergic inflammation focusing on them are under investigation. We extracted four isoflavonoids from sophorica japonica such as sophi, orobol, genistin and genistein which are known PTK antagonists. We documented that these iso-flavonoids except genistein had an antagonism on IL-5 and IL-3 in vitro eosinophil activation and also in allergic mouse model sensitized by OA(ovualbumin). Their common action is due to the common beta chains. GM-CSF also share common beta chains, through which it activates eosinophils. OBJECTIVES: From the above results, We observed the antagonistic effects of these compounds on GM-CSF using eosinophil activation in vitro. METHODS: LTC4 which was detected by RIA and ECP by UniCAP were activation markers. RESULTS: Among those compounds, sophi was the most potent antagonist on GM-CSF induced LTC4 release and even on degranulation and orobol and genistin also had antagonism on them but genistein an antagonist of PTK did not show any antagonistic effects. CONCLUSION: From these results, We concluded these three iso-flavonoids were GM-CSF antagonists and the mechanism might not be through PTK signaling.