TLC Bioautographic Assay Research Articles

Extensive phytochemical investigation of the polar constituents of Diospyros bipindensis Gürke traditionally used by Baka pygmies

The water maceration and methanolic extract of the stem barks of Diospyros bipindensis, which is a medicinal plant used in Cameroon by Baka pygmies, revealed a complex high-performance liquid chromatography (HPLC) profile primarily composed of coumarin and naphthoquinone glycosides. The methanolic and apolar extracts also exhibited significant antifungal activity on a TLC bioautography assay against Candida albicans. HPLC-microfractionation in 96-well plates combined with bioautography enabled the rapid localization of the antifungal compound that was identified by HPLC-PDA and HPLC-MS analysis as plumbagin. These on-line structural information were also used to dereplicate four known compounds. The isolation of the polar constituents from the methanolic extract enabled the identification of eleven other compounds also present in the traditional preparation, nine of which are reported for the first time. The structures of those compounds were elucidated by UV, NMR and HR-MS analysis.

Acetylcholinesterase inhibitors from Tetrapterys mucronata

In our search for acetylcholinesterase (AChE) inhibitors, the investigation of Tetrapterys mucronata Cav. (Malpighiaceae), a plant used in Brazil for its psychotropic properties, was pursued. The methanolic extract of the bark of T. mucronata presented an interesting AChE inhibition in a TLC bioautographic assay. In order to isolate the active compounds, bioassay-guided isolation was undertaken using HPLC-microfractionation in 96 well plates and agar-overlay bioautography to localize the active compounds. The analytical HPLC-PAD conditions were geometrically transferred to a preparative medium pressure chromatography (MPLC-UV) by chromatographic calculations for the efficient isolation of the active compounds at the milligram scale in one step. Using this approach, 22 compounds were isolated, six of them being new natural products, such as 5-hydroxy-2-(2,3-dihydroxy-1-(1 H-indol-3-yl)propyl)-1 H-indole-3-carboxamide and 3-(2-(dimethylamino)ethyl)-2-(2,3-dihydroxy-1-(1 H-indol-3-yl)propyl)-1 H-indol-5-ol. The structure of the isolated compounds was elucidated by classical spectroscopic methods including UV, 2D NMR and HR-MS and chemical derivatisation. Some of the isolated compounds presented a strong AChE inhibition on the bioautographic assay; however those compounds showed weak activity when it was measured in solution.

TLC-Bioautographic Evaluation of In Vitro Anti-tyrosinase and Anti-cholinesterase Potentials of Sandalwood Oil

Sandalwood oil, rich in sesquiterpenoid alcohols, has been used in traditional medicinal systems as a relaxant and coolant. Besides, sandalwood oil is used as an ingredient in numerous skin fairness enhancing cosmetics. However, there is no available information on biological activities that relate to the above applications. Hence, the anti-tyrosinase and anti-cholinesterase potentials of sandalwood oil were probed by both TLC-bioautographic and colorimetric methods. Results obtained from colorimetric assays indicated that sandalwood oil is a potent inhibitor of tyrosinase (IC50 = 171 microg mL(-1)) and cholinesterases (IC50 = 4.8-58 microg mL(-1)), in comparison with the positive controls used in the assays, kojic acid and physostigmine, respectively. The TLC-bioautographic assays indicated that alpha-santalol, the major constituent of the oil, is a strong inhibitor of both tyrosinase and cholinesterase. These in vitro results indicate that there is a great potential of this essential oil for use in the treatment of Alzheimer's disease, as well as in skin-care.

Evaluation of antimicrobial and phytochemical screening of Fennel, Juniper and Kalonji essential oils against multi drug resistant clinical isolates

ObjectiveThe inhibitory effects of essential oils including fennel, juniper and kalonji from Foeniculum Vulgare, Juniperus Osteosperma and Nigella Sativa on multi drug resistant clinical isolates were investigated. All the oils have been evaluated for phytochemical constituents, antibacterial activity and TLC bioautography assay. MethodsPreliminary phytochemical analysis was performed. The antibacterial potential of essential oils from fennel, juniper and kalonji fennel, juniper and kalonji was evaluated by agar well diffusion method against multi drug resistant clinical isolates. The antibacterial effect was investigated using the TLC-bioautographic method. ResultsPreliminary phytochemical analysis demonstrated the presence of most of the phytochemicals including saponins, cardiac glycosides, steroids, terpenoids, flavonoids and tannins. Antibacterial activity of essential oils was assessed on eight multi-drug resistant (MDR) clinical isolates from both Gram-positive and Gram-negative bacteria and two standard strains. All the oils tested showed significant to moderate antibacterial activity toward all tested strains except Acinetobacter sp and Staphylococcus aureus MRSA. The maximum zone of inhibition was found to be 25±0.12 mm for juniper oil followed by 21±0.085 mm for kalonji oil against Staphylococcus aureus 2. Thin layer chromatography and bioautography assay demonstrated well-defined growth inhibition zones against Staphylococcus aureus 2 and E. coli for juniper essential oil in correspondence with tannins observed at Rf values of 0.07 and 0.57. ConclusionsBased on the present study, the essential oils from juniper and kalonji possess antibacterial activity against several multi drug resistant pathogenic bacteria and thus can be used as a base for the development of new potent drugs and phytomedicine.

TLC Bioautographic Method for Detecting Lipase Inhibitors

Bioautographic assays using TLC play an important role in the search for active compounds from plants. A TLC bioautographic assay has previously been established for the detection of acetylcholinesterase inhibitors but not for lipases. Development of a TLC bioautographic method for detecting lipase inhibitors in plant extracts. After migration of the plant extracts, the TLC plate was sprayed with α-naphtyl acetate and enzyme solutions before incubation at 37°C for 20 min. Finally, the solution of Fast Blue B salt was sprayed onto the TLC plate giving a purple background colouration. Lipase inhibitors were visualised as white spots on the TLC plates. Orlistat (a known lipase inhibitor) inhibited lipase down to 0.01 µg. Methanolic extracts of Camellia sinensis (L.) kuntz and Rosmarinus officinalis L after migration on TLC gave enzymatic inhibition when applied in amounts of 82 and 56 µg, respectively. On the other hand the methanolic extract of Morus alba leaves did not exhibit any lipase inhibitory activity. The screening test was able to detect lipase inhibition by pure reference substances and by compounds present in complex matrices, such as plant extracts.

Acetylcholinesterase inhibitory activity of the total alkaloid from traditional Chinese herbal medicine for treating Alzheimer’s disease

Alzheimer’s disease (AD) is a progressive degenerative neurologic disorder resulting in impaired memory and behavior. A promising treatment strategy for AD has been the use of acetylcholinesterase (AChE) inhibitors. Several potent AChE inhibitors such as huperzine and galanthamine were originally isolated from plants, indicating that herbal medicines are a good source for the search of potential AChE inhibitors. In our study, to search for potential AChE inhibitors, the total alkaloidal extracts of 31 Chinese herbal medicines were tested for their AChE inhibitory activities by Ellman’s method and modified TLC bioautographic assay. The results showed that the alkaloidal extracts of Uncaria rhynchophylla (Rubiaceae), Nelumbo nucifera (Nymphaeaceae), Zanthoxylum nitidum (Rutaceae), Portulaca oleracea (Portulacaceae), and Pinellia ternata (Araceae) exhibited remarkable AChE inhibitory activities at the final concentration of 100 μg/ml. Their IC50 values were 10.8, 12.2, 17.4, 29.4, and 56.2 μg/ml, respectively. The rest of the alkaloidal extracts showed no or very weak AChE inhibitory activities at the same concentration. The results of this study indicate that the screening of traditional Chinese herbs for AChE inhibitory activity may provide useful lead compounds in the discovery of new drugs for the treatment of AD.

Skimmianine, a furoquinoline alkaloid from Zanthoxylum nitidum as a potential acetylcholinesterase inhibitor

Skimmianine (1), a newly discovered strong acetylcholinesterase (AChE) inhibitor, along with nine weakly or no active compounds, toddalolactone (2), dictamnine (3), γ-fagarine (4), magnolone (5), (−)-(S)-edulinine (6), zanthodioline (7), edulitine (8), 5,6,7-trimethoxycoumarin (9), and haplopine (10) have been isolated from Zanthoxylum nitidum (Z. nitidum). Skimmianine (1) inhibited 50% of AChE activity at the concentrations of 8.6 ± 0.7 μg/ml when the IC50 value of Physostigmine as a standard was 0.013 ± 0.002 μg/ml. Antiacetylcholinesterase activity of skimmianine (1) was also supported by TLC bioautographic assay. The structure activity relationship on the anti-acetylcholinesterase activity of the quinoline moiety is also discussed in this article.

Screening of Medicinal Plants from Iranian Traditional Medicine for Acetylcholinesterase Inhibition

To find new herbal compounds with an acetylcholinesterase (AChE) inhibitory effect, this study focused on herbal drugs and resins which have been used in Iranian traditional medicine for the treatment of cognitive disorders. Forty drugs were selected from authoritative written documents of Iranian traditional medicine. Each drug was extracted by accelerated solvent extraction using dichloromethane followed by methanol. The 80 extracts were screened for AChE inhibitory activity by a TLC bioautography method. The inhibiting effect of the 32 most active extracts was measured by a microplate colorimetric assay. Due to the best activity, the seeds of Peganum harmala L. were investigated in detail. From the TLC bioautography assay the alkaloids harmaline and harmine were identified as active compounds. This result was confirmed by means of HPLC-DAD. The IC(50) values were 41.2 μg/mL for the methanol extract, 95.5 μg/mL for the dichloromethane extract, 8.4 μg/mL for harmaline and 10.9 μg/mL for harmine. The concentrations of active compounds in the extracts were determined by a fast and precise HPLC method. As the amounts of harmaline and harmine in the extracts were correlated with the IC(50) values of the extracts, it can be concluded that these two alkaloids are responsible for the AChE inhibitory activity of P. harmala.

Chemical fingerprinting and bioactivity of Amazonian Ecuador Croton lechleri Müll. Arg. (Euphorbiaceae) stem bark essential oil: A new functional food ingredient?

Croton lechleri essential oil has been obtained by steam distillation of fresh stem bark from Amazonian Ecuador adult plants (yield: 0.61ml/kg [0.061%]; density: 1.01g/ml), and then chemically characterised by GC (Gas Chromatography) and GC–MS (gas chromatography–mass spectrometry). Seventy-four chemicals were detected and identified; the most abundant in descending order, were the sesquiterpenes sesquicineole (17.29%), α-calacorene (11.29%), 1,10-di-epi-cubenol (4.75%), β-calacorene (4.34%) and epi-cedrol (4.09%). Monoterpenes checked with a relative peak area higher than 2.0% were α-pinene (2.01%), p-cymene (2.61%), limonene (4.20%) and borneol (2.67%). The structure of the main chemicals were confirmed by GC–MS and 1H NMR analyses. Spectrophotometric 1,1-diphenyl-2-picrylhydrazyl (DPPH) and DPPH-(high performance) thin layer chromatography (DPPH-(HP)TLC) bioautographic assays showed a lower radical scavenging capacity (IC50) with respect to commercial thyme essential oil and BHA (butylated hydroxyl anisole), pointing out, however, that the C. lechleri essential oil fraction, characterised by α-calacorene, β-calacorene and δ-cadalene, was the most involved in the bioactivity. Similar results were obtained with β-carotene bleaching assay, where the IC50 values were 0.291±0.024mg/ml for C. lechleri essential oil, 0.164±0.013 and 1.34×10−4±10−5mg/ml for thyme essential oil and BHA, respectively. (HP)TLC-bioautographic assay performed with Gram positive and Gram negative bacteria revealed a minimum inhibitory concentration (MIC) values comprised between 0.10mg/ml (Escherichia coli) and 10.10mg/ml (for e.g. Pseudomonas aeruginosa), and the fraction mainly characterised by sesquicineole (97.38%) as the most involved in antibacterial capacity. Ames test employing Salmonella typhimurium TA98 and TA100 with and without a metabolic activation mixture (S9 mix) demonstrated the absence of mutagenicity of the C. lechleri essential oil between a concentration range of 10−2 and 100mg/plate. The same results were achieved by Saccharomyces cerevisiae D7 strain assay. An interesting mutagen-protective efficacy was evidenced by a 30% and 33% revertants reduction of TA98 strain treated with 2-aminoanthracene and nitrofluorene (2μg/plate), suggesting, above all, the possibility to employ C. lechleri essential oil as a new flavouring protective ingredient for foods or dietary supplements against potential mutagens formed during cooking and/or processing in general.

Free Radical Scavenging Activity and Characterization of Sesquiterpenoids in Four Species of Curcuma Using a TLC Bioautography Assay and GC-MS Analysis

The sesquiterpenoids are one of major groups of antioxidants in Curcuma besides curcuminoids. However, the real substances contributing to the antioxidant activity are still unknown. In this paper, the antioxidant activity of sesquiterpenoids in four species and two essential oils from Curcuma genus was determined and compared based on TLC separation and DPPH bioautography assay. Their antioxidant capacities were quantitatively evaluated using densitometry with detection at 530 nm (λreference = 800 nm) using vitamin C as reference. The results showed that Curcuma longa rhizomes had the highest antioxidant capacity while C. phaeocaulis presented the lowest one among the four species of Curcuma. Moreover, essential oil of C. wenyujin showed higher antioxidant potential than that of C. longa. The main TLC bands with antioxidant activity of the four species of Curcuma were collected and characterized using GC-MS, and thus curzerene, furanodiene, α-turmerone, β-turmerone and β-sesquiphellandrene were determined as major sesquiterpenoids with antioxidant activity in Curcuma.

Semisynthesis and Acetylcholinesterase Inhibitory Activity of Stemofoline Alkaloids and Analogues

Semisynthesis of the known Stemona alkaloids oxystemofoline (7) and methoxystemofoline (8) has been achieved starting from (11Z)-1',2'-didehydrostemofoline (6), which confirmed their structures and absolute configurations. The synthesis of (1'R)-hydroxystemofoline (9) helped establish this compound as a natural product from Stemona aphylla. (1'S)-Hydroxystemofoline (10) and a number of related analogues were also prepared. In a TLC bioautographic assay, 9 was found to be the most active acetylcholinesterase inhibitor, but it was not as active as galanthamine.

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.

Anticandidal pimaradiene diterpene from Phlomis essential oils

Abstract In the present study, it was aimed to investigate the phytochemical profile and antimicrobial effects of Phlomis lunariifolia Sm., Phlomis amanica Vierh., Phlomis monocephala P.H. Davis, Phlomis sieheana Rech. fil, Phlomis armeniaca Willd. essential oils collected from Turkey. The Phlomis essential oils were obtained from the aerial parts by hydrodistillation and were subsequently analyzed both by gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS). Chromatographic separations followed by structure identification of individual compounds of interest from Phlomis essential oils were conducted using 1D and 2D NMR, FT-IR, UV and HRMS techniques. In addition, antimicrobial studies using a microdilution assay and TLC bioautography were applied to the essential oils and the relevant components. The analysis of the essential oils led to the identification of 143 compounds, where an unknown volatile compound was detected as the major compound (22.8% and 12.7%) in the essential oils of P. amanica and P. monocephala, respectively. After chromatographic clean up, the isolation and characterization of this compound resulted in (−)-8(14),15-isopimaradien-11α-ol. The sesquiterpene germacrene-D was identified as the major constituent of P. lunariifolia (7.7%), P. sieheana (16.6%) and P. armeniaca (23.4%) oils. 4-Methoxycarbonyl-7-methyl cyclopenta[c]pyrane – a fulvoiridoid – was obtained by acid hydrolysis from iridoid ipolamiide which was shown to be present in the oils of P. armeniaca (1.4%) and P. sieheana (0.2%). Furthermore, Phlomis essential oils were investigated for their antifungal properties using a TLC bioautographic assay where the diterpene was shown as the active principle against Candida albicans and Candida tropicalis when compared with standard antifungal agents. Minimum inhibitory concentrations against various human pathogenic bacteria (from 125 to >1000 μg/ml), C. albicans and C. tropicalis (62.5–1000 μg/ml), were determined using a microdilution assay. The results obtained from this study suggest that essential oils and their individual compounds thereof may be potential resource and ingredients for pharmaceuticals or cosmetics with antimicrobial activity.

Antitumor and antifungal activities from red algae Bostrychia radicans and B. tenella (Rhodophyta)

Marine algae have been a rich source of active metabolites with antineoplastic, antimicrobial and antiviral effects [1]. Considering the potential of red algae, the antitumor and antifungal activities were evaluated in Bostrychia radicans (Mont.) Mont. (BRC–shore, BRM–mangrove) and Bostrychia tenella (J.V. Lamour.) J. Agardh (BTC–shore). Investigations were performed using the dichloromethane-methanol (2:1) extracts. These extracts were partitioned yielding hexane, dichloromethane, methanol and aqueous fractions. The antitumor assays were performed using the cell lines HCT-8 (colon); HL-60 (leukemia) and SF-295 (CNS) [2]. The most active fraction, BRC-H (hexane, 100µg/mL), showed inhibition tumor growing of 104.5±0.15% and 36.1±5.0% in HL-60 and HCT-8, respectively, while the hexane fraction obtained from mangrove species did not exhibit any antitumor potential. By means of spectroscopic methods and by comparison with literature data, the major components of BRC-H were determined as: heptadecane, hexadecanoic acid, oleanoic acid, cholesterol, trans-phytol, and others that are being investigated. Also, in a preliminary TLC bioautographic assay with Cladosporium cladosporioides and C. sphaerospermum only the hexane and dichloromethane fractions (200µg) from BRM displayed antifungal activities. However, B. tenella demonstrated interesting antifungal potential from hexane fraction. Also, from BTC initial extract were isolated two phenolic compounds, 1-methoxyphenethyl alcohol and a novel compound, potassium 4-(hydroxymethyl)-benzenosulfonate, but neither showed antifungal property. Thus, these results suggest that red algae are an interesting source of bioactive compounds. Further studies are in progress to determine the IC50 of bioactive fractions and compounds.

Rapid TLC/GC‐MS identification of acetylcholinesterase inhibitors in alkaloid extracts

Alkaloid extracts from 12 plant species of the families Amaryllidaceae, Fumariacae and Papaveraceae were studied with respect to their acetylcholinesterase inhibitory activity and alkaloid patterns. Fifty-three alkaloids were identified by GC-MS, including known acetylcholinesterase (AChE) inhibitors such as galanthamine, epigalanthamine, sanguinine and epinorgalanthamine in extracts of Amaryllidaceae plants and protopine in extracts of Fumariaceae and Papaveraceae plants. The galanthamine-containing extracts of the amaryllidaceous plants were found to be the most active while the extract of Corydalis bulbosa was the most active among the extracts of the tested plants from the Fumariaceae and Papaveraceae plants. TLC bioautographic assay, preparative TLC and GC-MS analysis were combined to identify the active compounds in the studied extracts. Galanthamine was isolated from the known AChE inhibitors in the extracts of Amaryllidaceae plants. Corydaline, bulbocapnine and stylopine were found to be active in the extracts of plant species of the families Fumariaceae and Papaveraceae. Available standards of deshydrocorydaline--a precursor of corydaline, corydaline and stylopine--were tested for AChE inhibitory activity. Deshydrocorydaline and corydaline showed potent inhibitory activity comparable with that of the positive control galanthamine.

New and Bioactive Aromatic Compounds fromZanthoxylum zanthoxyloides

In the course of a study of medicinal plants from Mali, the root bark of Zanthoxylum zanthoxyloides Lam. (Rutaceae) was investigated. The root bark of this plant is used as a toothbrush by West African people. Phytochemical investigation of the MeOH extract of Zanthoxylum zanthoxyloides Lam. (Rutaceae) led to the isolation of different biologically active compounds. The separation of acetylcholinesterase inhibitors was performed by centrifugal partition chromatography (CPC). The fractions were monitored by direct TLC bioautographic assays. The bio-guided isolation led to the isolation of a new peroxide derivative. In addition, LC/UV/MS analysis performed on the crude MeOH extract allowed on-line identification of some known compounds. The structures of the isolated compounds were elucidated by classical spectroscopic methods including UV, NMR, MS and HR-MS.

Flavonoids from the Stem ofEriophorumscheuchzeri

Activity-guided isolation of antifungal, antibacterial, and antioxidant compounds from the dichloromethane extract of the aerial parts of Eriophorum scheuchzeri led to the isolation of eight flavonoids. Three isoflavones and one flavanone are new natural compounds. The isolation of the bioactive compounds was performed by centrifugal partition chromatography. Fractions and pure compounds were monitored by a direct TLC bioautographic assay. Their structures were elucidated by spectroscopic and chemical methods.

Antifungal and antioxidant compounds from the root bark of Fagara zanthoxyloides.

Phytochemical investigation of the CH 2 Cl 2 extract of Fagara zanthoxyloides Lam. (Rutaceae) led to the isolation of eleven compounds. One phenylethanoid derivative is a new natural product. The isolation of the antifungal and the antioxidant compounds was monitored by direct TLC bioautographic assays. The structures of the isolated compounds were elucidated by classical spectroscopic methods including UV, NMR, MS and HR-MS.

TWO NEW ISOFLAVANOIDS FROM BOLUSANTHUS SPECIOSUS

. The structures of two new isoflavonoids from the combined ethyl acetate/methanolic extracts of the stem bark of Bolusanthus speciosus have been established as 4,7,2' -trihydroxy-4' -methoxyisoflavanol (1) and 5,7,3' ,4' -tetrahydroxy-5' -(2-epoxy-3-methylbutyl)isoflavanone (2). Five other known isoflavonoids, 5,7,3' -trihydroxy-4' -methoxy-5' -γγ; -dimethylallylisoflavanone, 5,7,2' -trihydroxy-4' -methoxy-6,5' -di)(γγ -dimethylallyl)isoflavanone, 5,7,2' ,4'-tetrahydroxy-8,5'2; -di(γγ -dimethylallyl)flavanone, 5,7,2' ,4' -tetrahydroxy-8,3' -di(γγ -dimethylallyl)-isoflavanone, and derrone were also isolated. Compound 2 showed moderate activity against gram positive bacteria and weak activity against gram negative bacteria, while compound 1 was weakly active against both organisms in a TLC bioautography assay. (Received March 22, 2001; revised August 13 2001) Bull.Chem.Soc.Ethiop. 2001, 15(2), 131-136

Further chromones from Eriosema tuberosum

Two new prenylated chromones, eriosematins D and E, have been isolated from a dichloromethane extract of the roots of Eriosema tuberosum. Their structures were elucidated by spectroscopic methods, including 2D NMR heteronuclear correlation spectra. In solution, both compounds were shown to be in equilibrium at room temperature; stable derivatives were obtained after acetylation. Both chromones exhibited antifungal activity against Cladosporium cucumerinum and Candida albicans in TLC bioautographic assays.