TLC Bioassay Research Articles

Inhibitory effect of Dalbergia latifoia extract on acetylcholinesterase enzyme by invitro methods

The present study was conducted to evaluate acetylcholinesterase (AChE) inhibitory effects of Dalbergia latifoia. The hexane, ethylacetate and ethanolic extract of Dalbergia latifoia were tested for their invitro anticholinesterase inhibitory effect by spectrophotometeric and by TLC bioassay method.The result revealed that ethanolic extract of Dalbergia latifoia showed better anticholinesterase inhibition compared to other extracts. The most active one was found to be ethanolic extract of Dalbergia latifoia having IC50 value at 81±1.56 µg/ml. Hexane extract of Dalbergia latifoia has not shown any anticholinesterase inhibitory effect. Ethylacetate extract of Dalbergia latifoia was found to have IC50 value at 625±2.65 µg/ml. TLC bioassay is an easier and rapid means for detection of enzyme inhibition. The hexane, ethylacetate and ethanolic extract of Dalbergia latifoia were tested for their anticholinesterase inhibition by TLC bioassay TLC bioassay is an easier and rapid means for detection of enzyme inhibition. The active spots appearedas white spots on yellow background. Ethanolic extract of Dalbergia latifoia showed more than one active spots compared to other extracts. The TLC assay also demonstrated AchE inhibitory activity for ethanolic extract of Dalbergia latifoia. Keywords: Dalbergia latifoia, acetylcholinesterase, Alzheimer’s disease, TLC bioautography

Antibacterial and Antioxidant Activities of Fungal Endophytes Isolated from Medicinal Plants in Simeulue Island, Aceh

Natural products from endophytic fungi have a wide range of medicinal applications, including antibacterial and antioxidant agents. The research aimed to evaluate the antibacterial and antioxidant activities of compounds derived from endophytic fungi isolated from medicinal plants collected from Simeulue Island by TLC dot-blot and microdilution assays. Eighty-one fungal extracts were screened for antibacterial and antioxidant activities. Antibacterial activity was evaluated against Gram-positive and negative bacteria, i.e., Staphylococcus aureus InaCC B-4 and Escherichia coli InaCC B-5, respectively. Antioxidant activity was carried out by using free radical scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH). The results of the TLC bioassay revealed that the fungal extracts have activities as antibacterial and antioxidant are 65 (against S. aureus), 57 (against E. coli), and 48 extracts (DPPH free radical scavenger). Two extracts, Phomopsis sp. 48BtSi-2.1 and Xylaria sp. 55DnSi-1.1, showed strong antibacterial activity against S. aureus with MIC value of 32 μg/ml. Furthermore, nine extracts (Schizophyllum sp. 20DnSi-1; Hyphomycetes 36BhSi-1.1; Phomopsis sp. 36DnSi-2.1; Schizophyllum sp. 39DnSi-1.1; Xylaria sp. 39RpSi-2.1; Phomopsis sp. 41BtSi-1.1; Phomopsis sp. 48BtSi-2.1; Lasiodiplodia sp. 48BtSi-3.1; Schizophyllum sp. 50DnSi-3) were strong activity against E. coli with MIC value ranges from 32 to 64 μg/ml. One extract, Xylaria sp. 04BtSi-2.2 has strong antioxidant activity as DPPH free radical scavenger with an IC50 value of 21.66 μg/ml (AAI value of >1).

Screening of acetylcholinesterase inhibitors in marine organisms from the Caribbean Sea

The acetylcholinesterase inhibitory activity of 89 organic extracts from marine organisms was evaluated through a TLC bioautography methodology. Extracts from soft corals (Eunicea and Plexaura) were the most active compared with extracts from sponges. The bioguided chemical study of the most active extract, obtained from Pseudoplexaura porosa, led to the isolation of a diterpene with spectroscopic properties consistent to those published to the cembrane Steylolide. However, further analysis by X-ray diffraction indicated that the compound was the 14-acetoxycrassine (1), correcting the structure reported to the Styelolide. Additionally, the acetylcholinesterase inhibitory activity of fourteen cembranoids (2–15) isolated from soft corals Eunicea knighti and Pseudoplexaura flagellosa was evaluated. Cembranoids 2, 3 and 4 were the most active compounds in the TLC bioassay. Then, the most promising cembranoids, 14-acetoxycrassine (1) and asperdiol (2), were tested quantitatively and they exhibited IC50 values of 1.40 ± 0.113 and 0.358 ± 0.130 μM, respectively.

Screening of antibacterial compounds in Thymus vulgaris L. tincture using thin-layer chromatography—direct bioautography and liquid chromatography—tandem mass spectrometry techniques

In this study, thin-layer chromatography—direct bioautography (TLC—DB) was used for guiding the isolation and identification of antibacterial constituents of Thymus vulgaris L. ethanol extract. This TLC—bioassay method enables the separation and detection of active components directly on the surface of chromatographic plates. They can be identified by comparison with reference substances or using physicochemical methods, preferably spectroscopic ones (liquid chromatography—tandem mass spectrometry [LC—MS/MS], in the presented paper). The described method belongs to the effect-directed analyses (EDA). Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, including methicillin-resistant Staphylococcus aureus as well as luminescent bacteria like Aliivibrio fischeri. Five fractions with the widest antimicrobial spectra were detected using TLC—DB, isolated by semi-preparative TLC and subjected to LC—MS/MS analyses. Finally, two bioactive components were tentatively identified, basing on their fragmentation pattern, as eriodictyol and 4,4′-dihydroxy-5,5′-diisopropyl-2,2′-dimethyl-3,6-bifenylodion.

The avocado defense compound phenol-2,4-bis (1,1-dimethylethyl) is induced by arachidonic acid and acts via the inhibition of hydrogen peroxide production by pathogens

The induction of defense compounds with arachidonic acid (AA) in avocado roots was analyzed in 6-month-old avocado seedlings. One organic extract was obtained 24 h after AA treatment that showed anti-oomycete activity. Two major regions of inhibition by TLC bioassay with Aspergillus were observed. One region had a high inhibitory effect on Aspergillus spore germination. This region was purified and analyzed by GC/MS spectrometry and the major compound was identified as phenol-2,4-bis (1,1-dimethylethyl) (PD). The antimicrobial activity of PD appeared to correlate with the antioxidative activity reported to this compound, because it was able to inhibit the ROS production in both Aspergillus and Phytophthora cinnamomi. This possibility was supported by an increase in the production of sporangia in Aspergillus in response to H2O2, also because the antioxidant ascorbic acid affects the Aspergillus growth concomitantly with a decrease in sporangia production. It is argued that the production of PD in avocado roots could be one of the mechanisms induced by AA that play an active role in the resistance against P. cinnamomi probably by regulating the production of H2O2.

Root Colonization and Quorum Sensing of the Antagonistic Bacterium Pseudomonas fluorescens 2112 involved in the Red-pepper Rhizosphere

Biofilm formation of multifunctional plant growth promoting rhizobacterium (PGPR), Pseudomonas fluorescens 2112 is necessary for P. fluorescens 2112 to have a positive impact on the rhizosphere of red-pepper. This study investigated whether signal molecules of the quorum sensing AHLs are produced in order to confirm biofilm formative ability. Through the use of Petri dish bioassays a blue circle formed evidence of AHLs. It was confirmed that P. fluorescens 2112 produced six-carbon-chain-long AHLs by TLC bioassay. The bacterial density of P. fluorescens 2112 on the top and bottom of pepper plant roots was estimated as and CFU/g root, respectively. P. fluorescens 2112 exist more with high-density of CFU/g soil at a depth of 1 cm but at a low-density of CFU/g soil at a depth of 5 cm, from the surface of rhizosphere soil. In addition, biofilm formation of P. fluorescens 2112 on the epidermises and the tips of the red-pepper roots were confirmed visually by SEM. Thus, the production of AHLs by P. fluorescens 2112 brings about quorum sensing signaling and the formation of biofilm on the roots which has a positive effect on economically important crops such as red-pepper by additionally producing a variety of antifungal substances and auxin.

Inhibitory effect of Anacycluspyrethrum extract on acetylcholinesterase enzyme by invitro methods

Aim: The present study was conducted to evaluate acetylcholinesterase (AChE) inhibitory effects of Anacyclus pyrethrum. Materials and Methods: The hexane, chloroform and ethanolic extract of Anacyclus pyrethrum were tested for their invitro anticholinesterase inhibitory effect by spectrophotometeric and by TLC bioassay method. Results: The result revealed that ethanolic extract of Anacyclus pyrethrum showed better anticholinesterase inhibition compared to other extracts. The most active one was found to be ethanolic extract of Anacyclus pyrethrum having IC50 value at 70±1.52 mg/ml. Hexane extract of Anacyclus pyrethrum has not shown any anticholinesterase inhibitory effect. Chloroform extract of Anacyclus pyrethrum was found to have IC50 value at 150±3.68 mg/ml and rivagistmine was found to have IC50 value to be 350±5.95 mg/ml. TLC bioassay is an easier and rapid means for detection of enzyme inhibition. The hexane, chloroform and ethanolic extract of Anacyclus pyrethrum were tested for their anticholinesterase inhibition by TLC bioassay. Conclusion: TLC bioassay is an easier and rapid means for detection of enzyme inhibition. The active spots appeared as white spots on yellow background. Ethanolic extract of Anacyclus pyrethrum showed more than one active spots compared to other extracts. The TLC assay also demonstrated AchE inhibitory activity for ethanolic extract of Anacyclus pyrethrum.

Inhibitory effect of Anacyclus pyrethrum extract on acetylcholinesterase enzyme by invitro methods

The present study was conducted to evaluate acetylcholinesterase (AChE) inhibitory effects of Anacyclus pyrethrum. The hexane, chloroform and ethanolic extract of Anacyclus pyrethrum were tested for their invitro anticholinesterase inhibitory effect by spectrophotometeric and by TLC bioassay method. The result revealed that ethanolic extract of Anacyclus pyrethrum showed better anticholinesterase inhibition compared to other extracts. The most active one was found to be ethanolic extract of Anacyclus pyrethrum having IC50, value at 70±1.52 μg/ml. Hexane extract of Anacyclus pyrethrum has not shown any anticholinesterase inhibitory effect. Chloroform extract of Anacyclus pyrethrum was found to have IC50 value at 150±3.68 μg/ml and rivagistmine was found to have IC50 value to be 350±5.95 μg/ml. TLC bioassay is an easier and rapid means for detection of enzyme inhibition. The hexane, chloroform and ethanolic extract of Anacyclus pyrethrum were tested for their anticholinesterase inhibition by TLC bioassay TLC bioassay is an easier and rapid means for detection of enzyme inhibition. The active spots appeared as white spots on yellow background. Ethanolic extract of Anacyclus pyrethrum showed more than one active spots compared to other extracts. The TLC assay also demonstrated AchE inhibitory activity for ethanolic extract of Anacyclus pyrethrum.

삼나무 정유의 표재성 진균에 대한 항진균 활성

본 연구에서는 표재성 진균에 의한 피부질환 개선용 소재로의 개발을 위해 삼나무 정유의 표재성 알레르기성 진균, Trichophyton schoenleinii, Trichophyton tonsurans, Trichophyton mentagrophytes (interdigitale), Trichophyton rubrum, Epidermophyton floccosum, Aspergillus fumigatus에 대한 항진균 활성을 평가하여 삼나무 정유의 표재성 진균에 의한 피부질환 개선 소재로의 개발 가능성을 평가하고자 하였다. 항진균 활성 평가는 삼나무 정유의 MIC (Minimum Inhibitory Concentration) 측정과 TLC bioassay, 한천희석법을 통해 측정하였다. 또한 활성 성분의 주성분을 확인하기 위해 GC/MS 분석을 실시하였다. 삼나무 정유 crude oil의 MIC는 모든 균에서 500 ppm 이하의 농도 값을 나타냈으며, TLC bioassay를 통해 spot 1에서 항진균 효과를 확인하였다. 한천희석법을 통한 항진균 활성은 A. fumigatus를 제외한 모든 진균에 대해 삼나무 정유 crude oil의 경우, 500 ppm 이상에서 100%의 활성을 나타냈고, 분획 D의 경우, 100 ppm에서도 100%의 활성을 나타냈다. TLC bioassay를 통해 항진균 활성을 나타낸 spot 1과 한천희석법을 통해 활성을 나타낸 분획 D를 GC/MS로 분석한 결과 주성분은 elemol, <TEX>${\gamma}$</TEX>-eudesmol, <TEX>${\beta}$</TEX>-eudesmol이었다. 따라서 elemol, eudesmol 등은 표재성 진균에 의한 피부질환의 개선 소재로의 이용 가능성이 충분하다고 사료된다. In this study, the antifungal activity of Cryptomeria japonica essential oil against superficial and allergic fungi, Trichophyton schoenleinii, Trichophyton tonsurans, Trichophyton mentagrophytes, Trichophyton rubrum, Epidermophyton floccosum, and Aspergillus fumigatus, was evaluated for determining the potential compound as dermatitis treatment. Minimum Inhibitory Concentration (MIC) measurement, TLC bioassay and agar dilution methods were used for determining the antifungal activity of crude essential oil and its fractions from C. japonica. Also, their major constituents were analyzed by GC/MS. The MICs were below 500 ppm at all superficial fungi, and spot 1 of C. japonica essential oil showed highly effective antifungal activity by TLC bioassay. In antifungal activity by agar dilution methods, crude oil showed high antifungal effect at more than 500 ppm and fraction D was significantly effective at even 100 ppm except for A. fumigatus. The major compounds of spot 1 and fraction D of C. japonica oil determined by GC/MS were elemol, <TEX>${\gamma}$</TEX>-eudesmol, and <TEX>${\beta}$</TEX>-eudesmol, which could be used as atopic dermatitis treatment material.

Acetylcholinesterase enzyme inhibitory potential of standardized extract of Trigonella foenum graecum L and its constituents

Ethno pharmacological approach has provided several leads to identify potential new drugs from plant sources, including those for memory disorders. Acetylcholinesterase inhibitors (AChEI) give a symptomatic relief to some of the clinical manifestations of the disease. The main objective of this study is to standardize the extract of Trigonella foenum graecum L with trigonelline by HPTLC method and determine the in vitro AChE inhibitory activity of Trigonella foenum graecum L and its constituents using galanthamine as a reference. Different concentrations of hydro alcoholic extract of Trigonella foenum graecum and trigonelline were subjected to HPTLC analysis using the mobile phase n propanol, methanol and water (4:1:2, v/v). The R(f) of trigonelline was found to be 0.43, and the correlation coefficient of 0.99 was indicative of good linear dependence of peak area on concentration. The concentration of trigonelline was found to be 13mgg(-1)w/w in the hydro alcoholic extract of Trigonella foenum graecum. The AChE inhibitory activity of crude fenugreek seed extracts, fractions and trigonelline was evaluated using Ellman's method in 96-well micro plate's assay and TLC bioassay detection. The ethyl acetate fraction of the alcohol extract (IC50 53.00 +/- 17.33microg/ml), and total alkaloid fraction (IC50 9.23+/-6.08microg/ml) showed potential AChE inhibition. Trigonelline showed IC50 233+/-0.12microM. Galanthamine was used as standard and it showed inhibition of acetyl cholinesterase with an IC50 value of 1.27+/-0.21microM.

Correlation between Growth Inhibitory Exhibition and Suspected Allelochemicals (Phenolic Compounds) in the Extract of Alfalfa (Medicago sativaL.)

Acidic fractions of the extracts from the three alfalfa cultivars Batasu, Rasen, and Yuba exhibited a varietial difference in the inhibitory effect on hypocotyls and radicle growth of alfalfa (cv. Nasuwakaba) and rice (cv. Koshihikari) seedlings. The extract from Rasen possessed the strongest inhibitory activity, and that of Batasu was the lowest. In a TLC bioassay with lettuce seedlings, inhibitory zones of the extracts were located at an Rf value of 0.6 -0.8, and maximum exhibitory zones at Rfs of 0.7-0.8. However, the extracts of Rasen and Yuba contained an additional inhibitory zone at Rf of 0.1-0.2. Inhibitory zones were analyzed by HPLC. Eight phenolic compounds were identified in the extracts from Rasen and Batasu, and six compounds in that from Yuba. However, the content of these phenolic compounds varied with the cultivar. The content of each phenolic compound was the highest in Rasen, followed by Yuba and Batasu, although the content of jft-hydroxybenzoic acid was equivalent in all cultivars. We suppose that the degree of inhibitory exhibition of allelopathy may be related to the presence and concentrations of allelochemicals (phenolic compounds), however, the allelopathic activity of the plant might be determined by interactions of all these compounds, not just a single chemical.

Antifungal characteristics of a fluorescent Pseudomonas strain involved in the biological control of Rhizoctonia solani

A plant growth-promoting isolate of a fluorescent Pseudomonas spp. EM85 was found strongly antagonistic to Rhizoctonia solani, a causal agent of damping-off of cotton. The isolate produced HCN (HCN +), siderophore (Sid +), fluorescent pigments (Flu +) and antifungal antibiotics (Afa +). Tn5:: lacZ mutagenesis of isolate EM85 resulted in the production of a series of mutants with altered production of HCN, siderophore, fluorescent pigments and antifungal antibiotics. Characterisation of these mutants revealed that the fluorescent pigment produced in PDA and the siderophore produced in CAS agar were not the same. Afa − and Flu − mutants had a smaller inhibition zone when grown with Rhizoctonia solani than the EM85 wild type. Sid − and HCN − mutants failed to inhibit the pathogen in vitro. In a pot experiment, mutants deficient in HCN and siderophore production could suppress the dampingoff disease by 52%. However, mutants deficient in fluorescent pigments and antifungal antibiotics failed to reduce the disease severity. Treatments with mutants that produced enhanced amounts of fluorescent pigments and antibiotics compared with EM85 wild type, exhibited an increase in biocontrol efficiency. Monitoring of the mutants in the rhizosphere using the lacZ marker showed identical proliferation of mutants and wild type. Purified antifungal compounds (fluorescent pigment and antibiotic) also inhibited the fungus appreciably in a TLC bioassay. Thus, the results indicate that fluorescent pigment and antifungal antibiotic of the fluorescent Pseudomonas spp. EM85 might be involved in the biological suppression of Rhizoctonia-induced damping-off of cotton.

Note Iridoid Glucosides, Phenylpropanoid Derivatives and Flavanoids from Bartsia alpina

Three iridoid glucosides, two phenylpropanoid derivatives and one flavonoid have been isolated from the methanolic whole plant extract of Bartsia alpina L. (Scrophulariaceae). Their structures were established by spectrometric (UV, D/CI and FAB mass spectrometry, 1 H and 13 C NMR) and chemical methods. Some of these compounds have radical scavenging activity against DPPH or antioxidant activity against ß-carotene on TLC bioassay.

Antimicrobial steroids from the fungus Fomitopsis pinicola

Phytochemical examination of the dichloromethane extract of the European fungus Fomitopsis pinicola led to the isolation of a new lanostanoid derivative identified from spectral and chemical evidences as 3α-(4-carboxymethyl-3-hydroxy-3-methylbutanoyloxy)-lanosta-8,24-dien-21-oic acid. In addition, seven known triterpenes, polyporenic acid C, 3α-acetyloxylanosta-8,24-dien-21-oic acid, ergosta-7,22-dien-3β-ol, 21-hydroxylanosta-8,24-dien-3-one, pinicolic acid A, trametenolic acid B and pachymic acid, were also isolated. Antimicrobial activity against Bacillus subtilis in a TLC bioassay was observed for five of the isolated steroids.

ANTIFUNGAL CONSTITUENTS OF CHENOPODIUM PROCERUM (CHENOPODIACEAE) HOCHST EX MOQ

Dried ground aerial parts were extracted successively with dichloromethane and methanol at room temperature. Antifungal activity was determined by a TLC bioassay using Cladosporium cucumerinum. Four compounds were isolated from a dichloromethane extract by different chromatographic techniques (silicagel CC, Sephadex LH-20 gel filtration, and low-pressure LC on Diol): the isoflavones irilin A and irilin B, the flavonone dihydrowogonin, and sesquiterpene pygmol. The latter three were antifungal. The minimal amount that inhibited the fungal growth of C. cucumerinum was 5 μ for these compounds. Four flavonol glycosides were obtained from the non-fungicidal extract by Sephadex LH-20 gel filtration and centrifugal partition chromatography. The structures of the compounds were elucidated by spectroscopic methods (UV, 1H NMR, 13CNMR, MS).

Antifungal compounds from the roots of mycotrophic and non‐mycotrophic plant species

SUMMARYThe roots of twelve plant spades From bath mycotrophic (hosts of YAM fungi) and non‐mycotropic (non‐hosts) families were examined for their ability to produce antifungal compounds. A TLC bioassay was used with the fungus Cladosporium cucumerimun Ellis &amp; Arch. Of the non‐mycotrophic species, five species from the Brassicaceae [Brassica kaber (DC.) Wheeler, Brassica napus L., Brassica campestris L.,Thlaspi arvense L., Rhaphanus raphasnistrum L.] had detectable antifungal compounds in extracts from roots, while two species from the Chenopodiaceae (Spinacea oleracea L., Beta vulgaris L.) and one species from the Amaranthaceae (Amaranthus retroflexus L.) did not. One mycotrophic species (Daucus carota L.) also had a detectable antifungal compound in root extracts, while three other mycotrophic species (Lactuca sativa LK., Abutilon theophrasti Medic., sorghum bicolor L.) did not. Chloroform extracts of B. kaber roots appeared to have the greatest quantity of extractable antifungal compounds as detarmined by the TLC bioassay. In a separate experiment, chloroform extracts from B. kaber roots greatly inhibited the germination of spores of Glomus etunicatum Becker and Gerd., while extracts from a number of other mycotrophic and non‐mycotrophic plant roots were only marginally inhibitory. Three antifungal compounds in chloroform extracts from B. kaber roots were shown to be derived from glucosinolates. The predominant antifungal compound was identified as +‐hydroxybenzyl isothiocyanate, the isothiocyanate produced by the action of myrosinase on glucosinalbin. The data provide evidence of a possible role for glucosinolates in determining the non‐mycotrophic status in B. kaber.

A New Phloroglucinol Derivative fromHypericum calycinumwith Antifungal andin vitroAntimalarial Activity1

The new phloroglucinol derivative 1 has been isolated from the light petroleum ether extract of the aerial parts of Hypericum calycinum. Its structure has been established by means of 1H- and 13C-NMR spectroscopy and by nOe, MHQC, and HMBC experiments on its monomethyl ether derivative 3. Compound 1 was fungicidal against Cladosporium cucumerinum in a TLC bioassay. In addition, this new phloroglucinol derivative was also found to exert an interesting antimalarial activity in an in vitro test system.

Antifungal diterpenic esters from the mushroom Boletinus cavipes

The structures of new antifungal diterpenic esters, isolated from Boletinus cavipes, have been established by chemical and spectral means. They were shown to be diterpenic esters of 16-hydroxygeranylgeraniol and mesaconic and/or fumaric acid. They inhibited Cladosporium cucumerinum spore formation in a TLC bioassay.

Antifungal Diterpenic Esters from the MushroomBoletinus cavipes

Abstract The structures of new antifungal diterpenic esters, isolated from Boletinus cavipes , have been established by chemical and spectral means. They were shown to be diterpenic esters of 16-hydroxygeranylgeraniol and mesaconic and/or fumaric acid. They inhibited Cladosporium cucumerinum spore formation in a TLC bioassay.

Piperine from an Ulocladium sp.

Piperine was isolated from the mycelium of a Ulocladium species and identified by spectroscopic means. The compound showed antifungal activity in the Cladosporium TLC bioassay.