Targeted metabolite analysis of Catharanthus roseus and its biological potential

Catharanthus roseus is nowadays one of the most studied medicinal plants. In this work, further knowledge on different parts of this species (leaves, stems, seeds and petals) was achieved, namely phenolics by HPLC-DAD and organic acids and amino acids by HPLC-UV. Also, the biological potential, expressed as acetylcholinesterase inhibitory activity was accessed and, in some parts, an acetylcholinesterase inhibitory capacity higher than 85% was found (IC₅₀ at 422, 442 and 2683 μg/mL in leaves, stems and petals, respectively). C. roseus aqueous extract revealed to be a rich source of phenolics, namely caffeoylquinic acids and flavonoids derivatives (up to 4127 mg/kg in stems, 4484 mg/kg in seeds, 8688 mg/kg in leaves and 41125 mg/kg in petals), organic acids (962, 6678, 25972 and 12463 mg/kg in seeds, petals, stems and leaves, respectively), such as citric acid (over 85% in some plant parts), and amino acids (31557, 39327, 50540 and 159697 mg/kg in stems, petals, seeds and leaves, respectively), of which arginine was a major compound.     

Liquid chromatography/tandem mass spectrometry study of anti-inflammatory activity of Plantain (Plantago L.) species

To evaluate anti-inflammatory activity of selected Plantago species (P. lanceolata L. and P. major L.) an optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken. By using intact cell system (platelets) as a source of COX-1 and 12-LOX enzymes and highly sensitive and specific LC–MS/MS technique for detection of main arachidonic acid metabolites formed by COX-1 and 12-LOX, this test provides efficient method for evaluation of anti-inflammatory potential of plant extracts and isolated compounds. Our results validated the well-known COX-1 inhibitory activity of P. lanceolata and P. major methanol extracts (concentration required for 50% inhibition (IC₅₀) was 2.00 and 0.65 mg/ml, respectively). Furthermore, 12-LOX inhibitory activity of examined extracts was reported for the first time (IC₅₀ = 0.75 and 1.73 mg/ml for P. lanceolata and P. major, respectively). Although renowned inhibitors, such as acetylsalicylic acid and quercetin showed higher activity, this study verifies P. lanceolata and P. major as considerable anti-inflammatory agents.     

Qualitative and quantitative analysis of steroidal saponins in crude extracts from Paris polyphylla var. yunnanensis and P. polyphylla var. chinensis by high performance liquid chromatography coupled with mass spectrometry

High performance liquid chromatography coupled with electrospray ionization multi-stage tandem mass spectrometry (HPLC–ESI-MSⁿ) and triple quadrupole mass spectrometric detection (HPLC–ESI-MS/MS), respectively, had been employed for the simultaneous identification and quantification of steroidal saponins in the rhizomes of Parispolyphylla var. yunnanensis and P. polyphylla var. chinensis, which are the qualified plants of “Chonglou” in Chinese. The HPLC experiments were performed by means of a reversed-phase C-18 column and a binary mobile phase system consisting of 0.1% aqueous formic acid and acetonitrile under gradient elution conditions. The characteristic fragmentation patterns of diosgenin- and pennogenin-type steroidal saponins were investigated using ESI-MSⁿ in negative ion mode. The MSⁿ data of the [M−H]⁻ ions provided structural information on the sugar sequence of the oligosaccharide chains and the aglycones of steroidal saponins. As a result, ten and seven saponins were determined in P. polyphylla var. yunnanensis and P. polyphylla var. chinensis, respectively, including four unknown compounds. One unknown compound was tentatively identified as diosgenin-3-O-α-l-rhamnopyranosyl(1 → 4) [α-l-rhamnopyranosyl(1 → 2)]-β-d-glucopyranoside and the aglycones of the other three new compounds were reported from Chonglou for the first time. The developed HPLC–ESI-MS/MS method was validated and found to be satisfactorily linear, selective and robust. The limits of detection (LODs) and quantitation (LOQs) ranged, respectively, from 0.5 to 10 ng/mL and 2 to 34 ng/mL depending on six various compounds. The intra- and inter-day precisions of the method were evaluated and were less than 5.0%. Recoveries ranged from 92% to 104% for all compounds. The established quality evaluation method was successfully used for simultaneous quantification of six predominant steroidal saponins in the rhizomes of these two Paris species.     

正交试验优选雪松松针中莽草酸的提取工艺

目的优选雪松松针中莽草酸的最佳提取工艺。方法采用L9（34）正交试验设计,以莽草酸的含量为指标,以液料比、浸泡时间、提取时间为因素,优化雪松松针中莽草酸的提取工艺。结果最佳提取工艺为加20倍量的水,浸泡0.5 h,加热回流2 h。在最佳工艺条件下,提取的莽草酸含量为3.58%。结论优选得到的提取工艺简单、稳定、可行。     

Rapid separation and characterization of active flavonolignans of Silybum marianum by ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry

Ultra-performance liquid chromatography (UPLC) interfaced with the electrospray ionization (ESI) tandem mass spectrometer (MSⁿ) was developed for the simultaneous determination of silychristins A (1) and B (2), silydianin (3), silybins A (4) and B (5), and isosilybins A (6) and B (7), major bioactive flavonolignans in silymarin, a herbal remedy derived from the milk thistle Silybum marianum. In this study, the seven major active flavonolignans including the diastereomers 1/2, 4/5, and 6/7 were completely separated using UPLC with an ACQUITY UPLC C₁₈ column and a MeOH/water/formic acid mobile phase system. The collision-induced dissociation (CID) MSⁿ spectra of these flavonolignans were studied systematically using ESI-MS. The results with the present methodology show that UPLC–MSⁿ can be useful for general screening of active natural products from plant extracts and for the specific quality control of silymarin.     

Metabolic profiling and biological capacity of Pieris brassicae fed with kale (Brassica oleracea L. var. acephala)

Phenolic and organic acid profiles of aqueous extracts from Pieris brassicae material and the host kale (Brassica oleracea L. var. acephala) leaves were determined by HPLC/UV–DAD/MSⁿ-ESI and HPLC–UV, respectively. The identified phenolics included acylated and nonacylated flavonoid glycosides, hydroxycinnamic acyl gentiobiosides, and sulphate phenolics. Kale exhibited the highest content (11 g/kg lyophilized extract), while no phenolics were identified in the butterflies or exuviae. Nine different organic acids were characterized in the materials, with kale showing the highest amount (112 g/kg lyophilized extract). With the exception of the exuviae extract, the rest were screened for bioactivity. Using spectrophotometric microassays, all exhibited antiradical capacity against DPPH and NO in a concentration-dependent way, whereas only kale and excrement extracts were active against superoxide. All displayed activity on intestinal smooth muscle, albeit with distinct relaxation–contraction profiles. Larvae and butterfly extracts were more efficacious for intestinal relaxation than was kale extract, whereas excrement extract evoked only contractions, thus evidencing their different compositions. Collectively, these results show that P. brassicae sequesters and metabolizes kale’s phenolic compounds. Moreover, the extract’s bioactivities suggest that they may constitute an interesting source of bioactive compounds whose complex chemical structures preclude either synthesis or isolation.     

Cholinesterase inhibitory effects of the extracts and compounds of Maclura pomifera (Rafin.) Schneider

We have investigated anticholinesterase potential of the methanol extracts from the leaf, wood, flower, twig, and stem bark of the female and male individuals and rhizodermis and fruit from the female tree of Maclura pomifera (Rafin.) Schneider (Moraceae) along with its major isoflavonoids; osajin and pomiferin as well as their semi-synthetic derivatives; iso-osajin and iso-pomiferin. Anticholinesterase activity was determined by Ellman method using ELISA microplate reader. Osajin and pomiferin had a noticeable inhibition of AChE with IC₅₀ values of 2.239 and 0.096 mM, respectively, while their iso-derivatives were found to display less inhibition towards AChE. The extracts and compounds did not inhibit BChE. The extracts were analyzed for osajin and pomiferin contents by LC–DAD–MS and only the fruits and female flowers contained osajin (fruit: 8.87%, female flowers: 0.19%, w/w) and pomiferin (fruit: 13.6%, female flowers: 0.36%, w/w).     

白背三七总黄酮含量季节性变化规律研究

目的:研究白背三七总黄酮含量的季节性变化规律,为适时采收药材提供依据。方法:对1年12个月采收的白背三七中的总黄酮进行了提取,以芦丁为对照品,用紫外分光光度法测定其含量。结果:白背三七地上部分中的总黄酮含量12月～次年4月较低,5～11月较高。结论:可以根据白背三七的生长周期和总黄酮变化规律,对白背三七进行适时扦插采收,提高白背三七产量。     

Simultaneous characterization and quantitation of 11 coumarins in Radix Angelicae Dahuricae by high performance liquid chromatography with electrospray tandem mass spectrometry

A high performance liquid chromatography with electrospray tandem mass spectrometry (HPLC–ESI-MS/MS) method has been developed to characterize and quantify 11 coumarin compounds in Radix Angelicae Dahuricae simultaneously. By using this HPLC–ESI-MS/MS method, all 11 coumarins were separated and determined within 10 min. These coumarins were detected by ESI⁺ ionization method and quantified by multiple reaction monitor (MRM). The linear regressions were acquired with r² > 0.995, respectively. The precision was evaluated by intra- and inter-day tests, and relative standard deviation (R.S.D.) values were reported within the range of 1.14–4.42% and 0.37–4.00%. The recovery studies for the quantified compounds were observed over the range of 92.1–105.6% with R.S.D. values less than 4.55%. It demonstrated that the method developed was successfully applied for identification and quantification of 11 coumarins in Radix Angelicae Dahuricae. The results showed that the contents of coumarins in Radix Angelicae Dahuricae were processed differently and varied significantly.     

Essential oils composition in two Rosmarinus officinalis L. varieties and incidence for antimicrobial and antioxidant activities

The essential oil composition of Rosmarinus officinalis var. typicus and var. troglodytorum endemic to Tunisia, and growing wild in different bioclimates, was determined by GC and GC–MS. Oils were assessed for their antimicrobial and antioxidant activity. A variation of the chemical composition attributed to varieties rather than to bioclimates was revealed. 1.8-Cineole (47.2–27.5%) and camphor (12.9–27.9%) were identified as the main constituents of var. typicus and var. troglodytorum, respectively. The principal component analysis performed on oil constituents for all the populations allowed the distinction of two distinct population groups in accordance to the varietal subdivision. Based on the determination of the diameter of inhibition and the determination of the minimum inhibitory concentration, a low to moderate antimicrobial activity according to oils was revealed against eight bacteria tested. However, oils from var. troglodytorum showed higher bactericidal effect than those from var. typicus. The oils’ antioxidant activity, determined by 1,1-diphenyl-1-picrylhydrazyl (DPPH) assay, ferric reducing (FRAP) assay and β-carotene bleaching test, was relatively high. The highest activity was found in oils from var. troglodytorum and in one population of var. typicus from the upper semi-arid bioclimate.     

Characterization of bioactive compounds from raw and ripe Mangifera indica L. peel extracts

Mango is one of the important tropical fruits in the world. As it is a seasonal fruit, it is processed for various products. During its processing, peel is one of the major byproducts, which is being wasted. Bioactive conserves were extracted using 80% acetone from peels of raw and ripe mango fruits and subjected to acid hydrolysis. The prominent phenolic compounds identified by HPLC were protocatechuic acid, gentisic acid and gallic acid. The phenolic acid derivatives present in acetone extracts of raw and ripe peels were tentatively identified by LC–MS. Gallic acid, syringic acid, mangiferin, ellagic acid, gentisyl-protocatechuic acid, quercetin were the phenolic compounds identified in both raw and ripe peels, while raw peel showed the presence of glycosylated iriflophenone and maclurin derivatives also. β-Carotene was the major carotenoid followed by violaxanthin and lutein. Thus, both raw and ripe mango peel extracts have different phenolic compounds and carotenoids, which will have various pharmaceutical applications.     

Anti-inflammatory and anti-ulcer activity of the extract from Alhagi maurorum (camelthorn)

Gastric hyperacidity, gastro inflammation and ulcer are very common diseases causing human suffering these days. Gastric irritation mechanism is still very poorly understood as mentioned in many scientific articles. Alhagi maurorum (camelthorn) is considered a medicinal plant with its prospective potent flavonoids. GC–MS spectrum has found three flavone structures (2-phenyl-1,4-benzopyrone derivatives) with rate more than 50% in the ethanolic plant extract. In rat experiment, ethanolic A. maurorum extract (oral daily 100 mg/kg body weight) and ranitidine the standard ulcer drug (oral daily 100 mg/kg body weight) were treated rats to protect against administration of aspirin ASP (oral 200 mg/kg body weight) for two times through the 10 days. Some rats were sacrificed after first and second aspirin administrations and the rest were sacrificed in the end of the experiment. Gastro fluid volume has been decreased in ASP group, and acid output was decreased for plant extract followed by ranitidine. Ranitidine and plant extract protect liver enzymes, oxidation status (MDA and GSH), fucosidase tumor marker and risk lipid ratio. No ulcer patterns have been shown in the histopathological study, but some inflammation in the gastric wall and vascular change dilatation of blood vessels were detected. More studies should be demonstrated potent natural plant extracts and their active components against gastro inflammation and ulcers.     

Plasma pharmacokinetics and tissue distribution study of cajaninstilbene acid in rats by liquid chromatography with tandem mass spectrometry

Cajaninstilbene acid (CSA; 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid) is a major active constituent of pigeonpea leaves, has been proven to be effective in clinical treatment of diabetes, hepatitis, measles and dysentery. A rapid and sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of CSA in rat plasma and various tissues (brain, heart, lung, liver, spleen, small intestine and kidney) of rat for the first time. Rat plasma and tissue distribution pre-treated by protein precipitation with acetoacetate was analyzed using LC–MS/MS with an electrospray ionization (ESI) interface, and isoliquiritigenin was used as an internal standard. Chromatographic separation was achieved on a HIQ Sil C₁₈ column with the mobile phase of water and methanol (9:91, v/v) containing 0.1% formic acid and resulted in a total run time of 10 min. The isocratic elution mode pumped at a flow rate of 1.0 mL/min. The lower limit of quantification (LLOQ) which was 10 ng/mL. The calibration curve was linear from 10 to 6000 ng/mL (R = 0.9967) for plasma samples and 10 to 6000 ng/mL (R ≥ 0.9974) for tissue homogenates. The intra- and inter-day assay of precision in plasma and tissues ranged from 0.6% to 6.1% and 1.5% to 6.6%, respectively, and the intra- and inter-day assay accuracy was 93.5–104.6% and 93.3–107.5%, respectively. Recoveries in plasma and tissues ranged from 95.0% to 106.8%. The method was successfully applied in pharmacokinetic and tissue distribution studies of CSA after oral administration to rats. The pharmacokinetics of CSA showed rapid absorption and elimination (T_max, 10.7 ± 0.31 min; t_1/2, 51.40 ± 6.54 min). After oral administration in rats, CSA was rapidly and widely distributed in tissues. High concentrations were found in liver and kidney indicating that CSA was possibly absorbed by liver and eliminated by kidney.     

Characterization of steroidal saponins in crude extracts from Dioscorea zingiberensis C. H. Wright by ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Steroidal saponins are the major bioactive constituents of Dioscorea zingiberensis C. H. Wright (D. zingiberensis). In this work, ultra-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS/MS) was applied to the separation and characterization of steroidal saponins in crude extracts from D. zingiberensis. The results showed that fragment ions from glycosidic and cross-ring cleavages gave a wealth of structural information related to aglycone skeletons, sugar types and the sequence of sugar units. According to the summarized fragmentation patterns, identification of steroidal saponins from D. zingiberensis could be fulfilled, even when reference standards were unavailable. As a result, a total of thirty-one saponins with five aglycone skeletons, including fourteen new trace saponins, were identified or tentatively elucidated in crude extracts from D. zingiberensis based on their retention times, the mass spectrometric fragmentation patterns, and MS and MS/MS data.     

Simultaneous determination of nine flavonoids in Polygonum hydropiper L. samples using nanomagnetic powder three-phase hollow fibre-based liquid-phase microextraction combined with ultrahigh performance liquid chromatography-mass spectrometry

A simple, inexpensive, and efficient nanomagnetic powder three-phase hollow fibre-based liquid-phase microextraction (HF-LPME) technique combined with ultrahigh performance liquid chromatography–mass spectrometry (UPLC–MS) was developed for the simultaneous analysis of nine flavonoids in Polygonum hydropiper L. samples. The final, optimised extraction conditions were as follows: an organic solvent of ethyl acetate, a donor phase of aqueous KH₂PO₄ at pH 3.0, an acceptor phase of aqueous NaHCO₃ at pH 8.5, a stirring rate of 1000 rpm, and an extraction time of 50 min. Under these conditions, analyte calibration curves were all linear, with correlation coefficients ≥0.9994. The relative standard deviation for all analytes in intra-day (0.8–2.2%) and inter-day (1.7–3.5%) precision tests was well within the acceptable ranges, as were the limits of quantitation (LOQ < 0.054 μg/L) and detection (LOD < 0.170 μg/L). Recoveries for all standard compounds were between 95.17% and 99.82%, with a RSD of no more than 2.3%. Correlative analyses demonstrated that the physicochemical parameters of the compounds themselves also influenced the extraction efficiency. This technology proved to be rapid, sensitive, and reliable for the quality control of P. hydropiper L. samples.     

Systematic screening and characterization of flavonoid glycosides in Carthamus tinctorius L. by liquid chromatography/UV diode-array detection/electrospray ionization tandem mass spectrometry

The traditional Chinese medicine (TCM) is a complex system, which always consists of numerous compounds with significant difference in the content and physical and chemical properties. In this paper, a screening method based on target molecular weights was developed to characterize the flavonoid glycosides in the flower of Carthamus tinctorius L. The screening tables of aglycone and glycan were designed, respectively, in order to select and combine freely. The multiple reaction monitoring (MRM) scan mode with higher sensitivity and selectivity was adopted in the screening, which benefit the characterization for the minor components. Seventy-seven flavonoid glycosides were screened out finally, and their structures were characterized by tandem mass spectrometric method in both positive and negative ion modes. The glycosylation mode, aglycone, sequence and/or the interglycosidic linkages of the glycan portion and glycosylation position were elucidated by the fragmentation rule in the MS. Numerous compounds screened out with this method showed the structure variety in secondary plant metabolites, and the purposeful screening systemically and subsequent structure characterization offered more information about the chemical constitutions of TCM.     

Analytical methods for determination of magnoflorine and saponins from roots of Caulophyllum thalictroides (L.) Michx. using UPLC, HPLC and HPTLC

Analytical methods including HPLC, UPLC and HPTLC are presented for the determination of major alkaloid and triterpene saponins from the roots of Caulophyllum thalictroides (L.) Michx. (blue cohosh) and dietary supplements claiming to contain blue cohosh. A separation by LC was achieved using a reversed phase column, PDA with ELS detection, and ammonium acetate/acetonitrile gradient as the mobile phase. Owing to their low UV absorption, the triterpene saponins were detected by evaporative light scattering. The eight triterpene saponins (cauloside H, leonticin D, cauloside G, cauloside D, cauloside B, cauloside C, cauloside A and saponin PE) and the alkaloid magnoflorine could be separated within 35 min using HPLC method and within 8.0 min using UPLC method with detection limits of 10 μg/mL for saponins and 1 μg/mL for magnoflorine. The detection wavelength was 320 nm for magnoflorine and ELS detection was used for the eight saponins. The methods were also successfully applied to analyze different dietary supplements. For the products claiming to contain blue cohosh, there was a significant variability in the amounts of triterpene saponins detected. Calculations based on the analysis results for dietary supplements showed that maximum daily intake of alkaloid and saponins vary with the form (solids/liquids) and recommended doses according to the products label. Intakes varied from 0.57 to 15.8 mg/day for magnoflorine and from 5.97 to 302.4 mg/day for total saponins. LC-mass spectrometry coupled with electrospray ionization (ESI) method is described for the identification and confirmation of nine compounds in plant samples and dietary products. A HPTLC method was also developed for the fast chemical fingerprint analysis of C. thalictroides samples.     

Immunostimulant Activity of a Novel Polysaccharide Isolated from Lactarius deliciosus (L. ex Fr.) Gray

More and more fungal polysaccharides have been reported to exhibit a variety of biological activities, including antitumor, antioxidant and immunostimulant activity. The non-starch polysaccharides have emerged as an important class of bioactive natural products. In this study, the immune activities of a novel polysaccharide (LDG-A) isolated from Lactarius deliciosus (L. ex Fr.) Gray were investigated at 20, 40 and 80 mg/kg dose levels. The inhibitory rate in mice treated with 80 mg/kg LDG-A can reach 68.422%, being the highest in the three doses, which may be comparable to mannatide. Histology of immune organs showed that the tissues were arranged in more regular and firm pattern, but the tumor tissue arranged looser in LDG-A group than those in control group. Meanwhile, there was no obvious damage to other organs, such as heart, lung, and kidney. The antitumor activity of the LDG-A was usually believed to be a consequence of the stimulation of the cell-mediated immune response because it can significantly promote the lymphocyte and macrophage cells in the dose range of 50-200 μg/ml and 100-400 μg/ml in vitro, respectively. The level of cytokines (IL-6, TNF-α, and NO) of macrophage cells induced by LDG-A treatment was similar to lipopolysaccharides at different concentrations. The expression of all these genes studied (TNF-α, IL-6, and iNOS mRNA) in the untreated macrophage was little, but increased dramatically in a dose-dependent manner in the LDG-A-treated cells. The results obtained in the present study indicated that the purified polysaccharide of L. deliciosus (L. ex Fr.) Gray is a potential source of natural immune-stimulating substances.