High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma

A simple HPLC method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of four active polyphenol components of hawthorn (Crataegus), chlorogenic acid, epicatechin, hyperoside and isoquercitrin, in rat plasma. Following extraction from the plasma samples with ethyl acetate–methanol (2:1, v/v), these four compounds were successfully separated using a C₁₈ column with a gradient elution of 5 and 25% acetonitrile in 25 mM phosphate buffer (pH 2.4). The flow-rate was set at 1 ml/min and the eluent was detected at 325 nm for chlorogenic acid, 278 nm for epicatechin, and 360 nm for both hyperoside and isoquercitrin. Narignin (0.82 μg) was used as the internal standard and was detected at 278 nm. The method is linear over the studied range of 0.16–40, 0.63–160, 0.13–32 and 0.13–30 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin, respectively. The correlation coefficient for each analyte was greater than 0.995. The intra-day and inter-day precision of the analysis was better than 4 and 7%, respectively. The extraction recoveries at low to high concentration were greater than 85% for both epicatechin and chlorogenic acid, and greater than 94% for both hyperoside and isoquercitrin. The detection limits were 0.04, 0.20, 0.03 and 0.03 μg/ml for chlorogenic acid, epicatechin, hyperoside and isoquercitrin. The developed method was used to analyze the plasma concentrations of the four analytes after the intravenous administration of hawthorn polyphenol extract to rats.     

Anticonvulsant, antidepressant-like activity of Abelmoschus manihot ethanol extract and its potential active components in vivo

Depression is the most common psychiatric comorbidity in patients with epilepsy. Searching for antiepileptic (anticonvulsant) and antidepressant-like medicines from natural products is very important for the treatment of this disease. The flower of Abelmoschus manihot (Linn.) Medicus has been reported to have neuroprotective effect against cerebral ischemia injury. In order to further explore the activity of Abelmoschus manihot on the central nervous system, the anticonvulsant and antidepressant-like effects of Abelmoschus manihot ethanol extract (AMEE) as well as its potential active components in vivo was investigated in the present study. It was found that AMEE could protect mice against PTZ-induced clonic convulsions and mortality. AMEE could also decrease immobility time in the FST in mice. Furthermore, the potential active components of AMEE in rat brain were identified by ultra performance liquid chromatography-mass spectrometer (UPLC-MS). Five parent components including isoquercitrin, hyperoside, hibifolin, quercetin-3′-O-glucoside, quercetin and three metabolites were detected in rat brain after administration of AMEE. In conclusion, eight flavonoids were identified in rat brain after administration of AMEE; meanwhile, these flavonoids might represent the potential bioactive components of AMEE and contribute to its anticonvulsant and antidepressant-like activity in vivo.     

Identification of the major constituents of Hypericum perforatum by LC/SPE/NMR and/or LC/MS

The newly established hyphenated instrumentation of LC/DAD/SPE/NMR and LC/UV/(ESI)MS techniques have been applied for separation and structure verification of the major known constituents present in Greek Hypericum perforatum extracts. The chromatographic separation was performed on a C18 column. Acetonitrile-water was used as a mobile phase. For the on-line NMR detection, the analytes eluted from column were trapped one by one onto separate SPE cartridges, and hereafter transported into the NMR flow-cell. LC/DAD/SPE/NMR and LC/UV/MS allowed the characterization of constituents of Greek H. perforatum, mainly naphtodianthrones (hypericin, pseudohypericin, protohypericin, protopseudohypericin), phloroglucinols (hyperforin, adhyperforin), flavonoids (quercetin, quercitrin, isoquercitrin, hyperoside, astilbin, miquelianin, I3,II8-biapigenin) and phenolic acids (chlorogenic acid, 3-O-coumaroylquinic acid). Two phloroglucinols (hyperfirin and adhyperfirin) were detected for the first time, which have been previously reported to be precursors in the biosynthesis of hyperforin and adhyperforin.     

Antilipoxygenase activity of compounds from Hypericum perforatum

The main biologically active constituents of Hypericum species are flavonoids (quercetin, isoquercitrin, hyperoside, rutin), biflavonoids and naphthodianthrones (hypericin, pseudohypericin). Lipoxygenase is the key enzyme in the biosynthesis of leukotriens, which have been postulated to play an important role in the pathophysiology of several inflammatory and allergic diseases. This work deals with the investigation of potential antilipoxygenase activity of different compounds and extracts isolated from Hypericum perforatum L. The highest inhibitory effect was exhibited by flavonoid derivative hyperoside (IC₅₀ 5.768 × 10⁻⁶ M). Acetone and ethanolic extracts caused also an inhibition of lipoxygenase. On the basis of inhibitory effect of compounds tested we assume that the most of them may be involved in the antiinflammatory principles of Hypericum perforatum L.     

Anti-inflammatory and spasmolytic activity of extracts from Droserae herba.

An ethanolic extract of Drosera madagascariensis inhibited human neutrophil elastase with an IC50 of 9.4 microg/ml. The naphthoquinones present in the extract were not responsible for this effect, but flavonoids like quercetin (IC50 0.8 microg/ml), hyperoside (IC50 0.15 microg/ml) and isoquercitrin (IC50 0.7 microg/ml) contributed to inhibition of the enzyme. In guinea-pig ileum the extract (0.5-1 mg/ml) induced a spasmolytic effect via affecting cholinergic M3 receptors and histamine H1 receptors, respectively. At contractile prostanoid receptors of guinea-pig trachea the Drosera extract was not effective.     

艾纳香化学成分的研究

从艾纳香(Blumea balsamifera DC.)中分离得到12个化合物,通过理化性质和波谱数据分析分别鉴定为;商路素(1),花椒油素(2),2,4-二羟基-6-甲氧基苯乙酮(3),5,7-二羟基色原酮(4),金丝桃苷(5),异槲皮苷(6),3′,4′,5,7-四羟基-3-甲氧基黄酮(7),槲皮素(8),槲皮素-3′-甲氧基-3-O-β-D-半乳吡喃糖苷(9),4′,5,7-三羟基-3,3′-二甲氧基黄酮(10),3,5,7-三羟基-3′,4′-二甲氧基黄酮(11),木犀草素(12).其中,化合物3-7和9- 11为首次从该属植物中分离得到.     

Isolation and Characterisation of Two Quercetin Glucosides with Potent Anti-Reactive Oxygen Species (ROS) Activity and an Olean-12-en Triterpene Glucoside from the Fruit of Abelmoschus esculentus (L.) Moench

Abelmoschus esculentus (Okra) is used in the traditional treatment of cancer, hyperlipidaemia and hyperglycaemia. We, therefore, investigated its composition and potential cytotoxic or antioxidant properties that might underlie its phytotherapeutic applications. Its methanolic fruit extract yielded compounds 1 , 2 and 3 , identified through NMR, UV and MS analyses as olean-12-en-3-O-β-d -glucopyranoside, isoquercitrin (quercetin glucoside) and 5,7,3′,4′-tetrahydroxy-flavonol-3-O-[β-d -glucopyranosyl-(1→6)]-β-d -glucopyranoside (quercetin diglucoside), respectively. Following 48 h exposure, oleanene glucoside was mildly toxic to the HeLa and the MRC5-SV2 cancer cells, isoquercitrin was not toxic except at 100 μg/ml in HeLa, and quercetin diglucoside elicited no toxicity. In a 2′,7′-dichlorofluorescein diacetate (DCFDA) assay of intracellular levels of reactive oxygen species (ROS), hydrogen peroxide increased ROS levels, an effect not affected by oleanene glucoside but protected against by isoquercitrin and quercetin diglucoside, with IC₅₀ values, respectively, of 2.7±0.5 μg/ml and 1.9±0.2 μg/ml (3 h post-treatment) and 2.0±0.8 μg/ml and 1.5±0.4 μg/ml (24 h post-treatment.) This is the first report of this oleanene skeleton triterpenoid in the plant. The work provides some insight into why the plant is included in remedies for cancers, cardiovascular complications and diabetes, and reveals it as a potential source of novel therapeutics.     

Chemical Characterization and Multidirectional Biological Effects of Different Solvent Extracts of Arum elongatum: in Vitro and in Silico Approaches

Arum elongatum (Araceae) is widely used traditionally for the treatment of abdominal pain, arterial hypertension, diabetes mellitus, rheumatism and hemorrhoids. This study investigated the antioxidant properties, individual phenolic compounds, total phenolic and total flavonoid contents (HPLC/MS analysis), reducing power and metal chelating effects of four extracts obtained from A. elongatum (ethyl acetate (EA), methanol (MeOH), methanol/water (MeOH/water) and infusion). The inhibitory activity of the extracts were also determined against acetylcholinesterase, butyrylcholinesterase, tyrosinase, amylase and glucosidase enzymes. The MeOH/water extracts contained the highest amount of phenolic contents (28.85 mg GAE/g) while the highest total flavonoid content was obtained with MeOH extract (36.77 mg RE/g). MeOH/water demonstrated highest antioxidant activity against DPPH⋅ radical at 38.90 mg Trolox equivalent per gram. The infusion extract was the most active against ABTS⁺⋅ (133.08 mg TE/g). MeOH/water extract showed the highest reducing abilities with the CUPRAC value of 102.22 mg TE/g and the FRAP value of 68.50 mg TE/g. A strong metal chelating effect was observed with MeOH/water extract (35.72 mg EDTAE/g). The PBD values of the extracts ranged from 1.01 to 2.17 mmol TE/g. EA extract displayed the highest inhibitory activity against AChE (2.32 mg GALAE/g), BChE (3.80 mg GALAE/g), α-amylase (0.56 mmol ACAE/g) and α-glucosidase (9.16 mmol ACAE/g) enzymes. Infusion extract was the most active against tyrosinase enzyme with a value of 83.33 mg KAE/g. A total of 28 compounds were identified from the different extracts. The compounds present in the highest concentration were chlorogenic acids, 4-hydroxybenzoic acid, caffeic acid, p-coumaric acid, ferulic acid, isoquercitrin, delphindin 3,5-diglucoside, kaempferol-3-glucoside and hyperoside. The biological activities of A. elongatum extracts could be due to the presence of compounds such as gallic acid, chlorogenic acids, ellagic acid, epicatechin, catechin, kaempferol, 4-hydroxybenzoic acid, caffeic acid, p-coumaric acid, ferulic acid, quercetin, isoquercitrin, and hyperoside. Extracts of A. elongatum showed promising biological activities which warrants further investigations in an endeavor to develop biopharmaceuticals.     

Biologically active substances isolated from Hypericum attenuatum Choisy

Four dianthrones, two oxymethylanthraquinones, two reductive forms of anthraquinones and five flavonoids have been isolated from the plant source Hypericum attenuatum Choisy (Guttiferae) firstly investigated for these substances contents. Dianthrones pseudohypericin, protohypericin, protopseudohypericin, oxymethylanthraquinones frangulaemodin, frangulin, reductive anthraquinones emodin anthrone, emodin anthranol, and flavonoids quercetin, quercitrin, isoquercitrin, rutin have been identified for the first time. High hyperoside contents in herb of Hypericum attenuatum is established.     

Screening for in vitro metabolites of Abelmoschus manihot extract in intestinal bacteria by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry

Abelmoschus manihot has drawn much attention recently due to its potential beneficial health effects after oral administration. However, the metabolic fate of A. manihot in intestinal flora is not well understood. In this paper, we describe a strategy using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF MS) with automated data analysis software (MetaboLynx?) for fast analysis of the metabolic profile of flavonoids from A. manihot in intestinal flora. The human and rat incubated samples collected 72 h in the anaerobic incubator were analyzed by UPLC-Q-TOF MS within 10 min. A total of 14 metabolites were identified in human and rat incubated solution compared with blank samples. The results indicated that hydrolysis, hydroxylation and acetylation were the major metabolic pathways of flavonoids in A. manihot extract in vitro. MS(E) was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of metabolites. This work demonstrated the potential of the UPLC-Q-TOF MS approach using Metabolynx for fast and automated identification of metabolites of natural product in intestinal flora.     

不同成熟度老鹰茶中酚类化合物含量及抗氧化活性研究

为了探究两种不同成熟度老鹰茶中酚类化合物含量及抗氧化活性的差异,以对其进行辨识及质量评价,该研究利用液相色谱-串联质谱(LC-MS/MS)法测定老鹰茶中15种酚类化合物,采用DPPH自由基清除率、ABTS⁺自由基清除率、Fe³⁺还原能力评价两种茶叶抗氧化能力,再通过数据统计分析探讨两种老鹰茶酚类化合物含量及抗氧化活性的差异,并进一步探索老鹰茶中不同酚类化合物对于抗氧化的贡献。结果表明:(1)嫩叶茶中儿茶素、对香豆酸、异槲皮苷、金丝桃苷、烟花苷、紫云英苷、山奈酚、槲皮素、阿福豆苷含量显著高于老叶茶,其中儿茶素、异槲皮苷、紫云英苷平均含量比老叶茶分别高1 039.43、169.12、257.35 mg·100 g^-1。聚类分析(HCA)、主成分分析(PCA)、正交偏最小二乘判别分析(OPLS-DA)均可将二者区分。(2)方差分析(ANOVA)结果显示在抗氧化能力上,二者在DPPH自由基清除率、ABTS⁺自由基清除率、Fe³⁺还原能力之间具有显著性差异,嫩叶茶优于老叶茶。(3)偏最小二乘回归分析(PLSR)法提示老鹰茶中的异槲皮苷、儿茶素、紫云英苷、绿原酸、金丝桃苷、对香豆酸、山奈酚是其发挥抗氧化效能的主要酚类化合物。该研究结果可为老鹰茶的质量控制及应用推广提供一定的参考。     

Studies on the Chemical Components of Oxytropis ochrocephala Bunge

Oxytropis ochrocephala Bunge has been characterized as one of the most destructive plants to livestock in the North-Western China. Its chemical components have not been studied. We have been isolated four flavonoids from the aerial parts of Oxytropis ochrocephala Bunge. They have been identified as quercetin, rhamnocitrin, isoquercitrin and rhamnocitrin-3-glucoside by UV, IR, 1HNMR and MS spectra of these flavonoids. Their acetyl derivatives have been prepared and their 1HNMR and MS spectra are fully in accord with this conclusion. The rhamnocitrin-3-glucoside has been found first of all in Oxytropis species. It is perhaps valuable for chemotaxonomy of Oxytropis genus.     

In vitro degradation of the flavonol quercetin and of quercetin glycosides in the porcine hindgut

The present study investigated the microbial degradation of the plant flavonol quercetin and its naturally occurring glycosides isoquercitrin and rutin in the porcine hindgut. The experiments were carried out with the semicontinuous colon-simulation technique. The fluid and particle phase of pig hindgut contents from freshly slaughtered animals were used for the in vitro incubations. Following a five-day equilibration period, quercetin, isoquercitrin or rutin were administered to fermentation vessels and their turnover rate was determined. None of the flavonols affected parameters of microbial fermentation like pH, redox potential or VFA production. The turnover rate for isoquercitrin was seven times higher than the turnover for the fermentation fluid. The turnover rates for quercetin and rutin were four and twofold higher than fluid turnover, respectively. After administration of isoquercitrin or rutin, their aglycone quercetin was detected as an intermediary metabolite. Under sterile conditions using autoclaved incubation fluids and hindgut contents, turnover rates for quercetin and rutin were still higher than the fluid turnover in the fermentation vessels. This indicates a certain chemical instability of the flavonols and/or adsorption to ingesta particles. Thus, flavonols are subjected to microbial metabolism in the porcine hindgut. The glycosidic structure strongly influences the rate of metabolism.     

Comparison of chemical composition of Hypericum perforatum and H. maculatum in Estonia

Of numerous species belonging to the medicinally important genus Hypericum, only H. perforatum L. and H. maculatum Crantz grow widely in Estonia. A comparative biochemical systematics study of hypericins, hyperforins and other phenolics within Hypericum spp. growing in Estonia was performed. For comprehensive metabolomic investigation, 42 samples of H. perforatum and 16 samples of aerial parts of H. maculatum were collected in two consecutive years from various locations; methanolic extracts were prepared from airdried leaves and flowers. The concentrations of a quinic acid derivative, caffeic acid glucoside, vanillic acid glucoside, neochlorogenic acid, chlorogenic acid, catechin, epicatechin, myricetin glucoside, hyperoside, isoquercitrin, rutin, quercetin pentoside, quercitrin, kaempferol glucoside, kaempferol rutinoside, quercetin, hyperforin, adhyperforin, protopseudohypericin, pseudohypericin, and hypericin were determined by LC-DAD-MS/MS. All the aforementioned compounds were detected in both species, although some at very different levels – H. maculatum contained rutin and hyperforins only in trace amounts and overall tended to contain more phenolic compounds. The level of total hypericins was the same for both species. These results constitute a further contribution to the systematic knowledge about the Hypericum spp. Results of principal component analysis (PCA) demonstrated distinct between-years and between-species diversity in the chemical composition of the plants studied. Between-years diversity in Hypericum spp. has not been addressed before.     

Secondary metabolites during ontogenetic phase of reproductive structures in Hypericum maculatum

The distribution patterns of flavonoids hyperoside, isoquercitrin, quercitrin, quercetin, I3,II8-biapigenin and naphtodianthrones hypericin and pseudohypericin were studied in reproductive structures during ontogenetic phase of flowering in Hypericum maculatum Crantz. Considerable differences in the content of these secondary metabolites, in the particular flower parts were found. The content of all the metabolites studied is stable during the whole period of flowering in green flower parts (sepals). In petals, stamens and pistils their content undergoes considerable change associated with the biological functions of particular metabolites. The most conspicuous changes during ontogenetic phase of flowering were the decrease of hyperoside and isoquercitrin content in petals (average content in buds 1.589 mg g⁻¹ dry weight, average content in overblown flowers 0.891 mg g⁻¹ dry weight), the decrease of the I3,II8-biapigenin content in stamens (in buds 1.189 mg g⁻¹ dry weight, in overblown flowers 0.319 mg g⁻¹ dry weight), and the increase of hypericin and pseudohypericin content in both petals (total average content of hypericins in the buds 0.547 mg g⁻¹ dry weight; in overblown flowers 0.792 mg g⁻¹ dry weight) and stamens (in buds 0.189 mg g⁻¹ dry weight; in overblown flowers 0.431 mg g⁻¹ dry weight). Hypericins are absent in the pistil. The flavonoids hyperoside and isoquercitrin, the content of which decreased during ontogenetic phase of flowering, reach the highest contents in the pistil.     

In vitro degradation of the flavonol quercetin and of quercetin glycosides in the porcine hindgut

Abstract

The present study investigated the microbial degradation of the plant flavonol quercetin and its naturally occurring glycosides isoquercitrin and rutin in the porcine hindgut. The experiments were carried out with the semicontinuous colon-simulation technique. The fluid and particle phase of pig hindgut contents from freshly slaughtered animals were used for the in vitro incubations. Following a five-day equilibration period, quercetin, isoquercitrin or rutin were administered to fermentation vessels and their turnover rate was determined. None of the flavonols affected parameters of microbial fermentation like pH, redox potential or VFA production. The turnover rate for isoquercitrin was seven times higher than the turnover for the fermentation fluid. The turnover rates for quercetin and rutin were four and twofold higher than fluid turnover, respectively. After administration of isoquercitrin or rutin, their aglycone quercetin was detected as an intermediary metabolite. Under sterile conditions using autoclaved incubation fluids and hindgut contents, turnover rates for quercetin and rutin were still higher than the fluid turnover in the fermentation vessels. This indicates a certain chemical instability of the flavonols and/or adsorption to ingesta particles. Thus, flavonols are subjected to microbial metabolism in the porcine hindgut. The glycosidic structure strongly influences the rate of metabolism.     

微生物糖基转移酶催化合成槲皮素糖苷及其抗炎活性评价

利用微生物来源的糖基转移酶GT-1、GT-2和BTGT-1对槲皮素进行糖基化修饰。研究发现这3种酶均能够催化以槲皮素和UDP-葡萄糖为底物生成槲皮素-3-O-β-D-葡萄糖（异槲皮苷）,GT-1、 GT-2和BTGT-1催化反应的产物生成率分别为5.33%、15.18%和63.82%。通过对槲皮素、异槲皮苷和槲皮素-N-乙酰-D-氨基葡萄糖进行水溶性以及体外细胞抗炎活性检测,发现槲皮素经糖基化后其水溶性得到较大提高,异槲皮苷和槲皮素-N-乙酰-D-氨基葡萄糖水溶性分别是槲皮素的13.8倍和15.4倍。同等浓度下槲皮素糖苷对RAW264.7 细胞的毒性作用低于槲皮素,且3种化合物在一定浓度范围内对LPS诱导RAW264.7细胞释放NO、 IL-1β、IL-6 都有显著的抑制作用,且呈剂量依赖性,研究表明3种化合物都具有一定的抗炎作用。     

Determination of baicalin in rat cerebrospinal fluid and blood using microdialysis coupled with ultra-performance liquid chromatography-tandem mass spectrometry

An in vivo microdialysis sampling method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed for continuous simultaneous monitoring of unbound baicalin in rat blood and brain. Microdialysis probes were inserted into the jugular vein and brain cerebrospinal fluid (CSF) of Sprague-Dawley rats then, following administration of baicalin at doses of 24mg/kg via the candal vein, samples were collected every 20min and injected directly into the UPLC-MS/MS system. In vitro recoveries of the probes were 19.26% and 18.38%, while in vivo recoveries of the probes were 15.0% and 17.52% for blood and brain, respectively. This improved method offers a rapid quantitative procedure for the determination of baicalin with a retention time of only 1.6min. The lower limit of quantification (LLOQ) and the lower limit of detection (LLOD) based on a signal-to-noise ratio of 5 were 2.37 and 0.1ng/ml for anticoagulant citrate dextrose (ACD) solution, and 1.185 and 0.3ng/ml for artificial cerebrospinal fluid (aCSF), respectively. The pharmacokinetics results indicated that baicalin could pass through the blood-brain barrier (BBB) and was detectable in brain dialysate. These in vivo microdialysis-based measurements provide a technique for simple sampling and rapid sensitive analysis of unbound baicalin in rat blood and CSF and for further application in pharmacokinetic studies.     

高效液相色谱法同时测定荷叶中6种黄酮类成分

为了探索同时测定荷叶茶及饮片中6种黄酮类成分(芦丁、金丝桃苷、紫云英苷、槲皮素、山奈素和异鼠李素)含量的方法,本研究以8种不同的荷叶样本为材料,采用高效液相色谱分析法对6种黄酮类成分进行了同时测定。样品经过前处理,以0.5%甲酸-水(A)和0.1%甲酸-乙腈(B)作为流动相进行梯度洗脱,流速为1mL/min,柱温为25℃,进样量为20μL,经Agilent TC-C18(2)(150mm×4.6mm,5μm)色谱柱分离,于360nm波长处检测,结果显示,芦丁、金丝桃苷、紫云英苷、槲皮素、山奈酚、异鼠李素6种黄酮类成分分别在1.6~160、1.8~180、2.16~216、1.4~140、2.12~212、1.6~160μg/mL浓度范围内有良好的线性关系(R20.9992),其检测稳定性、重复性、日内精密度、日间精密度以及加样回收率的RSD均小于2%。进一步用该方法对不同来源的8个荷叶样本进行检测,结果显示荷叶样本中6种黄酮类成分含量以槲皮素最高,并且以样本G荷叶茶(购自G公司,批号为130802)的槲皮素含量最高。本研究建立的同时测定6种荷叶黄酮类成分含量的方法快速、准确,可为荷叶有效成分的检测和质量控制提供技术支撑。