Analysis of five pharmacologically active compounds from the Tibetan medicine Elsholtzia with micellar electrokinetic capillary chromatography

A high performance capillary electrophoresis method with diode array detector detection for the determination of five bioactive ingredients in Tibetan medicine Elsholtzia, namely quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid, has been developed. The effects of several factors, such as the acidity, concentration of running buffer, separation voltage, temperature, and SDS concentration were investigated. The optimal conditions were 44 mmol/L boric acid running buffer (pH 8.5), 45 mmol/L SDS, 16 KV voltage, 20 degrees C, and 10.0% (V/V) of acetonitrile. Under the optimum conditions, five components could be separated with a good baseline resolution within 17 min. The calibration curves showed good linear relationship over the concentration range of 5 x 10(-4)similar to 0.1 mg/mL for quercetin, rutin, saussurenoside, kaempferol, and 1 x 10(-3) similar to 0.1 mg/mL for oleanolic acid. The average recoveries of the method and RSD were ( 99.2%, 3.2%) for quercetin, (102.1%, 2.1%) for rutin, (99.4%, 1.5%) for saussurenoside, (98.9%, 1.8%) for kaempferol, and (99.0%, 2.9%) for oleanolic acid, respectively. The detection limits (S/N = 3) were 1.1 x 10(-4) mg/mL for quercetin, 2.6 x 10(-4) mg/mL for rutin, 1.8 x 10(-4) mg/mL for saussurenoside, 2.9 x 10(-4) mg/mL for kaempferol, and 6.3 x 10(-4) mg/mL for oleanolic acid, respectively. The method was simple, rapid, and reproducible and could be applied for the determination of quercetin, rutin, saussurenoside, kaempferol, and oleanolic acid in Tibetan medicine Elsholtzia, and the assay results were satisfactory.

Determination of five pharmacologically active compounds extracted from Rhodiola for natural product drug discovery with HPLC-APCI-MS

A rapid and sensitive liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (HPLC-APCI-MS) assay for the determination of five pharmacologically active compounds (PAC) extracted from the traditional Chinese medicine, Rhodiola , namely salidroside, tyrosol, rhodionin, gallic acid, and ethyl gallate has been developed. In this method, PAC could be baseline separated and detected with DAD at 275 nm. The validation of the method, including sensitivity, linearity, repeatability, and recovery, was examined. The linear calibration curves were acquired with correlation coefficient >0.999 and the limits of detection LOD (at a signal-to-noise ratio=3:1) were between 0.058 and 1.500 mu mol/L. It was found, that the amounts of PAC varied with different species of Rhodiola . The established method is rapid and reproducible for the separation of five natural pharmacologically active compounds from extracts of Rhodiola with satisfactory results.

Analysis of five pharmacologically active compounds from Rhodiola for natural product drug discovery with capillary electrophoresis

A rapid capillary electrophoresis method for the separation of five natural pharmacologically active compounds from extracted Rhodiola, namely salidroside, tyrosol, rhodionin, gallic acid and ethyl gallate has been developed. The separation of five natural pharmacologically active compounds was carried out in a fused-silica capillary with 14 mM boric acid, 30 mM SDS and 2.5% acetonitrile, adjusted to pH 10.7 with NaOH. Applied potential was 21 kV. The temperature of the capillary was maintained at 25 degreesC by the instrument thermostating system, with the correlation coefficients of 0.9805-0.9989 for migration time, and relative standards of < 3.52% for peak areas. The established method is rapid and reproducible for the separation of five natural pharmacologically compounds from extracts of Rhodiola with satisfactory results.

Methylxanthines are the psycho-pharmacologically active constituents of chocolate

Rationale: Liking, cravings and addiction for chocolate ("chocoholism") are often explained through the presence of pharmacologically active compounds. However, mere "presence" does not guarantee psycho-activity. Objectives: Two double-blind, placebo-controlled studies measured the effects on cognitive performance and mood of the amounts of cocoa powder and methylxanthines found in a 50 g bar of dark chocolate. Methods: In study 1, participants (n=20) completed a test battery once before and twice after treatment administration. Treatments included 11.6 g cocoa powder and a caffeine and theobromine combination (19 and 250 mg, respectively). Study 2 (n=22) comprised three post-treatment test batteries and investigated the effects of "milk" and "dark" chocolate levels of these methylxanthines. The test battery consisted of a long duration simple reaction time task, a rapid visual information processing task, and a mood questionnaire. Results: Identical improvements on the mood construct "energetic arousal" and cognitive function were found for cocoa powder and the caffeine+theobromine combination versus placebo. In chocolate, both "milk chocolate" and "dark chocolate" methylxanthine doses improved cognitive function compared with "white chocolate". The effects of white chocolate did not differ significantly from those of water. Conclusion: A normal portion of chocolate exhibits psychopharmacological activity. The identical profile of effects exerted by cocoa powder and its methylxanthine constituents shows this activity to be confined to the combination of caffeine and theobromine. Methylxanthines may contribute to the popularity of chocolate; however, other attributes are probably much more important in determining chocolate's special appeal and in explaining related self-reports of chocolate cravings and "chocoholism".

Capillary electrophoretic analysis of pharmacologically active xanthone compounds from Swertia przewalskii pissjauk extract

Pharmacologically active xanthone compounds isolated from Swertia przewalskii pissjauk were well separated by capillary electrophoresis (CE) within 5 min, using a running buffer of 25 mM disodium tetraborate at pH 9.0. Quantitative determination was shown to be possible because regression equations revealed a linear relationship between the peak area of each constituent and its concentration, with correlation coefficients of 0.9972-0.9994. The relative standard deviations were between 0.44%-0.73% for migration times and 2.52%-4.28% for peak areas. The dissociation constant of 1,7-O-beta-D-glucopyranosyl-8-hydroxy-3,7-dimethoxyxanthone, 1,8-dihydroxy-3, 7-dimethoxy-xanthone and 1,7-dihydroxy-3,8-dimethoxyxanthone were also measured by the CE method, giving a value of 9.04, 8.94, and 8.59, respectively.

Screening and analysis of biologically active compounds in Angelica sinensis by molecular biochromatography

A novel strategy for the screening and analysis of biologically active compounds in traditional Chinese medicine by molecular biochromatography is proposed. Molecular biochromatography with human serum albumin (HSA) immobilized on silica as stationary phase was used to screen and analyse the bioactive compounds in the typical Chinese medicine of Angelica sinensis (Oliv.) Diels. Ten peaks showed retention on this column, which is based on their affinity for HSA. Ferulic acid and liguistilide were identified as the principal active components, which agrees very well with the results in the literature. A quality control method was also developed based on the simultaneous determination the concentrations of ferulic acid and liguistilide in solutions of Angelica sinensis (Oliv.) Diels extracted with water and methanol. It was observed that the concentrations of ferulic acid and liguistilide in solution extracted with methanol were 2 and 53 times higher, respectively, than those with water. It was shown that molecular biochromatography is an effective way of analysing and screening biologically active compounds in traditional Chinese medicine.

Synthetic approaches to syn-diol containing biologically active compounds /

The deoxy derivative of pancratistatin 1.10 was prepared in good yield through the use of a [4+2] Diels-Alder cycloaddition and Bischler-Napieralski cyclization approach. The Bischler-Napieralski cyclization was shown to yield two additional side products 2.9, 2.10, however, under slightly modified hydrolysis conditions, the tetracyclic product 2.11 was obtained exclusively in greater than 84% yield. Initial screening of the di-hydroxylatgd derivative, and the other complementary pair analogue 1.10' previously prepared in our laboratories gave interesting results. Both of these compounds were shown to exhibit cytostatic activity; the mono-alcohol was marginally active while the di-hydroxylated analogue proved to be more potent although one to two magnitudes less potent than pancratistatin itself Human tumour cell line assay results indicated that the di-hydroxylated derivative exhibited selective cytotoxic inhibition in the following cell lines: non-small cell lung cancer line NCI-H226 (ED50 - 0.65 ^g/mL), leukemia cell lines CCRF-CEM (ED30 = 0.55 Hg/mL) and HL-60(TB) (ED50 = 0.89^ig/mL). Our results demonstrated that the pharmacophore is not a mono-alcohol, and that the minimum pharmacophore contains the hydroxyl group at the C4 position in addition to either, or both, of the hydroxyl groups present at C2 and C3.' The minimum pharmacophore has been narrowed to only three possibilities which are current synthetic targets in several research groups. The controlled Grignard addition to the tartaric acid derived bis-Weinreb amide 1.25 afforded a direct entry to a host of 1,4-diflferentiated tartaric acid derived intermediates (2.12-2.18). This potentially usefiil methodology was demonstrated through the efficient synthesis of the naturally occurring lactone 2.23, which bears the inherent syn-dio\ subunit. Based on this result, a similar approach to the synthesis of syn-dio\ bearing natural products looks very promising? A direct 2,3-diol desymmetrization method using TIPS-triflate was shown to be effective on the selective differentiation of Z,-methyl tartrate (and diisopropyl tartrate). The mono-silyl-protected intermediates 2.31 also proved to be useful when they were selectively differentiated at the 1,4-carboxyl position (2.35, 2.36) through the use of a borohydride reducing agent. Furthermore, the mono-silyl-protected derivative underwent periodate cleavage affording two synthetically useful a,P-unsaturated esters 2.43, 2.44, with one of esters being obtained via a silyl-migration method.''

Comparison of two extraction methods for evaluation of volatile constituents patterns in commercial whiskeys: Elucidation of the main odour-active compounds

An analytical procedure based on manual dynamic headspace solid-phase microextraction (HS-SPME) method and the conventional extraction method by liquid–liquid extraction (LLE), were compared for their effectiveness in the extraction and quantification of volatile compounds from commercial whiskey samples. Seven extraction solvents covering a wide range of polarities and two SPME fibres coatings, has been evaluated. The highest amounts extracted, were achieved using dichloromethane (CH2Cl2) by LLE method (LLECH2Cl2)(LLECH2Cl2) and using a CAR/PDMS fibre (SPMECAR/PDMS) in HS-SPME. Each method was used to determine the responses of 25 analytes from whiskeys and calibration standards, in order to provide sensitivity comparisons between the two methods. Calibration curves were established in a synthetic whiskey and linear correlation coefficient (r ) were greater than 0.9929 for LLECH2Cl2LLECH2Cl2 and 0.9935 for SPMECAR/PDMS, for all target compounds. Recoveries greater than 80% were achieved. For most compounds, precision (expressed by relative standard deviation, R.S.D.) are very good, with R.S.D. values lower than 14.78% for HS-SPME method and than 19.42% for LLE method. The detection limits ranged from 0.13 to 19.03 μg L−1 for SPME procedure and from 0.50 to 12.48 μg L−1 for LLE. A tentative study to estimate the contribution of a specific compound to the aroma of a whiskey, on the basis of their odour activity values (OAV) was made. Ethyl octanoate followed by isoamyl acetate and isobutyl alcohol, were found the most potent odour-active compounds.

Microwave-Mediated Hetero Diels-Alder reaction: Synthesis of biologically active compounds

Heterocyclic compounds represent almost two-thirds of all the known organic compounds: they are widely distributed in nature and play a key role in a huge number of biologically important molecules including some of the most significant for human beings. A powerful tool for the synthesis of such compounds is the hetero Diels-Alder reaction (HDA), that involve a [4+2] cycloaddition reaction between heterodienes and suitable dienophiles. Among heterodienes to be used in such six-membered heterocyclic construction strategy, 3-trialkylsilyloxy-2-aza-1,3-dienes (Fig 1) has been demonstrated particularly attractive. In this thesis work, HDA reactions between 2-azadienes and carbonylic and/or olefinic dienophiles, are described. Moreover, substitution of conventional heating by the corresponding dielectric heating as been explored in the frame of Microwave-Assisted-Organic-Synthesis (MAOS) which constitutes an up-to-grade research field of great interest both from an academic and industrial point of view. Reaction of the azadiene 1 (Fig 1) will be described using as dienophiles carbonyl compounds as aldehyde and ketones. The six-membered adducts thus obtained (Scheme 1) have been elaborated to biologically active compounds like 1,3-aminols which constitutes the scaffold for a wide range of drugs (Prozac®, Duloxetine, Venlafaxine) with large applications in the treatment of severe diseases of nervous central system (NCS). Scheme 1 The reaction provides the formation of three new stereogenic centres (C-2; C-5; C-6). The diastereoselective outcome of these reactions has been deeply investigated by the use of various combination of achiral and chiral azadienes and aliphatic, aromatic or heteroaromatic aldehydes. The same approach, basically, has been used in the synthesis of piperidin-2-one scaffold substituting the carbonyl dienophile with an electron poor olefin. Scheme 2 As a matter of fact, this scaffold is present in a very large number of natural substances and, more interesting, is a required scaffold for an huge variety of biologically active compounds. Activated olefins bearing one or two sulfone groups, were choose as dienophiles both for the intrinsic characteristic flexibility of the “sulfone group” which may be easily removed or elaborated to more complex decorations of the heterocyclic ring, and for the electron poor property of this dienophiles which makes the resulting HDA reaction of the type “normal electron demand”. Synthesis of natural compounds like racemic (±)-Anabasine (alkaloid of Tobacco’s leaves) and (R)- and (S)-Conhydrine (alkaloid of Conium Maculatum’s seeds and leaves) and its congeners, are described (Fig 2).

Stereoselective synthesis of heterocycles as intermediates of biologically active compounds

The aim of this thesis was to investigate the synthesis of enantiomerically enriched heterocycles and dehydro-β-amino acid derivatives which can be used as scaffolds or intermediates of biologically active compounds, in particular as novel αvβ3 and α5β1 integrin ligands. The starting materials of all the compounds here synthesized are alkylideneacetoacetates. Alkylidene derivates are very usefull compounds, they are usually used as unsaturated electrophiles and they have the advantage of introducing different kind of functionality that may be further elaborated. In chapter 1, regio- and stereoselective allylic amination of pure carbonates is presented. The reaction proceeds via uncatalyzed or palladium-catalyzed conditions and affords enantiopure dehydro-β-amino esters that are useful precursor of biologically active compounds. Chapter 2 illustrates the synthesis of substituted isoxazolidines and isoxazolines via Michael addition followed by intramolecular hemiketalisation. The investigation on the effect of the Lewis acid catalysis on the regioselectivity of the addition it also reported. Isoxazolidines and isoxazolines are interesting heterocyclic compounds that may be regarded as unusual constrained -amino acids or as furanose mimetics. The synthesis of unusual cyclic amino acids precursors, that may be envisaged as proline analogues, as scaffolds for the design of bioactive peptidomimetics is presented in chapter 3. The synthesis of 2-substituted-3,4-dehydropyrrole derivatives starting from allylic carbonates via a two step allylic amination/ring closing metathesis (RCM) protocol is carried out. The reaction was optimized by testing different Grubbs’ catalysts and carbamate nitrogen protecting groups. Moreover, in view of a future application of these dehydro-β-amino acids as central core of peptidomimetics , the malonate chain was also used to protect nitrogen prior to RCM. Finally, chapter 4 presents the synthesis of two novel different classes of integrin antagonists, one derived from dehydro-β-amino acid prepared as described in chapter 1 and the other one has isoxazolidines synthesized in chapter 2 as rigid constrained core. Since that these compounds are promising RGD mimetics for αvβ3 and α5β1 integrins, they have been submitted to biological assay. and to interpret on a molecular basis their different affinities for the αvβ3 receptor, docking studies were performed using Glide program.

Original analytical methods for the determination of psychoactive compounds in complex matrices

This thesis work aims to develop original analytical methods for the determination of drugs with a potential for abuse, for the analysis of substances used in the pharmacological treatment of drug addiction in biological samples and for the monitoring of potentially toxic compounds added to street drugs. In fact reliable analytical techniques can play an important role in this setting. They can be employed to reveal drug intake, allowing the identification of drug users and to assess drug blood levels, assisting physicians in the management of the treatment. Pharmacological therapy needs to be carefully monitored indeed in order to optimize the dose scheduling according to the specific needs of the patient and to discourage improper use of the medication. In particular, different methods have been developed for the detection of gamma-hydroxybutiric acid (GHB), prescribed for the treatment of alcohol addiction, of glucocorticoids, one of the most abused pharmaceutical class to enhance sport performance and of adulterants, pharmacologically active compounds added to illicit drugs for recreational purposes. All the presented methods are based on capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) coupled to various detectors (diode array detector, mass spectrometer). Biological samples pre-treatment was carried out using different extraction techniques, liquid-liquid extraction (LLE) and solid phase extraction (SPE). Different matrices have been considered: human plasma, dried blood spots, human urine, simulated street drugs. These developed analytical methods are individually described and discussed in this thesis work.