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  • Application of TLC Bioautography for Natural Bioactive Screening

  • Photo Vishwa Patel* Photo Niyati Acharya

  • 1Assistant Professor, Department of Pharmaceutical analysis, Sardar Patel College of Pharmacy, Bakrol. 
    2Head of Department of Pharmacognosy, Institute of pharmacy, Nirma University.
     

Abstract

Bioautography is a mean of targeted-directed isolation of active molecules on chromatogram. Bioautography is technique to detect substances affecting the growth rates to test organisms in complex mixtures and matrices. The antimicrobial, antioxidant and enzyme inhibitory activities can be performed on TLC bioautography. Bioautography methods are divided into three categories. 1)Agar diffusion or contact bioautography 2) Immersion or agar-overlay bioautography 3) Direct bioautography. High throughput method enabling analyses of many samples in parallel and the comparison of their activity. Both screening and semi-quantitative analysis is possible. The targeted compounds can be identified using spectroscopic methods, that can be performed directly on a TLC plate. This review summarizes known TLC-bioautography methods and their applications for determining the presence of enzyme inhibitors in extracts, preliminary screening, natural products possessing these biological activities and for the bioactivity-directed fractionation and isolation of active components from complex extracts. It also indicates the current state and perspective of the development of TLC-bioautography and its possible future applications.

Keywords

Thin layer Chromatography-Direct Bioautography, Screening natural products, Agar diffusion, Immersion bioautography, Direct bioautography.

Introduction

Planar chromatographic analysis hyphenated with the biological detection method is known as Bioautography [1].It is effective and inexpensive technique for the phytochemical analysis of plant extracts to identify bioactive lead/ scaffolds. It can thus be performed both in highly developed laboratories as well as in small laboraties which have maximum access to sophisticated equipment [2]. In 1964, Goodall and Levi introduced paper chromatography (PC)- based bioautography for the first time to estimate the purity of penicillin [3]. In 1961, Fisher and Lautner and Nicolaus et.al introduced thin layer chromatography (TLC) based bioautography [4].The first review on bioautography was written by Betina in 1973 [5]. TLC can separate many samples in parallel and has an open layer that allows for solvent evaporation, it appears to be the perfect technique for hyphenation with bioautographic detection. Besides classical TLC, High performance thin-layer chromatography (HP-TLC), Over pressured- layer Chromatography (OPLC) and planar electro- chromatography (PEC) can also be easily linked to bioautography [6-12]. Thin-layer chromatography-direct bioautography (TLC-DB) is one of three variants of a TLC bioautography method (the others are contact and agar overlay bioautography) in which separation given assay visualization are performed directly on a TLC plate [13-15].  The major application of bioautography are the fast screening of a large number of samples for bioactivity like antibacterial, antifungal, antioxidant, enzyme inhibition, antimicrobial etc. In this review, the techniques and application of bioautography are discussed in detail with suitable examples.

Bioautography Methods:

Three bioautographic methods are 1) Agar diffusion or contact bioautography 2) Immersion or agar overlay bioautography 3) Direct TLC bioautography.

(Table:1 Types of Bioautography)

Bioautography

Basic Procedure

Specification

Agar diffusion

or

Contact bioautography

 

Inoculation with microbial strains.

Allowing diffusion into agar media.

Incubation.

Applying for chromatography silicic acid Fiber sheets and Chroma AR still helped to avoid these shortcomings.

Immersion

or

Agar-overlay bioautography

 

Solidification.

Incubation.

Staining with tetrazolium salt.

 

A combination of direct and contact bioautography is agar overlay.
It is highly recommended to use agar overlay when direct bioautography cannot be completed.

TLC- Direct bioautography

 

Dipping of chromatogram in microbial strains.

Spraying with tetrazolium salt incubation under humid condition for 48hr.

Indicate antimicrobial leads.

 

Direct bioautography is usually perform without agar gel.

Plant extracts can be quickly screened chemically and biologically using TLC-direct bioautography.
"Agar spray assay" is a recently discovered bioautographic technique that can be thought of as an agar variant of direct bioautography.

Figure 1: Scheme of bioautographic methods. (HP-TLC) (High Performance Thin layer chromatography)

2.1 Agar diffusion or contact bioautography [16-20]

The technique with the least usage is agar diffusion. It entails the antimicrobial agent being transferred by diffusion from the chromatogram (PC or TLC) to an agar plate that has already been inoculated with the pathogen under investigation.
The agar plate is incubated and the chromatogram is taken out after a few minutes or hours to allow for diffusion. The areas where the antimicrobial chemicals come into touch with the agar layer are where the growth inhibition zones develop.
The growth requires an incubation period of 16 to 24 hours, however, this can be shortened to 5 to 6 hours by spraying 2,6-dichlorophenol-indophenol or 2,3,5-tetrazolium chloride. Antimicrobials had to be diluted and lost during the process of moving them from chromatographic plates to agar, which followed the same general procedure. An other unique instance involves the bioautographic identification of 6-aminopenicilanic acid, a very weak antibiotic that needs to be transformed into benzyl penicillin by phenyl acetylation. This is achieved by chromatographic plates or paper with acetyl chloride under mildly alkaline conditions prior to bioautography.  Microbiologists are accustomed to using this method to find antibiotics in bacteria, and several techniques have been applied to enhance its efficacy.

2.2 Immersion or agar overlay bioautography [21-25]

Combining contract and direct bioautography is known as agar overlay. Using this technique, a melted, seeded agar media is applied on the chromatogram. The growth or inhibition bands are visible after solidification, incubation, and staining (often with tetrazolium dye).  An agar solution containing the red-colored bacterium Serratia marcescens can be used to identify gram-negative bacteria. After an overnight incubation period at room temperature, the red-colored gel takes on a white or pale yellow, indicating inhibitory zones. Through the use of a dehydrogenase activity, microbes transform the tetrazolium salt (MTT) into corresponding highly colored formazan, allowing other colorless zones of microbial growth inhibition to be seen. The agar overlay assay has been applied to bacteria, including Bacillus subtilis, E. coli, Pseudomonas, and Staphylococcus aureus, as well as yeast, and Candida albicans.

2.3 TLC-DB (Thin layer chromatography-direct bioautography) [26-28]

A suspension of microorganisms growing in nutrient broth is applied to a designed TLC plate (TCL-DB of antimicrobials), which is subsequently incubated in a humid environment.
The bacteria grows straight on a TLC plates surface, avoiding antimicrobial areas. 3-{4,5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide, or MTT, is the most widely used tetrazolium dye. Addition of detergent (e.g., Triton X-100) to the dye can boost the reduction of tetrazolium salts many times. The purple formazan that results from the dehydrogenases of live microorganisms transforms tetrazolium salt. Inhibition zones, which are cream-colored patches that occur on a purple backdrop, indicate the presence of antimicrobial drugs.
Gram-positive Bacillus subtilis Aerobic Bacteria and Gram-negative Escherichia coli are the most commonly utilized test bacteria. Additionally, TLC can be hyphenated with bioluminescence. Detection mostly employing Photobacterium phosphoreum, also known as luminous Vibrio phosphoreum, and Aliivibrio fischeri, formerly known as Vibrio fischeri. The metabolism of bacteria is disrupted by antibacterial agents and other bacterially toxic substances (such as pesticides, heavy metals, and mycotoxines), which ultimately results in the cessation of the bacteria's luminescence. The TLC plate was quickly immersed in a bacterial broth suspension that had been infected with luminous bacteria, and it was then viewed at 490 nm using a luminograph or CCD (cooled charged coupled device) camera. Toxic chemicals are indicated by variations in the emission of blue-green light (darker or brighter zones), which are linked to disruptions in essential cell activities. The technique can be regarded as a direct bioautography even though there are no alterations in bacterial growth.

Application Of Bioautography

The majority of applications for thin-layer chromatography bioautography (TLC-B) use microorganisms, such as fungi and bacteria, as testing organisms. The development of novel antibiotics is a particularly crucial goal in light of the growing microbial resistance to conventional antibiotics in both medicine and veterinary practice.

Ref. No

Name of plant material/extracts

Activity reported/bacterial strain

Chromatographic condition

Bioassay

Conclusion

 

[29]

Saraca indica linn

(Acetone extract)

Family: Caesalpiniaceae

Activities:

Vibriocidal activity

Antibacterial activity

Mobile phase: methanol: water (70:30 v/v)

HPLC/TLC bioautography

 

The aqueous extracts of A. Sativum were the most efficacious, although the acetone extracts of S. indica and D. stramonium were more potent in most cases.

  

Stationary phase: Silica gel 60 F254 TLC plate

  

Datura stramonium linn

(Acetone extract)

Family: Solanaceae

Bacteria:

Gram-negative bacteria

Vibrio cholerae

Vibrio parahaemolyticus

  

Column: Reverse phase Lichrosphere C18 column (250 mm × 4.6 mm,5 μm)

  

Wavelength: UV 310 nm

  

Allium sativum Linn

(Aqueous extract)

Family: Liliaceae

  

Flow rate : 0.7 ml/min

  

[30]

Erycibe obtusifolia

(E.obtusoifolia)

(Crude extracts)

Family: Convolvulaceae

Activities:

Antioxidant activity

Xanthine oxidase inhibitor

Antifungal activity

Mobile phase: 0.1% formic acid

Stationary phase: Silica gel 60 F254 TLC plate

DART-MS/TLC bioautography

 

Effective treatment for gout and disorders associated with oxidative stress is provided by E. obtusifolia.

  

Column: XBridge C18 column (19 ×150 mm,5 μm)

  

Fungus:

Penicillium italicum

Wave length: UV 290 nm

  

 

Flow rate: 1 mL/min.

  

[31]

Rumex crispus L.

(R. crispus)

Rumex sanguineus L. (R. sanguineus)

Family: Polygonaceae

 

Activity:

Anti-A. baumanni activity

Antimicrobial activity

Mobile phase: Ethyl acetate:toluene:formic acid:water  (80:10:5:5 v/v/v/v)

HPTLC-bioautography/LC-DAD-MS

 

The application of R. sanguineous and R. crispus extracts for wound healing.

 

  

Stationary phases: Silica gel 60 F254 aluminum plates

  

Bacteria:

Gram negative bacteria

Acinetobacter baumannii

Klebsiella pneumoniae  Emodin

E-coli

  

Column: Zorbax Eclipse XDB-C18 column (50 mm×4.6 mm,1.8 μm)

  

Wave length: UV 366 nm

  

Flow rate: 0.8 ml/min

  

[32]

Phlomis tuberosa

(P. tuberosa)

Family:

Lamiaceae

Activity:

α-glucosidase inhibitory activity

 

Mobile phase: ethyl acetate:methanol:water (15:2:1 v/v/v)

SEP BOX/TLC bioautography

 

P. tuberosa compounds showed much greater α-glucosidase inhibitory capabilities, suggesting that they could be used as substitute medications for diabetes mellitus.

  

Stationary phase: Silica gel 60 F254 TLC plates

  

Column: Agilent Zorbax SB-C18 column (250 mm× 4.6 mm, 5 μm)

  

Wave length: UV 366 nm

  

Flow rate: 1 mL/min

  

[33]

Philippine Piper betle Linn

(Ethanol extract)

Family: Piperaceae

Activities:

Antibacterial activity

Antimicrobial activity

Mobile phase: ethyl acetate: n-hexane (7 :3 v/v)

GC-MS/ TLC bioautography

The potential for P. betle to yield innovative therapeutic antibacterial compounds that can treat particular illnesses or ailments. phytochemical examination of P. betle's secondary metabolites, which include saponins, alkaloids, terpenoids, phenolic acids, and flavonoids.

  

Stationary phase: silica gel  60 F254 TLC plates

  

Gram-positive MDR bacteria: Methicillin resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE)

  

Column:Elite-5 MS capillary column 30mm×0.25 mm, 25 ????m

  

Wave length: for six bands: UV 254 nm

  

Gram-negative MDR bacteria: Carbapenem-resistant Enterobacteriaceae (CRE) Klebsiella pneumoniae and metalloid-????-lactamase (M????L)

  

For eight bands: UV 366 nm

  

Flow rate:1 mL/min

  

[34]

Onopordon  Macrocephalum

Or

Scotch thistle

(Methanolic extract)

Family: Asteraceae

Activities:

Antioxidant activity

Antimicrobial activity

Antibacterial activity

Mobile phase: ethyl acetate:methanol:water:glacial acetic acid:formic acid (63:4:5:2:1v/v/v)

TLC bioautography

 

This plant is used in traditional medicine to treat a number of ailments linked to liver disorders and bacterial infections.

  

Stationary phase: silica gel 60 F254  TLC plates

  

Bacteria:

Gram- positive bacteria

Bacillus cereus

Staphylococcus aureus

  

Wave length: UV 245nm and 365nm

 

  

[35,36]

Green Tea (Camellia)

(Methanol, ethanol & DMSO extracts)

Family:

Theaceae

Activities:

Antibacterial activity

Antioxidant activity

Mobile phase: Chloroform:ethyl acetate:acetic acid (50:50:1)

TLC bioautography

 

The biologically active substances, including glycosides, alkaloids, phenols, and flavonoids. It has been demonstrated that polar solvents with bioactive components have antioxidant and antibacterial properties.

  

Stationary phase:

Silica gel 60F 264  TLC plate

  

Bacteria:

Gram-positive

cocci

S.aureus

S. pyogene

  

Column: Zorbax Eclipse XDB-C18 column (50 mm×4.6 mm,1.8 μm)

  

Wavelength:

UV 360 nm

  

Gram-negative

E. coli

S. marcescens

  

Flow rate:1 mL/min

  

[37]

Ficus carica Linn

(Ethyl Acetate Extract)

Family: Moraceae

 

Activities:

Antibacterial activity

Mobile phase: chloroform:methanol (7: 3)

TLC-DB

 

Ethyl acetate has been used in numerous studies on the isolation of antibiotics. This is because ethyl acetate makes it simple to isolate antibiotics, which are typically semipolar.

  

Bacteria:

Staphylococcus aureus

Escherichia coli

Stationary phase: silica gel F254 TLC plate

  

Wave length: UV 254 and 366 nm

  

[38,39]

Justicia wynaadensis (Nees) T.Anders

(methanol extract)

Family: Acanthaceae

Activities:

Antibacterial activity

Antimicrobial activity

Mobile phase: petroleum ether :ethyl acetate (7:3)

TLC/GC-MS

 

The antibacterial properties of J. wynaadensis's methanolic extract against Klebsiella pneumoniae may be ascribed to fatty acids including stearic acid, myristic acid, palmitic acid, and linoleic acid as well as volatile components like phytol.

  

Stationary phase: Silica gel 60F254 TLC plate

 

  

Bacteria:

Gram negative bacteria

Klebsiella pneumoniae

  

[40]

Salvia officinalis Linn.

(S. officinalis L)

(Sage Extract)

Family: Lamiaceae

Activities:

Antibacterial activity

Antioxidant activity

Mobile phase: chloroform:diethyl ether:methanol (30:10:1 v/v)

LC-MS-MS/TLC-DB

 

Sage extract, a natural antioxidant utilized in the food sector, has excellent antioxidant qualities. Salvigenin and cirsimaritin are two examples of flavonoids that have anti-inflammatory, analgesic, and antioxidant qualities.

  

Stationary phase: aluminum-backed TLC Si60 F254 plates

  

Bacteria: Staphylococcus aureus

Bacillus subtilis

methicillin-resistant S. aureus (MRSA)

  

Column: Rt-β-DEXm (Restek) capillary column, 30 mm, 0.25mm

  

Wave length: UV 365nm

  

Flow rate:0.5 mL/min

  

[41]

Acacia Senegal

(Crude extract)

Family:

Fabaceae

Activity:

Antioxidant activity

Antibacterial activity

Mobile phase:

Chloroform

TLC-DB

 

Phytochemical examination to look for cardiac glycosides, phenols, flavonoids, and saponins.The plant was applied externally to sooth irritated areas and was used to treat diabetes.

  

Stationary phase:

Silica gel 60F254 TLC plate

 

  

Bacteria:

E-coil

S.pyogenes

V. Cholera

  

[42]

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

[43]

Vetiver Zizanoides

(ethanol extract)

Family:

Poaceae

Phragmites karka.

(diethyl ether extract)

Family:

Poaceae

Manuka

(Leptospermum scoparium)

leaf and branch extracts

Activities:

Antimicrobial activity

Mobile phase:

toluene: ethyl acetate (93:7 v/v)

TLC Bioautography

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TLC Bioautography & HR-ESI-MS, and -MS/MS

Vetiver zizanoides and Phragmites karka solvent extracts have the potential to be employed as antimicrobial drugs against infectious agents and to treat a variety of infectious disorders.

TLC-bioautography protocol for assessing the antibacterial ability of Leptospermum scoparium. The minimum effective dose (MED) was observed to be 0.29–0.59 µg/cm2 against S. aureus and 2.34–4.68 µg/cm2 against E. coli. The enhanced protocol demonstrated suitability for both S. aureus and E. coli.

  

Bacteria:

Gram positive bacteria

S. aureus

Stationary phase:

pre-coated aluminium silica gel G 25 plates

  

Column: Elite-5 MS 30×mm×0.25 mm, 25 ????m capillary column

  

Gram negative bacteria

Salmonella paratyphi

Klebsiella pneumonie

Fungus:

Spergillus niger

Candidas albicans

Activities:

Antimicrobial activity

  

Wave length: UV 235nm

  

Flow rate:1 mL/min

  

Column: GOLD C18 Column, 100 × 2.1 mm, particle size 1.9 µm

Solvent A water/0.1% formic acid,

Solvent B acetonitrile/0.1% formic acid

Inject volume- 5 µL

  

Bacteria:

Gram positive bacteria

S. aureus and E. coli

  

CONCLUSION

In spite of wide employment of sophisticated chromatographic techniques coupled with on-line bioassays, bioautography is still proving its worth as a simple and inexpensive tool for simultaneous chemical-biological screening of natural sources. In other word, it offers the simplest mean of bioassay guided lead discovery from natural products. For the natural product the separation process is not easy, and if separated the amount is very less in maximum cases, so it is necessary to develop a process which can detect lead in a small amount and biological activity can also be measured successively. The standard experimental procedures are required for TLC antioxidant and antimicrobial assays. Some new enzymes should be attempted and adopted on TLC bioautography. The existing TLC methods for enzyme inhibition need more application studies to assess their screening capacity in the discovery of active compounds. The GC-MS or LC-MS approaches have gradually been coupled to TLC bioautography for fast structural characterization of active compounds. Considering these problems, we can say that bioautographic detection technique would create a new era in separation science.

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Reference

  1. Hostettmann K, Terreaux C, Marston A, Potterat O. The role of planar chromatography in the rapid screening and isolation of bioactive compounds from medicinal plants. JPC. Journal of planar chromatography, modern TLC. 1997;10(4):251-7.
  2. Goodall RR, Levi AA. A microchromatographic method for the detection and approximate determination of the different penicillins in a mixture. Nature. 1946 Nov 9;158(4019):675-6
  3. Fischer R, Lautner H. On the paper chromatographic detection of penicillin preparations. Archiv der Pharmazie und Berichte der Deutschen Pharmazeutischen Gesellschaft. 1961 Jan 1;294:1-7.
  4. BJ N, Coronelli C, Binaghi A. Microbiological determination of antibiotics by thin layer chromatograms. Experientia. 1961 Oct 1;17:473-4.
  5. Betina V. Bioautography in paper and thin-layer chromatography and its scope in the antibiotic field. Journal of Chromatography A. 1973 Apr 11;78(1):41-51.
  6. Heinemann B, Howard AJ, Hollister ZJ. Application of paper chromatograms to the study of inducers of λ bacteriophage in Escherichia coli. Applied Microbiology. 1967 Jul;15(4):723-5.
  7. Bohlin L, Bruhn JG, editors. Bioassay methods in natural product research and drug development. Springer Science & Business Media; 1999 Jan 31.
  8. Wagman GH, Bailey JV. Use of silicic acid—glass fiber sheets for bioautography of antimicrobial substances. Journal of Chromatography A. 1969 Jan 1;41:263-4.
  9. Rios JL, Recio MC, Villar A. Screening methods for natural products with antimicrobial activity: a review of the literature. Journal of ethnopharmacology. 1988 Jul 1;23(2-3):127-49.
  10. Sherma J. Planar chromatography. Analytical chemistry. 1992 Jun 1;64(12):134-47.
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Vishwa Patel
Corresponding author

Assistant Professor, Department of Pharmaceutical analysis, Sardar Patel College of Pharmacy, Bakrol.

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Niyati Acharya
Co-author

Head of Department of Pharmacognosy, Institute of pharmacy, Nirma University.

Vishwa Patel*, Niyati Acharya, Application of TLC Bioautography for Natural Bioactive Screening, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 5, 1376-1386 https://doi.org/10.5281/zenodo.15378848

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