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.
Aspergillus niger/genetics* ; beta-Glucosidase/chemistry* ; Scopoletin ; Polysorbates ; Coumarins

This study aimed to investigate the effects of hypoxia on RhoA/Rho-kinase (ROCK) signaling pathway and autophagy in pulmonary artery smooth muscle cells (PASMCs), and to explore the underlying mechanism of Umbelliferone (Umb) in ameliorating chronic hypoxic pulmonary hypertension. PASMCs were cultured from Sprague-Dawley (SD) rats and randomly divided into control group, hypoxia group, hypoxia + Umb intervention group and normoxia + Umb intervention group. Alpha smooth muscle actin (α-SMA) and LC3 were assessed by immunofluorescence staining. Protein expression of RhoA, ROCK2, p-MYPT1, LC3-II, Beclin-1, p62, C-Caspase 3, Bax and Bcl-2 was analyzed by Western blotting. In in vivo study, SD rats were divided into control group, hypoxia group and hypoxia + Umb intervention group. Weight ratio of the right ventricle (RV)/left ventricle plus septum (LV+S) was detected, and pulmonary arterial morphological features were examined by HE staining. The results indicated that compared with the control group, the LC3-II/LC3-I ratio and expression of Beclin-1 were significantly increased, while p62 expression was significantly decreased, and the expressions of RhoA, ROCK2 and p-MYPT1 were significantly increased in PASMCs of hypoxia group (P < 0.05). The changes of LC3-II/LC3-I ratio, the expressions of Beclin-1, p62, RhoA, ROCK2 and p-MYPT1 in PASMCs were reversed by Umb treatment (P < 0.05). Consistently, the pulmonary arterial wall was thickened and the RV/(LV+S) ratio was increased in hypoxic rats, which were significantly improved by Umb treatment (P < 0.05). These results suggest that Umb can improve hypoxia-induced pulmonary hypertension by inhibiting the RhoA/ROCK signaling pathway and autophagy in PASMCs.
Animals ; Autophagy ; Beclin-1/pharmacology* ; Hypertension, Pulmonary/etiology* ; Hypoxia/complications* ; Myocytes, Smooth Muscle/metabolism* ; Pulmonary Artery ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Umbelliferones/pharmacology* ; rho-Associated Kinases/pharmacology*

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 μmol·Lin vivo and 100 μmol·Lin vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.
Alkaloids ; chemistry ; pharmacology ; Animals ; Benzodioxoles ; chemistry ; pharmacology ; Benzyl Compounds ; chemistry ; pharmacology ; Cholecalciferol ; chemistry ; pharmacology ; Flavonoids ; chemistry ; pharmacology ; Humans ; Kaempferols ; chemistry ; pharmacology ; Melanins ; genetics ; metabolism ; Monophenol Monooxygenase ; genetics ; metabolism ; Pigmentation ; drug effects ; Piperidines ; chemistry ; pharmacology ; Polyunsaturated Alkamides ; chemistry ; pharmacology ; Purines ; chemistry ; pharmacology ; Scopoletin ; chemistry ; pharmacology ; Vitiligo ; drug therapy ; enzymology ; metabolism ; Zebrafish

PURPOSE: The objective of this study was to evaluate the relaxant effect of scoparone from Artemisia capillaris on rabbit penile corpus cavernosum smooth muscle (PCCSM) and to elucidate the mechanism of action of scoparone for the treatment of erectile dysfunction (ED). MATERIALS AND METHODS: PCCSM that had been precontracted with phenylephrine was treated with 3 Artemisia herbs (A. princeps, A. capillaris, and A. iwayomogi) and 3 fractions (n-hexane, ethyl acetate, and n-butanol) with different concentrations (0.1, 0.5, 1.0, and 2.0 mg/mL). Four components (esculetin, scopoletin, capillarisin, and scoparone) isolated from A. capillaris were also evaluated. The PCCSM was preincubated with Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). Cyclic nucleotides in the perfusate were measured by a radioimmunoassay. The interactions of scoparone with udenafil and rolipram were also evaluated. RESULTS: A. capillaris extract relaxed PCCSM in a concentration-dependent manner. Scoparone had the highest relaxant effect on PCCSM among the 4 components (esculetin, scopoletin, capillarisin, and scoparone) isolated from the ethyl acetate fraction. The application of scoparone on PCCSM pretreated with L-NAME and ODQ led to significantly less relaxation. Scoparone also increased the cyclic guanosine monophosphate (cGMP) levels in the perfusate in a concentration-dependent manner. Furthermore, scoparone enhanced udenafil- and rolipram-induced relaxation of the PCCSM. CONCLUSIONS: Scoparone relaxed the PCCSM mainly by activating the nitric oxide-cGMP signaling pathway, and it may be a new promising treatment for ED patients who do not completely respond to udenafil.
Artemisia* ; Coumarins ; Erectile Dysfunction ; Guanosine Monophosphate* ; Guanosine* ; Humans ; Male ; Muscle, Smooth ; NG-Nitroarginine Methyl Ester ; Nitric Oxide ; Nucleotides, Cyclic ; Penile Erection* ; Phenylephrine ; Phosphodiesterase 5 Inhibitors ; Radioimmunoassay ; Relaxation ; Rolipram ; Scopoletin

Fourteen compounds were isolated from the stem of Angelica polymorpha. On the basis of spectral data, these compounds were identified as isoimperatorin (1), phellopterin (2), bergapten (3), xanthyletin (4), cnidilin (5), geijerine (6), (−)-3'-acetyl hamaudol (7), 7-demethylsuberosine (8), dehydrogeijerin (9), (−)-hamaudol (10), (+)-visamminol (11), divaricatol (12), scopoletin (13), and decursidate (14), respectively. Among them, compounds 4 - 6, 8, 9, 13, and 14 were isolated for the first time from A. polymorpha. Dehydrogeijerin (6) and geijerin (9) were isolated for the first time from genus Angelica. All isolates tested for inhibitory activity against acetylcholinesterae. Compounds 1 to 13 showed acetylcholinesterase inhibitory activity with IC₅₀ values ranging from 1.4 to 37.5 µM.
Acetylcholinesterase* ; Angelica* ; Cholinesterase Inhibitors* ; Chromones ; Coumarins ; Scopoletin

OBJECTIVETo investigate the dual role of daphnetin in suppressing high mobility group box-1 protein (HMGB1) release and blocking HMGB1-induced inflammatory response.

METHODSMurine macrophage RAW264.7 cells were cultured in the presence of daphnetin, lipopolysaccharide (LPS), or both. HMGB1 release from the cells was determined using ELISA, and phosphorylations of JAK1/2 and of STAT1 were detected by Western blotting. Human monocytic THP-1 cells exposed to daphnetin, rhHMGB1, or both were examined for NO production using a NO detection kit, for the release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) using ELISA, and for expressions of iNOS, COX-2 and phosphorylated p38, ERK, and JNK with Western blotting.

RESULTSDaphnetin dose-dependently reduced the release of HMGB1 in RAW264.7 cells and suppressed rhHMGB1-induced iNOS and COX-2 expressions and release of TNF-α, IL-6, PGE2, and NO in THP-1 cells. Western blotting revealed that daphnetin significantly down-regulated the phosphorylations of JAK-STAT1 pathway in LPS-stimulated RAW264.7 cells but did not suppress the phosphorylations of MAPKs signaling pathway induced by rhHMGB1 in THP-1 cells.

CONCLUSIONDaphnetin can reduce the release of HMGB1 and suppress HMGB1-induced inflammatory response. In RAW264.7 cells, daphnetin inhibited LPS induced HMGB1 release is at least partly mediated by suppressing JAK-STAT1 signaling pathway activation.

Animals ; Cell Line ; Cyclooxygenase 2 ; metabolism ; Dinoprostone ; metabolism ; HMGB1 Protein ; metabolism ; Humans ; Inflammation ; metabolism ; Interleukin-6 ; metabolism ; Janus Kinase 1 ; metabolism ; Lipopolysaccharides ; Macrophages ; drug effects ; Mice ; Monocytes ; drug effects ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type II ; metabolism ; RAW 264.7 Cells ; STAT1 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism ; Umbelliferones ; pharmacology

The stem barks, heartwoods, and leaves of Acer tegmentosum (Aceraceae) are widely used in Korea to treat hepatic or cerebral disorders mainly due to alcohol poisoning. This study was aimed to analyze phenolic substances in A. tegmentosum. Quantitative analysis of the three phenolic substances (salidroside, (+)-catechin and scopoletin) was performed by HPLC and the identification of volatile phenolic substances were done by GC-MS. The contents of the three compounds in the three MeOH extracts were higher in the stem bark (salidroside: 80.22 mg/g, (+)-catechin: 23.31 mg/g, and scopoletin: 9.45 mg/g) compared to the heartwoods and leaves. And GC-MS analysis of the stem bark extract demonstrated that p-tyrosol is a main substance of twenty-one compounds identified.
Acer* ; Aceraceae ; Catechin ; Chromatography, High Pressure Liquid* ; Korea ; Phenol* ; Poisoning ; Scopoletin

OBJECTIVEAn accurate and reliable analytical method for-simultaneous determination of six active components (scopolin, chlorogenic acid, scopoletin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C) in plants of Erycibe was developed.

METHODScopolin, chlorogenic acid, scopoletin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C in the samples were well separated in analytical HPLC by gradual elution with methanol-0.1% formic acid solution. The chromatographic condictions: Agilent Poroshell 120 EC-C18 column, flowing rate being 1 mL x min(-1), detecting wavelength at 345 nm.

RESULTGood linearities of scopolin, chlorogenic acid, scopoletin, isochlorogenic acid A, isochlorogenic acid B and isochlorogenic acid C were in the range of 0.026 8-2.68, 0.027 0-2.70, 0.008 1-0.81, 0.018 8-1.88, 0.017 6-1.76, 0.019 6-1.96 μg, respectively (r > 0.999 6). The average recoveries of the six components were 98.1%, 98.7%, 100.8%, 100.4%, 99.7%, 101.1%; the relative standard deviations were 2.67%, 2.86%, 2.62%, 1.98%, 2.76%, 2.19%.

CONCLUSIONThe method is simple, feasible and reproducible and can be used for the quality control of plants of Erycibe.

China ; Chlorogenic Acid ; analogs & derivatives ; analysis ; Chromatography, High Pressure Liquid ; methods ; Convolvulaceae ; chemistry ; Coumarins ; analysis ; Drugs, Chinese Herbal ; analysis ; Glucosides ; analysis ; Scopoletin ; analysis

This study is aimed to establish a high-performance liquid chromatography (HPLC) method for simultaneous determination of skimmin, scopolin and umbelliferone in Saussurea hieracioides. Samples were analyzed on a Wondasil C18-WR column (4.6 mm x 250 mm, 5 microm) with methanol (A) and water containing 0.1% phosphate (B) as mobile phases for gradient elution at a flow rate of 1.0 mL x min(-1). The detection wavelength and column temperature were set at 325 nm and 35 degrees C, respectively, and the sample size was 10 microL. The results showed that skimmin, scopolin and umbelliferone were simultaneously achieved within 40 min under the above conditions. A good linearity was observed in the range of 0.18-5.6 microg (r = 1.000 0), 0.060-1.8 microg (r = 0.999 9), 0.032-0.97 microg (r = 0.999 8) for skimmin, scopolin and umbelliferone, respectively, with the average recoveries of 99.16% (RSD = 0.41%), 100.3% (RSD = 0.79%), 102.2% (RSD = 0.87%). The method is simple, accurate and reproducible and can be used for the quality control of S. hieracioides.
Chromatography, High Pressure Liquid ; Coumarins ; analysis ; Glucosides ; analysis ; Medicine, Tibetan Traditional ; Reproducibility of Results ; Saussurea ; chemistry ; Umbelliferones ; analysis

The objective of this study is to develop a sensitive and reliable high-performance liquid chromatography mass spectrometry (LC-MS) method for simultaneous determination of artemisinin, arteannuin B, artemisic acid, and scopoletin, and study the pharmacokinetics of the four constituents in mouse serum after oral administration of the four components to mice. The analytical column used was Agilent Zorbax SB-C18 (2.1 mm x 150 mm, 5 mm). The mobile phase was acetonitrile: 0.5% acetic acid (60: 40) and the flow rate was 0.3 mL x min(-1). The temperature of the column was 40.0 degrees C. In this condition, we established an analysis method to simultaneously determine the four components. A sensitive and specific liquid chromatography-mass spectrometric (LC-MS) method was developed and validated for the determination of artemisin in derivatives in mice plasma. The method we established has a linear range of 5-3 000 μg x L(-1) with a good sensitivity and specificity for all of the four components. This method is simple, rapid, accurate and suitable for the determination of the content of the four compounds.
Animals ; Artemisinins ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; instrumentation ; methods ; Dose-Response Relationship, Drug ; Male ; Mice ; Reproducibility of Results ; Scopoletin ; blood ; pharmacokinetics ; Spectrometry, Mass, Electrospray Ionization ; methods