Zijia Zhang

Isolation of antioxidants from Psoralea corylifolia fruits using high-speed counter-current chromatography guided by thin layer chromatography-antioxidant autographic assay.

A combinative method using high-speed counter-current chromatography (HSCCC) and thin layer chromatography (TLC) as an antioxidant autographic assay was developed to separate antioxidant components from the fruits of Psoralea corylifolia. Under the guidance of TLC bioautography, eight compounds including five flavonoids and three coumarins were successfully separated from the fruits of P. corylifolia by HSCCC with an optimized two-phase solvent system, n-hexane-ethyl acetate-methanol-water (1:1.1:1.3:1, v/v/v/v). The separation produced 5.91mg psoralen, 6.26mg isopsoralen, 3.19mg psoralidin, 0.92mg corylifol A, and 2.43mg bavachinin with corresponding purities of 99.5, 99.8, 99.4, 96.4, and 99.0%, as well as three sub-fractions, in a single run from 250mg ethyl acetate fraction of P. corylifolia extract. Following an additional clean-up step by preparative TLC, 0.4mg 8-prenyldaidzein (purity 91.7%), 4.18mg neobavaisoflavone (purity 97.4%) and 4.36mg isobavachalcone (purity 96.8%) were separated from the three individual sub-fractions. The structures of the isolated compounds were identified by (1)H NMR and (13)C NMR. The results of antioxidant activity estimation by electron spin resonance (ESR) method showed that psoralidin was the most active antioxidant with an IC50 value of 44.7microM. This is the first report on simultaneous separation of eight compounds from P. corylifolia by HSCCC.

Acetylcholinesterase inhibitive activity-guided isolation of two new alkaloids from seeds of Peganum nigellastrum Bunge by an in vitro TLC- bioautographic assay

Acetylcholinesterase inhibitors (AChEIs) currently form the basis of the newest drugs available for the treatment of Alzheimer’s disease. For the aim of screening effective AChEIs, the methanol extracts of the seeds of genus Peganum were found to show significant inhibitory activity of acetylcholinesterase enzyme (AChE) using an in vitro TLC-bioautographic assay. In further studies to seed of P. nigellastrum Bunge, activity-guided fractionation led to the isolation of two new alkaloids nigellastrine I (9) and nigellastrine II (10), and along with eight known alkaloids, vasicinone (1), vasicine (2), harmine (3), deoxyvasicinone (4), deoxyvasicine (5), harmaline (6), harmol (7), harman (8), in which harmol and harman were first isolated from species P. nigellastrum Bunge. As active constituents, all compounds showed good inhibitory activities against AChE. The results of in vitro semi-quality TLC-bioautographic assay showed that harmine, harmaline and harmol displayed a similar AChE inhibitive activities comparing to galanthamine. These results indicated that these alkaloids in P. nigellastrum Bunge could be a potent class of AChEIs.

Identification of acetylcholinesterase inhibitors from seeds of plants of genus Peganum by thin-layer chromatography-bioautography

In traditional Chinese medicine, plants of the genus Peganum have been used to treat cough, hypertension, diabetes, asthma, jaundice, lumbago, and many other ailments. In this study, seeds of the plants of genus Peganum, including P. harmala Linn., P. multisectum (Maxim) Bobr, P. nigellastrum Bunge, and Peganum variety were collected from different provinces in China. A simple, rapid, and effective thin-layer chromatographic (TLC) fingerprint combined with bioautographic technique has been established for the identification of acetylcholinesterase (AChE) inhibitors from these seeds. The methanol extracts of seeds were separated on silica gel plates with ethyl acetate-methanol-ammonia 10:1.5:0.5 (v/v) as mobile phase, and then the plates were inspected under UV 366 nm and visualized by spraying with both Dragendorff’s and vanillin-sulfuric acid reagents as well as by bioautographic assay. Moreover, the limits on AChE inhibitive activity of harmine and harmaline were found to be 0.01 µg, in comparison to that of galanthamine of also 0.01 µg. The TLC fingerprints combined with the bioautographic method could distinguish the seeds of the different species of genus Peganum investigated. Moreover, harmine and harmaline displayed similar AChE inhibition compared to galanthamine.

Quick identification of xanthine oxidase inhibitor and antioxidant from Erycibe obtusifolia by a drug discovery platform composed of multiple mass spectrometric platforms and thin-layer chromatography bioautography.

As a final step of the purine metabolism process, xanthine oxidase catalyzes the oxidation of hypoxanthine and xanthine into uric acid. Our research has demonstrated that Erycibe obtusifolia has xanthine oxidase inhibitory properties. The purpose of this paper is to describe a new strategy based on a combination of multiple mass spectrometric platforms and thin-layer chromatography bioautography for effectively screening the xanthine oxidase inhibitory and antioxidant properties of E. obtusifolia. This strategy was accomplished through the following steps. (i) Separate the extract of E. obtusifolia into fractions by an autopurification system controlled by liquid chromatography with mass spectrometry. (ii) Determine the active fractions of E. obtusifolia by thin-layer chromatography bioautography. (iii) Identify the structure of the main active compounds with the information provided by direct analysis in real time mass spectrometry. (iv) Calculate the IC50 value of each compound against xanthine oxidase using high-performance liquid chromatography. Using the caulis of E. obtusifolia as the experimental material, seven target peaks were screened out as xanthine oxidase inhibitors or antioxidants. Our screening strategy allows for rapid analysis of small molecules with almost no sample preparation and can be completed within a week, making it a useful assay to identify unstable compounds and provide the empirical foundation for E. obtusifolia as a natural remedy for gout and oxidative-stress-related diseases.

UPLC-MS/MS determination and gender-related pharmacokinetic study of five active ingredients in rat plasma after oral administration of Eucommia cortex extract

ETHNOPHARMACOLOGICAL RELEVANCE Eucommiae cortex (EC), the bark of Eucommia ulmoides Oliv., has been traditionally used to treat many diseases in China for more than 2000 years. The pharmacological effects are primarily attributed to the presence of lignans, iridoids and phenolics, which are main active ingredients in EC. AIM OF THE STUDY First, to investigate the active ingredients that can be absorbed into the rat plasma according to which ingredients exhibit significant correlation of drug concentration-time curve. Second, to establish an efficient ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of ingredients absorbed in rat plasma. Finally, to investigate gender effect on the pharmacokinetics of the ingredients absorbed in male and female rats plasma after oral administration with EC extract. MATERIALS AND METHODS 18 ingredients from EC were detected by UPLC-MS/MS, 9 out of 18 ingredients were absorbed into rat plasma. And 5 ingredients exhibit significant correlation of drug concentration-time curve. They were pinoresinol di-O-β-d-glucopyranoside (PDG), geniposide (GE), geniposidic acid (GA), aucubin (AN) and chlorogenic acid (CA). The analytes were extracted from rat plasma via a simple protein precipitation procedure and osalmid was used as the internal standard. Chromatographic separation was achieved on a Waters ACQUITY HSS T3 column (2.1mm×100mm, 1.8μm) using a gradient elution program with acetonitrile and 0.1% formic acid water as the mobile phase, with a flow rate of 0.3mLmin(-1). The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reactions monitoring (MRM) mode in a positive ion mode via electrospray ionization (ESI). The transition monitored were /z 683.00[M+H](+)→235.10 for PDG, / z 389.00[M+H](+)→208.80 for GE, m/z 375.00[M+H](+)→194.79 for GA, m/z 364.00[M+NH4](+)→148.81 for AN, m/z 355.10[M+H](+)→162.84 for CA and m/z 230.03[M+H](+)→120.77 for internal standard. RESULTS The developed method showed good linearity over a wide concentration range, the lower limits of quantification and higher accuracy and precision for determination of the 5 analytes. Then the method was applied to study the pharmacokinetics in rats, and the results indicated that there were significant differences in pharmacokinetic parameters of the analytes between the male and female rats, and absorptions of these analytes in male group were all significantly higher than those in female group. CONCLUSION This study established an efficient, sensitive and selective UPLC-MS/MS method for simultaneous determination of the five ingredients in rat plasma, and it could be successfully applied to the comparative pharmacokinetic studies in male and female rats after oral administration with EC extract.

Comparison of active constituents, acute toxicity, anti-nociceptive and anti-inflammatory activities of Porana sinensis Hemsl., Erycibe obtusifolia Benth. and Erycibe schmidtii Craib.

ETHNOPHARMACOLOGICAL RELEVANCE Erycibe obtusifolia and Erycibe schmidtii, which belong to the same genus as Erycibe, are widely used in traditional medicine for the treatment of joint pain and rheumatoid arthritis (RA). Porana sinensis has become a widely used substitute for Erycibe obtusifolia and Erycibe schmidtii as they have declined in the wild. In the present work, the content of the main active components, the acute toxicity, the anti-nociceptive and anti-inflammatory activities of Porana sinensis, Erycibe obtusifolia and Erycibe schmidtii were compared, and the mechanisms of anti-nociceptive and anti-inflammatory activities were discussed. MATERIALS AND METHODS A quantitative HPLC (high performance liquid chromatography) method was first developed to compare the content of the main active components (scopoletin, scopolin and chlorogenic acid). The anti-inflammatory and anti-nociceptive activities of 40% ethanolic extracts of the three plants were compared using the models of xylene-induced ear edema, formalin-induced inflammation, carrageenan-induced air pouch inflammation, acetic acid-induced writhing and formalin-induced nociception. The acute toxicity of the 40% ethanolic extracts of the three plants was studied. RESULTS The assay suggested a large content of scopoletin, scopolin and chlorogenic acid in the three plants. The 40% ethanolic extracts of the three plants were almost non-toxic at the dose of 5g/kg and all of them showed significant anti-inflammatory effects in the tests of xylene-induced ear edema and formalin-induced inflammation. In the carrageenan-induced air pouch inflammation test, the synthesis of PGE2 was significantly inhibited by all the extracts. They significantly inhibited the number of contortions induced by acetic acid and the second phase of the formalin-induced licking response. Naloxone was not able to reverse the analgesic effect of these extracts. CONCLUSION The study identifies the similarity of the three plants in their main active components as well as acute toxicity, anti-nociceptive and anti-inflammatory activities. It supports the use of Porana sinensis as a suitable substitute, but further studies are needed to confirm this.

Different fingerprinting strategies to differentiate Porana sinensis and plants of Erycibe by high‐performance liquid chromatography with diode array detection, ultra high performance liquid chromatography with tandem quadrupole mass spectrometry, and chemometrics

Plants of Erycibe are widely used in traditional Chinese medicine for the treatment of joint pain and rheumatoid arthritis. With the reduction of Erycibe resources in the wild, Porana sinensis has been widely used as a substitute. However, it is important to understand the chemical distinctions between the two kinds of plants and identify their individual chemical markers. In this study, multiwavelength chromatographic fingerprint and precursor ion fingerprint techniques were used in conjunction with chemometric tools to fingerprint and thus differentiate between plant samples. The similar results obtained from different fingerprints prove the reliability of the two fingerprints. Results obtained from principal component analysis and orthogonal projection to latent structures discriminant analysis identified similarities between the chemical components of P. sinensis and plants of Erycibe. However, concentrations of 4-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid were higher in P. sinensis than in plants of Erycibe, suggesting that P. sinensis may be more effective in medical treatments of some diseases than Erycibe.

Toxicology and the chemical foundation of plants of Erycibe

Erycibe is a relatively small genus in the family Convolvulaceae with over 10 identified species. Some Erycibe plant species are purportedly toxic at high doses. However, few toxicology studies have been conducted on those species. In this study, the toxicity of 40% ethanolic extracts of Erycibeobtusifolia, Erycibeschmidtii, and Erycibeellipptimba was evaluated. E. ellipptimba has been reported to be more toxic due to containing larger amounts of Baogongteng C, an alkaloid with known toxicity. Thus, E. ellipptimba was chosen for further toxicology study here. An HPLC-MS method was developed to identify the main components and determine the percentages of Baogongteng C in total alkaloid of E. ellipptimba (EWA). The toxicity of total alkaloid and Baogongteng C was evaluated and compared. The results indicated that Baogongteng A and Baogongteng C are the major toxic chemical compounds of the Erycibe species tested. The results also suggest EWA is cholinergic. Finally, in a subacute toxicity study of EWA, alterations observed with high dosage suggest that the liver and kidney could be the target organs of toxicity.

Direct Analysis in Real‐time Mass Spectrometry for Rapid Identification of Traditional Chinese Medicines with Coumarins as Primary Characteristics

INTRODUCTION The increasing popularity of traditional Chinese medicines (TCMs) necessitates rapid and reliable methods for controlling their quality. Direct analysis in real-time mass spectrometry (DART-MS) represents a novel approach to analysing TCMs. OBJECTIVE To develop a quick and reliable method of identifying TCMs with coumarins as primary characteristics. METHODOLOGY DART-MS coupled with ion trap mass spectrometry was employed to rapidly identify TCMs with coumarins as primary characteristics and to explore the ionisation mechanisms of simple coumarins, furocoumarins and pyranocoumarins in detail. With minimal sample pretreatment, mass spectra of Fraxini Cortex, Angelicae Pubescentis Radix, Peucedani Radix and Psoraleae Fructus samples were obtained within seconds. The operating parameters of the DART ion source (e.g. grid electrode voltage and ionisation gas temperature) were carefully investigated to obtain high-quality mass spectra. The mass spectra of samples and DART-MS/MS spectra of marker compounds were used to identify sample materials. RESULTS Successful authentication was achieved by analysing the same materials of different origins. Some simple coumarins, furocoumarins and pyranocoumarins can be directly detected by DART-MS as marker compounds. CONCLUSION Our results demonstrated that DART-MS can provide a rapid and reliable method for the identification of TCMs containing different configurations of coumarins; the method may also be applicable to other plants. Copyright