Samiuela Lee

Validation of a method for the simultaneous determination of four schisandra lignans in the raw herb and commercial dried aqueous extracts of Schisandra chinensis (Wu Wei Zi) by RP-LC with DAD.

A rapid and specific reversed-phase high performance liquid chromatography (RP-LC) method with photodiode array detection (DAD) was developed and validated for the determination of four common schisandra lignans, schisandrin (1), schisandrol B (2), deoxyshisandrin (3) and gamma-schisandrin (4), in raw herb materials and commercial dried aqueous extracts of Schisandra chinensis (Wu Wei Zi). The extraction solvent and extraction method were optimised where it was found that a 4 h Soxhlet extraction using methanol was successful at extracting >99.5% of each of the schisandra lignans analysed from the raw herb material. The sample preparation process for the dried aqueous extract samples involved sonication using methanol for 2 x 30 min. The herb and extract solutions were separated on a Varian Microsorb-MV 100-5 C18 column using a gradient mixture of 0.1% aqueous phosphoric acid and acetonitrile. Subsequent detection and quantitation of the schisandra lignans was performed at 210 nm. The correlation coefficients of the linear regression analysis performed on these calibration curves were >0.9996 for all four schisandra lignans assayed. The detection limits and quantification limits ranged from 0.12 to 0.57 and 0.41 to 1.89 mg g(-1), respectively. The mean recoveries of the various analytes ranged from 92.20 to 107.01%. The method was used to investigate the levels of the four mentioned components in herb samples and dried aqueous extracts. The identities of the chromatographic peaks were confirmed by (+) electrospray ionisation LC-MS/MS.

The determination of glycyrrhizic acid in Glycyrrhiza uralensis Fisch. ex DC. (Zhi Gan Cao) root and the dried aqueous extract by LC–DAD

A rapid, sensitive and specific reversed phase high-performance liquid chromatographic (LC) method with photodiode array detection (DAD) has been developed for the determination of glycyrrhizic acid in both the raw herb and a commercially prepared dried aqueous extract of Glycyrrhiza uralensis Fisch. ex DC. root (Zhi Gan Cao, liquorice). It was determined that extracting the raw herb in aqueous methanol (50:50 v/v) by sonication for 2 x 30 min was the most efficient sample preparation. Baseline resolution of the glycyrrhizic acid peak was achieved on a Varian Polaris RP C18-A (250 mm x 4.6 mm, 5 microm packing) column using an isocratic mobile phase consisting of 0. v aqueous phosphoric acid and acetonitrile in the ratio 60:40 v/v. Chromatograms were monitored between 200 and 400 nm for peak purity assessments, with quantitation performed at 254 nm. Glycyrrhizic acid calibration curves in the concentration range of 14-558 microg/ml were prepared on the day of analysis. Curve fitting was by the least-squares method, with correlation coefficients of >0.9998 obtained each time. The average recovery at three spike levels (50, 100, 200%) was of 95.91+/-1.05% and 98.36+/-3.45% (+/-S.D., n=7) for the spiked raw herb and dried aqueous extract respectively. The limit of detection and limit of quantitation was 0.52 and 1.72 mg/g respectively for the raw herb, and 0.75 and 2.51 mg/g respectively for the dried aqueous extract. Identity confirmation of the chromatographic peak was achieved by (-) electrospray ionisation tandem mass spectrometry. The concentration of glycyrrhizic acid in the root and dried aqueous extract was found to be 31.1+/-0.2 and 40.4+/-0.3mg/g (+/-S.D., n=7) respectively.

Anti-inflammatory activity of cinnamon (C. zeylanicum and C. cassia) extracts – identification of e-cinnamaldehyde and o-methoxy cinnamaldehyde as the most potent bioactive compounds

Chronic inflammation is a contributing factor in many age-related diseases. In a previous study, we have shown that Sri Lankan cinnamon (C. zeylanicum) was one of the most potent anti-inflammatory foods out of 115 foods tested. However, knowledge about the exact nature of the anti-inflammatory compounds and their distribution in the two major cinnamon species used for human consumption is limited. The aim of this investigation was to determine the anti-inflammatory activity of C. zeylanicum and C. cassia and elucidate their main phytochemical compounds. When extracts were tested in LPS and IFN-γ activated RAW 264.7 macrophages, most of the anti-inflammatory activity, measured by down-regulation of nitric oxide and TNF-α production, was observed in the organic extracts. The most abundant compounds in these extracts were E-cinnamaldehyde and o-methoxycinnamaldehyde. The highest concentration of E-cinnamaldehyde was found in the DCM extract of C. zeylanicum or C. cassia (31 and 34 mg g(-1) of cinnamon, respectively). When these and other constituents were tested for their anti-inflammatory activity in RAW 264.7 and J774A.1 macrophages, the most potent compounds were E-cinnamaldehyde and o-methoxycinnamaldehyde, which exhibited IC₅₀ values for NO with RAW 264.7 cells of 55 ± 9 μM (7.3 ± 1.2 μg mL(-1)) and 35 ± 9 μM (5.7 ± 1.5 μg mL(-1)), respectively; and IC₅₀ values for TNF-α of 63 ± 9 μM (8.3 ± 1.2 μg mL(-1)) and 78 ± 16 μM (12.6 ± 2.6 μg mL(-1)), respectively. If therapeutic concentrations can be achieved in target tissues, cinnamon and its components may be useful in the treatment of age-related inflammatory conditions.

The Saccharomyces cerevisiae transcriptome as a mirror of phytochemical variation in complex extracts of Equisetum arvense from America, China, Europe and India

BackgroundPattern-oriented chemical profiling is increasingly being used to characterize the phytochemical composition of herbal medicines for quality control purposes. Ideally, a fingerprint of the biological effects should complement the chemical fingerprint. For ethical and practical reasons it is not possible to test each herbal extract in laboratory animals or humans. What is needed is a test system consisting of an organism with relevant biology and complexity that can serve as a surrogate in vitro system. The purpose of this study was to test the hypothesis that the Saccharomyces cerevisiae transcriptome might be used as an indicator of phytochemical variation of closely-related yet distinctly different extracts prepared from a single species of a phytogeographically widely distributed medicinal plant. We combined phytochemical profiling using chromatographic methods (HPTLC, HPLC-PDA-MS/MS) and gene expression studies using Affymetrix Yeast 2.0 gene chip with principal component analysis and k-nearest neighbor clustering analysis to test this hypothesis using extracts prepared from the phytogeographically widely distributed medicinal plant Equisetum arvense as a test case.ResultsWe found that the Equisetum arvense extracts exhibited qualitative and quantitative differences in their phytochemical composition grouped along their phytogeographical origin. Exposure of yeast to the extracts led to changes in gene expression that reflected both the similarities and differences in the phytochemical composition of the extracts. The Equisetum arvense extracts elicited changes in the expression of genes involved in mRNA translation, drug transport, metabolism of energy reserves, phospholipid metabolism, and the cellular stress response.ConclusionsOur data show that functional genomics in S. cerevisiae may be developed as a sensitive bioassay for the scientific investigation of the interplay between phytochemical composition and transcriptional effects of complex mixtures of chemical compounds. S. cerevisiae transcriptomics may also be developed for testing of mixtures of conventional drugs (“polypills”) to discover novel antagonistic or synergistic effects of those drug combinations.

Choosing chemical markers for quality assurance of complex herbal medicines: Development and application of the herb MaRS criteria

With increasing use of herbal medicines for chronic or serious illness, relevant quality assurance methods are essential for making claims of therapeutic benefit. Adequate demonstration of safety and efficacy based on chemical composition and ensuring consistency between manufactured batches is critical. To date, there has been no uniform standard approach or detailed framework provided to industry for selecting relevant chemical markers used to standardize herbal products. We developed the Herbal Marker Ranking System (Herb MaRS) providing guidance on prioritizing the selection of chemical markers for quality control of complex multi‐herb mixtures, while also taking into account the bioactivity in relation to the symptoms of the disease and its concentration in the formula. We apply the Herb MaRS evaluation criteria to a seven‐herb formulation for the treatment of irritable bowel syndrome with constipation. Our ranking scale accommodates the clinical and pharmacological use of the formulation and its claimed indications.

Using GenBank® for Genomic Authentication: A Tutorial

The GenBank(®) database is perhaps one of the most important repositories of genetic information. A researcher working in the field of genomic authentication must therefore be equipped with the skills needed to competently access the required information from this database whilst ultimately contributing their own data to it. This chapter presents a practical guide to using GenBank(®) to search for sequences, search and align unknown sequences using BLAST, and uploading and maintaining your own sequences. This chapter also details some other software helpful in sequence manipulation.

The Liquid Chromatographic Determination of Chlorogenic and Caffeic Acids in Xu Duan (Dipsacus asperoides) Raw Herb

A validated analytical method is reported for the analysis of chlorogenic and caffeic acids in Xu Duan (Dipsacus asperoides) in the dried raw herb. The ground samples were extracted by ultrasonication in water and the extract was analysed by LC-PDA with identity confirmation by (