水曲柳HPLC指纹图谱研究

目的:建立水曲柳药材的高效液相(HPLC)指纹图谱,为水曲柳的鉴别及质量控制提供依据。方法:对不同产地、不同采集时间的水曲柳药材皮中的香豆素成分进行指纹图谱研究。结果:HPLC指纹图谱中峰的分离度均好,共得到10个共有峰;10批样品的相似度较好。结论:所建立的指纹图谱可用于水曲柳药材的鉴定及质量评价。     

The simultaneous determination of coumarins in Angelica gigas root by high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry

An high performance liquid chromatography-diode array detector coupled with electrospray ionization/mass spectrometry (HPLC-DAD/MS) based method has been developed for the simultaneous determination of nine coumarin compounds, nodakenin (1), peucedanone (2), marmesin (3), decursinol (4), 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-enyl)coumarin (5), demethylsuberosin (6), decursin (7), decursinol angelate (8) and isoimperatorin (9) in the Korean medicinal herb, Cham-Dang-Gui, the dried root of Angelica gigas (Umbelliferae). The methanol extracts were analyzed by HPLC using a reversed-phase C18 column (5 microm, 4.5 mm x 250 mm) using a gradient acetonitrile-water solvent system at a flow rate of 1.0 ml/min. The analysis of six coumarins (1, 3, 4 and 6-8) with DAD was done at 330 nm and showed excellent linearity (r(2)=0.998-0.999) in a range of 0.2-250 microg/ml for all the compounds. The average recoveries (n=3) were between 96.5% and 110.8%. Identification of each peak was also discussed with the electrospray ionization multi-stage tandem mass spectroscopy (ESI-MS(n)). The amount of these coumarin compounds was evaluated in A. gigas samples. Meanwhile, three coumarins (2, 5 and 9) could not been quantified by DAD because these peaks were overlapped with others. Determination of these compounds could be successfully accomplished with the HPLC-ESI/MS in selected ion monitoring/selected reaction monitoring mode.     

Simultaneous analysis of coumarins and secoiridoids in Cortex Fraxini by high-performance liquid chromatography-diode array detection-electrospray ionization tandem mass spectrometry

A high-performance liquid chromatography-diode array UV detection-electrospray ionization tandem mass spectrometry (HPLC-DAD-ESI-MS) method was developed and validated for the simultaneous analysis of seven major constituents in Cortex Fraxini, including esculin, esculetin, fraxetin, fraxin, escuside, oleuropein and ligustroside. The contents of the seven constituents were determined by using HPLC-DAD, and the chemical structures of these constituents were identified by using HPLC-DAD-ESI-MS method. The separation was performed on an Agilent Zorbax SB-C18 column (250 mm x 4.6 mm i.d., 5 microm) with gradient elution of acetonitrile and 0.3% aqueous acetic acid within 40 min. Detection was performed at 254 nm. The calibration curves showed good linearity (r2>0.9992). The limits of detection (LOD) ranged from 1.07 to 3.19 ng/ml and limits of quantification (LOQ) ranged from 2.79 to 12.75 ng/ml, respectively. The intra- and inter-day precision was less than 5% and the accuracy was ranging from 96.49% to 103.55%. The recovery of the assay was in the range of 97.61-104.36%. The method was successfully applied to the quantification of the seven constituents in different samples of Cortex Fraxini. The results indicated that the developed method could be considered to be a simple, rapid and reliable method for the quality evaluation of Cortex Fraxini.     

Structure–activity relationship of (−) mammea A/BB derivatives against Leishmania amazonensis

To study the structure–activity relationship of coumarin (−) mammea A/BB isolated from the CH₂Cl₂ extract of Calophyllum brasiliense leaves, we evaluated the antileishmanial activity of natural, synthetic and derivatives of this coumarin, against promastigote and intracellular amastigote forms of Leishmania amazonensis, and their cytotoxicity to J774G8 murine macrophages. The derivatives were obtained by hydrogenation and methoxylation reactions. The compound structures were elucidated on the basis of spectroscopic data. The compounds 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), 7-hydroxy-5-methoxy-8-(2-methylbutanoyl)-6-(3-methylbut-2-en-1-yl)-4-phenylcoumarin (4) and 5,7-dimethoxy-8-(1-methoxy-2-methylbutyl)-6-(3-methylbut-2-en-1-yl)-4 phenylcoumarin (6) were more biologically active than the compound (−) mammea A/BB (1) (7.4 μM), with IC₅₀ values from 0.9, 2.4 and 1.9 μM respectively; compound (3) displayed the highest activity. The compounds mammea B/BB (2), 5,7-dimethoxy-8-(2-methylbutanoyl)-6-(3-methylbut-2-en-1-yl)-4-phenylcoumarin (5) and 5,7-dihydroxy-4-phenylcoumarin (7) were less active than (−) mammea A/BB (1), with IC₅₀ of 30.1, 15.1 and 60.2 μM respectively; compound (7) showed the lowest antileishmanial activity of all. Compounds (1), (3), (4) and (6) were active not only against promastigote forms of L. amazonensis, but also against intracellular amastigote forms with IC₅₀ of 14.3, 0.6, 34.0 and 22.2 μM, respectively. Interestingly, compound (3) showed the most antileishmanial activity of all. This study demonstrated that several aspects of the structure were important for antileishmanial activity.     

The standardized extract of Loeselia mexicana possesses anxiolytic activity through the γ-amino butyric acid mechanism

Ethnopharmacological relevance

Loeselia mexicana (Lam.) Brand has been used in Mexican Traditional Medicine to treat “espanto” or “susto” (fear), which is a culturally affiliated syndrome whose symptomatology comprises loss of appetite, difficulty in sleeping, and also nausea and fatigue, with a sensation of fear or risk - real or imagined - to external stimuli.

Aim of the study

The anxiolytic effect of the standardized methanol extract of Loeselia mexicana, with regard to its content of coumarin daphnoretin, was researched utilizing the elevated plus maze (EPM) in order to demonstrate whether the biological effect produced by the plant is antagonized by drugs that block γ-amino butyric acid (GABA)ergic transmission.

Materials and methods

The methanolic extract of Loeselia mexicana (LmMeOH) was tested at different doses on the EPM and then the interaction of this extract was evaluated in the same model with different GABAergic drugs, such as flumazenil (FLU) 10 mg/kg, bicuculline (BIC) 5 mg/kg, pentylenetetrazole (PTZ) 10 mg/kg, and picrotoxin (PTX) 2 mg/kg. The effect of all of these treatments was evaluated by means of the open field test (OFT). Coumarin content was measured by the high performance liquid chromatography (HPLC) technique.

Results

The 200- and 400-mg/kg doses of methanolic extract containing 3.14 and 6.28 mg of daphnoretin, respectively, induced an anxiolytic effect in the EPM without modification of the spontaneous motor activity. The anxiolytic activity of 200 mg/kg of methanolic extract in EPM-exposed mice was antagonized by PTX, BIC, and FLU, but not by PTZ.

Conclusion

The data presented here indicate that the Loeselia mexicana Brand methanolic extract possesses a significant anxiolytic effect that appears to be mediated in part by activation of the GABAergic system.     

当归属植物传统药用、香豆素类成分及药理毒理学研究进展

当归属作为伞形科中的一个属，全世界已发现约110种。多种当归属植物作为传统药用植物，具有祛风除湿、补血活血的药用价值。香豆素类成分作为当归属植物的主要有效成分之一，是发挥疗效的主要药效物质基础。因此，本文系统的综述了当归属植物传统药用、香豆素类化学成分、药理活性和毒理学等方面研究进展，分析当归属的研究现状，为今后当归属植物的开发利用与进一步的研究提供科学依据和方向。     

Comprehensive chemo-profiling of coumarins enriched extract derived from Aegle marmelos (L.) Correa fruit pulp,as an anti-diabetic and anti-inflammatory agent

Aegle marmelos (L.) Correa is an Indian medicinal plant known for its vast therapeutic activities. In Ayurveda, the plant is known to balance “vata,” “pitta,” and “kapha” dosh. Recent studies suggest anti-inflammatory, anti-microbial, and anti-diabetic potential but lack in defining the dosage over the therapeutic activities. This study aims to determine the chemical profile of Aegle marmelos fruit extract; identification, enrichment, and characterization of the principal active component(s) having anti-inflammatory and anti-diabetic potential. Targeted enrichment of total coumarins, focusing on marmelosin, marmesin, aegeline, psoralen, scopoletin, and umbelliferone, was done from Aegle marmelos fruit pulp, and characterized using advanced high-throughput techniques. In vitro and in silico anti-diabetic and anti-inflammatory activities were assessed to confirm their efficacy and affinity as anti-diabetic and anti-inflammatory agents. The target compounds were also analysed for toxicity by in silico ADMET study and in vitro MTT assay on THP-1 and A549 cell lines. The coumarins enrichment process designed, was found specific for coumarins isolation as it resulted into 48.61% of total coumarins enrichment, which includes 31.2% marmelosin, 8.9% marmesin, 4% psoralen, 2% scopoletin, 1.7% umbelliferone, and 0.72% aegeline. The quantification with HPTLC and qNMR was found to be correlated with the HPLC assay results. The present study validates the potential use of Aegle marmelos as an anti-inflammatory and anti-diabetic agent. Coumarins enriched from the plant fruit have good therapeutic activity and can be used for Phytopharmaceutical ingredient development. The study is novel, in which coumarins were enriched and characterized by a simple and sophisticated methodology.