OPLC (Overpressured layer chromatography) separations were performed by Personal OPLC BS50 system (OPLC-NIT, Budapest, Hungary). Method of Bioassay: The dried chromatoplates were dipped into the soil bacteria Bacillus subtilis cell suspension (10 s). After 1 hour incubation the antibacterial assay was visualized with aqueous solution of dye reagent MTT (Sigma Aldrich Ltd., Budapest) (dipping into for 5 s). In BioArena, studying the influence of L-arginine (Sigma) or Cu(II) (Reanal, Budapest) on the antibacterial activity of oil components we dissolved them in the bacterial cell suspension before inoculation. For luminescent detection the adsorbent layers dipped into luminescence gene tagged Arabidopsis pathogen Pseudomonas maculicola cell suspension and were placed in a glass cage keeping them wet in a closed air atmosphere. Bioautograms were acquired under a computer- controlled cooled CCD camera (IS-4000; Alpha Innotech, San Leandro, USA) and documented immediately after the inoculation. References Móricz Á.M., Adányi N., Horváth E., Ott P.G., Tyihák E., J. Planar Chromatogr. 21 (2008) 417-422. Móricz Á.M., Ott P.G., Otta K.H., Tyihák E., Nat. Prod. Commun. 6 (2011) 657-660. Mincsovics E., Garami M., Kecskés L., Tapa B., Végh Z., Kátay Gy., Tyihák E., J. AOAC Int. 82 (1999) 587–598. Mincsovics E., Sárdi É., Velich I., Kátay Gy., and Tyihák E., J. Planar Chromatogr. 15 (2002) 280-285. Tyihák E., Móricz Á.M., Ott P.G., Thin layer chromatography in Phytochemistry. (eds.: M. Waksmundzka-Hajnos, J. Sherma, T. Kowalska) CRC Press (2008) pp. 193-213. Potential applicability of modern bioautography (BioArena) in the study of plant ingredients Ágnes M. Móricz 1 , Péter G. Ott 1 ,Emil Mincsovics 2 , Ernı Tyihák 1 1 Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, H-1525 Budapest, Hungary 2 OPLC-NIT Co., Ltd, Budapest, Hungary TLC- and OPLC-bioautography Direct bioautography, the combined application of planar layer liquid chromatographic separation (TLC, HPTLC, OPLC) and post-chromatography bioassay, enables the detection of antimicrobial components e.g. of plant extracts. In the course of the biological detection the adsorbent layer after development is dipped into or sprayed with given cell suspension and afterwards the bioautogram is visualized. Visualization of the bioautogram is usually performed by the use of tetrazolium salts (see bioautograms using B. subtilis). The living cells reduce the yellow tetrazolium salts to bluish formazan, so the antimicrobial active compounds appear as clear spots/zones against a darker background. Using the emitted light as signal, the performance of biodetection is very easy. The image of the bioautogram (see the use of luminescent gene tagged P. maculicola) can be directly recorded by a cooled camera in a dark box. Because of the overloaded condition there is no characteristic differences in the separation efficiency comparing the use of TLC and HPTLC adsorbent layer. However the biological detection is more sensitive in the case of HPTLC layer, which may be the result of the differences between TLC and HPTLC in thickness, binding material, pH and/or the concentration of trace elements. Infusion OPLC, 20x20 cm TLC/HPTLC layer, (dried 130 0 C/3 h), 5MPa, 300/450 μL/min, chloroform, total volume 5540/4250 μL, 918/572 s. a - standards, 3 μg of each thymol and carvacrol b - Thymus essential oil (16 μg) Merck TLC layer Merck HPTLC layer a b λ=254 nm Bacillus subtilis Pseudomonas maculicola thymol carvacrol a b a b a b λ=254 nm Bacillus subtilis Pseudomonas maculicola thymol carvacrol a b a b λ=365nm λ=254 nm B. subtilis 3 6 9 μL of plant extract B. subtilis λ=365nm λ=254 nm 3 6 9 μL of plant extract The influence of the quality of the adsorbent layer on the biological detection Conventional TLC/HPTLC chromatography with chloroform-acetone 9:1 (v/v). OPLC 50 system Overpressured Layer Chromatography (OPLC) 0 5 10 15 20 25 30 35 40 45 0 5 10 15 20 TLC-layer-OPLC HPTLC-layer-OPLC 3 μm-OPLC E. Mincsovics, 1998 development distance (cm) H (μm) In OPLC system the mobile phase migrates through the entire layer, being under homogeneous pressure (50 bar), with constant velocity that is achieved by the application of a pump system. The forced flow leads to a faster separation and makes possible the longer development distance, increasing zone capacity. Constant velocity results in almost constant average theoretical plate height (H) in the whole development distance. The OPLC system has a high flexibility. The principal operation steps as sample application, separation, detection and isolation can be freely combined. The fully off-line process (spotting/streaking sample application & separation & in situ densitometric evaluation) started with dry non-segmented adsorbent layer corresponds to TLC. The fully on-line OPLC (injection & flow-cell detection) performed on a conditioned adsorbent layer is analogous with HPLC and at the same time single sample can be processed. Isolation of important components can be carried out off-line by elution from the chromatographic spots/bands scraped off or on-line by collecting their peaks after flow-cell detection using an overrun. 1. TLC - bioautography UV (365 nm) Psmlux 2. OPLC with on-line detection the fractionation of chamomile flower extract chamomile root leaf flower 3. Checking of the fractions 4. GC-MS analysis of the fractions O O H 3 C O O H 3 C O O H 3 C O herniarin cis-, trans-spiroethers Identification of antibacterial components of plant extract using OPLC with on-line detection and GC-MS extract BioArena investigations The chromatographic BioArena means the coordination of the operating steps in biological detection of potential ingredients as well as the unlimited use of biochemicals/chemicals for interactions with the cells in chromatographic spots/bands. BioArena, beyond the detection of antimicrobial components, is also appropriate for examination of the mechanism of cell proliferation inhibition and/or promotion effects. The influence of different endogenous and/or exogenous substances on the bioactivity of separated compounds can be examined by dissolving substances in the cell suspension just before inoculation. After biological detection there is a possibility of in situ quantitative densitometric evaluation in addition to in situ and ex situ qualitative and quantitative investigations, for example other chromatographic separations, IR, FT-IR, FT-Raman, NMR spectroscopy, LC/GC–MS, and MALDI-MS. The antibacterial activity of O. onites components and aflatoxins (AFs) was investigated against Bacillus subtilis in BioArena. In the presence of formaldehyde (HCHO) capturer (e.g. L- arginine) their antimicrobial activity (the inhibition zones) was decreased comparing with the control layer. If HCHO generator and transporter Cu(II) ions or HCHO precursor N G - monomethyl-L-arginine (MMA) were dissolved in the culture medium the antibacterial effect were increased characteristically (not shown all results). The FT-Raman spectra, obtained in situ around the AFB1-containing spots in bacterium-free and inoculated TLC layers, indicates an excess of HCHO formation by demethylation of AFB1 at its methoxy group in the presence of bacterial cells (the intensity of the δCH 3 band of AFB1 (1386 cm –1 ) was reduced by 50%). It seems that these compounds generate antimicrobial activity through HCHO and its reaction products. Surface enhanced FT-Raman spectra of (a) the aflatoxin B1 spot in bacteria free TLC layer and (b) the aflatoxin B1 spot in TLC layer inoculated with Psm cell suspension The influence of MMA on the antibacterial effect of aflatoxins and its densitometric confirmation Control 1 mg/mL N G -monomethyl- -L-arginine 164% 121% 178% 143% development distance (mm) Detector signal (λ=590 nm) „Off-line” OPLC, 20x20 cm TLC layer, (dried 130 o C, 3 h), 5MPa, 400 μL/min, chloroform-acetone 9:1 (v/v), total volume 4500 μL, 685 s. Infusion OPLC, 20x20 cm TLC layer, (dried 130 o C/3 h), 5MPa, 400 μL/min, dichloromethane, total volume 4847 μL, 738 s. Infusion OPLC, 20x20 cm TLC layer, (dried 130 o C/3 h), 5MPa, 400 μL/min, dichloromethane, total volume 4847 μL, 738 s. 1 3 1 2 3 1 2 3 1 2 3 1 2 3 A B under UV λ=254 nm vanillin- sulphuric acid reagent Control 4 mg/mL L-arginine 3 mg/100mL CuSO 4 x5H 2 O The effect of L-arginine and Cu(II) ions on the antibacterial effect of Origanum onites oil components 1. Standards, 10 μg of each thymol (A) and carvacrol (B); 2-3. Origanum oils (30 μg of each) 1 3 1 2 3 1 2 3 1 2 3 1 2 3 A B under UV λ=254 nm vanillin- sulphuric acid reagent Control 4 mg/mL L-arginine 3 mg/100mL CuSO 4 x5H 2 O
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OPLC (Overpressured layer chromatography) separations were performed by Personal OPLC BS50 system (OPLC-NIT, Budapest, Hungary).Method of Bioassay: The dried chromatoplates were dipped into the soil bacteria Bacillus subtilis cell suspension (10 s). After 1 hour incubation the antibacterial assay was visualized with aqueous solution of dye reagent MTT (Sigma Aldrich Ltd., Budapest) (dipping into for 5 s). In BioArena, studying the influence of L-arginine (Sigma) or Cu(II) (Reanal, Budapest) on the antibacterial activity of oil components we dissolved them in the bacterial cell suspension before inoculation. For luminescent detection the adsorbent layers dipped into luminescence gene tagged Arabidopsis pathogen Pseudomonas maculicola cell suspension and were placed in a glass cage keeping them wet in a closed air atmosphere. Bioautograms were acquired under a computer-controlled cooled CCD camera (IS-4000; Alpha Innotech, San Leandro, USA) and documented immediately after the inoculation.
ReferencesMóricz Á.M., Adányi N., Horváth E., Ott P.G., Tyihák E., J. Planar Chromatogr. 21 (2008) 417-422.Móricz Á.M., Ott P.G., Otta K.H., Tyihák E., Nat. Prod. Commun. 6 (2011) 657-660. Mincsovics E., Garami M., Kecskés L., Tapa B., Végh Z., Kátay Gy., Tyihák E., J. AOAC Int. 82 (1999) 587–598.Mincsovics E., Sárdi É., Velich I., Kátay Gy., and Tyihák E., J. Planar Chromatogr. 15 (2002) 280-285.Tyihák E., Móricz Á.M., Ott P.G., Thin layer chromatography in Phytochemistry. (eds.: M. Waksmundzka-Hajnos, J. Sherma, T. Kowalska) CRC Press (2008) pp. 193-213.
Potential applicability of modern bioautography (BioArena)
in the study of plant ingredientsÁgnes M. Móricz1, Péter G. Ott1,Emil Mincsovics2, Ernı Tyihák1
1 Plant Protection Institute, Hungarian Academy of Sciences, P.O. Box 102, H-1525 Budapest, Hungary 2 OPLC-NIT Co., Ltd, Budapest, Hungary
TLC- and OPLC-bioautographyDirect bioautography, the combined application of planarlayer liquid chromatographic separation (TLC, HPTLC, OPLC) and post-chromatography bioassay, enables thedetection of antimicrobial components e.g. of plantextracts. In the course of the biological detection theadsorbent layer after development is dipped into orsprayed with given cell suspension and afterwards thebioautogram is visualized.Visualization of the bioautogram is usually performed by
the use of tetrazolium salts (see bioautograms using B. subtilis). The living cells reduce the yellow tetrazoliumsalts to bluish formazan, so the antimicrobial activecompounds appear as clear spots/zones against a darkerbackground.Using the emitted light as signal, the performance of biodetection is very easy. The image of the bioautogram(see the use of luminescent gene tagged P. maculicola) can be directly recorded by a cooled camera in a dark box.Because of the overloaded condition there is no characteristic differences in the separation efficiencycomparing the use of TLC and HPTLC adsorbent layer. However the biological detection is more sensitive in thecase of HPTLC layer, which may be the result of thedifferences between TLC and HPTLC in thickness, bindingmaterial, pH and/or the concentration of trace elements.
Infusion OPLC, 20x20 cm TLC/HPTLC layer, (dried 130 0C/3 h), 5MPa, 300/450 µL/min, chloroform, total volume
5540/4250 µL, 918/572 s. a - standards, 3 µg of each thymol and carvacrol
b - Thymus essential oil (16 µg)
MerckTLClayer
Merck HPTLC
layer
a b
λ=254 nmBacillus subtilis
Pseudomonasmaculicola
thymol
carvacrol
a b a b
a b
λ=254 nmBacillus
subtilis
Pseudomonas
maculicola
thymolcarvacrol
a b a b
λ=365nm λ=254 nm B. subtilis
3 6 9 µL of plant extract
B. subtilisλ=365nm λ=254 nm
3 6 9 µL of plant extract
The influence of the quality of the adsorbent layer on the biological detection
Conventional TLC/HPTLC chromatographywith chloroform-acetone 9:1 (v/v).
OPLC 50 system
Overpressured Layer Chromatography (OPLC)
0
5
10
15
20
25
30
35
40
45
0 5 10 15 20
TLC-layer-OPLC
HPTLC-layer-OPLC
3 µµµµm-OPLC
E. Mincsovics,
1998development distance (cm)
H (µµµµm)
In OPLC system the mobile phase migrates through the entirelayer, being under homogeneous pressure (50 bar), withconstant velocity that is achieved by the application of a pumpsystem. The forced flow leads to a faster separation and makespossible the longer development distance, increasing zonecapacity. Constant velocity results in almost constant averagetheoretical plate height (H) in the whole development distance.
The OPLC system has a highflexibility. The principal operationsteps as sample application, separation, detection and isolationcan be freely combined. The fullyoff-line process (spotting/streakingsample application & separation & in situ densitometric evaluation) started with dry non-segmentedadsorbent layer corresponds toTLC. The fully on-line OPLC (injection & flow-cell detection) performed on a conditionedadsorbent layer is analogous withHPLC and at the same time singlesample can be processed. Isolationof important components can be carried out off-line by elution fromthe chromatographic spots/bandsscraped off or on-line by collectingtheir peaks after flow-cell detectionusing an overrun.
1. TLC - bioautography
UV (365 nm) Psmlux2. OPLC with on-line detection
the fractionation of chamomile flower extract
chamomile
root leaf flower
3. Checking of the fractions
4. GC-MS analysis of the fractions
O
O
H3C
O O
H3C
OO
H3C
O
herniarin
cis-, trans-spiroethers
Identification of antibacterial components of plant
extract using OPLC with on-line detection and GC-MS
extract
BioArena investigationsThe chromatographic BioArena means the coordination of the operating steps in biological detection of potential ingredients as well as the unlimited use of biochemicals/chemicals forinteractions with the cells in chromatographic spots/bands. BioArena, beyond the detection of antimicrobial components, is also appropriate for examination of the mechanism of cellproliferation inhibition and/or promotion effects. The influence of different endogenous and/or exogenous substances on the bioactivity of separated compounds can be examined bydissolving substances in the cell suspension just before inoculation. After biological detection there is a possibility of in situ quantitative densitometric evaluation in addition to in situ and ex situ qualitative and quantitative investigations, for example other chromatographic separations, IR, FT-IR, FT-Raman, NMR spectroscopy, LC/GC–MS, and MALDI-MS.The antibacterial activity of O. onites components and aflatoxins (AFs) was investigated against Bacillus subtilis in BioArena. In the presence of formaldehyde (HCHO) capturer (e.g. L-arginine) their antimicrobial activity (the inhibition zones) was decreased comparing with the control layer. If HCHO generator and transporter Cu(II) ions or HCHO precursor NG-monomethyl-L-arginine (MMA) were dissolved in the culture medium the antibacterial effect were increased characteristically (not shown all results). The FT-Raman spectra, obtained insitu around the AFB1-containing spots in bacterium-free and inoculated TLC layers, indicates an excess of HCHO formation by demethylation of AFB1 at its methoxy group in thepresence of bacterial cells (the intensity of the δCH3 band of AFB1 (1386 cm–1) was reduced by 50%). It seems that these compounds generate antimicrobial activity through HCHO and its reaction products.
Surface enhanced FT-Raman spectra of (a) the aflatoxin B1 spot in bacteria free TLC
layer and (b) the aflatoxin B1 spot in TLC layer inoculated with Psm cell suspension
The influence of MMA on the antibacterial effect of aflatoxins and its densitometric confirmation
Control1 mg/mL
NG-monomethyl--L-arginine
164% 121%
178% 143%
development distance (mm)
Det
ecto
rsi
gnal
(λ=
590
nm)
„Off-line” OPLC, 20x20 cm TLC layer, (dried 130 oC, 3 h), 5MPa, 400 µL/min, chloroform-acetone 9:1 (v/v), total volume 4500 µL, 685 s. Infusion OPLC, 20x20 cm TLC layer, (dried 130 oC/3 h), 5MPa,
400 µL/min, dichloromethane, total volume 4847 µL, 738 s. Infusion OPLC, 20x20 cm TLC layer, (dried 130 oC/3 h), 5MPa, 400 µL/min, dichloromethane, total volume 4847 µL, 738 s.
1 3 1 2 3 1 2 3 1 2 3 1 2 3
A
B
under UV
λ=254 nmvanillin-
sulphuric
acid reagent
Control4 mg/mL
L-arginine
3 mg/100mL
CuSO4x5H2O
The effect of L-arginine and Cu(II) ions on theantibacterial effect of Origanum onites oil components
1. Standards, 10 µg of each thymol (A) and carvacrol (B); 2-3. Origanum oils (30 µg of each)
1 3 1 2 3 1 2 3 1 2 3 1 2 3
A
B
under UV
λ=254 nmvanillin-
sulphuric
acid reagent
Control4 mg/mL
L-arginine
3 mg/100mL
CuSO4x5H2O
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