Expression of β-glucosidase An-bgl3 from Aspergillus niger for conversion of scopolin / 生物工程学报

Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However, interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding β-glucosidase An-bgl3 derived from Aspergillus niger were carried out. The expression product was purified and characterized with further structure-activity relationship between it and β-glucosidase analyzed. Subsequently, its ability for transforming scopolin from plant extract was studied. The results showed that the specific activity of the purified β-glucosidase An-bgl3 was 15.22 IU/mg, the apparent molecular weight was about 120 kDa. The optimum reaction temperature and pH were 55 ℃ and 4.0, respectively. Moreover, 10 mmol/L metal ions Fe2+ and Mn2+ increased the enzyme activity by 1.74-fold and 1.20-fold, respectively. A 10 mmol/L solution containing Tween-20, Tween-80 and Triton X-100 all inhibited the enzyme activity by 30%. The enzyme showed affinity towards scopolin and tolerated 10% methanol and 10% ethanol solution, respectively. The enzyme specifically hydrolyzed scopolin into scopoletin from the extract of Erycibe obtusifolia Benth with a 47.8% increase of scopoletin. This demonstrated that the β-glucosidase An-bgl3 from A. niger shows specificity on scopolin with good activities, thus providing an alternative method for increasing the extraction efficiency of scopoletin from plant material.

Studies on chemical constituents of Chloranthus fortunei / 中草药

Objective: To study the antitumor constituents from Chloranthus fortunei. Methods: Various chromatographic techniques and spectroscopic methods were applied to investigate the chemical constituents from C. fortunei, and some of the compounds were screened for their antitumor activities by MTT method. Results: Sixteen compounds were obtained from the whole plants of C. fortunei and identified as rosmarinic acid (1), 2’-hydroxy-4,3’,4’,6’-tetramethoxychalcone (2), flavokawain A (3), cycloshizukaol A (4), atractylenolide III (5), 4β-hydroxy-8,12-epoxyeudesma-7,11-diene-1,6-dione (6), (8α)-6,8-dihydroxycadina-7 (11),10 (15)-dien-12-oic acid γ-lactone (7), curcolonol (8), 11-hydroxyldrim-8,12-en-14-oic acid (9), friedelin (10), isovanillic acid (11), 6β-hydroxystigmast-4-en-3-one (12), 3,4-dihydroxybenzoic acid (13), shikimic acid (14), scopolin (15) and N-acetyltyramine 1-O-β-D-glucoside (16). Compounds 4 and 5 showed weak cytotoxicity with IC50 ranged from 46 to 85 μmol/L. Conclusion: Compounds 2, 10, 11, and 13-15 are obtained from the genus Chloranthus for the first time and compounds 1-3 and 6-16 are isolated from C. fortunei for the first time. Some sesquiterpenoids from C. fortunei exhibited weak antitumor activities.

In vivo metabolism of three coumarins from Chimonanthi Radix based on UHPLC-QTOF-MS/MS / 中国中药杂志

Scopolin (SC-1), scopoletin (SC-2) and isofraxidin (IS-1) are the main active constituents in Chimonanthi Radix. However, the in vivo metabolism of SC-1, SC-2 and IS-1 have not been comprehensively clarified. In this study, the in vivo metabolic profiles of these three coumarins in the rat plasma, urine and feces were analyzed. Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS/MS) method was applied to characterize the prototypes and metabolites of SC-1, SC-2 and IS-1 in rat feces, urine, and plasma after intravenous administration. A total of 11 metabolites of the three parent compounds were tentatively identified. The main metabolic pathways were analyzed by identification of metabolites, and it was found that these three coumarins underwent multiple in vivo metabolic reactions including glucuronidation, sulfonation, isomerism and reduction. In this study, the analysis of metabolites of three coumarins basically demonstrated their in vivo metabolic process, providing basis for the further pharmacokinetics and pharmacological evaluations of SC-1, SC-2 and IS-1.

Determination of lobetyolin, scopolin, scopoletin, quercetin, kaempferol and isorhamnetin in Shenqi Jiu by HPLC with gradient elution method / 国际药学研究杂志

Objective To develop an HPLC method with gradient elution for determination of lobetyolin, scopolin, scopoletin, quercetin, kaempferol and isorhamnetin in Shenqi Jiu. Methods A Shiseido C18 column (4.6 mm×250 mm, 5μm) was adopted, the mobile phase was acetonitrile (A)-0.5% acetic acid solution (B) with gradient elution at a flow rate of 1.0 ml/min, the column temperature was set at 30 ℃, the sample quantity was 20 μl, and the detection wavelengths were 269 (lobetyolin), 346 (scopolin and scopoletin) and 360 nm (quercetin, kaempferol and isorhamnetin). Results The linear ranges of above mentioned 6 ingredients in order fell within the range of 4.59-91.80 (r=0.9999), 2.62-52.40 (r=0.9996), 1.95-39.00 (r=0.9998), 3.34-66.80 (r=0.9995), 2.30-46.00 (r=0.9991), and 2.86-57.20 μg/ml (r=0.9999), respectively. The average recoveries and RSD (n=9) were 98.27% (1.33%), 97.62% (1.48%), 99.17% (0.99%), 98.50% (1.37%), 97.35% (1.35%), and 98.86% (1.70%), respectively. Conclusion The established HPLC gradient elution method can simultaneously determine the above mentioned 6 ingredients Shenqi Jiu. The method is simple, accurate, with good reproducibility. The method is helpful for the quality control of Shenqi Jiu.

Determination of lobetyolin,scopolin,scopoletin,quercetin,kaempferol and isorhamnetin in Shenqi Jiu by HPLC with gradient elution method / 国际药学研究杂志

Objective To develop an HPLC method with gradient elution for determination of lobetyolin,scopolin,scopole?tin,quercetin,kaempferol and isorhamnetin in Shenqi Jiu. Methods A Shiseido C18 column(4.6 mm×250 mm,5μm)was adopted, the mobile phase was acetonitrile(A)-0.5%acetic acid solution(B)with gradient elution at a flow rate of 1.0 ml/min,the column tem?perature was set at 30℃,the sample quantity was 20μl,and the detection wavelengths were 269(lobetyolin),346(scopolin and sco?poletin)and 360 nm(quercetin,kaempferol and isorhamnetin). Results The linear ranges of above mentioned 6 ingredients in order fell within the range of 4.59-91.80(r=0.9999),2.62-52.40(r=0.9996),1.95-39.00(r=0.9998),3.34-66.80(r=0.9995),2.30-46.00 (r=0.9991),and 2.86-57.20μg/ml(r=0.9999),respectively. The average recoveries and RSD(n=9)were 98.27%(1.33%),97.62%(1.48%),99.17%(0.99%),98.50%(1.37%),97.35%(1.35%),and 98.86%(1.70%),respectively. Conclusion The established HPLC gradient elution method can simultaneously determine the above mentioned 6 ingredients Shenqi Jiu. The method is simple,ac?curate,with good reproducibility. The method is helpful for the quality control of Shenqi Jiu.

Study on chemical constituents from whole herbs of Xanthium mongolicum / 中草药

Objective: To study the chemical constituents in the whole herbs of Xanthium mongolicum. Methods: The chemical constituents were isolated and purified by chromatography on silica gel column and HPLC, and their structures were elucidated by spectral analysis. Results: Seventeen compounds were isolated and identified as lasidiol p-methoxybenzoat (1), β-selinene (2), xanthatin (3), xanthinosin (4), luteone (5), daucosterol (6), 4β,5β-epoxy xanthatin-1α,4α-endoperoxide (7), (6S,9R)-vomifoliol (8), dehydrovomifoliol (9), 3(Z)-hexenyl-β-D-glycoside (10), 4-oxo-bedfordia acid (11), 11α,13-dihydro-8-epi-xanfbut (12), scopolin (13), pinoresinol (14), β-sitosterol (15), quercetin (16), and methyl p-hydroxybenzonate (17). Conclusion: The compounds 2, 8, 9, 10, and 17 are isolated from the plants of Xanthium L. for the first time and the compounds 1, 5, 7, 11 and 13 are isolated from the whole herbs of X. mongolicum for the first time.