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.

A new lignan from Artemisiae Annuae Herba / 中草药

Objective: To study the constituents from the dried aboveground part of Artemisia annua. Methods: The chemical constituents were isolated by various chromatographic techniques and their structures were elucidated by spectroscopic analyses and comparison of NMR data with those reported in literatures. Results: Six compounds were obtained and characterized as artelignan (1), scopoletin (2), scoparone (3), chrysosplenol B (4), jaceidin (5) and mikanin (6). Conclusion: Compound 1 is identified as a new compound named artelignan.

Cellular Uptake and Metabolism of Soluplus-based Scopoletin Micelles in Caco-2 Cells / 中国药学杂志

OBJECTIVE: To develop an analytical method for the quantification of scopoletin (Sco) and investigate the cellular uptake and metabolism of polyvinylcaprolactam-polyvinyl acetate-polyethylene glycol (soluplus)-based Sco micelles (Sco-Ms) in Caco-2 cells, as well as exploring the possible mechanisms involved in the oral absorption of Sco-Ms.METHODS: Combined with enzymatic hydrolysis for pretreatment, a liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-MS/MS) method was developed for the quantification of Sco and its corresponding metabolite. Then, cellular uptake efficiency and metabolic rate of Sco were calculated.RESULTS: This method was proven to be linear over the concentration range of 5-1 000 ng•mL-1. Cellular uptake of Sco-Ms increased significantly compared with that of free Sco at various time points. Meanwhile, Sco-Ms inhibited the enzymatic degradation of Sco. Sco-Ms were primarily internalized into enterocytes via macropinocytosis and clathrin-dependent pathways.CONCLUSION: Enhanced cellular uptake and decreased metabolic rate are pivotal mechanisms by which Sco-Ms promotes oral absorption of Sco.

A new sesquiterpenoid from Artemisia lavandulaefolia / 中草药

Objective To study the chemical constituents from the aerial parts of Artemisia lavandulaefolia. Methods The constituents were isolated and purified by various column chromatographic techniques, and their structures were identified by spectroscopic analysis. Results Eleven compounds were isolated from 95% ethanol aqueous extract of A. lavandulaefolia and the structures were identified as (4R,5R,7R,10R)-4-hydroxy-eudesma-2,11-dien-1-one (1), 5-epi-eudesma-4(15)-ene-1β,6β-diol (2), eudesma-4(15)-ene-1β,6α-diol (3), methyl 3-oxo-eudesma-1,4,11(13)-trien-12-oate (4), 11β,13-dihydrosantamarin (5), anthemidin (6), scopoletin (7), paeonol (8), ethyl caffeate (9), espeletone (10), and (1R*,2S*,3S*,4S*)-mentha-1,2,3,4-tetrol (11). Conclusion Compound 1 is a new compound named artemilavanone A, and compounds 2-11 are isolated from this plant for the first time.

Chemical constituents from leaves of Adinandra nitida (II) / 中草药

Objective: To study the chemical constituents from the ethyl acetate extract of the leaves of Adinandra nitida. Methods: Several column chromatography including silica gel, Sephadex LH-20 gel, and ODS were applied in isolation and purification. The structures were elucidated on the basis of physicochemical properties and spectral data from 1H-NMR, 13C-NMR, ESI-MS and so on. Results: Nine flavonoids, three phenolic acids, and two other compounds were isolated and determined as apigenin (1), naringenin (2), luteolin (3), chrysoeriol (4), (-)-epicatechin (5), quercetin (6), quercitrin (7), camellianin A (8), camellianin B (9), p-methoxyphenol (10), protocatechuic acid (11), 3,3',4,4'-tetrahydroxybiphenyl (12), scopoletin (13), and palmitic acid (14). Conclusion: Compounds 3, 10-14 are firstly isolated from the plant of A. nitida and compounds 3, 10, 12, and 13 are obtained from Theaceae for the first time.

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.

Improvement in Quality Control Methods for Belladonna Oral Solution / 中国药师

Objective:To improve the identification and determination methods in the original quality standard for belladonna oral solution.Methods:Belladonna oral solution was identified by a specific chromatogram of HPLC,and scopoletin and hyoscyamine sulfate in belladonna oral solution were detected by dual wavelength spectrophotometry.The detection of fingerprints was performed on a Waters SunFire C18 (250 mm×4.6mm,5 μm) column.The mobile phase was methanol-0.05% phosphoric acid solution at a flow rate of 1.0 ml·min-1.The detection wavelength was 344 nm and the column temperature was 30℃.The detection of scopoletin and hyoscyamine sulfate was performed on the same C18 column.The mobile phase was 10 mmol·L-1 heptanesulfonate sodiumat (pH value was 3.3 adjusted by glacial acetic acid)-absolute ethanol-acetonitrile (68.75∶6.25∶25) at a flow rate of 1.0 ml·min-1.The detection wavelengths were 344 nm and 210 nm,and the column temperature was 30℃.Results:The specific chromatogram of belladonna oral solution was accordance with that of belladonna tincture raw material.The retention time and relative peak area of each characteristic peak and reference peak all met requirements of Chinese Pharmacopoeia.Scopoletin and hyoscyamine sulfate were completely separated from the other compositions under the above mentioned conditions.The calibration curves were linear within the range of 5.168-103.360 μg·ml-1 (r=1.000 0) for scopoletin and 50.560-758.400 μg·ml-1 (r=0.999 9) for hyoscyamine sulfate.The average recovery was 101.79% (RSD=1.05%,n=6) and 100.92% (RSD=0.97%,n=6),respectively.Conclusion:After the quality control method improvement,the identification shows high specificity and the quality of belladonna oral solution can be better controlled by the two selected index components.The method is easy and accurate,which can provide a reliable way to control the quality.

Chemical Constituents of Chimonanthus nitens Oliv / 中国药学杂志

OBJECTIVE: To study the chemical constituents from the leaves of Jiangxi genuine medicinal material Chimonanthus nitens Oliv. METHODS: The compounds were isolated and purified by silica gel column chromatography, Sephadex LH-20, ODS column chromatography, semi-preparative HPLC, and so on. Their structures were elucidated on the basis of physiochemical properties and spectral data. RESULTS: Ten compounds were isolated and elucidated as uracil (1), 6, 7-dimethoxycoumarin (2), 6, 7, 8-trimethoxycoumarin (3), p-hydroxybenzoic acid ethyl ester (4), 1∶1 mixture of two diastereomers identical with (3RS, 6RS)-2, 6-dimethyl-octa-1, 7-dien-3, 6-diol (5 and 6), kaempferol (7), isofraxidin (8), scopoletin (9), and loliolide (10). CONCLUSION: Compounds 1, 3-6 and 10 are isolated from this plant for the first time and compounds 1, 4-6 are isolated from this genus for the first time.

Chemical constituents of Ajuga ovalifolia var. calantha / 中草药

Objective To investigate the chemical constituents of Ajuga ovalifolia var. calantha. Methods The 95% ethanol extract from the whole herb of A. ovalifolia var. calantha was separated and purified by column chromatography over silica gel, Sephadex LH-20, ODS, and RP-HPLC. Their chemical structures were identified on the basis of physicochemical characteristics and spectral analyses. Results Thirteen compounds were isolated and identified as (16S)-12,16-epoxy-11,14-dihydroxy-17 (15→16)-abeo-abieta- 8,11,13-trien-7-one (1), ajuforrestin B (2), ajudecumin A (3), 14,15-dihydroajugapitin (4), cyasterone (5), β-sitosterol (6), acacetin (7), apigenin (8), luteolin (9), scopoletin (10), isoscopoletin (11), 4-hydroxy-3-methoxy-benzaldehyde (12), and acetovanillon (13). Conclusion Compounds 1 and 10-13 are reported from the genus Ajuga Linn., and compounds 2-5 and 7 are isolated from this plant for the first time. Single crystal X-ray diffraction experiment is carried out for compound 1, and the crystal structure data of single crystal X-ray diffraction of compound 1 is obtained for the first time.

Quality standard for Weigela japonica var.sinica / 中成药

AIM To establish the quality standard for Weigela japonica Thunb.var.sinica (Rehd.) Bailey (W.j.).METHODS TLC was adopted in this medicinal material's qualitative identification after morphological identification and microscopic identification.The contents of water,total ash,acid-insoluble ash and extracts were detected according to Chinese Pharmacopoeia methods.Then the contents of scopoletin and total coumarins were determined by HPLC and UV,respectively.RESULTS The morphologies and microscopic characters of W.j.could be distinguished from other same generic plants.The clear TLC spot displayed good resolution.The contents of water,total ash,acid-insoluble ash,water-soluble extract and acid-soluble extract were no more than 12.0％,no more than 2.0％,no more than 0.5％,no less than 5.0％ and no less than 4.5％,respectively.Scopoletin and total coumarins showed good linear relationships within the ranges of 1.25-40.0 μg/mL(r =0.999 7)and 2.0-64.0 μg/mL (r =0.999 9),whose average recoveries were 98.19％ (RSD =0.90％) and 99.21％ (RSD =2.5％),respectively.CONCLUSION This accurate and reliable method can be used for the quality control of W.j..

Determination of chemical consituents in Lycii Fructus from Qaidam Basin / 中成药

AIM To determine the contents of chemical consituents in Lycii Fructus from Qaidam Basin.METHODS Spectrophotometry was adopted in the content determination of polysaccharides,total flavonoids and carotenoid.HPLC was applied to the content determination of betaine and scopoletin.Kjeldahl method was used for the content determination of protein.Then principal component analysis was performed.RESULTS The contents of carotenoid,betaine and scopoletin in samples from six growing areas showed obvious differences (P ＜ 0.05),while those of polysaccharides,total flavonoids and protein exhibited no obvious differences (P ＞ 0.05).The contents of various constituents in samples at three picking time also had no obvious differences (P ＞ 0.05).The comprehensive score of principal components of samples from Delingha City was the highest,followed by that from Ulan County.CONCLUSION The quality of Lycii Fructus from Qaidam Basin from Delingha City is the best.

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.

Effects of arteannuin B, arteannuic acid and scopoletin on pharmacokinetics of artemisinin in mice

Objective To explore the effects of arteannuin B, arteannuic acid and scopoletin on the pharmacokinetics of artemisinin in mice. Methods Artemisinin and a combination of artemisinin, arteannuin B, arteannuic acid and scopoletin were administered together to mice via oral administration. Blood samples were collected at different time intervals and pretreated by liquid–liquid extraction. The contents of four compounds in mouse plasma were determined by a validated HPLC-MS/MS method. Results Compared to single artemisinin group, the C

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.

Effects of arteannuin B, arteannuic acid and scopoletin on pharmacokinetics of artemisinin in mice

OBJECTIVE@#To explore the effects of arteannuin B, arteannuic acid and scopoletin on the pharmacokinetics of artemisinin in mice.@*METHODS@#Artemisinin and a combination of artemisinin, arteannuin B, arteannuic acid and scopoletin were administered together to mice via oral administration. Blood samples were collected at different time intervals and pretreated by liquid-liquid extraction. The contents of four compounds in mouse plasma were determined by a validated HPLC-MS/MS method.@*RESULTS@#Compared to single artemisinin group, the Cmax values from the combination group rose from 947 ng/mL to 1254 ng/mL. AUC(0-t) (2371 h ng/mL) was significantly higher than that from single artemisinin group (747 h ng/mL). The peak time lag and the CL values reduced at a proportion of 66%.@*CONCLUTIONS@#Arteannuin B, arteannuic acid and scopoletin can markedly affect the pharmacokinetics of artemisinin.

Enhanced Accumulation of Scopoletin in Cell Suspension Culture of Spilanthes acmella Murr. Using Precursor Feeding

In this study, the various concentrations of casein hydrolysate (25, 50, 75, 100 mg/L) and L-phenylalanine (50, 100, 150, 200 µM/l) were incorporated in MS containing 15 µM BA plus 5 µM 2,4-D for enhancement of secondary metabolites in cell culture of Spilanthes acmella. The presence of casein hydrolysate in the nutrient medium improved the growth of cell biomass and the production of scopoletin. The addition of casein hydrolysate up to 75 mg/L stimulated the accumulation of scopoletin, but increasing excess 75 mg/L the level of casein hydrolysate reduced the production of scopoletin. The addition of L-phenylalanine in the nutrient medium was found to be more effective for production of secondary metabolite in S. acmella. The addition of 50 µM/L of L-phenylalanine in the medium increased scopoletin content to 27.12 ± 0.58 µg/g dry weight, compared to the scopoletin content of control at 7.89 ± 0.61 µg/g dry weight. The highest accumulation of scopoletin was observed in the 100 µM/L L-phenylalanine in cell suspension, which was 4.51 times more than the control. As a result, using moderate concentration of L-phenylalanine was ideal for the production of scopoletin. In general, casein hydrolysate was more effective than L-phenylalanine for production of scopoletin and growth of cell biomass in the cell culture of S. acmella.

Rapid validated high performance thin layer chromatography method for simultaneous estimation of mangiferin and scopoletin in Canscora decussata (South Indian Shankhpushpi) extract

AbstractMangiferin (polyphenolic xanthone) and scopoletin (phenolic coumarin) are well-studied biological markers present in Canscora decussata(Roxb.) Roem. & Schult., Gentianaceae. The objective set for the present studies is to establish and develop a new, simple, selective, sensitive, and precise high performance thin layer chromatography method for the simultaneous estimation of mangiferin and scopoletin in hydroalcoholic extract of C. decussata. The thin layer chromatographic separation of these biomarkers was carried out on aluminum plate pre-coated with silica gel 60F254, eluted with ethyl acetate:acetic acid:formic acid:water (10:0.5:0.5:1.5). The plate was then dried and densitometric scanning was performed at 254 nm using a Camag TLC scanner III. The system was found to give compact spots for mangiferin (RF 0.22) and scopoletin (RF 0.78). A good relationship of linear precision between the concentrations (100–600 ng/spot) and peak areas was obtained with correlation coefficient (r) of 0.9979 (mangiferin) and 0.9962 (scopoletin), respectively. The limits of detection and limit of quantification were determined to be 46 and 94 ng/spot for mangiferin and 31 and 78 ng/spot for scopoletin respectively. The percentage of recovery was found from 99.91 to 99.94% for mangiferin and 99.75 to 99.86% for scopoletin. Results obtained from recovery studies showed excellent reliability and reproducibility of the method. Present communication on validated high performance thin layer chromatography method may provide a new, selective, sensitive, and precise method to estimate mangiferin and scopoletin as phytomarkers in the hydroalcoholic extract of C. decussata used in Ayurvedic formulations.

Study on the Quality Standard of Root of Urtica dioica and Different Harvest Periods / 中国药房

OBJECTIVE:To study the quality standard of root of Urtica dioica and investigate the sample’s content in different harvest periods. METHODS:TLC was conducted to identify the scopoletin in the root of U. dioica with the developing agent of hexane-dichloromethane- ethyl acetate- formic acid (6∶10∶7∶1.2,V/V/V/V);alcohol-hot dipping was used to determine the con-tent;and the root of U. dioica in different harvest periods were comparatively researched. RESULTS:The lactone thin-layer chro-matogram feature was obvious,alcohol-hot dipping showed the contents of root of u. dioica was relatively high(RSD≤1%),the ex-tract content of root of U. dioica was the highest from late fall to early spring,and the stage was suitable excavation period. CON-CLUSIONS:The established quality standard can be used for accurately qualitative and quantitative,and studying different harvest periods is conducive to effectively control the quality of root of U. dioica.

Design, synthesis and antiproliferative activity of scopoletin derivatives / 中国药科大学学报

@#To explore novel scopoletin derivatives with more potent antiproliferative activity, a series of compounds were designed and synthesized(10a-10i)by linking benzamide to scopoletin′s framework. Their structures were determined by ESI-MS, IR and 1H NMR. The antiproliferative activity of the synthetic derivatives against human cancer cells MCF-7, A549 and HCT-116 was evaluated by MTT assay. Preliminary experimental results showed that compound 10g was the most potent one, whose antiproliferative activity was stronger than that of the positive control scopoletin and sunitinib.

Chemical constituents of Carthamus tinctorius / 中草药

Objective: To investigate the chemical constituents of Carthamus tinctorius. Methods: Compounds were isolated by a combination of various column chromatography over silica gel, Sephadex LH-20, and pre-HPLC and their structures were identified by spectral analytical methods of MS and NMR and/or comparison with literature data. Results: Twenty compounds were isolated from the ethanol extract of C. tinctorius. Their structures were identified as kaempferol-3-O-β-D-glucosyl-(1→2)-β-D-glucoside (1), scutellarein (2), hexacosanoic acid (3), (2S)-1-O-heptatriacontanoyl glycerol (4), 4,4-dimethyl heptanedioic (5), 5,7,4'-trihydroxy-6- methoxyflavone-3-O-β-D-rutinoside (6), n-tetratriacont-20,23-dienoic acid (7), vanillic acid (8), gallic acid (9), tetrephthalic acid mono-[2-(4-carboxy-phenoxycarbonyl)-vinyl] ester (10), esculetin (11), 6-hydroxyapigenin-6-O-β-D-glucoside-7-O-β-D-glucuronide (12), quercetin-3,7-di-O-β-D-glucoside (13), 6-methoxykaempferol (14), syringing (15), E-1-(4'-hydroxypheny)-but-1-en-3-one (16), ursolic acid (17), 1-hexadecanoyl propan-2,3-diol (18), citrostadienol (19), and scopoletin (20). Conclusion: Compounds 1, 5, 7, 10, 18, and 19 are isolated from the plants of Carthamus L. for the first time, and compounds 2-4, 6, 8, 9, 11, 17, and 20 are obtained from C. tinctorius for the first time.