Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Summary

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper ammonia-producing bacteria (HAB) native to the bovine rumen.

Abstract

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth. The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions. This paper describes standard protocols for TLC and contact bioautography with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light. Zones are cut out and incubated face down onto agar inoculated with the test microorganism. Inhibitory bands are visualized by staining the agar plates with tetrazolium red. The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen. The TLC methods apply to many types of plant extracts and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Introduction

Assaying for antimicrobial compounds in plants requires separating the components of a plant extract, exposing a test microorganism to those components, and determining whether the microorganism’s growth is inhibited by any of the compounds. Separations by paper or thin-layer chromatography (PC or TLC) are convenient because many compounds can be separated on a planar surface. Separation is based on polarity, with some compounds binding tightly to the adsorbent (cellulose in the case of PC, and a variety of adsorbents in the case of TLC) and migrating less than others¹. Figure 1 provides an example of the relative positions of polar and non....

Protocol

1. Preparation of Plant Extract

1. See Kagan and Flythe¹⁰ for extraction of phenolic compounds from Trifolium pratense cv. Kenland. 
2. To extract other compounds in other plants, check the phytochemical analysis literature for plant- or metabolite-specific extraction methods (many are described), or look for protocols such as those of Khurram et al.^7,8 which  isolate many compounds with a wide range of polarities.

2. Preparation of Thin-layer Plates

1. Clean TLC plates by developing in one or more polar, neutral solvents, in order to move adsorbed contaminant....

Results

Representative silica TLC separations of red clover (Trifolium pratense cv. Kenland) extracts, containing phenolic compounds, are shown in Figure 2. Separation of red clover extract in ethyl acetate-hexane (9:1, v/v), over 8.5 cm, resulted in five bands, one incompletely resolved from the origin (Figure 2A). However, Figure 2B demonstrates that about twice as many bands were revealed when a different sample of red clover extract (from the same cultivar, but grow.......

Discussion

This protocol describes a simple method for separating an extract into subsets of compounds and assaying those subsets by contact bioautography. The method is quite similar to one used by Chomnawang et al.¹⁵ to screen for plant metabolites inhibitory to gonorrhea-causing bacteria. The type of bioautography employed to screen for antimicrobial plant compounds depends on many factors, including the test microorganism, the laboratory setup, and the preferences of the person(s) performing the bioassa.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Silica F254 TLC plates, aluminum-backed, 0.2 mm thickness, 20 cm x 20 cm	EMD Chemicals	5554/7	These plates are coated with silica that contains an indicator fluorescing at 254 nm.  Compounds absorbing at that wavelength appear dark on a fluorescent green background.  Alternative sources include Analtech, Selecto Scientific, Fluka.  Adsorbents other than silica may be needed.  Plastic-backed plates may be suitable, depending on the solvents to be used.
Sharp, heavy-duty scissors	any sewing supply company	similar to Fiskars 175800-1002	For cutting TLC plates.  A paper cutter with a sharp blade can be used as well.  Do not inhale silica dust.
Drying oven at 100 °C (mechanical convection)	Thermo Scientific	PR305225M	Quincy Lab, Inc, Chicago, IL (www.quincylab.com); Cascade Technical Sciences, Hillsboro, OR (www.cascadetek.com)
TLC chamber	Kimble Chase	416180-0000	Alternative sources:  Aldrich. Pyrex beakers or preserving jars can be used for small plates (i.e. 5 cm x 10 cm).  Cover with aluminum foil (jar lids may contain material extractable by solvent vapors).
50 µl Syringe with flat needle tip	Hamilton	80965	For loading amounts of standard or sample exceeding 5-10 µl.  Alternative sources are equivalent.
Micropipettes	Drummond	2-000-001	For loading small amounts of standards or samples.  Alternative sources:  VWR.  Also, Pasteur pipets can be stretched to a thinner diameter with a butane torch.
Filter paper (#1 grade)	Whatman	1001 917	Serves as a chamber wick.  Other grades of filter paper are OK.  This size can be trimmed for the chambers holding 20 cm x 20 cm plates.
Beaker tongs	Fisher Scientific	15-186	For putting plates in and out of a large TLC chamber.  Alternate sources: VWR
Flat-edge forceps	Fisher Scientific	10-275	For putting plates in and out of a small chamber. Alternate sources: VWR
Small portable UV lamp with 4 W or 6 W bulbs for short- and long-wave UV light illumination (254 and 365 nm, respectively)	Ultraviolet Products	95-0271-01	Alternate sources: Spectronics Corporation (www.spectroline.net)
Viewing cabinet for use with hand-held UV lamp	Ultraviolet Products	Chromato-Vue C-10E	UV-active bands are more easily circled if plates can be set in here.  Alternate sources: Spectronics Corporation.
Photodocumentation system with overhead UV lamp and visible lamp	Kodak	Gel Logic 200	Alternate sources: Ultraviolet Products (www.uvp.com).  See protocol for homemade alternative.
Anaerobic Chamber, Type A, Vinyl	Coy	7150000	This chamber is appropriate for anaerobic bacteria, like Clostridium sticklandii, as described.  However, growth conditions must be tailored to organism used in the assay.  A biosafety cabinet and other precautions should be taken if pathogenic organisms are used. Alternate sources: Anaerobe Systems, BioRad, Plas Labs, others
Tetrazolium red	Sigma-Aldrich	T8877	Alternate sources: MP Biomedicals, Santa Cruz Biotechnology, Alfa Aesar
Ingredients for HAB media
Pyridoxamine · 2HCl	Sigma-Aldrich	P9380	For this and for all the other reagents in this table, alternative sources are equivalent.
Riboflavin	Sigma-Aldrich	R4500
Thiamine HCl	Sigma-Aldrich	T3902
Nicotinamide	Sigma-Aldrich	N3376
Calcium D-Pantothenate	Sigma-Aldrich	C8731
Lipoic Acid	Sigma-Aldrich	T5625
p-Aminobenzoic acid	Sigma-Aldrich	A9878
Folic acid	Sigma-Aldrich	F8798
Biotin	Sigma-Aldrich	B4639
Cobalamine	Sigma-Aldrich	C3607
Pyridoxal HCl	Sigma-Aldrich	P9130
Pyridoxine	Sigma-Aldrich	P5669
EDTA	Sigma-Aldrich	E6758
Iron sulfate · 7H2O	Sigma-Aldrich	F8263
Zinc sulfate · 7H2O	Sigma-Aldrich	Z0251
Manganese chloride · 4H2O	Sigma-Aldrich	M8054
Boric acid	Sigma-Aldrich	B6768
Cobalt chloride · 6H2O	Sigma-Aldrich	C8661
Copper chloride · 2H2O	Sigma-Aldrich	459097
Nickel chloride · 6H2O	Sigma-Aldrich	203866
Sodium molybdate · 2H2O	Sigma-Aldrich	331058
Trypticase (Pancreatic digest of casein)	Thermo Fisher	B11921
Potassium phosphate monobasic anhydrous	Thermo Fisher	P284
Sodium carbonate · H2O	Thermo Fisher	S636
Agar	Thermo Fisher	50841063
Magnesium sulfate · 6H2O	Thermo Fisher	7791-18-6
Calcium chloride · 2H2O	Thermo Fisher	BP510
Cysteine HCl	Thermo Fisher	19464780
Potassium phosphate dibasic anhydrous	Thermo Fisher	P290
Sodium chloride	Thermo Fisher	BP358