Conventional and modern bioassays—detection, isolation, identification

Chapter

6

Conventional and modern bioassays— detection, isolation, identification Á.M. Móricz, P.G. Ott Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary

CHAPTER OUTLINE

6.1 6.2 6.3 6.4

Introduction 347 Principle and History of Planar Layer Chromatography—Biological Detection 349 Classification and Comparison of Methods for Detection of Antimicrobials 353 The Basic Elements of Direct Bioautography (DB) 357 6.4.1 Conventional and Forced-Flow Layer Separation Systems: Advantages of OPLC to TLC/HPTLC in DB 357 6.4.2 Detection Methods in DB 358

6.5 Practice of DB 363 6.5.1 Optimization of Layer Liquid Systems 363 6.5.2 Use of Optimum Conditions for Test Cell System 364 6.5.3 Influencing Factors in DB 368

6.6 Applications of Planar Layer Chromatography-DB 371 6.6.1 Test Organisms for DB 371 6.6.2 Detection of Biologically Active Compounds with OPLC-DB 372 6.6.3 Quantification of Bioactive Compounds Using Planar Layer Chromatography Coupled with Bioassay 378 6.6.4 Bioassay-Guided Separation, Detection, and Isolation Using DB 381

6.7 Future Development and Application Potential of Planar Layer Chromatography-Bioassay 386 References 387

6.1 INTRODUCTION To fight against various diseases, there is an increasing demand for effective compounds applicable in human and animal medicine as well as in plant protection. This is especially true for the antimicrobials, because the origin of

Forced-Flow Layer Chromatography. http://dx.doi.org/10.1016/B978-0-12-420161-3.00006-X Copyright # 2016 Elsevier Inc. All rights reserved.

347

348 CHAPTER 6 Conventional and Modern Bioassays

a broad range of diseases is infection, and the incidence of (multi)drug resistance in pathogens is increasing against the widely used antimicrobials [1]. The destruction of pathogens in animals is the result of the synchronized work of specialized cells of the immune system where cellular mobility is pivotal. However, in plants and microorganisms there is neither such cellular specialization nor mobility, so the cell wall-isolated cells are doomed to produce an arsenal of secondary metabolites that contribute to the success of disease control. Therefore, it is not surprising that modern drug development uses the microbes and the plant kingdom as an unsurpassed source of compounds having diverse chemical structure and large-scale beneficial effects, e.g., antibacterial [2–4]. There are roughly 300,000 plant species believed to exist, and only a minor fraction of these have been chemically investigated [5]. In the search for new biologically active agents, the detection of the desired bioactivity is crucial. For this purpose there are scads of biotests; however, most of them are suitable to demonstrate the effect of only crude extracts or their fractions or the isolated pure components. So we can test a component only after its isolation process, which is usually time-consuming and expensive. This is the case with the generally used in vitro tests, such as the antimicrobial susceptibility tests [6,7], the diffusion and the dilution methods. With the introduction of planar layer chromatography hyphenated with detection of biological activity, there is a possibility of investigating various bioeffects of separated matrix components in situ in the adsorbent layer, which means that the time and effort to isolate the components is spared. Cell suspensions and/or chemical reagents are applied for these coupled detection methods that belong to effect-directed analysis (EDA), a promising tool for the determination of major active components in environmental matrices. Direct bioautography (DB) [8–11], a combination of a microbial detection and a planar layer chromatographic technique, is mainly applied for the visualization of antifungal, antibacterial, and antiyeast properties of analyzed substances. With this biotest there is a high-throughput, reliable method in our hands. This tool has also the requisites of biomonitoring systems that are suitable in bioassay-guided isolation processes. Obviously, adsorbents within closed chromatographic columns are inaccessible to seeding with microbes, so they cannot be used for DB. This chapter summarizes the advance of planar layer chromatographybioassay developments covering methodological solutions, with special emphasis on the DB and highlighting the advantage of the use of forcedflow planar layer chromatographic separation techniques in this system.

6.2 Principle and History of Planar Layer Chromatography—Biological Detection 349

6.2 PRINCIPLE AND HISTORY OF PLANAR LAYER CHROMATOGRAPHY—BIOLOGICAL DETECTION Planar layer chromatography can be coupled with various effect-directed analyses resulting in an in situ detection system that enables a relatively cheap and fast screening of many samples in parallel. The main advantage of this method is the possibility of the analysis of single components in various matrices. The first biodetection combined with paper chromatography was bioautography, published in 1946 by Goodall and Levi [8]. They separated different penicillins on filter paper impregnated by potassium phosphate buffer (pH ¼ 6-7) with water-saturated ether as the mobile phase. After elimination of the solvent, the developed strips were placed on the surface of agar medium previously inoculated with Bacillus subtilis for 3-4 h at 0-5 °C, ensuring the diffusion of penicillins into the agar layer. The elliptical inhibition zones were revealed after an overnight incubation at 37-38 °C. The identification of the penicillins was based on the comparison of the migration distances and their ratio using test substances. This method was applied also for quantitative estimation, calculating with the following equation: diameter of zone ¼ a + b  log ðunits in zoneÞ;

where a and b are constants. This contact bioautographic process provides a direct, analytical method for both qualitative and quantitative evaluation of the composition of penicillin mixtures. However, it is time-consuming (72 h) and still involves cumbersome apparatus and technique. A simpler and faster direct bioautographic method was introduced in 1958 [12] and further developed in 1961 [13] for the detection of fungitoxic substances. The compounds were separated on a filter paper, which was sprayed with a conidial suspension of Glomerella cingulata. After incubation the zones of active compounds are clearly visible as spots without fungal growth (lack of fungal hyphae). Because of the disadvantages of the paper chromatography, such as the high time requirement and low separation efficiency, there was a demand to displace the paper chromatography with thin-layer chromatography (TLC) in the bioautographic system. In 1961 the first combinations of layer chromatography with a bioassay were used for the detection of antibiotics and carried out as the agar overlay method [14,15], in which the developed adsorbent layer was covered with seeded agar containing a vital dye and after incubation the clear zones against the colorful background indicated the presence of antibiotics.

350 CHAPTER 6 Conventional and Modern Bioassays

The layer chromatography was first coupled with DB in 1970 [16] utilizing Cladosporium cucumerinum, and later on many others, such as Aspergillus niger, Ascochyta pisi, Botrytis cinerea, Colletotrichum lindemuthianum, Fusarium culmorum, Penicillium expansum, and Glomerella cingulata. Based on the series of experiments it was found that the direct spraying of the adsorbent layer with cell suspension is very useful: easy to perform, rapid, and gives reliable results. Apart from detection of microbiologically active compounds (DB), planar layer chromatography can be hyphenated with specific detection of antioxidant, estrogenic, and various enzyme inhibition activities as well as with immunostaining [17,18]. These are considered to be biotests; however, except for DB and planar yeast estrogen screen (pYES), the bioactivity response is obtained by the use of chemicals or isolated biological agents (e.g., enzyme) and is not originated from a living organism. For example, antioxidant (free radical or superoxide scavenging) tests can be performed with the use of relatively stable free radicals, like 2,2-diphenyl-1-picrylhydrazyl (DPPH•) or 2,20 -azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS+); or with unstable β-carotene or crocin that are easily oxidized [19–21]. Overpressured layer chromatography (OPLC)-DPPH• have been applied for visualization of antioxidant activity [22]. The purple DPPH• is a stable free radical that is neutralized, thereby decolorized in the presence of antioxidants by electrons or hydrogen atom transfers (Figure 6.1a). So the radical trapping components on the developed layer dipped into or sprayed with methanol solution of DPPH• appear as bright spots against a darker background (Figure 6.1b).

N+ O–

O

N

N+

+

N

O

–O

O–

O–

O

N

N

N

O

–O

+ R•

O

N

+

N+

2,2-Diphenyl-1-picrylhydrazyl (DPPH•, free radical, purple)

(a)

H

+RH

+

O–

N+ O

2,2-Diphenyl-1picrylhydrazine (yellow)

(b)

n FIGURE 6.1 Detection of antioxidant activity in the adsorbent layer by reduction of DPPH. (a) The chemical

reaction involved, (b) a representative result (the bright zones indicate the free radical scavenging activity). (Figure 6.1a reproduced from Ref. [18] with permission.)

6.2 Principle and History of Planar Layer Chromatography—Biological Detection 351

The beneficial role of several enzymes in the therapy of various diseases has been revealed, which demanded the development of in vitro detection of the inhibitors or activators of the enzymes. This was the case with the acetyl- and butyryl-cholinesterase (AChE and BChE) inhibitors that may prevent the progress of Alzheimer’s disease, retarding the drop of the acetylcholine level in the brain of the patients [18,23]. Planar layer chromatography was first coupled with AChE inhibition assay in 1964 [24]. This method utilized human plasma as the source of the enzyme and alkaline bromothymol blue to indicate acetic acid (acidic condition) produced from the reaction between the enzyme and acetylcholine. After application of the human plasma, acetylcholine and indicator to the layer and incubation, the AChE inhibitors appeared as yellow spots against the blue background. Later on two further assays were developed directly applying the isolated AChE and BChE enzymes [25–27]. In both cases the chromatoplate is filled up with the solution of the enzyme in a buffer to prevent coagulation of the enzyme. After a short 15-20 min incubation in a humid environment at the temperature of the human body (37 °C), the active chromatographic spots are visualized. In the first method [26] the remaining free enzyme reacts with acetylothiocholine substrate and the originated product thiocholine is stained with Ellman’s reagent. The inhibition zones are revealed as white spots against the yellow background. In the other assay [25,27] a similar procedure is used but 1-naphtylacetate is the substrate, 1-naphtol is formed, and fast blue B salt is the dyeing reagent, creating a bluish background (Figure 6.2). An analogous method can be used for the screening of α- and β-glucosidase inhibitors in the adsorbent layer. The developed layer is covered with the solution of α- or β-glucosidase enzymes and incubated in a humid chamber at 37 °C. The subsequent visualization is carried out respectively by the application of 2-naphthyl-α-D-glucopyranoside or 2naphthyl-β-D-glucopyranoside and fast blue B salt; the appearing clear spots show the active zones [27]. These glucosidase inhibitors are potential antidiabetic, antiobesity, antiviral, antiadhesive, antibacterial, or antimetastatic agents and also have therapeutic value in type 2 diabetes [28,29]. Recently much scientific attention has been focused on the estrogenic endocrine disrupting chemicals that alter the hormonal and homeostatic systems. These xenoestrogens occur naturally as phytoestrogens, flavonoids, zearalenone, lignans, etc. or are produced synthetically, e.g., plasticizers, pesticides, polychlorinated biphenyls [30]. Their structure is similar to estrogen, so they are able to bind and activate estrogen receptors, giving a comparable response in the absence of estrogen. To detect estrogenic compounds in the adsorbent layer a bioassay utilizing transgenic yeast cells was introduced and named pYES [31–35]. In yeast cells there is no estrogen

352 CHAPTER 6 Conventional and Modern Bioassays

OH

OCOCH3 Acetylcholinesterase

1-Naphthyl acetate

+

CH3COOH

1-Naphthol

– 2Cl

H3CO

+

+

NLN

NLN OCH3 Fast blue B salt

OH

H3CO NKN

OH NKN OCH3 Azo dye (purple)

(a)

(b)

n FIGURE 6.2 (a) Reaction of acetylcholinesterase with 1-naphthyl acetate to give 1-naphthol and subsequent formation of a purple azo dye with a diazonium salt.

(b) A representative result (the white zones indicate the acetylcholinesterase inhibitors). (Figure 6.2a reproduced from Ref. [18] with permission.)

receptor. A DNA sequence of the human estrogen receptor was stably integrated into and expressed from the main chromosome of the yeast Saccharomyces cerevisiae. The yeast also received an expression plasmid containing the reporter gene lacZ driven by a promoter containing estrogen response element (ERE). Upon ligand binding to it, the receptor interacts with ERE to cause lacZ (β-galactosidase) expression [36,37]. The activity of the formed β-galactosidase can be measured, e.g., by photometry with the use of colorless o-nitrophenyl-β-D-galactopyranoside [38] or yellow chlorophenol red-β-Dgalactopyranoside [37] as substrates, that, after hydrolyzation, become yellow and red, respectively. A further development of this method is proposed to utilize 4-methylumbelliferyl-β-D-galactopyranoside (MUG) as substrate, obtaining a more characteristic fluorescent signal [31]. The bioassay is carried out as follows [35]: the chromatoplate is covered with cell suspension and put into incubator (3 h, 100% humidity at 30 °C); the yeast cells are grown on it; the substrate is applied by dipping or spraying and the layer is incubated again at 37 °C for 1 h; the reaction is stopped by dipping the layer into the solution of glycine. The detection can be performed by fluorescent measurement. The schematic of the process is shown in Figure 6.3 [32].

6.3 Classification and Comparison of Methods for Detection of Antimicrobials 353

Rf

0.7

t

Silica matrix

Gal-O

O

UV -li gh

0.8

CH3 O

OH

0.9

CH3

0.6 HO

0.4

OH HO

0.5

0.3

+ ERE HO

0.2 0.1

betaGalactosidase

O

O

Fluorescence

lacZ

Yeast cell

n FIGURE 6.3 The steps of pYES. (Reprinted (adapted) with permission from [32]. Copyright (2004) American

Chemical Society.)

6.3 CLASSIFICATION AND COMPARISON OF METHODS FOR DETECTION OF ANTIMICROBIALS Commonly used antimicrobial tests can be categorized into the following three groups [39]: diffusion methods, dilution methods, and bioautography. These screening methods give a simple “yes/no” response for the presence or absence of antimicrobials in effective concentrations, like antibacterial, antifungal, antitumor, or antiprotozoae components in the given matrix. Diffusion and dilution methods are used as routine antimicrobial susceptibility tests; however, bioautography provides additional information pointing to the active substances previously separated. There are other antibiotic tests using radionuclide method, fluorometry, gas pressure method, and flow cytometry, but in this chapter they will not be discussed. In the diffusion methods [9,11,40,41], which are commonly used for the determination of in vitro antimicrobial activity of preferably polar components in solid media, the sample of different concentrations is applied to filter paper disks, put on, or pipetted directly into stainless steel or porcelain cylinders placed on the surface of the agar layers previously inoculated with the test organism or the sample solution is applied into a few millimeter wide wells previously cut into the seeded agarose medium [42,43]. The sample can diffuse from the sources into the agar plate and the inhibition of germination and growth is recognizable as clear zones without bacterial or fungal growth covering the background (Figures 6.4 and 6.5). The diameter of these zones increases with antibiotic concentration. A gradient method, the so-called Etest, is performable with the use of a filter paper strip

354 CHAPTER 6 Conventional and Modern Bioassays

CH

Con

n FIGURE 6.4 Results of agar disk diffusion test using cured composites (CH and Con) and Streptococcus

mutans. (Reproduced from Ref. [43] with permission.)

NZ

NA

P

M

n FIGURE 6.5 Agar-well diffusion showing the inhibitory effect of NA, nisin A; NZ, nisin Z; P, pediocin PA-1;

and M, mutacin B-Ny266 on Bifidobacterium bifidum ATCC 15696. Concentrations of nisin A, nisin Z, pediocin PA-1, and mutacin B-Ny266 were 93, 126, 140, and 400 mg/mL, respectively. (Reproduced from Ref. [42] with permission.)

impregnated with dried antibiotic concentration gradient [6,40], resulting in an drop-shaped inhibition zone (Figure 6.6) [44], so making possible the determination of minimal inhibitory concentration (MIC). The dilution methods [9,40,45] are generally suitable for the determination of the MIC of the analyzed sample and can be carried out in solid as well as in

6.3 Classification and Comparison of Methods for Detection of Antimicrobials 355

liquid phases. Various amount of sample is dissolved in agar medium before solidification or in cell suspension (broth dilution assay). The agar plate is inoculated and after incubation the MIC is determined as the lowest concentration where visible cell growth is inhibited (Figure 6.7) [46]. In the dilution assay the cells can be grown either in tubes containing a minimum volume of 2 mL (macrodilution) or in smaller volumes using microtitration plates (microdilution). After incubation the cell concentration is measured as

Etest macromethod

Etest GRD (glycopeptide resistance detection)

n FIGURE 6.6 Detection of heterogeneous glycopeptide-intermediate phenotype in methicillin-resistant Staphylococcus aureus strain #25. VA, vancomycin; TP, teicoplanin. (Reproduced from Ref. [44] with permission.)

n FIGURE 6.7 (a) Determining of the MIC and minimum fungicidal concentration (MFC) of fluconazole on

potato dextrose agar (PDA) after 3 days of incubation. Note the significant inhibition of the growth at 4.0 μg/mL in comparison with the drug-free control and the complete inhibition of fungal growth at 8.0 μg/mL. (b) Reading of the MIC and MFC of caspofungin on PDA after 3 days of incubation. There was no inhibition (significant or complete) in any concentration tested. (Reproduced from Ref. [46] with permission.)

356 CHAPTER 6 Conventional and Modern Bioassays

turbidity or absorbance with the help of vital dye reagent [6,7]. It follows from the application of usually serial twofold dilutions that the MIC is not an absolute value but is between the lowest concentration that inhibits the cell growth and the next lower concentration. Thus, the MIC determined by dilution method may be considered to have an inherent variation of the dilution series. Bioautography also belongs to in vitro antimicrobial susceptibility/ resistance detection methods. It consists of a planar layer separation and a biological detection step. The separation can be carried out by, for example, TLC, high-performance thin-layer chromatography (HPTLC), or OPLC, but column chromatography cannot be applied. After the elimination of the mobile phase, the chromatoplate is transferred for the biological detection. Based on the different methods of biodetection, bioautography can be classified as contact, immersion or agar overlay, and direct [39]. The first bioautography method introduced was the contact one [8], when the developed, dried chromatogram is placed onto the surface of an inoculated agar layer for hours to make possible the diffusion of the separated compounds into the agar [9,47]. After appropriate incubation the antimicrobial zones appear as clear zones indicating the lack of cell growth (Figure 6.8). During agar overlay bioautographic experiments, the chromatogram is immersed into inoculated agar, or the agar is poured onto it, and the contact between the separated substances with the microbial cells takes place [11,18]. The antimicrobial effect becomes visible after a suitable incubation time (Figure 6.9). In both contact and immersion bioautographic methods, the use of vital dyes is usually not necessary, but can enhance the visibility of the inhibition zones. Several different approaches have been elaborated for the bioautography tests, which has resulted in the introduction of DB, summarized in more papers (e.g., [9–11,48]). In this further developed method the microbial

(a)

(b)

(c)

n FIGURE 6.8 The steps of the contact bioautography. (a,b) The developed, dried chromatoplate is placed onto

the surface of the inoculated agar plate to let the separated compounds diffuse from the adsorbent to the agar plate. (c) After incubation clear zones (lack of cell growth) indicate the presence of antimicrobial active compound.

6.4 The Basic Elements of Direct Bioautography (DB) 357

(a)

(b)

(c)

n FIGURE 6.9 The steps of the agar overlay bioautography. (a,b) Inoculated agar is evenly distributed over the developed chromatographic layer. (c) After incubation, spots with lack of cell growth indicate the locations of the antimicrobial substances.

detection is performed directly, in situ in the adsorbent layer—that is, the whole process including separation and biotest is accomplished in one chromatoplate without the use of an agar layer. For this purpose, a developed chromatographic layer is filled up with liquid cell suspension and later on the bioautogram is visualized. Thus the basic issue associated with the limited diffusion of lipophilic compounds to the agar is solved. DB, as a combination of planar layer chromatography with in vitro bioassay, is a very useful tool to search for and/or monitor active components of complex mixtures, where (1) planar layer chromatography is a flexible, high-throughput, open system that enables the elimination of the disturbing mobile phase from the adsorbent layer between the development and the biological detection, as well as making possible the parallel developments of the sample and the use of a large number of more or less specific reagents for the chemical characterization of the compounds of interest [49]; (2) biological detection method is faster (0.5-2.5 h including the separation as well) than the other previously mentioned tests and give information not only about the crude sample but also its separated components.

6.4 THE BASIC ELEMENTS OF DIRECT BIOAUTOGRAPHY (DB) DB is the combination of planar chromatographic separation with in vitro bioassay to notice antimicrobial compounds in various complex matrices. The biological detection can be performed immediately after the spot-wise application of the sample to the adsorbent layer without chromatographic development. This process is named the dot-blot test, with which the antibacterial activity of the whole sample can be assessed, but not its components, so it is suited only for preliminary investigation.

6.4.1 Conventional and forced-flow layer separation systems: Advantages of OPLC to TLC/HPTLC in DB OPLC coupled with effect-directed analyses is an especially beneficial construction, because the use of forced-flow technique gives better separation and more compact spots [22,48,50,51], making possible more selective and

358 CHAPTER 6 Conventional and Modern Bioassays

more sensitive detection of biologically active compounds. Comparing conventional TLC and OPLC separation of thyme [52] and oregano [53] essential oil components using the same mobile phase systems, the efficient separation of active components was achieved only by OPLC, which was confirmed by direct bioautographic detections as well (Figure 6.10).

6.4.2 Detection methods in DB The biological detection can be divided into two steps: (1) wetting of the developed, dried chromatoplate with cell suspension; and (2) visualization of the inhibition zones [9–11]. After ascertaining that the mobile phase has been totally eliminated from the adsorbent layer, the chromatoplate can be dipped into or sprayed with cell suspension. Thus, the cells (the diameter of the bacterium cell is between 0.2 and 2 μm) are adsorbed on the layer and the ˚ ¼ 6 nm) are filled up with liquid nutripores of the adsorbent (usually 60 A ent medium. The dipping, using house-made or commercially available chambers or devices (Figure 6.11), is preferred to obtain a homogenous suspension film on the surface of the adsorbent layer. However, it has to be kept in mind that the separated components can leave the adsorbent layer and be dissolved into the cell suspension. In this case the sensitivity of the test is decreased; moreover, the cell suspension is not suitable for further dipping because there is a risk that the dissolved component will alter the result. To avoid cross-contamination, a single rolling device was developed for covering the chromatoplate with cell suspension (Figure 6.12) [54]. Generally, the bioautogram (chromatoplate filled up with cell suspension) is put into a chamber providing 100% humidity at an appropriate temperature to ensure an environment (condition) where the cells can live and multiply directly on the adsorbent bed. After suitable incubation the bioautogram is visualized by dipping in or spraying with aqueous solution of vital dye. The generally used yellow tetrazolium salts (e.g., MTT—3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) are reduced by the dehydrogenases of living cells to bluish/purple formazans (Figure 6.13), so the inhibition zones of the active compounds appear as clear spots against a colorful background. However, it has to be noted that the vital dye is a co-factor in the system that can influence the result. One of the recent developments of bioautography is aimed at eliminating the use of the vital dye and shorter assay time. Key to this alternative was the introduction of luminescent bacterium strains as test organisms, such as the naturally luminescent Vibrio fischeri [55] (renamed to Aliivibrio fischeri), which is frequently found in symbiotic relationships with marine animals like squids and some fishes. These bacteria emit

6.4 The Basic Elements of Direct Bioautography (DB) 359

50

l/c

l/d

Scan (mm)

l/b

50

l/a

A B

0

1

2

1

2

105

105

0

C

Detector signal (λ = 275 nm)

ll/a

ll/b

ll/c

ll/d

A

Scan (mm)

50 0

C

1

2

1

2

105

105

0

50

B

Detector signal (λ = 275 nm) n FIGURE 6.10 Separation and biological detection of the main components of Thymus vulgaris L. essential oil by (I) TLC and (II) OPLC. Lane 1, standards, 10 μg each of thymol (A), carvacrol (B), and linalool (C). Lane 2, Thymus vulgaris L. oil, 25 μg. (a) densitometric evaluation of lane 1 before biological detection; (b) densitometric evaluation of lane 2 before biological detection; (c) the developed layer under UV illumination (254 nm) before biological detection; (d) The bioautogram obtained by use of luminescent Pseudomonas syringae pv. maculicola; the emitted light was visualized immediately after inoculation by use of a cooled camera with 15 min exposure time. (Reproduced from Ref. [52] with permission.)

360 CHAPTER 6 Conventional and Modern Bioassays

4

3

2

1

(a)

(b)

n FIGURE 6.11 House-made (a) and commercially available (b; Camag, Muttenz, Switzerland)

immersion devices. 1, Glass sheet; 2, silicone tube with wire; 3, clips; 4, forceps. 2 4

5

1

6

3

n FIGURE 6.12 Schematic of the rolling device: 1, Plastic-wrapped roll; 2, pad; 3, cutting board; 4, stop

bar; 5, HPTLC plate; and 6, forerun. (Reproduced from Ref. [54] with permission.)

N

Reductase H+, 2e–

N N

N+ N

N N

N H N N

S S

MTT yellow

(a)

MTT-formazan violet

(b)

n FIGURE 6.13 Detection of antimicrobial activity in the adsorbent layer by reduction of tetrazolium salt MTT

to MTT-formazan with bacterial dehydrogenase. (a) The chemical reaction involved, (b) a representative bioautogram with the use of Bacillus subtilis as test organism (bright spots indicate the inhibiting effect). (Figure 6.13a reproduced from Ref. [18] with permission.)

6.4 The Basic Elements of Direct Bioautography (DB) 361

bluish-greenish light (λ ¼ 490 nm) that is dependent on high cell density; this so-called quorum sensing is regulated by a cell-permeable N-(3-oxohexanoyl)-L-homoserine lactone [56,57]. Actually the light is a side-product of a chemical reaction, in which the oxidation of a long-chain aliphatic aldehyde is catalyzed by the luciferase enzyme and the excess free energy is liberated in this form (Figure 6.14). Recently the use of engineered (genetically modified) luminescent bacterial cells was established for different in vitro as well as in vivo biotests (e.g., [58,59]). The emission of light by these transgenic luminescent bacteria is controlled by simple, well-defined constitutive promoters, reflecting cellular energy status more directly than that of naturally luminescent bacteria. Gram-negative Arabidopsis pathogen Pseudomonas syringae pv. maculicola chromosomally tagged with luxCDABE gene from Photorhabdus luminescens [58] and the Gram-positive soil bacterium Bacillus subtilis, plasmid-tagged with luxABCDE have been applied for DB [60]. These bacteria emit light continuously, dependent only on metabolic activity, thus viability. The method in which DB is carried out with luminescent bacteria is also called TLC-bioluminescence [25,55]. This method is very fast and simple and comprises the planar layer chromatographic development, the immersion of the developed plate into the suspension containing luminescent cells and the documentation of the bioautogram by taking a photo with a charge Luciferase RCOOH + FMN + H2O + hv

RCHO + FMNH2 + O2

(a) OO

P

O- OH

O

OH

CH C H2

CH CH

H2C H3C

N

H3C

N H

R = C8 – C16

OH H N

O NH

O

(b)

Reduced flavin mononucleotid FMNH2

(c)

n FIGURE 6.14 Detection of antimicrobial activity in the adsorbent layer by the use of a luminescent

bacterium strain. (a) The chemical reaction involved in bioluminescence; (b) the structure of FMNH2; (c) a representative bioautogram with the use of Pseudomonas syringae pv. maculicola as test organism (black spots indicate the inhibiting activity).

362 CHAPTER 6 Conventional and Modern Bioassays

coupled device (CCD) camera. The camera should be sensitive and a cooled one, to enhance the signal-to-noise ratio. The dipped layer has to be kept damp and therefore it is put, e.g., into an air-tight transparent glass cage [60]. The presence of bioactive compounds reduces the intensity of the emitted light, marked by dark spots in the bioautogram (Figure 6.14c). The detection of the anti-quorum sensing-active compounds in the developed adsorbent bed can be carried out by the use of violacein inhibitory assay. The purple violacein is the quorum sensing-controlled product of Chromobacterium violaceum bacterium. So the bright zones (lack of the characteristic color) can indicate the components responsible for the inhibition of quorum sensing [61]. Direct bioautographic antifungal tests are carried out in a similar way to the antibacterial test [16,62–64]. The fungal spore suspension is applied to the developed, dried adsorbent layer and put into a moisture chamber for the fungal growth to take place. After 3-4 days incubation, in many cases the mycelium of the test organism is visible, so the inhibition zones can be seen as spots free of fungal growth (Figure 6.15). Otherwise, if Botrytis cinerea Penicillium expansum Rhizopus stolonifer

Surfactine RF = 0.75 Iturin RF = 0.4 Bacillomycin RF = 0.3 Fengycin RF = 0.1

1

2

1

2

1

2

n FIGURE 6.15 TLC-bioautography shows the absence of mycelial growth of Botrytis cinerea, Penicillium

expansum and Rhizopus stolonifer. The spot at RF ¼ 0.3 corresponded to bacillomycin produced by Bacillus subtilis UMAF6614 (1, standard), and the spot at RF ¼ 0.4 corresponded to iturin A produced by Bacillus amyloliquefaciens PPCB004 (2). (Reproduced from Ref. [64] with permission.)

6.5 Practice of DB 363

(a)

H

D

Ac

M

(b)

H

D

Ac

M

n FIGURE 6.16 TLC plates indicating anti-Candida activity of Curtisia dentata extracts. The extracts (loaded

from left to right) were n-hexane (H), dichloromethane (D), acetone (Ac), and methanol (M). The plates were sprayed with Candida albicans culture (a) or vanillin-sulfuric acid (b). (Reproduced from Ref. [63] with permission.)

the fungi are not characterized by pigmented hyphae, spores, or conidia, or if the contrast needs to be enhanced, vital dyes are used to stain the cells (Figure 6.16), as in the case of Candida albicans assay, which requires only 2 h incubation time before staining [63].

6.5 PRACTICE OF DB 6.5.1 Optimization of layer liquid systems The aim of planar layer chromatography in the direct bioautographic system is, on the one hand, separation of the sample components from each other and from the matrix components; and on the other hand to prepare a chromatogram that is suited for biological detection. In DB bacterial cells attach to the adsorbent, so the condition of all the types of adsorbents needs optimization to allow life and growth of the test microorganisms on it. An early study advised against cellulose, polyamide, and alumina [65]. A more detailed report showed that none of the four adsorbents (silica gel, polyamide, alumina, cellulose) could detect activity of all selected antibiotics. During the long incubation time the adsorbent layers wet with aqueous cell suspension can loosen [9,66]. To avoid adsorbent detachment, preconditioning of the TLC plates (e.g., heating at 120 °C for 2 h, then cooling for 1 h in the desiccator) has been recommended [9,66]. Preconditioning, however, was found unnecessary in other studies [67,68]. Precleaning of the chromatographic plates with appropriate solvent is vital [69]. The binders used in adsorbents should also be taken into account because they can also have undesirable biological activity.

364 CHAPTER 6 Conventional and Modern Bioassays

One of the earliest problems found for DB was remnants of solvent/mobile phase, which should be minimized. Therefore, special emphasis has to be placed on the selection of the mobile phase. Volatility is advantageous [65]. Tetrahydrofuran was found inappropriate itself, but other common solvents/mobile phases can be used [9]. If in doubt, the use of solvent controls is advised [70]. The quality as well as stability must be taken care of. The most common problems are impurities or deterioration products in the solvents (e.g., chloroform converts to phosgene and ethers to peroxide). The chemicals used as stabilizers (e.g., ethanol or amylene in the case of chloroform) are also able to disrupt not only the separation but the biological detection as well. To avoid the influence of the solvent system on the growth of the microorganism, it has to be totally eliminated from the adsorbent after the development, e.g., by air flow. The most common critical point in the solvent selection is the use of acid or base [10,16]. Basically, their application is not preferred. Otherwise, the elimination of ammoniac is relatively easy, by extended air stream or by the use of vacuum. It has to be noted that the evaporation of formic and acetic acids from the adsorbent is not easy and the low pH caused is not suited to the microorganisms generally used in DB. The RP-18W stationary phase was intended to be used for bioassay; however, it has a natural acidic character that inhibits the cell growth. This disturbing effect was eliminated by dipping the developed plate into highcapacity basic buffer before the performance of the biological detection [35]. This process may provide a chance to set the appropriate pH of the chromatoplate developed with acidic or basic eluent as well.

6.5.2 Use of optimum conditions for test cell system DB brings the test microbes in direct proximity to the adsorbent as well as the substances separated in the adsorbent, so diffusion is minimal compared with assays using agar layers. Consequences are the highest assay sensitivity so that both hydrophilic and lipophilic substances can be examined, and that conditioning microbial life has some special aspects beyond traditional culturing ones. Here we will focus on optimizing DB for bacterial test organisms, but the principles are the same for fungi. Choice of test organism: Selection of the test microbe(s) is primarily governed by the purpose of the study and should cover the widest possible range of characteristics that can account for differences in chemical sensitivity. Such a characteristic is the basic cell wall structure (Gram stainability). Ecological traits, e.g., affinity to different environmental niches, to oxygen, to other, for example, host organisms (pathogenicity), also bring substantial variance in chemical sensitivity and may have relevance to the substance examined. It is wise to try strains from standard collections for the sake

6.5 Practice of DB 365

of comparability. Detection and cultivation methods should suit the microbe’s biology and vice versa. For some popular bacteria (the Gramnegative Escherichia coli, the Gram-positive Bacillus subtilis) optimization of DB has been carried out [67,68,71,72]. A list of employed bacterial species excelling in cultivability and chemical sensitivity can be found under Section 6.6.1 in Table 6.2. Physiology of test organism: Test cells on the TLC plate should be in sufficient number in a properly active physiological state, the product of which gives a visible signal, allowing distinction between treatment (substance separated in the chromatographic layer) and control (the background). DB, more than other tests, requires high metabolic activity because of the relatively small room the chromatoplate can afford for the biomass. (The high requirement can be a disadvantage, as there may well be situations when cells with attenuated vigor or specific state are to be studied. Nevertheless, DB is worth a try even in these cases.) A detecting reagent (dye) or natural or transgenic bioluminescence assists during DB to make growth and/or metabolic activity visible. As always, detection influences the process to be detected. The popular tetrazolium salts and bioluminescence both need electron donors, such as nicotinamide adenine dinucleotide hydrogen (NADH) and flavine adenine dinucleotide hydrogen (FADH), consuming considerable energy. While natural bioluminescence remains physiological, the dyes can be detrimental for some strains. Bioluminescence enables continuous, real-time monitoring, while the dye only reports from a limited time window, usually a couple of minutes, because formazan is quite stable. Luminescence detection does not need rewetting of the plate. Thus, optimizing of DB takes much less effort and time with bioluminescence. Transgenic luminescence has the added benefit over the natural one in that it is not dependent on evolutionally wired control mechanisms, e.g., quorum sensing, that can make interpretation of toxicity difficult. The growth phase was the earliest physiological parameter examined, and authors repeatedly state that cells on the TLC plate must be in the logarithmic growth phase featuring a homogeneous, metabolically active population for best display of sensitivity [69,70,89,90]. Homogeneous population results in sharp inhibition zone boundaries. Botz and co-workers correlated the early logarithmic phase (when cells are at the beginning of division) with a high amount of adenosine triphosphate (ATP) and stainability with tetrazolium dyes [71,72]. The best stage of growth shows high ATP content, without protein degradation [69]. We illustrate the effect of growth phase in Figure 6.17. Quantity is also needed from the quality. How to best achieve this in practice? First, a liquid culture should be set up and allowed to grow until the

366 CHAPTER 6 Conventional and Modern Bioassays

0.1

0.5

1 μg

Ochratoxin A

(a)

0.1

0.5

1 μg

0.1

Ochratoxin A

(b)

0.5

1 μg

Ochratoxin A

(c)

0.1

0.5

1 μg

Ochratoxin A

(d)

n FIGURE 6.17 The antibacterial effect of ochratoxin A on Pseudomonas savastanoi pv. phaseolicola bacterial

cells in different growth phases. The growth status of applied cells in layers: (a) Lag (before log), (b) late logarithmic, (c) stationary, (d) late stationary, before death.

desired phase, usually around 106 to 107 cells/mL. Older cell population can cause less sharp inhibition zones [68,72]. The developed TLC plate is dipped in this suspension. Excess liquid is removed and the damp plates are incubated in a high humidity. Cells brought onto the developed TLC plate encounter a new environment. Time is needed for them to adhere, acclimate (continue metabolic activity) and/or divide to an appreciable extent. If bioluminescent strains are used, the plates are simply monitored over time. Detection is dependent on the performance of the cooled CCD camera. Rather specific for natural luminescence is its quorum sensing control that boosts light emitting at higher cell densities. So it may be rewarding to wait until the late logarithmic phase, when this happens. In the case of a dye, the seeded TLC plates undergo an incubation period before staining. The length of this period must be determined well in advance, as progress in cell number and/or metabolic activity is too problematic to follow in real time on the plate. The optimum incubation time for Gram-positive spore-forming test bacteria was between 8 and 12 h while for Gram-negative enterobacteria it is between 3 and 6 h [69]. Too short a time decreases sharpness of inhibition zones (but they are bigger); after too long a time overgrowth of these zones occurs [68,91]. When higher cell densities (late logarithmic phase cells, about 109 cells/mL) are used for dipping, shorter times and so a higher assay sensitivity may be realized, such as for the biodetection of trans-resveratrol [91] and ochratoxin A (Figure 6.18).

6.5 Practice of DB 367

0.1

(a)

0.5 Ochratoxin A

1 μg

0.1

(b)

0.5

1 μg

Ochratoxin A

n FIGURE 6.18 Effect of incubation time on the size of inhibition zones. (a) 1 h, (b) 18 h incubation before

staining.

In some applications like BioArena a long (several days) poststaining incubation period was found useful [91]. In this case, maintaining sterility on the TLC plate is important, while if monitoring plates for less than one day it is not. Increasing the viscosity of the liquid medium, e.g., with 0.5% agarose, can help retain more medium, and bacterial cells in it, on the chromatoplate after immersion. However, too high a viscosity can negate the profit that DB offers [67,77,80]. Principally, any medium that is able to furnish the test species with nutrients can be used. Culture media should be from the same batch throughout the bioassay, since such media are frequently prepared from digests which are intrinsically variable [70]. We observed that some complex media components like proteose peptone can deteriorate within a month, even when held in a fridge. Bacterial growth in such a medium is unaffected but it will not perform well in DB. We encourage investigators to experiment with test strains, materials, and conditions in their specific systems. This approach could bring new scientific discoveries, as the possible applications are far more than the realized ones. There is a lot more to be learned about optimum conditions. If a quick result is needed, rely on one of the established procedures, especially those involving optimization measures. These procedures can only serve as guidelines as even seemingly slight variations may lead to very different results. DB is very sensitive to variations of any condition. Standardization of the procedure is pivotal to keep data comparable to other data.

368 CHAPTER 6 Conventional and Modern Bioassays

6.5.3 Influencing factors in DB We have described the influence of the separation systems and the condition of the cell suspension applied. It was stated that forced-flow layer liquid chromatographic separation has an advantage, providing better resolution and more compact chromatographic spots that enable the detection of the separated bioactive substances in lower concentration. The growth phase and the concentration of the cell suspension are also fundamental to obtaining suitable results. Apart from these, the seeding method in the bioassays also affects the background of the bioautogram and the compactness of the inhibition zones, and thus the sensitivity of the method. Comparing the spraying and the dipping processes, the dipping one gives more homogeneous background and is more intensive as well (Figure 6.19). Using dipping or a rolling device makes no significant difference in the background; however, the rolling process provides more compact, narrower inhibition zones without any tailing and higher signal-to-noise ratios (Figure 6.20) [54]. Most recently a new methodological improvement was published to make the zones obtained by bioassays sharper [35]. In the pYES test applying a silica gel adsorbent layer, the diffusion during the necessarily long incubation time causes wide (diffuse) fluorescence spots. To eliminate this disturbing zone-broadening effect, the use of a wettable, but diffusion controlled, adsorbent layer is introduced, namely HPTLC RP-18W (Figure 6.21). With

A B

C

1

(a)

2

1

2

(b)

n FIGURE 6.19 Antibacterial activity of thyme essential oil components against Bacillus subtilis. The

bioautograms were obtained by (a) spraying, (b) dipping of the layers into cell suspension and MTT vital dye solution. The incubation time was 2 h. Lane 1, standards, 5 μg each of thymol (A), carvacrol (B), and linalool (C). Lane 2, Thymus vulgaris L. oil, 25 μg. Chromatographic conditions: OPLC separation on normal particle size silica gel layer with chloroform, external pressure 50 bar, flow rate 400 μL/min, initial flash volume 450 μL, separation volume 4900 μL, and total development time, 746 s.

6.5 Practice of DB 369

n FIGURE 6.20 Typical BioLuminizer images of octhilinone (top) and methylparaben (bottom) HPTLC

plates after dipping (left) and rolling (right). Applied amount of octhilinone from left to right: 3.0, 2.5, 2.0, 1.5, and 1.0 μg; applied amount of methylparaben from left to right: 2.0, 1.5, 1.0, 0.7, 0.5, and 0.1 μg. (Reproduced from Ref. [54] with permission.) Amount (pg/zone) 1250 – 25 15 × 10

3 – 0.3 × 103

1250 – 25 3 – 1.25 × 103

62.5 × 10

1250 × 103 – 25 × 103 312.5 × 103 – 6.25 × 103

n FIGURE 6.21 pYES after 24 h of aqueous cultivation (cell growth) and incubation, both on the plate.

Yeast cells grew on the HPTLC RP-18W plate and reacted with the six endocrine disrupting compounds (E2, 17 β-estradiol; E1, estrone; EE2, 17 α-ethinylestradiol; E3, andestriol; BPA, bisphenol A; NP, 4-n-Nonylphenol) applied in descending concentrations on the plate; sharp-bounded, blue fluorescent 4-methylumbelliferone zones were obtained after 24 h. (Reproduced from Ref. [35] with permission.)

the improved procedure, much lower detection and quantification limits (LOD and LOQ) could be reached. The most sensitive detection was achieved in the case of 17 β-estradiol (E2) with LOD ¼ 0.5 pg/zone and LOQ ¼ 1 pg/zone. Although this adsorbent layer was chiefly meant to solve the issue in the case of the pYES assay, its usability was established also for

370 CHAPTER 6 Conventional and Modern Bioassays

DB after a neutralization process with a high-capacity buffer (Figure 6.22). While the RP layers are too acidic (pH ¼ 4.8) for the growth of microorganisms, their neutralization is therefore needed to adjust the appropriate pH before DB. While performing planar layer chromatography-bioassay, the possible pitfalls should be kept in mind. It has been reported that the inhibition effect of some natural compounds—for example, the stilbene trans-resveratrol, the flavonoid genistein, and other compounds—which interfere with cell growth was underestimated, because of the reduction of tetrazolium salts by the investigated compounds. It means that a false negative result may be obtained (Figure 6.23) [92–94]. As opposed to the MTT assays, in AChE and BChE inhibiting as well as antioxidant tests, false positive results were observed in several cases. Nonactive simple aldehydes and amines may give inhibition zones in cholinesterase tests [95], and in DPPH• assay, compounds with hydrophobic character may appear as clear white zones similar to the yellow spots of the antioxidants [19]. BioArena is the further development of DB to examine the mechanism of the antimicrobial effect [10,89]. Therefore, its aim is to search for such

n FIGURE 6.22 General applicability of RP-18W plate proven by transfer to HPTLC-Bacillus subtilis bioassay. Chromatogram of plant sample extracts obtained after plate neutralization, 3 h incubation with Bacillus subtilis suspension and 10 min MTT substrate reaction; the same zone sharpness was obtained as for HPTLC-pYES; colorless zones (marked *) indicate antibiotics. (Reproduced from Ref. [35] with permission.)

6.6 Applications of Planar Layer Chromatography-DB 371

n FIGURE 6.23 The arrow shows a typical false negative result in antibacterial assay using MTT vital dye reagent for the visualization.

co-factors (chemicals) that can alter (decrease or enhance) the bioactivity of the separated components (for further details see Chapter 8). Based on the essence of the BioArena system, we have to note that all endogenous as well as exogenous substances, including even the broth constituents can influence the sensitivity of the method. The best practice is to optimize the system with appropriate positive and negative controls.

6.6 APPLICATIONS OF PLANAR LAYER CHROMATOGRAPHY-DB 6.6.1 Test organisms for DB In DB assays only those microorganisms can be applied that can live and multiply in the chromatographic adsorbent layer. A wide range of bacteria fulfill this requirement; however, even so there are some favored ones like Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Aliivibrio fischeri. Among fungi the use of spore-producing ones is widespread, such as Aspergillus, Colletotrichum, Cladosporium strains, and the yeasts Candida albicans and Saccharomyces cerevisiae [96,97]. Tables 6.1 and 6.2 contain the fungal and bacterial strains that have been introduced for DB. It has to be noted that the TLC-agar overlay bioautographic method is also very popular for screening the antimicrobial components [111].

372 CHAPTER 6 Conventional and Modern Bioassays

Table 6.1 The Fungal Strains Utilized in Planar Layer Chromatography-DB Adsorbent Layer

Strains

Detection Agent

Reference

Silica gel

Alternaria alternata Ascochyta pisi Aspergillus cellulosae Aspergillus flavus Aspergillus fumigatus Aspergillus niger

ws ws Iodine vapor ws INT ws Iodine vapor ws ws ws MTT INT TTC ws ws ws ws ws ws ws ws INT MTT ws MTT ws MTT INT ws ws ws INT

[98] [16] [99] [100] [63] [16,101] [99] [100] [100] [16,64] [97,102,103] [63] [104] [105] [16] [106] [105] [53,62] [53,62] [53,62] [16] [63] [107] [16] [101,108] [109] [103] [63] [110] [16,64] [64] [63]

Aspergillus nomius Aspergillus parasiticus Botrytis cinerea Candida albicans

Cladosporium cladosporioides Cladosporium cucumerinum Cladosporium herbarum Cladosporium sphaerospermum Colletotrichum acutatum Colletotrichum fragariae Colletotrichum gloeosporioides Colletotrichum lindemuthianum Cryptococcus neoformans Fusarium culmorum Fusarium oxysporium Fusarium sambucinum Magnaporthe grisea Microsporum canis Penicillium digitatum Penicillium expansum Rhizopus stolonifer Sporothrix schenckii

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; TTC, 2,3,5-triphenyltetrazolium chloride; ws, without staining; INT, p-iodonitrotetrazolium violet.

6.6.2 Detection of biologically active compounds with OPLC-DB Bacteria, fungi, and yeast have been introduced as test microorganisms in the OPLC-DB system. The antifungal components of essential oils of Scaligeria tripartita [112], Origanum onites [53] and Angelica sinensis [113] as

6.6 Applications of Planar Layer Chromatography-DB 373

Table 6.2 The Bacterial Strains Utilized in Planar Layer Chromatography-DB Adsorbent Layer

Strain

Detection

Reference

RP-18W Cellulose

Bacillus subtilis Bacillus subtilis Escherichia coli Bacillus subtilis Escherichia coli Bacillus subtilis Escherichia coli Aliivibrio fischeri Aliivibrio fischeri (also referred as Vibrio fischeri) Bacillus cereus Bacillus subtilis

MTT MTT, INT, and TNBT

[35] [65] [65] [65] [65] [65] [65] [73] [25,55] [74] [60,67,71] [75] [60] [76] [63] [77] [78,79] [78] [78] [63,75] [68,72,77,80,81] [80] [82] [83] [84] [75] [81] [81] [81] [81] [63] [77,80] [85] [60] [66] [86] [63,87] [77,81,83] [83] [83] [88]

Neutral alumina Polyamide Cyanopropyl Silica gel

Bacillus subtilis (transgenic) Enterobacter cloacae Enterococcus faecalis Enterococcus faecalis Erwinia amylovora Erwinia carotovora Erwinia carotovora subsp. atroseptica Escherichia coli Klebsiella pneumoniae Listeria monocytogenes Methicillin-resistant Staphylococcus aureus (MRSA) Micrococcus kristinae Micrococcus luteus Mycobacterium aurum Mycobacterium avium Mycobacterium microti Mycobacterium scrofulaceum Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Pseudomonas syringae pv. maculicola (transgenic) Pseudomonas savastanoi pv. phaseolicola Salmonella paratyphi Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Xanthomonas euvesicatoria

MTT, INT, and TNBT MTT, INT, and TNBT Luminescence Luminescence MTT MTT INT Luminescence TTC INT MTT MTT MTT MTT INT MTT MTT TTC MTT No data INT MTT MTT MTT MTT INT MTT INT Luminescence MTT TTC INT MTT MTT MTT MTT

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; TTC, 2,3,5-triphenyltetrazolium chloride; ws, without staining; INT, p-iodonitrotetrazolium violet; TNBT, tetranitro blue tetrazolium.

374 CHAPTER 6 Conventional and Modern Bioassays

well as root extract of Diospyros virginiana [114] were investigated by OPLC-DB using plant pathogenic Colletotrichum acutatum, Colletotrichum fragariae, and Colletotrichum gloeosporioides species. In the cases of these strains, after appropriate incubation time the inhibition zones, the spots without fungal growth, are clearly visible. For appropriate separation of S. tripartita root, stem, leaf, and fruit essential oil components, a fine particle silica gel 60F254 20 cm  20 cm aluminum sheet sealed at all four edges was used for the double infusion OPLC developments (Figure 6.24) [112]. The conditions were as follows: 5 MPa external pressure; 300 μL flash and 4400 μL total volume of solvent; 500 μL/min flow rate; 534 s elution time. The mobile phases were n-hexane-Et2O (95:5, v/v) and n-hexane, successively. Based on the direct bioautographic results, the oils contained more active components and root oil was the most active. The presence of antifungal components in A. sinensis essential oil was confirmed with OPLC-DB [113]. The separation was carried out on 10 cm  20 cm fine particle size silica gel 60F254 plates with n-hexane-ethyl acetate 9:1 (v/v) using the following conditions: 5 MPa external pressure; 150 μL flash and 1800 μL total volume of solvent; 150 μL/min flow rate; 730 s elution time. Two active compounds at RF ¼ 0.29 and 0.35 were identified with standards as (Z)-ligustilide and apiol, which showed a nonselective antifungal effect against the three Colletotrichum strains. For antifungal [53] DB assay the components of O. onites essential oil were separated by infusion OPLC on 10 cm  20 cm normal particle size silica

SSL 2 mL

Second run with hexane UV254 nm

First run with hexane-Et2O (95:5, v/v) UV254 nm

(a)

(b)

SSL 4 mL

SF 2 mL

SF 4 mL

SR 2 mL

SR NE231C NE231C 4 mL 2 mL 4 mL

Second run with hexane vanillin-sulfuric acid + heating, visible

(c)

n FIGURE 6.24 Chromatogram of off-line OPLC separation of Scaligeria oils under UV and visible with (a) hexane-Et2O, (b) hexane, and (c) visualized by vanillin-sulfuric acid reagent. 2 and 4 μL of 16, 18, and 16 mg/mL with 10 mm band size of SSL, SF, and SR oils were applied at 27 mm measured from the bottom edge of the adsorbent layer. SSL, Scaligeria tripartita stems and leaves oil; SF, S. tripartita fruits oil; SR, S. tripartita roots oil; NE231C, reference oil of Salvia recognita. (Reproduced from Ref. [112] with permission.)

6.6 Applications of Planar Layer Chromatography-DB 375

gel 60F254 layer with toluene-ethyl acetate 99:1 (v/v). The separation conditions were set as: 5 MPa external pressure; 300 μL flash and 2085 μL total volume of solvent; 250 μL/min flow rate; 850 s elution time. Both major components thymol and carvacrol displayed antifungal activity against the organisms tested. For antibacterial DB test of O. onites oil [115] the OPLC development was carried out on 20 cm  20 cm aluminum backed, normal particle silica gel 60F254 plates. Single infusion OPLC development was performed with dichloromethane as a mobile phase. The external pressure was 50 bar, the rapid mobile phase flush was 450 μL, the mobile phase flow rate was 400 μL/min, and 5330 μL total volume was admitted during 743 s development time. The same thymol and carvacrol gave antibacterial zones against Bacillus subtilis soil bacterium, made visible with the use of MTT vital dye reagent. In this paper, BioArena (the further development of DB) investigations of the two active components were also demonstrated to confirm the possible role of formaldehyde in the antibacterial activity of thymol and carvacrol (Figure 6.25). Antibacterial activity of aflatoxin B1, B2, G1, and G2 separated by OPLC was established against Pseudomonas savastanoi pv. phaseolicola (Figure 6.26) [66]. The separation was achieved on 20 cm  20 cm normal particle size silica gel 60F254 with the mobile phase chloroform-acetone 9:1 (v/v). The OPLC parameters were set as: 5 MPa external pressure; 300 μL flash and 5000 μL total volume of solvent; 400 μL/min flow rate; 685 s elution time. After the visualization of the bioautogram with MTT, the

A B

C 1

(a)

3

1

(b)

2

3

1

(c)

2

3

1

(d)

2

3

1

2

3

(e)

n FIGURE 6.25 The effect of formaldehyde capturer L-arginine and formaldehyde inducer Cu(II) ions on

the antibacterial effect of O. onites oil components, separated by infusion OPLC, against Bacillus subtilis soil bacterium. Lane 1, standards, 10 μg each of thymol (A), carvacrol (B), and linalool (C). Lane 2, Origanum oil, 15 μg, Lane 3, Origanum oil, 30 μg. Visualization: (a) vanillin-sulfuric acid reagent (exactly the same conditions as for b except for the oil sample application and the visualization); (b) UV 254 nm; (c) control (only cell suspension); (d) 4 mg/mL L-arginine in the cell suspension; (e) 3 mg CuSO45H2O per 100 mL cell suspension. (Reproduced from Ref. [115] with permission.)

376 CHAPTER 6 Conventional and Modern Bioassays

chromatographic zones of all four aflatoxins appeared as clear spots against the bluish background. The same test organism was applied to study the antibacterial effect of wine ingredients [116] as well as ascorbigen and methylascorbigen (Figure 6.27) [117,118] separated with OPLC. Wine components and ascorbigens were separated on 20 cm  20 cm silica

Aflatoxin B1 B2 G1 G2 n FIGURE 6.26 Inhibitory effect of aflatoxins on the growth of Pseudomonas savastanoi pv. phaseolicola. The four aflatoxins (2 μg of each were applied to the plates) were, from top to bottom, aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2. (Reproduced from Ref. [66] with permission.)

MeAG

AG

1

(a)

2

3

4

1

(b)

2

3

4

1

2

3

4

(c)

n FIGURE 6.27 The effect of arginine (Arg) and reduced glutathione (GSH), on the antimicrobial activity of

ascorbigen (AG) and methylascorbigen (MeAG). (a) Control, Pseudomonas savastanoi pv. phaseolicola; (b) Arg; (c) GSH. 1, AG and MeAG; 2, HPLC fraction of the broccoli extract; 3, fraction of the broccoli extract; 4, broccoli extract. (Reproduced from Ref. [117] with permission.)

6.6 Applications of Planar Layer Chromatography-DB 377

gel 60F254 layer with chloroform-methanol 80:8 and 9:1 (v/v) at external pressure, 5.0 MPa; flow rate, 250 and 200 μL/min; eluent volume, 9800 and 6500 μL; eluent flush, 450 and 350 μL; separation time, 2370 and 1967 s, respectively. Apart from trans-resveratrol, other wine ingredients also showed antibacterial effects against Pseudomonas savastanoi pv. phaseolicola. trans-Resveratrol as well as ascorbigen and 10 -methylascorbigen were tested against Erwinia amylovora after OPLC development (with previously described separation methods) [79]. The developed chromatoplates were dipped into cell suspension and after 2 h incubation the bioautograms were stained with MTT. All investigated compounds gave inhibition zones (Figure 6.28). OPLC separated essential oil components of thyme were investigated against Bacillus subtilis and transgenic luminescent Pseudomonas syringae pv. maculicola [52,119]. Infusion OPLC conditions were: 20 cm  20 cm normal particle size silica gel 60F254 layer, chloroform, mobile phase; external pressure, 50 bar; flow rate, 400 μL/min; initial flash volume, 450 μL; separation volume, 4900 μL; and total development time, 746 s. The separated thymol and carvacrol showed a strong antibacterial effect, while linalool showed a weak antibacterial effect against both strains (Figure 6.29). The difference between the two visualization methods can be seen. Generally the visualization of a bioautogram is performed with vital dye; as opposed to this, utilizing luminescent bacteria, the bioautogram can be documented by taking a photo of it with longer exposure time. In the first case, the inhibition zones are bright against the colorful background, while in bioluminescence the black zones, which show the lack of emitted light,

MeAG

AG

0.5 2

5 μg

AG and MeAG

0.1

0.5

1 μg

trans-Resveratrol

n FIGURE 6.28 Effect of ascorbigen (AG) and methylascorbigen (MeAG) and trans-resveratrol on Erwinia

amylovora cells. (Reproduced from Ref. [79] with permission.)

378 CHAPTER 6 Conventional and Modern Bioassays

t c

l

1

(a)

2

1

(b)

2

1

2

(c)

n FIGURE 6.29 Detection of Thymus vulgaris L. essential oil components separated by OPLC. Lane 1,

standards, 10 μg each of thymol, carvacrol, and linalool. Lane 2, Thymus vulgaris L. oil, 40 μg. t, Thymol; c, carvacrol; l, linalool. (a) The developed layer after vanillin-sulfuric acid detection; (b) bioautogram obtained by the use of luminescent Pseudomonas syringae pv. maculicola (black zones indicate the inhibition); (c) bioautogram obtained by the use of Bacillus subtilis (bright zones indicate the inhibition). (Reproduced from Ref. [52] with permission.)

indicate the inhibition effect. In this system the advantage of OPLC was shown, because when using the same mobile phase in conventional TLC the separation of thymol and carvacrol is insufficient (Figure 6.10). The antibacterial chamomile components separated by infusion OPLC were detected against the naturally luminescent marine bacterium Aliivibrio fischeri [120]. The active components cis- and trans-spiroethers, alphabisabolol, and herniarin giving inhibition zones in the biotest (Figure 6.30) were separated on a 20 cm  20 cm normal particle size silica gel 60F254 layer with chloroform. The OPLC conditions were as follows: 50 bar external pressure, 400 μL rapid mobile phase flush, 400 μL/min mobile phase flow rate, 4414 μL mobile phase, and 672 s development time.

6.6.3 Quantification of bioactive compounds using planar layer chromatography coupled with bioassay Planar layer chromatography coupled with bioactivity detection is mainly used as a qualitative screening method; however, its suitability for quantitative determination of active components has already been established as well. The antimicrobial compounds can be quantitatively evaluated after direct bioautographic visualization. A logarithmic relationship was found between the amount of the applied aflatoxin B1 and the peak area of the inhibition zone against Pseudomonas savastanoi pv. phaseolicola obtained by densitometry at 590 nm (Figures 6.31 and 6.32) [89].

cs

ts

(c)

ab h

(a)

1

2

3

4

1 (b)

2

3

4

(d)

n FIGURE 6.30 Detection and identification of ingredients of 50% aqueous ethanol chamomile extract separated by infusion OPLC; 1-6 μL 50% aqueous

ethanol chamomile (Herbaria) extract; 2-3 μg (–)-alpha-bisabolol (ab); 3-3 μg herniarin (h); 4-6 μL 50% aqueous ethanol chamomile extract + 1.5 μg of each (–)-alpha-bisabolol and herniarin test substances. (a) The developed layer visualized with vanillin-sulfuric acid reagent. (b) Bioautogram using Vibrio fischeri (dark spot ¼ inhibition zone). TIC chromatograms of (c) 5 g of dried powered chamomile flower (JuvaPharma) and (d) isolated chamomile components extracted from the chromatographic bands at RF ¼ 0.5 with their mass spectra obtained by SPME-GC-MS analysis. Peaks at different tR were identified as follows: 15.8 min, trans-beta-farnesene; 17.9 min, spathulenol; 19.0 min, bisabolol-oxide B; 19.4 min, (–)-alpha-bisabolol; 20.0 min, herniarin; 20.3 min, bisabolol-oxide A; 22.3 min, cis-spiroether (cs); 22.5 min, trans-spiroether (ts). (Reproduced from Ref. [120] with permission.)

(a)

(b) 0.125

0.25

0.5

0.75

1 μg

Aflatoxin B1 n FIGURE 6.31 Aflatoxin B1 spots (0.125, 0.25, 0.5, 0.75, and 1 μg) before biological detection (a) under UV

light (λ ¼ 365 nm) and after biological detection (b) in visible light. Aflatoxin was developed by OPLC on TLC silica gel layer with chloroform-acetone 9:1 (v/v). (Reproduced from Ref. [89] with permission.)

380 CHAPTER 6 Conventional and Modern Bioassays

Parabens were quantified by exploiting their antibacterial activity against luminescent Vibrio fischeri [73]. Recording the light intensity from the separated layer immersed into the cell suspension (bioautogram) by CCD camera, the spots of parabens were revealed as black spots against a bright background. The evaluation of the bioautogram showed correlation between the amount of the parabens and their peak area (Figures 6.33 and 6.34) [73].

Detector signal (l = 590 nm)

2,900,000 2,400,000 1,900,000 y = 1E + 06 ln(x) + 3E + 06 R2 = 0.9969

1,400,000 900,000 400,000 0

0.125 0.25 0.375 0.5 0.625 0.75 0.875 Amount of AFB1 (mg)

1

1.125

n FIGURE 6.32 Logarithmic correlation between the amount of AFB1 and the peak area obtained by

densitometric evaluation (λ ¼ 590 nm) after biological detection (bioautogram in Figure 6.31b was evaluated). (Reproduced from Ref. [89] with permission.) 0.15 0.13 0.11

(1/R)–1

0.09 0.07 0.05 0.03 0.01 –0.01 –0.03 0

10

20 30 40 50 Separation distance (mm)

60

70

n FIGURE 6.33 Densitogram of a 100 ng butyl-, propyl-, ethyl-, and methylparaben separation (from

left to right) over 70 mm separation distance, measured at 255 nm. The front signal is at 65 mm separation distance. TLC separation was performed on cyanopropyl HPTLC plate using water-acetonitrile-dioxaneethanol-NH3 (25%) (8:2:1:1:0.05, v/v) as mobile phase. (Reproduced from Ref. [73] with permission.)

6.6 Applications of Planar Layer Chromatography-DB 381

1.3 1.2

(1/R)–1

1.1 1 0.9 0.8 0.7 0.6

0

10

30 40 20 Separation distance (mm)

50

60

n FIGURE 6.34 Densitogram of a 300 ng butyl-, propyl-, ethyl-, and methylparaben separation (from left to

right), measured with Vibrio fischeri bacteria. Ethyl- and methylparaben show the largest signal. (Reproduced from Ref. [73] with permission.)

The range of linearity was achieved by extending Kubelka-Munk expression for data transformation. Antioxidant compounds were quantitatively determined by a validated method that combines HPTLC separation and the DPPH• test. The purple 2,2-diphenyl-1-picrylhydrazyl (DPPH•) is a stable free radical that is neutralized, thereby decolorized in the presence of an antioxidant. The bright spots against a colorful background were densitometrically evaluated and quantification was carried out based on a linear calibration curve obtained by the use of authentic compounds (Figures 6.35 and 6.36) [121]. The fluorescence measured in the pYES test showed a dose-dependent response; however, repeating the analyses four times on four different plates and days, a high standard deviation was obtained [35] (Figure 6.37). It confirmed the necessity of continuous calibration in the biological test systems for trustworthy results.

6.6.4 Bioassay-guided separation, detection, and isolation using DB There is a high demand for bioactive chemicals; therefore, researchers embarked on the biological screening of natural sourced matrices, such as plant extracts, for new agents. For in vitro detection of components having the desired antimicrobial, antioxidant, enzyme inhibitor, etc. effect, there

382 CHAPTER 6 Conventional and Modern Bioassays

Magnolol Honokiol

1

2

3

4

5

6

7

8

9

(a)

(b)

n FIGURE 6.35 TLC chromatogram (a) and scanning profile (b) of Cortex Magnoliae officinalis samples

from different locations in China. 1-9, Samples from various locations in China. Separation was carried out on silica gel HPTLC plate with toluene-methanol 20:2 (v/v). (Reproduced from Ref. [121] with permission.)

y = 38212x + 4527.9 R2 = 0.9989

45,000 40,000

Peak area

35,000

Honokiol

30,000 25,000 20,000

y = 9288.6x + 553.97 R 2 = 0.9939

15,000 10,000

Magnolol

5000 0

0

0.2

0.4

0.6 0.8 Concentration (μg)

1

1.2

n FIGURE 6.36 Linear calibration curves of magnolol and honokiol, based on their antioxidant

effect. (Reproduced from Ref. [121] with permission.)

are several tests that are easy to perform. However, the isolation of the effective substance is an issue and is usually carried out through several fractionation and purification steps. Thus, to reach a pure form can take a long time and much money. Therefore there is a tendency to focus on the isolation of only the active compounds, discarding the inactive extracts, fractions, and compounds during the isolation process [119]. For this purpose we need to biomonitor every step in the procedure—that is, check the extracts, fractions, and purified compounds for the desired activity (Figure 6.38).

6.6 Applications of Planar Layer Chromatography-DB 383

HPTLC-pYES fluorescence signal, peak area (AU)

30,000

E2

EE2

E1

E3

BPA

NP

25,000

20,000

15,000

10,000

5000

0 1.00E–01 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 Amount of EDC (pg/zone)

n FIGURE 6.37 Mean HPTLC-pYES dose-response curves (n ¼ 4) for six endocrine disrupting compounds

(E2, 17 β-estradiol; E1, estrone; EE2, 17 α-ethinylestradiol; E3, andestriol; BPA, bisphenol A; NP, 4-nNonylphenol). Response was measured fourfold on four different plates and days. (Reproduced from Ref. [35], supplement with permission.) Biological activity-guided isolation

Preparation and screening of the extracts

Further fractionation of active fractions

Further purification of active fractions/compounds

Continuous bio-monitoring (desired effect)

Fractionation of active extract

Needed: : Fast Effective Reliable High-throughput Screening method

Further characterization, determination of the pure active compounds

n FIGURE 6.38 A scheme of the steps of a bioassay-guided isolation and characterization.

Planar layer chromatography coupled with EDA is very suitable as an in vitro biomonitoring tool owing to its reliability, simplicity, and costeffectiveness, as well as high-throughput capability. An important point in these processes is the characterization and identification of the active compounds. The chromatographic techniques make possible the spectroscopic and spectrometric investigation of the individual

384 CHAPTER 6 Conventional and Modern Bioassays

separated compounds. Opposite to column chromatographic techniques, layer chromatography enables the characterization of the substances also in situ in the adsorbent layer. Spectroscopic characterization can be carried out by recording UV, infrared (IR), Fourier transform IR (FT-IR), Raman, and FT-Raman spectra of the chromatographic zones. The use of surfaceenhanced Raman spectroscopy (SERS) was reported for identification of compounds in their TLC spots [122] as well as for confirmation of demethylation of aflatoxin B1 in the presence of Pseudomonas savastanoi pv. phaseolicola [123]. The compounds separated by TLC can be directly investigated with mass spectrometry without their elution [124]. For this purpose molecules from the adsorbent layer have to be desorbed and ionized and led into the mass spectrometer (MS). The desorption and the ionization can be achieved by means of a laser beam (e.g., matrix-assisted laser desorption and ionization (MALDI, [125]), laser ablation-inductively coupled plasma (LA-ICP) [126], liquid or gas jet (e.g., desorption electrospray ionization (DESI) [127]), excited gas beam (e.g., direct analysis in real time (DART) [128]) or acoustic desorption combined with secondary ionization (e.g., laserinduced acoustic desorption (LIAD) with electrospray ionization (ESI) [129]). The variety of possible techniques and methods used for characterization of interesting compounds separated by TLC may be increased by their elution that enables ex situ investigations. Forced-flow layer chromatographic techniques (OPLC and RPC) ensure the overrun of the substances, and thus their on-line coupling with MS [130,131]. The components can also be eluted from the layer by scraping off and extracting the adsorbent or with a commercially available, manually operated, pneumatically driven device (thin-layer chromatography-mass spectrometry (TLC-MS) Interface) [132], which can transfer the eluate directly into MS or the eluate can be collected for further investigations (e.g., liquid chromatography-tandem mass spectrometry (LCMS/MS) or gas chromatography–mass spectrometry (GC-MS)). In the cases of TLC and off-line OPLC the separated components remain in the adsorbent layer. Applying the sample in a wide band (up to 18 cm), a narrow lane of the developed layer can be used for biodetection to indicate the location of zones of active compounds. These zones can be scraped off from the rest of the native plate and eluted (Figure 6.39). Using this activity-guided process the isolation of antibacterial chamomile components separated by off-line OPLC was achieved. The zones showing an inhibiting effect against Aliivibrio fischeri were eluted and identified by solid-phase microextractiongas chromatography-mass spectrometry (SPME-GC-MS) as cis- and transspiroethers, alpha-bisabolol, and herniarin [120]. As a flexible tool, OPLC is especially usable for an isolation process enabling the extension of an off-line separation procedure to on-line detection and fractionation [51]. The off-line separation can be combined with

6.6 Applications of Planar Layer Chromatography-DB 385

Scraping at appropriate RF and eluting the component

Bioassay P. maculicola

n FIGURE 6.39 An effect-directed isolation process using off-line separation method. Biological detection was

carried out with Pseudomonas syringae pv. maculicola plant pathogen bacterium.

biological detection, and the RF value of active components predicts their retention time in the on-line fractionation, which can also be guided by flow-cell detection as well. The adaptation of the off-line process to on-line is very easy: the development must just be continued to let the components overrun, leaving the adsorbent layer. The isocratic on-line OPLC method was used for the isolation of antibacterial thyme essential oil components. Preliminary off-line OPLC-DB showed three components having inhibiting activity against the growth of Bacillus subtilis Gram-positive soil bacterium and transgenic luminescent Pseudomonas syringae pv. maculicola Gram-negative plant pathogenic bacterium. OPLC separations were performed on an analytical TLC layer with chloroform [119]. An infusion (off-line) method (4 mm bands, external pressure: 50 bar, flow rate: 400 μL/min, initial flash volume: 450 μL, separation volume: 4380 μL and total development time: 668 s) was extended to on-line providing the isolation of substances of interest. For isolation one sample was applied in a 16 cm wide band and the mobile phase flow rate was increased to 1 mL/min. The first part of the isolation process was carried out in infusion mode (with closed mobile phase outlet), and the mobile phase outlet was opened after 4 min to let the overrun eluent flow introduce into the detector. The UV active thymol and carvacrol were collected based on a detector signal at λ ¼ 260 nm, while the retention time of the non-UV active linalool was calculated from its retention factor obtained from off-line OPLC. Bioassay-guided fractionation and purification of ingredients of Diospyros virginiana acetone root extracts were performed by isocratic OPLC separation with on-line detection and fraction collection [114]. Extract and fractions were tested against Colletotrichum spp., and mild polar compounds appear to be responsible for antifungal activity. Two new and nine known

386 CHAPTER 6 Conventional and Modern Bioassays

natural compounds possessing antifungal property were isolated and identified. Further in vitro antimicrobial tests showed that 7-methyljuglone and isodiospyrin is promising against Phomopsis obscurans, which causes leaf blight and fruit rot in strawberries, resulting in the loss of the fruit. The elution of the compounds with low RF would occur only after a long development time that would cause a loss in separation efficiency. Therefore, in such cases a step-wise gradient elution can be carried out, which was applied for the fractionation of chamomile flower extracts [133,134]; 50% ethanol extract was applied to normal particle size silica gel layer in a 16 cm wide band and was fractionated by chloroform and the mixtures of chloroform and acetone with 1 mL/min flow rate. The fractions were collected according to the signal of a flow-cell UV detector [133]. The bioassay-guided isolation of the active components from methanol extract was performed after sample clean-up in situ in the adsorbent bed after sample application [134]. In both clean-up and separation processes the mobile phases used were hexane-chloroform-acetonitrile mixtures. The fractions collected and the compounds remaining on the layer were tested against Bacillus subtilis and/or Pseudomonas syringae pv. maculicola and/or Aliivibrio fischeri; the active ones were further analyzed by SPME-GC-MS and/ or LC-MS/MS and/or OPLC-TLC-MS interface-MS. OPLC separations can be scaled-up utilizing 0.5 mm thick preparative layer [135]. In these studies the active components found were identified as chamomile oil components, coumarins, flavonoids, and flavonoid glucosides. In all the previously mentioned OPLC processes the development was started with a dry stationary phase—that is, the sample application was carried out off-line by a sample applicator or microliter syringe. However, the OPLC ensures a fully on-line process similar to HPLC. In this case, the sample is applied by an injector and the chromatographic development is performed in an adsorbent bed saturated with the mobile phase and the overrun separated compounds can be detected and collected on-line. Using this mode, red wine components were isolated and tested against the bean pathogen Pseudomonas savastanoi pv. phaseolicola [136].

6.7 FUTURE DEVELOPMENT AND APPLICATION POTENTIAL OF PLANAR LAYER CHROMATOGRAPHY-BIOASSAY In the last decade the main new developments in planar layer chromatography were to introduce new types of stationary phases and to expand as well as to improve the hyphenated methods/techniques applicable. Hyphenations in layer chromatography, namely its combination with bioassay methods,

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388 CHAPTER 6 Conventional and Modern Bioassays

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