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PubMed | CAMAG Laboratory, University of Geneva, National and Kapodistrian University of Athens and University of Innsbruck
Type: Journal Article | Journal: Planta medica | Year: 2015

A simple and rapid high-performance thin-layer chromatography-based autographic assay was established to screen plant extracts for the presence of tyrosinase-inhibiting substances. Three mobile phases were selected for the chromatographic separation of different types of extracts. After development, the plate was sprayed with the substrate solution Levodopa followed by a solution of the enzyme tyrosinase. Several known tyrosinase inhibitors were tested simultaneously as positive controls. They were detected as white spots with white light in remission from the plate as well as with white light transmitted through the plate. Some of the investigated extracts included spots showing a different behaviour; some lipophilic substances appeared as white spots in white light remission but were black in white light transmission. This behaviour, which could lead to false-positive results, was due to poor wettability of the corresponding spots. False-positive results were eliminated by adding Triton X-100 to the Levodopa solution and drying the plate after 10 minutes incubation with a molecular sieve. Tyrosinase inhibitors can be clearly identified as white spots against a dark background in white light remission as well as in white light transmitted through the plate. The established high-performance thin-layer chromatography autographic assay was validated and can be used as a standard method for the detection of tyrosinase inhibitors in plant extracts without causing false-positive results.


Li Z.,Albert Ludwigs University of Freiburg | Merfort I.,Albert Ludwigs University of Freiburg | Reich E.,CAMAG Laboratory
Journal of AOAC International | Year: 2010

Due to their complexity, multicomponent herbal drugs pose enormous analytical challenges for quality control (QC). Although they may have traditionally been used for hundreds of years, the information about their chemical composition is often still limited. Selecting suitable markers to monitor the identity and potency of the mixture is, therefore, difficult. There is also the possibility of natural variability for each plant. This paper illustrates a pragmatic and practical approach to QC of a multicomponent herbal drug by HPTLC. Cangzhu Xianglian San (CXS), composed of the herbal drugs Coptis rhizome, Aucklandia root, and Atractylodes rhizome (30 + 20 + 60, w/w/w), is used as an example. A characteristic fingerprint can be generated for CXS with toluene-ethyl acetate-methanol-isopropanol-water (60 + 30 + 20 + 15 + 3, v/v/v/v/v) mobile phase on HPTLC silica gel 60 conditioned with ammonia. While the corresponding monograph of the Chinese Veterinary Pharmacopoeia focuses only on the detection of berberine, one of the principal components of Coptis rhizome, the proposed method of identification determines the presence of all three components in the drug after derivatization with anisaldehyde reagent. The same method can also be used to quantitatively determine the content of berberine by scanning densitometry. This paper provides details about the validation of the qualitative and quantitative determinations.


Walworth M.J.,Oak Ridge National Laboratory | Walworth M.J.,University of Tennessee at Knoxville | Stankovich J.J.,Oak Ridge National Laboratory | Van Berkel G.J.,Oak Ridge National Laboratory | And 4 more authors.
Analytical Chemistry | Year: 2011

An aerosol application procedure involving one or more commercially available silicone-based products was developed to create hydrophobic surfaces that enable analysis of otherwise wettable, absorbent surfaces using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system. The treatment process resulted in a hydrophobic surface that enabled formation of the requisite probe-to-surface liquid microjunction for sampling and allowed efficient extraction of the analytes from the surface, but did not contribute significant chemical background in the mass spectra. The utility of this treatment process was demonstrated with the treatment of wettable high-performance thin layer chromatography plates, post-plate development, and their subsequent analysis with the sampling probe. The surface treatment process for different surface types was described and explained and the effectiveness of the treatment and subsequent analysis was illustrated using alkaloids from goldenseal (Hydrastis canadensis) root separated on a normal phase silica gel 60 F254S plate and peptides from protein tryptic digests separated on a ProteoChrom HPTLC Silica gel 60 F254S plate and a ProteoChrom HPTLC Cellulose sheet. This simple surface treatment process significantly expands the analytical surfaces that can be analyzed with the liquid microjunction surface sampling probe, and therefore, also expands the analytical utility of this liquid extraction based surface sampling approach. © 2010 American Chemical Society.


Rueda D.C.,University of Basel | Zaugg J.,University of Basel | Quitschau M.,University of Basel | Reich E.,CAMAG Laboratory | And 2 more authors.
Planta Medica | Year: 2012

In a twomicroelectrode voltage clamp assay using Xenopus laevis oocytes, a petroleum ether extract prepared from a commercial sample of the traditional Chinese herbal drug labelled as Chaihu (Bupleurum chinense DC. roots) enhanced the Iby 156% ±22% when tested at 100μg/mL. By means of HPLC-based activity profiling combined with high-resolution LC-MS and microprobe NMR, the germacranolide aristolactone (1) was identified as one of the main active compounds (EC56.02μM±5.09μM). However, aristolactone has been previously reported only from the genus Aristolochia (Aristolochiaceae), suggesting a possible adulteration. With the aid of a validated HPTLC protocol for detection of aristolochic acids and with reference samples, the commercial sample was confirmed to be a mixture of Aristolochia manshuriensis root and Bupleurum chinense root. This finding was corroborated by macroscopic inspection of the drug. This case of adulteration with a highly nephrotoxic drug raises concerns about adequate quality control of TCM drugs commercialized in Europe. © Georg Thieme Verlag KG Stuttgart - New York.


Adhami H.-R.,University of Vienna | Scherer U.,Northwestern University | Kaehlig H.,University of Vienna | Hettich T.,Northwestern University | And 3 more authors.
Phytochemical Analysis | Year: 2013

Introduction In the search for new natural compounds with acetylcholinesterase (AChE) inhibitory activity this study focused on galbanum, the oleo gum-resin from Ferula gummosa Boiss., which had shown AChE inhibitory activity in a screening. Objective The isolation of bioactive compounds from plant extracts usually is laborious and time consuming. In an approach to accelerate the characterisation of compounds with AChE inhibitory activity, the potential of a combination of HPTLC bioautography with HPTLC-MS/NMR for the fast identification of active compounds in galbanum was studied. Method Pre-fractionation of the dichloromethane extract was performed by vacuum liquid chromatography. The resulting fractions were separated by HPTLC and active zones determined by bioautography. A TLC-MS interface was used to elute the single zones from the plates directly into a mass spectrometer. The interface was also used to extract the two major active zones from HPTLC plates for off-line one- and two-dimensional NMR and quadrupole time of flight (QTOF) MS. Results The isolated compounds were identified as 7-{[(2E)-3,7-dimethylocta-2,6-dien-1-yl] oxy}-2H-chromen-2-one (auraptene) and 7-{[(1R,4aR,6S,8aS)-6-hydroxy-5,5,8a- trimethyl-2-methylenedecahydronaphthalen-1-yl]methoxy}-2H-chromen-2-one (farnesiferol A). This is the first report of these substances in F. gummosa. Their median inhibitory concentration (IC50) values for AChE inhibition were determined as 47 and 17 μg/mL in comparison with physostigmine as a positive control (IC50: 0.8 μg/mL) and their concentrations in galbanum were quantified by HPLC as 3.5% and 7.9%, respectively. Conclusion The study showed that HPTLC-MS/NMR can be considered as a fast and high-confidence method for dereplication of natural compounds. From the correlation of the concentration of the elucidated compounds and their IC50 values for AChE inhibition it can be concluded that auraptene and farnesiferol A are contributing to this activity of galbanum. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.


Senol F.S.,Gazi University | Ankli A.,CAMAG Laboratory | Reich E.,CAMAG Laboratory | Orhan I.E.,Gazi University
Food Technology and Biotechnology | Year: 2016

Inhibitory activity of thirty-one ethanol extracts obtained from albedo, flavedo, seed and leaf parts of 17 cultivars of Citrus species from Turkey, the bark and leaves of Olea europaea L. from two locations (Turkey and Cyprus) as well as caffeic acid and hesperidin was tested against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), related to the pathogenesis of Alzheimer's disease, using ELISA microtiter assays at 500 μg/mL. Metal-chelating capacity of the extracts was also determined. BChE inhibitory effect of the Citrus sp. extracts was from (7.7±0.7) to (70.3±1.1) %, whereas they did not show any inhibition against AChE. Cholinesterase inhibitory activity of the leaf and bark ethanol extracts of O. europaea was very weak ((10.2±3.1) to (15.0±2.3) %). The extracts had either no or low metal-chelating capacity at 500 μg/mL. HPTLC fingerprinting of the extracts, which indicated a similar phytochemical patt ern, was also done using the standards of caffeic acid and hesperidin with weak cholinesterase inhibition. Among the screened extracts, the albedo extract of C. limon 'Interdonato', the flavedo extracts of 'Kara Limon' and 'Cyprus' cultivars and the seed extract of C. maxima appear to be promising as natural BChE inhibitors.


PubMed | CAMAG Laboratory and Gazi University
Type: Journal Article | Journal: Food technology and biotechnology | Year: 2016

Inhibitory activity of thirty-one ethanol extracts obtained from albedo, flavedo, seed and leaf parts of 17 cultivars of


Kaale E.,Muhimbili University of Health and Allied Sciences | Risha P.,Muhimbili University of Health and Allied Sciences | Reich E.,CAMAG Laboratory | Layloff T.P.,Management science for Health
Journal of AOAC International | Year: 2010

Two laboratories extensively investigated the use of HPTLC to perform assays on lamivudine-zidovudine, metronidazole, nevirapine, and quinine composite samples. To minimize the effects of differences in analysts' technique, the laboratories conducted the study with automatic sample application devices in conjunction with variable-wavelength scanning densitometers to evaluate the plates. The HPTLC procedures used relatively innocuous, inexpensive, and readily available chromatography solvents used in the Kenyon or the Global Pharma Health Fund Minilabs® TLC methods. The use of automatic sample applications in conjunction with variable- wavelength scanning densitometry demonstrated an average repeatability or within-laboratory RSD of 1.90%, with 73% less than 2% and 97% at 2.60% or less, and an average reproducibility or among-laboratory RSD of 2.74%.


Do T.T.K.,CAMAG Laboratory | Theocharis G.,CAMAG Laboratory | Reich E.,CAMAG Laboratory
Journal of AOAC International | Year: 2015

An HPTLC method is proposed to permit effective screening for the presence of three phosphodiesterase type 5 inhibitors (PDE5-Is; sildenafil, vardenafil, and tadalafil) and eight of their analogs (hydroxyacetildenafil, homosildenafil, thiohomosildenafil, acetildenafil, acetaminotadalafil, propoxyphenyl hydroxyhomosildenafil, hydroxyhomosildenafil, and hydroxythiohomosildenafil) in finished products, including tablets, capsules, chocolate, instant coffee, syrup, and chewing gum. For all the finished products, the same simple sample preparation may be applied: ultrasound-assisted extraction in 10 mL methanol for 30 min followed by centrifugation. The Rf values of individual HPTLC bands afford preliminary identification of potential PDE5-Is. Scanning densitometry capabilities enable comparison of the unknown UV spectra with those of known standard compounds and allow further structural insight. Mass spectrometric analysis of the material derived from individual zones supplies an additional degree of confidence. Significantly, the proposed screening technique allows focus on the already known PDE5 Is and provides a platform for isolation and chemical categorization of the newly-synthesized analogs. Furthermore, the scope could be expanded to other therapeutic categories (e.g., analgesics, antidiabetics, and anorexiants) that are occasionally coadulterated along with the PDE5-Is. The method was successfully applied to screening of 45 commercial lifestyle products. Of those, 31 products tested positive for at least one illegal component (sildenafil, tadalafil, propoxyphenyl hydroxyhomosildenafil, or dimethylsildenafil).


PubMed | CAMAG Laboratory, ISA University, CIBLOT, Montpellier University and Université des Sciences et Techniques de Masuku
Type: | Journal: BMC complementary and alternative medicine | Year: 2016

Diabetes mellitus is a metabolic disorder which is rising globally in rich and developing countries. In the African region this rate is the highest, with 20 million diagnosed diabetics. Despite a noticeable progress in the treatment of diabetes mellitus by synthetic drugs, the search for new natural anti-diabetic agents is going on. Nauclea diderrichii (De Wild.) Merr. (ND) and Sarcocephalus pobeguinii Hua ex Pellegr. (SP) are used as traditional medicines in Gabon for the treatment of different diseases, especially in the case of diabetes. The aim of this study was to evaluate the antidiabetic potential of these two medicinal plants traditionally used in Gabon.Pharmacological (inhibitory action on and -glucosidases) and toxicological (effect on human T cell proliferation) studies were conducted on aqueous extracts of ND (leaves and bark) and SP (bark) collected in Gabon. All raw extracts were analyzed by HPTLC and their content in phenolic compounds was determined by using standard method. The most active extracts were submitted to preparative HPLC in order to evidence the most efficient subfractions by biological evaluation.The results showed that two extracts from ND were potent -glucosidase inhibitors, the leaf extract being more active that the bark extract: the first one was more than 60 fold more active than Acarbose, which is an oral medication used to treat type 2 diabetes; the extract from SP bark was less efficient. The HPLC subfractions of the extracts of ND leaves and SP bark were tested in the same experimental conditions. In each case, the most active subfractions still show very potent inhibitory effect on -glucosidase (80-90% inhibition at 0.1 mg/mL). The most efficient extract, from ND leaves, was also characterized by the highest percentage of phenolic compounds, which suggests a relationship between its inhibitory potential on -glucosidase and its content in phenolic compounds. Conversely, only a moderate inhibitory activity of the three extracts was observed on -glucosidase.These results clearly indicated that active compounds present in N. diderrichii and S. pobeguinii leaves or/and bark were selective and highly potent inhibitors of -glucosidase and validate their popular use for the treatment of diabetes.

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