HPTLC – Bioautographic methods for selective detection of the antioxidant and α-amylase inhibitory activity in plant extracts

Graphical abstract


Sample preparation
Myrmecodia platytyrea subsp. antoinii (Becc.) Huxley & Jebb tubers (commonly referred to a ant plant) were collected from West Papua, Indonesia. Plant tubers were cut into small pieces, dried and ground into powder. Fat components were removed from 800 g of sample with n-hexane in a defatting step. The defatted plant material was sequentially extracted using solvents with increasing polarity (dichloromethane, ethyl acetate, ethanol, and finally methanol). The solvents from each extract were then removed using a rotary evaporator under reduced pressure at 40 C. Final extracts for analysis were prepared as 1.0 mg/mL solutions. A 1.0 mg/mL standard solution of stigmasterol in absolute ethanol and a 0.4% w/v DPPH solution in methanol were prepared. An anisaldehyde reagent solution was freshly prepared by combining 1 mL anisaldehyde with a refrigerated solution of glacial acetic acid/concentrated sulfuric acid in methanol in the ratio of 0.5:50:1. A 1% w/v amylase solution was prepared by diluting approximately 1.25 mL (1 g) of α-amylase from Bacillus licheniformis liquid (Cat. No. A4862, Sigma-Aldrich, Denmark) with distilled water to 100 mL. The enzyme stock solution was then refrigerated at 4 C until required.

High-performance thin-layer chromatography
High-performance thin-layer chromatography (HPTLC) plates were pre-washed before use with a Multiple Development Chamber (AMD2, CAMAG). For the DPPH assay, a two-step (40: 80) gradient elution method was used with 100% methanol over a 40 mm developing distance in the first step, and n-hexane, ethyl acetate, acetic acid (20:9:1) over a 80 mm developing distance in the second step. For the stigmasterol and α-amylase inhibitory activity, a single step elution using a n-hexane, ethyl acetate, acetic acid (20:9:1) mobile phase, over a 80 mm developing distance was sufficient [1].
Derivatization was achieved by dipping a HPTLC plate into the derivatizing agent for 1 s using the Chromatogram Immersion Device (CAMAG, Muttenz, Switzerland). Plates derivatized with DPPH solution, were stored in the dark for 30 min and then photographed. Plates derivatized with anisaldehyde-sulfuric acid were heated at 110 C for 10 min and then photographed. Images of the plates were captured with a TLC-visualizer (CAMAG, Muttenz, Switzerland) [1].

Method validation for stigmastrol quantification
Stigmasterol was well resolved in sample extracts on the developed plates after derivatization with anisaldehyde/sulfuric acid. It was observed as a purple zone under visible light or as a peak in HPTLC densitometric chromatograms at hRF = 66. The methods for stigmasterol quantification by digital image analysis and TLC/HPTLC densitometry were validated for linearity, specificity, repeatability, limit of detection and limit of quantification (Table 1). Quantitative analysis of chromatogaphic plates was performed with digital image analysis software Sorbfil TLC Videodensitometer (Sorbpolymer, Krasnodar, Russia) and with CAMAG TLC/HPTLC scanner 4 controlled by winCATS 3.1 software (CAMAG, Muttenz, Switzerland). The plates were scanned in automatic mode at a wavelength of 550 nm, using a slit width of 0.3 mm and a slit length of 4.0 mm. The working range was assessed by plotting chromatographic peak areas against standard concentration (mg/band). Linear ranges were established using a least squared regression analysis. Specificity was assessed by the ability to separate samples. Repeatability was assessed by applying three repetitions of each standard at three concentrations within the calibration curve. Variance between repetitions was expressed as a relative standard deviation (%RSD). The sensitivity of the established method was estimated in terms of the limit of quantitation (LOQ) and limit of detection (LOD). LOQ and LOD were calculated using equations LOD = 3 Â Sd/B and LOQ = 10 Â Sd/B, where Sd is the standard deviation of the peak areas of the standards (n = 3), taken as a measure of noise, and B is the slope of the corresponding calibration curve.

Antioxidant activity
The antioxidant activity of M. platytyrea extracts was assessed with a direct HPTLC-DPPH assay coupled with digital image analysis. Plates were developed with a two-step (40: 80) gradient elution method. Methanol was used in the first step to spread/stretch the bands of the polar compounds present in the sample and move them from the start. In this instance, this spreading out of the zones of more polar compounds, mostly polyphenolics with free radical scavenging activity, is desirable. By stretching out the yellow zones, saturation of the zone on the plate is avoided and better (more Table 1 Statistical parameters for stigmasterol determinations after post-chromatographic derivatization with anisaldehyde/sulfuric acid, using digital image analysis and the densitometric method at 550 nm (n = 3).

Standard
Analysis precise) quantification of the total free radical scavenging activity in the reaction with DPPH in the sample is achieved. The second elution step with the less polar mobile phase (n-hexane, ethyl acetate, acetic acid (20:9:1)) was used to better separate the less polar compounds. Antioxidant activities, in terms of free radical scavenging activity of the separated compounds that are present in the samples, appeared as yellowish zones against the purple background on the plate. Total antioxidant activity was expressed as the sum or total area of the yellow zones for each sample extract ( Table 2).
α-Amylase inhibitory activity assay α-Amylase inhibitory activity was estimated using the starch test with iodine solution as an indicator [2]. The developed plate was saturated with enzyme solution, and then incubated for 30 min at 37 C in a humid chamber, in order for the primary reaction between the enzyme and any inhibitors present in a sample to react. After incubation, the plate was dipped in a 1% w/v starch solution, incubated for 10-20 min for enzyme substrate reaction, washed with Gram's Iodine solution (detection solution), and then photographed. Starch produces a dark blue color on the HPTLC plate in the presence of iodine. A blue area around the zones indicates reduced or α-amylase inhibitory activity in the sample (Fig. 1). Therefore, plant extracts, positive for α-amylase inhibitors have blue zones after iodine staining at the positions of separated zones on the HPTLC plate, indicating the separated zone that is responsible for the inhibitory activity in the corresponding extract. The blue zone comes from the starch-iodine complex formed from starch that was not hydrolyzed by the amylase due to enzyme inhibition by the compound(s) in the extract sample. Ethanol and ethyl acetate extracts were found to have higher antioxidant activities due to the presence of polyphenolic antioxidants that are highly soluble in alcohol ( Table 2). The dichloromethane extract does not have significant antioxidant activity, but shows significant α-amylase inhibitory activity. The ethyl acetate extract has significant antioxidant and anti-hyperglycemic activities.
Relative α-amylase inhibitory activity was also determined densitometrically by comparing the area size of the peaks in densitometric chromatograms obtained at 550 nm that correspond to the blue Table 2 Comparison of total area size (in pixels) for the antioxidant activity (yellow zones after derivatization with DPPH ), area size of zones corresponding to stigmasterol in the extracts, and total area size of blue zones for α-amylase inhibitory activity from digital plate image analysis.  zones. The sum of the peak area size of the blue zones in the methanol extract was used as a standard reference to compare the total area size of blue zones in the remaining extracts (i.e. relative α-amylase inhibitory activity) ( Table 3). Relative α-amylase inhibitory activity was highly correlated with the stigmasterol concentration in the sample extracts (R = 0.95). The HPTLC method for α-amylase inhibitory activity, based on the blue starch-iodine complex reaction to visualize the hydrolysis of starch by enzyme that is pre-absorbed on the developed plate, is simple, rapid, and does not require special equipment or instrumentation. It is highly suitable for high throughput screening of plant samples for the presence of components that inhibit α-amylase activity.

Additional information
The management of diabetes and its related complications is a global problem. One of the therapeutic approaches for treating diabetes is the inhibition of α-amylase, an exoenzyme that hydrolyses starch to release glucose [3]. Since diabetic complications results from the oxidative stress and formation of hyperglycemia-derived oxygen free radicals that lead to oxidative degradation of glycated proteins [4,5], an antioxidant therapy combined with hypoglycemic drugs is recommended in order to avoid complications. The use of general antioxidants, like Vitamins C and E, has failed to demonstrate any beneficial effect in clinical trials [6]. Instead, the use of drugs with modest antioxidant activity has been shown to improve many of the problems associated with diabetic complications [7]. Myrmecodia, or ant plant, is a tropical woody plant widely distributed in equatorial region of the world, traditionally used in Irian Jaya as an alternative treatment for diabetes, as the substances produced by ants can reduce blood sugar levels. Strong antioxidant and antimicrobial activities have been observed in bioactive flavonoids and phenolic compounds isolated from the crude ethyl acetate extract from a related species Hydnophytum formicarum Jack. Stigmasterol was isolated from the crude hexane and dichloromethane extracts of this plant [8]. The antibacterial activities of stigmasterol and β-sitosterol [9][10][11], and the potent antioxidant, hypoglycemic, and thyroid inhibiting properties of stigmasterol have been previously reported [12,13]. a Inhibitory activity in the methanol extract is used as a standard reference unit to express the relative α-amylase inhibitory activity in other extracts; R = correlation coefficient.