A rapid MALDI MS/MS based method for assessing saffron (Crocus sativus L.) adulteration.

We report on a sensitive and fast quantitative MALDI-MS/MS method used to assess saffron authenticity by direct analysis through the determination of picrocrocin as the saffron authenticity marker, and using curcumin as the non-isotopic isobaric internal standard. The internal standard curcumin yielded good linearity (R2 = 0.994), and with confidence intervals at 95% for intercept. The detectable maximum adulteration percentage (99.0%) was estimated interpolating the limit of detection (LOD) for the isobaric internal standard in linear regression. The LOD was 47.63 ppm, and LOQ was 56.53 ppm. Good accuracy and precision were obtained for all concentrations. The capability of the MS approach to monitor analytes in a specific, selective fashion was used to obtain a semi-quantitative adulteration percentage and to establish the adulterant by additional experiments. The detection of gardecin and its derivatives in commercial samples indicated that Gardenia jasminoides Ellis was used as the adulterant.

Ecotype Variation of Methyl Eugenol Content in Tea Tree (Melaleuca alternifolia and Melaleuca linariifolia).

Methyl eugenol is a natural phenylpropanoid compound found in a wide range of plants used for food, flavouring, cosmetics, and health-care. As a suspected rodent carcinogen, methyl eugenol may also be harmful to humans when present in significant concentrations. Consequently, its level has been restricted in some foodstuffs and cosmetics for some markets. In order to assess the potential to breed uniformly low methyl eugenol cultivars for an essential oil crop, tea tree, the source of 'Oil of Melaleuca, terpinene-4-ol type', we examine levels in individual trees (n = 30) from two geographic regions and six terpene chemotypes. Overall, methyl eugenol levels were low in this species (Mean [SD] 354 [239] ppm, n = 30), much lower than levels predicted to be of toxicological concern. Within each chemotype, there was a lack of evidence for correlations between terpenoid constituents and methyl eugenol levels. Further support for the independence of methyl eugenol and terpene biosynthesis was evident from similar mean levels in selected (Mean [SD] 586 [339] ppm, n = 12) and undomesticated Melaleuca alternifolia trees (Mean [SD] 480 [299] ppm, n = 5) with terpinen-4-ol type oils. By contrast, methyl eugenol level varied by geographic origin and chemotype. Trees from the upland region, where there is a prevalence of terpinolene type trees, had lower average methyl eugenol levels than trees from the coastal region, where there is a prevalence of terpinen-4-ol and 1,8-cineole type trees.

Quantitative analysis of safranal in saffron extract and nanoparticle formulation by a validated high-performance thin-layer chromatographic method.

INTRODUCTION: Safranal is an effective anticonvulsant shown to act as an agonist at GABA(A) receptors. Nose to brain delivery via nanoparticle formulation might improve its brain delivery. A selective and sensitive analytical method is required for evaluation of safranal-based novel drug delivery systems. OBJECTIVE: To develop and validate a high-performance thin-layer chromatographic (HPTLC) method for the quantitative analysis of safranal as bulk, in saffron extract and in developed safranal-loaded nanoparticle formulation. METHODOLOGY: Chromatographic separation was achieved on silica gel pre-coated TLC aluminium plates 60F-254, using n-hexane:ethyl acetate (9 : 1, v/v) as the mobile phase. Quantitative analysis was carried out by densitometry at a wavelength of 310 nm. The method was validated and applied to detect related impurities, to analyse safranal in saffron extract and to evaluate safranal-loaded nanoparticles. RESULTS: Compact spots of safranal were observed at R(f) value 0.51 +/- 0.02. The method was linear (r = 0.9991) between 0.5 and 5.0 ng/spot. The intra- and inter-day precisions were 1.08-2.17 and 1. 86-3.47%, respectively. The limit of detection was 50 ng/spot and the limit of quantification was 150 ng/spot. The method proved to be accurate (recovery 97.4-102.0%) and was selective for safranal. Evaluation of safranal-loaded nanoparticle formulation demonstrated drug loading of 23.0%, encapsulation efficiency of 42.0% and sustained drug release following biphasic pattern. CONCLUSION: The present method is useful for the quantitative and qualitative analysis of safranal and safranal-loaded nanoparticle formulation. It provides significant advantages in terms of greater specificity and rapid analysis.

Stability of active ingredients of traditional Chinese medicine (TCM).

Studies on stability of active ingredients are fundamental and critical for the rational development of Traditional Chinese Medicine (TCM) in view of its modernization and worldwide use. The stability of both active and marker constituents of plants used in TCM is reviewed for the first time. More than 100 papers, mostly written in Chinese, have been reviewed. Studies concerning plant constituents were analyzed according to their chemical classification of active ingredients. In addition, several crude drugs of animal origin are also reported. Stability of active ingredients is summarized during extraction and/or storage of the herbal drug preparations, and under stress conditions (pH, temperature, solvents, light, and humidity) and in the presence of preservatives, antioxidants, and metals.

[Chemical constituents analysis of Mgrtol standardized by GC-FTIR].

The chemical constituents of the Mgrtol standardized were analyzed by capillary gas chromatography-Fourier transform infrared spectroscopy (GC-FTIR). The relative content of each component was determined by area nomalization method. Twelve peaks were separated and were all identified by FTIR through search method. The results showed that the Mgrtol standardized mainly consists of 3 kinds of single terpenes, including 1,8-cineole, D-limonen and alpha-pinene, which account for 14.70%, 36.83% and 46.35% respectively, totaling to 97.88% of the entire compositions of the Mgrtol standardized. The results are the same as those analyzed by GC-MS. Comparing with GC-MS, the isomers of organic compounds separated by gas chromatography could be determined by GC-FTIR. So this method has wider application value. The results also provided a scientific proof for the further development of the plant.

Separation and quantification of terpenoids of Boswellia serrata Roxb. extract by planar chromatography techniques (TLC and AMD).

An high-performance TLC (HPTLC) method for the separation of boswellic acids, the active constituents in Boswellia serrata extract, has been developed and TLC of these compounds on silica by automated multiple development (AMD) using solvent gradients was performed. Enhancement of the separation of boswellic acids on HPTLC plates was carried out by AMD chromatography. Densitometric analysis of the developed plate was carried out to quantify the four boswellic acids. 11-Keto-beta-boswellic acid (KBA) and acetyl-11-keto-beta-boswellic acid (AKBA) were quantified by densitometric scanning of the developed plate at 254 nm. beta-Boswellic acid (BA) and acetyl-beta-boswellic acid (ABA) were quantified after derivatization with anisaldehyde sulfuric acid reagent at 560 nm. The AMD system provided a clean separation according to polarity for each of the four groups studied and good results were obtained. The proposed HPTLC method for the simultaneous quantification of the major boswellic acids BA, ABA, KBA, and AKBA was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control and for the quantification of these compounds in plant materials. The study of market products revealed significant variations in the content of these pharmacologically active compounds in commercial samples.

Quantitative analysis of the major constituents of St John's wort with HPLC-ESI-MS.

A method was developed to profile the major constituents of St John's wort extracts using high-performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI-MS). The objective was to simultaneously separate, identify and quantify hyperforin, hypericin, pseudohypericin, rutin, hyperoside, isoquercetrin, quercitrin and chlorogenic acid using HPLC-MS. Quantification was performed using an external standardisation method with reference standards. The method consisted of two protocols: one for the analysis of flavonoids and glycosides and the other for the analysis of the more lipophilic hypericins and hyperforin. Both protocols used a reverse phase Luna phenyl hexyl column. The separation of the flavonoids and glycosides was achieved within 35 min and that of the hypericins and hyperforin within 9 min. The linear response range in ESI-MS was established for each compound and all had linear regression coefficient values greater than 0.97. Both protocols proved to be very specific for the constituents analysed. MS analysis showed no other signals within the analyte peaks. The method was robust and applicable to alcoholic tinctures, tablet/capsule extracts in various solvents and herb extracts. The method was applied to evaluate the phytopharmaceutical quality of St John's wort preparations available in the UK in order to test the method and investigate if they contain at least the main constituents and at what concentrations.

Enrichment of hyperforin from St. John's wort (Hypericum perforatum) by pilot-scale supercritical carbon dioxide extraction.

St. John's Wort (Hypericum perforatum L.) was extracted with supercritical carbon dioxide using a pilot batch extraction plant. The effects of pressure, temperature, flow rate and extraction time were examined with respect to extraction yield and hyperforin content. Supercritical carbon dioxide showed a high selectivity for phloroglucinols. Extracts were analyzed using an isocratic HPLC method with a mixture of hyperforin/adhyperforin as an external standard. Within the studied range of extraction pressure (90-150 bar) and extraction time (1-5 h), extraction at 90 bar for 3 h and 120 bar for 1 h provided higher hyperforin content (up to 35%) in the resulting extracts. An increase in extraction temperature showed a negative effect, leading to increased degradation of hyperforin into orthoforin. When the total mass of carbon dioxide passing the extraction vessel was kept constant, changes in mass flow rate did not affect the extraction result.

Structural investigations of isomeric oxidised forms of hyperforin by HPLC-NMR and HPLC-MSn.

The prenylated phloroglucinol hyperforin, thought to be an essential component for the anti-depressant activity of St. John's Wort (Hypericum perforatum), is unstable. The facile oxidative degradation of hyperforin poses serious problems for standardisation, and may also dramatically affect the pharmacological activity of the extracts. Hyperforin was dissolved in hexane and stored at room temperature for 3 days and yielded various closely related degradation products which, although difficult to isolate on the preparative scale, have been analysed by on-flow and stop-flow HPLC-NMR and HPLC-MS/MS. From on-line spectroscopic data, and with the aid of complementary in-mixture standard NMR two-dimensional correlation experiments, the different oxidised forms of hyperforin were found to be phloroglucinol derivatives in which a hydroxy-dihydrofuran ring is formed involving the enol OH at C-7 or C-9 (tautomeric form) and the prenyl chain at C-8 of the core nucleus of hyperforin. The strategy followed for the on-line identification of these constituents is discussed.

Liquid chromatography/electrospray mass spectrometry of bioactive terpenoids in Ginkgo biloba L.

Standardized extracts of Ginkgo biloba leaves are mainly used in the treatment of peripheral and celebral circulation disorders, and also as a remedy against asthma, coughs, bladder inflammation, blenorrhagia and alcohol abuse. The leaf extracts contain biflavones, flavonol glycosides and terpene lactones. This paper reports a method based on liquid chromatography coupled with electrospray mass spectrometry for the analysis of terpenoids in G. biloba extracts. This method allows the rapid isocratic separation of underivatized ginkgolides (GA, GB, GC and GJ) and bilobalide at very low levels (10 pg on the column) and their quantitative detection by external standardization with relative standard deviations of 3 and 5% for intra- and inter-day analyses, respectively.

Antifertility effects of neem (Azadirachta indica) oil in male rats by single intra-vas administration: an alternate approach to vasectomy.

An alternate approach to vasectomy for long-term male contraception following a single intra-vas application of a traditional plant (Azadirachta indica) product having immunomodulatory properties is described. Male Wistar rats of proven fertility were given a single dose (50 microliters) of neem oil in the lumen of the vas deferens on each side; control animals received the same volume of peanut oil. Animals were put on continuous mating 4 weeks after the treatment, with females of proven fertility. While the control animals impregnated the female partners, all males treated with neem oil remained infertile throughout the 8 months of observation period. Epididymal and vas histology were normal without any inflammatory changes or obstruction. The intra-vas administration of neem oil resulted in a block of spermatogenesis without affecting testosterone production; the seminiferous tubules, although reduced in diameter, appeared normal and contained mostly early spermatogenic cells. No anti-sperm antibody could be detected in the serum. Unilateral administration of neem oil in the vas resulted in a significant reduction of testicular size and spermatogenic block only on the side of application; the draining lymph node cells of the treated side also showed enhanced proliferative response to in vitro mitogen challenge. These results indicate that the testicular effects following intra-vas application of neem oil may possibly be mediated by a local immune mechanism.