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Snezana Agatonovic-Kustrin, a professor in pharmaceutical chemistry at Monash University, in Kuala Lumpur, Malaysia, spoke to The Column about the development of a method to evaluate antidiabetic and antioxidant activity in marine algae using high-performance thin-layer chromatography (HPTLC)-direct bioautography.
Snezana Agatonovic-Kustrin, a professor in pharmaceutical chemistry at Monash University, in Kuala Lumpur, Malaysia, spoke to The Column about the development of a method to evaluate antidiabetic and antioxidant activity in marine algae using high-performance thin-layer chromatography (HPTLC)-direct bioautography. - Interview by Kate Mosford
Q. You recently developed a method using high-performance thin-layer chromatography (HPTLC)-direct bioautography to evaluate antidiabetic and antioxidant activity in marine algae (1). What are the possible benefits of brown and green algae for diabetes prevention and management?
A: There is great interest in functional foods because they have the potential to improve and maintain human health. Therefore, they also have the potential to provide many growth opportunities for the food industry. Functional foods contain components that are either not present or are present at lower concentrations in similar conventional foods consumed as part of a usual diet. There is an emerging trend to use functional foods for the prevention of cardiovascular disease and diabetes. As the most ancient members of the plant kingdom, marine algae are well known sources of bioactive compounds, with a range of different biological activities. Marine sources have received great attention in the search for natural bioactive compounds and as a source of functional foods. Marine algae is increasingly being consumed for functional benefits beyond the traditional considerations of nutrition and health benefits. Marine algae or seaweed is the secret ingredient to a healthy heart. It is well known that people from Okinawa, Japan, have the longest lifespan. It is also known that they consume the most seaweed of all the world’s population (2).
There have only been a few studies previously investigating alpha amylase inhibitory activity in marine algae (3–6). In these studies, they applied the alpha amylase test on the whole sample without chromatographic separation, and hypothesized that polyphenolic-rich extracts are responsible for antidiabetic activity. In our study, we reported on the alpha amylase inhibitory activity of natural products separated from selected marine algae extracts (1). For this work, an efficient experimental protocol for a HPTLCâalpha amylase enzyme inhibition assay was developed. After chromatographic separation of extracts and application of the alpha amylase essay test, we established that terpenoids are actually responsible for this activity.
Q. Can you describe the principles behind high-performance thin-layer chromatography (HPTLC)?
A: Planar chromatography and, of course, thin-layer chromatography (TLC) have been traditionally used for the qualitative analysis of mixtures, to characterize and track components visually, or as an initial separation technique. With technical improvements in recent years, highâperformance thin-layer chromatography (HPTLC) has emerged as the most advanced form of TLC. This technique involves automation of a number of different steps, an increase in plate resolution, and the ability to allow both qualitative and quantitative analysis.
HPTLC plates offer a higher speed of separation and better resolution (clearer sample separation) than TLC plates because of smaller stationary phase particle size and associated larger surface area. The use of HPTLC plates-in combination with automated sample applicators, development chambers that enable gradient elution, high-resolution cameras, and computing software-allows for more control over experimental conditions and better analytical capabilities.
Q. What is HPTLC-direct bioautography and what benefits does it offer the analyst?
A: Bioautography combines chromatographic separation with biochemical assays, making it an incredibly effective method for in vitro screening for potential drug leads in complex samples such as plant extracts. The technical improvements in instrumentation and automatization of HPTLC, combined with biochemical (enzymatic) derivatization and direct hyphenation with spectroscopic techniques, enables parallel bio-profiling, bioâdetection, and characterization of biologically-active compounds in a sample.
Q. What were the main obstacles you encountered developing this method and how did you overcome them?
A: The main obstacle was to effectively optimize experimental conditions and accurately and quantitatively compare alpha amylase inhibitory activity in different extracts.
Q. What are the advantages of using HPTLC over existing methods?
A: HPTLC has the ability to run many samples in parallel on the same plate, uses small volumes of solvent, is costâeffective, provides rapid analysis, gives an instant visual result, and is very userâfriendly compared to high performance liquid chromatography (HPLC) or gas chromatography (GC). Results can be presented as chromatograms (peaks) and also as plate images. HPTLC also offers the option of multiple detection methods, before and after specific derivatization on the same plate. In column chromatography, separated compounds are identified by their Rf value. In TLC chromatography, compounds are identified by their Rf value and by their colour or fluorescence under UV–vis light, UV 366 nm and UV 254 nm, before and after derivatization. An automated multiple development (AMD) procedure in HPTLC allows gradient elution, which in many cases can significantly improve component separation. Planar layer chromatography provides further advantages, such as minimal sample preparation. Crude extracts can be applied on the plate (spotted or sprayed as bands) without losing components during extraction or sample pretreatment. This approach also offers parallel profiling of samples on the same plate. Different effect-directed (bio) assays can be run because mobile phase can be removed after plate development, enabling direct post-chromatographic assays to be performed without much effort. Thus, various microchemical (derivatization reagent), biochemical (enzymatic), and biological (cell-based) assays can be performed directly in situ on the chromatographic plate. On the other hand, HPLC provides higher separation, but operates with solvents that are often toxic to biological cells and to enzymes, making it more difficult or impossible for the tests previously discussed to be performed.
Q. Do you think there are any misconceptions surrounding HPTLC that prevent it being more commonly used?
A: Yes, there are many misconceptions about HPTLC and TLC. TLC and HPTLC are seen as being “not reproducible” and only as a good “separation tool” for “semiquantitative determinations”. However, modern TLC and HPTLC have the ability to perform quantitative analysis and furthermore, offer the possibility to incubate enzymes and
viable cells directly on to the plate and to run bioassays. Furthermore, they can also be coupled on-line with spectroscopic methods for detection and identification, as well as with other analytical and preparative separation techniques.
Q. What are you working on next?
A: My current interest is in investigating cardiovascular effects of selected marine algae. We are investigating the interaction of bioactive compounds from marine algae extracts, previously separated on chromatographic plate, with nitric/nitrous acid. In this work, a polar silica (silicon dioxide) stationary phase acts as a support to bring phenols close to nitrates by forming a ternary complex through hydrogen bonding so that they react together.
Snezana AgatonovicâKustrin received her Ph.D. in pharmaceutical chemistry and drug analysis in 1993, her Master of Science degree in pharmaceutical chemistry in 1988, and her Bachelor of Science in pharmacy in 1984. She has over 30 years’ experience and background as an academic in different pharmacy disciplines worldwide. She has taught in a wide range of pharmacy subject areas (both at bachelor and master degree levels). She has been a pioneer in chemometry and experimental design, artificial neural network modelling, computational pharmacokinetic studies, and quantitative structure–activity relationship (QSARs) models. Her current research interest is in natural products and food chemistry, marine drugs, HPTLC bioassay guided highâthroughput screening, HPTLC effect-directed screening, drug quality control, and colloidal drug delivery systems.