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Journal of Chromatography A, 1530 (2017) 197–203

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

High-performance thin-layer chromatography HPTLC-direct


bioautography as a method of choice for alpha-amylase and
antioxidant activity evaluation in marine algae
Snezana Agatonovic-Kustrin a,b,∗ , David W. Morton b
a
School of Pharmacy, Monash University Malaysia, Jalan Lagoon Selatan, Bandar Sunway, 47500, Selangor Darul Ehsan, Malaysia
b
School of Pharmacy and Applied Science, La Trobe Institute for Molecular Sciences, La Trobe University, Edwards Rd, Bendigo, 3550, Australia

a r t i c l e i n f o a b s t r a c t

Article history: High-Performance Thin-layer chromatography (HPTLC) combined with DPPH• free radical method and
Received 8 October 2017 ␣-amylase bioassay was used to compare antioxidant and antidiabetic activities in ethanol and ethyl
Received in revised form acetate extracts from 10 marine macroalgae species (3 Chlorophyta, 4 Phaeophyta and 3 Rhodophyta)
13 November 2017
from Blue Lagoon beach (Malaysia). Samples were also evaluated for their phenolic and stigmasterol con-
Accepted 13 November 2017
tent. On average, higher antioxidant activity was observed in the ethyl acetate extracts (55.1 mg/100 g
Available online 14 November 2017
gallic acid equivalents (GAE) compared to 35.0 mg/100 g GAE) while, as expected, phenolic content was
higher in ethanol extracts (330.5 mg/100 g GAE compared to 289.5 mg/100 g GAE). Amounts of fucox-
Keywords:
Antidiabetic
anthin, stigmasterol and ␣-amylase inhibitory activities were higher in ethyl acetate extracts. Higher
HPTLC enzyme inhibition is therefore related to higher concentrations of triterpenes and phytosterols (Note:
Marine algae these compounds are more soluble in ethyl acetate). Ethyl acetate extracts from Caulerpa racemosa and
Triterpenes Padina minor, had the highest ␣-amylase inhibitory activity, and also showed moderately high antioxi-
Phytosterols dant activities, stigmasterol content and polyphenolic content. Caulerpa racemose, being green algae, does
not contain fucoxanthin, while Padina minor, being brown algae, contains high amounts of fucoxanthin.
Therefore, it is very unlikely that fucoxanthin contributes to ␣-amylase inhibitory activity as previously
reported.
Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved.

1. Introduction there has been a considerable focus on marine algae as a source


of bioactive compounds with therapeutic activity. The antidiabetic
Diabetes is a worldwide health challenge and economic burden activity of bioactive compounds from marine algae has been pre-
to health care providers, due to our modern lifestyles and increased viously associated with the antioxidant properties of compounds
consumption of carbohydrate. One of the therapeutic targets in the such as polyphenols and bromophenols [2,3]. Thus, the majority
management of diabetes is the inhibition of ␣-amylase, a diges- of studies have focussed on phenolic compounds with ␣-amylase
tive enzyme secreted from the pancreas and salivary gland [1]. inhibitory activity. Within this group, flavonoids have shown the
Pancreatic ␣-amylase is involved in the breakdown of starch into highest activities which is thought to be related to the number of
disaccharides and oligosaccharides and release of glucose, which is hydroxyl groups in the molecule [4]. Brown algae are well known
then absorbed into the bloodstream. The inhibition of ␣-amylase for a large variety of bioactive substances, such as fucoxanthin,
reduces the breakdown of starch in the gastro-intestinal tract and polyphenolic compounds, and phytosterols [5]. Fucoxanthin rich
subsequent glucose absorption. This may also lead to a reduction extracts from the brown algae Sargassum hemiphyllum have been
in postprandial hyperglycemia levels. shown to strongly inhibit ␣-amylase, ␣-glucosidase, sucrase, and
Plants present an important source of chemical compounds with maltase activities, and also to stimulate insulin secretion in vitro
potential ␣-amylase inhibitory activity. Due to their great diversity, [6]. Fucoxanthin is a major carotenoid present in the chloroplasts
of brown algae, followed by violoxanthin, and then ␤-carotene
[7,8]. Fucoxanthin has attracted a lot of interest due to its reported
anti-cancer [9], anti-obesity, anti-inflammatory effects [10,11],
∗ Corresponding author at: School of Pharmacy, Monash University Malaysia, Jalan anti-diabetic, anti-angiogenic [12], antioxidant [13,14], and cancer
Lagoon Selatan, Bandar Sunway, 47500, Selangor Darul Ehsan, Malaysia. chemo protective activity. It is a structurally unique molecule due to
E-mail address: snezana.agatonovic@monash.edu (S. Agatonovic-Kustrin).

https://doi.org/10.1016/j.chroma.2017.11.024
0021-9673/Crown Copyright © 2017 Published by Elsevier B.V. All rights reserved.
198 S. Agatonovic-Kustrin, D.W. Morton / J. Chromatogr. A 1530 (2017) 197–203

the presence of an unusual allenic double bonded carbon (C C C), from Sigma-Aldrich (Munich, Germany), anisaldehyde from ACROS
a 5,6-monoepoxide, and 9 conjugated double bounds [10,15]. organics (New Jersey, USA). All these chemicals were analytical
Phytosterols, are a group of compounds related to cholesterol reagent grade.
that are present in all plants [16]. They are usually found in plant cell
membranes, where they play important roles, similar to cholesterol 2.2. Sample collection and extraction
in humans. Phytosterols are recognized as important components
of healthy diets due to their ability to reduce cholesterol absorption Eight fresh algae samples (samples 1–8) were collected in
and low-density lipoprotein (LDL) concentrations in the blood [17]. approximate 0.2–0.5 kg wet weight quantities from Blue Lagoon
A diet rich in phytosterols is recommended by the EFSA (European beach, Teluk Kemang, Port Dickson, Malaysia, and transported in
Food Safety Authority) and the FDA (The US Food and Drug Admin- sea water in cooled insulated containers to our laboratory (Table 1).
istration) as part of a dietary strategy to reduce the risk of coronary Two commercially dried algae samples (sample 9 and 10) were
heart disease [18,19]. The most frequently found phytosterols are purchased from the local shop in Kuala Terengganu (Table 1).
␤-sitosterol, campesterol, and stigmasterol [20]. Research on stig- Within twenty-four hours of collection, fresh algae samples were
masterol has shown it has antioxidant, hypoglycemic and thyroid thoroughly rinsed three times with filtered seawater. Samples
inhibiting properties [21]. were then photographed, divided into 50–200 g portions, frozen at
Due to the increase in the number of patients suffering from −80 ◦ C and then lyophilised in a LAB CONCO freeze-drier (Kansas
diabetes and the limited number of anti-diabetic drugs on the mar- City, Missouri). Freeze dried samples were ground to a fine powder.
ket, the search for new compounds, especially from marine sources, Approximately 5 g of finely ground sample was extracted 5 times by
has attracted much interest recently. Uncontrolled high blood sugar vigorous shaking for 15 min with 50 mL of solvent (either ethanol
(≥ 7.0 mmol/L of fasting plasma glucose or 11.1 mmol/L of plasma or ethyl acetate), in sealed glass stoppered conical flasks and fil-
glucose) [22] can lead to a number of diabetes complications tered. The resulting solvents were combined and concentrated
that result in high morbidity and mortality rates. Hyperglycaemia to approximately 10 mL using a Büchi rotary evaporator Model
induces excess generation of highly reactive free radicals due to R-200 (Labortechnik AG, Switzerland) , transferred into 25 mL vol-
auto-oxidation of glucose, resulting in oxidative stress, which fur- umetric flasks and adjusted to volume with solvent. Commercial
ther aggravates the development and progression of diabetes and samples were ground and extracted following the same procedure
its complications [23]. However, clinical trials with classic antiox- as fresh samples. All extracts were stored at 4 ◦ C to minimize degra-
idants have not shown any benefit for diabetic patients [24]. Since dation. Samples species were identified based on morphological
oxidative stress plays a major role in the development of dia- characteristics and compared with descriptions of the samples that
betes complications, an antioxidant therapy in combination with were collected from the same location and classified previously by
anti-diabetic drugs may be a good approach to prevent diabetic Asmida et al. [27] and confirmed by comparison with the AlgaeBase
complications. database [28].
Since algae live in habitats that can vary significantly, they have
developed diverse defence strategies to protect them from harsh 2.3. High-performance thin-Layer chromatography
environmental conditions, such as extensive light and oxygen, that
leads to the formation of free radicals and other strong oxidizing Plates were pre-washed before use with a blank run of methanol
agents. The absence of oxidative damage in their structural compo- and activated by drying in an oven at 100 ◦ C for 30 min. Sample
nents and their resistance to oxidation during storage suggest the extracts (20 ␮L) were sprayed on the HPTLC-plates as 6 mm bands
presence of powerful antioxidative defence systems [25,26]. by using the Automatic TLC Sampler 4 (ATS 4, CAMAG, Muttenz,
Therefore, we wanted to evaluate and to characterise antidia- Switzerland), 8 mm from the lower edge, with 14 mm distance
betic and antioxidant activities in marine algae extracts in terms from each side, and a minimum distance of 2 mm between each
of ␣-amylase inhibition and free radical scavenging activity. We track. For a five-point calibration, 1.0–20.0 ␮L/band of the 1 mg/mL
also wanted to compare the activities of ethanol and ethyl acetate standard solutions were applied. TLC plates were developed in an
algal extracts (as phytosterols are more soluble in ethyl acetate) to Automated Multiple Development Chamber (AMD2, CAMAG, Mut-
establish if ␣-amylase inhibitory activity is related to fucoxanthin tenz, Switzerland) with a mobile phase of n-hexane, ethyl acetate,
content, antioxidant potential, and/or phytosterol content. Combi- acetic acid (20:9:1) over a 80 mm developing distance.
nation of bioassays with chromatography (direct bioautography)
enables rapid characterisation and identification of compounds 2.3.1. Post chromatographic derivatization
in complex raw samples according to their activity profile. Plate A 0.4% DPPH• solution was prepared in methanol (Merck,
chromatograms are directly immersed into an enzyme solution, Darmstadt, Germany), stored at 2–8 ◦ C, and protected from light.
incubated up to several hours, and then the activity profile of Neutralized ferric chloride solution was prepared by adding dilute
the enzyme-substrate reaction can be visualized, recorded and sodium hydroxide solution to freshly prepared 2% ferric chloride
assessed. solution in methanol, drop by drop until ferric hydroxide starts to
precipitate. The solution was then filtered to remove the precipitate
and the clear filtrate was used for derivatization [29]. The anisalde-
2. Material and methods hyde reagent solution was freshly prepared by mixing anisaldehyde
with a refrigerated solution of glacial acetic acid/concentrated sul-
2.1. Chemicals used phuric acid in methanol (a ratio of 0.5:10:5:85). The colourless
solution was stored in refrigerator (if color develops the reagent
2,2-Di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH• ) free radi- must be discarded).
cal, iron(III) chloride (97%), fucoxanthin (98%), gallic acid (97%), Derivatization was achieved by dipping (immersing) a HPTLC
stigmasterol (95%) were purchased from Sigma-Aldrich (Munich, plate into the derivatizing agent for 1 s and with an immer-
Germany). HTLC separations were performed on 20 × 10 cm nor- sion speed of 5 cm/s using the Chromatogram Immersion Device
mal phase Silica gel 60 F254 HPTLC glass plates (Merck, Darmstadt, (CAMAG, Muttenz, Switzerland). Plates derivatized with DPPH•
Germany). solution, were stored in dark for 30 min and then photographed.
Acetic acid, and methanol were purchased from Merck (Darm- Plates derivatized with anisaldehyde-sulfuric acid and with ferric
stadt, Germany), n-hexane from BDH (Poole, England), ethyl acetate chloride were heated at 110 ◦ C for 10 min. Images of the plates were
S. Agatonovic-Kustrin, D.W. Morton / J. Chromatogr. A 1530 (2017) 197–203 199

Table 1
Algae samples used in this work.

Sample Number Species Type Algae type and track no. in chromatogram

1 Gracilaria arcuata Red algae Tracks 1 & 11


2 Sargassum polycystum Brown algae Tracks 2 & 12
3 Euchema cottoni Red algae Tracks 3 & 13
4 Padina pavonica Brown algae Tracks 4 & 14
5 Dictyota dichotoma Brown algae Tracks 5 & 15
6 Padina minor Brown algae Tracks 6 & 16
7 Caulerpa brachypus Green algae Tracks 7 & 17
8 Caulerpa racemosa Green algae Tracks 8 & 18
9 Laminaria sp. Green algae Tracks 9 &19
10 Kappaphycus sp. Red algae Tracks 10 & 20

captured with a TLC-visualiser (CAMAG, Muttenz, Switzerland) 3. Results and discussion


equipped with a 12-bit charged couple device (CCD) digital camera
and winCATS software (CAMAG, Muttenz, Switzerland). WinCATS Polyphenolic compounds such as flavonoids, phenolic acids and
image capturing parameters were fixed to ensure high quality carotenoids are considered as major contributors to the antioxidant
images and reproducibility between plates. Quantitative HPTLC activity of marine algae. The antioxidant activities of polyphenols
analysis was performed from TLC plate images with digital image are attributed to their redox properties, which allow them to act as
analysis software Sorbfil TLC Videodensitometer (Sorbpolymer, reducing agents, hydrogen donators and singlet oxygen quenchers,
Krasnodar, Russia) and with CAMAG TLC scanner III controlled by as well as their metal chelating abilities [3,25]. Metal ions, such
winCATS 3.1 software (CAMAG, Muttenz, Switzerland) at 470 nm as iron or copper, can act as catalysts for the initial formation of
for starch-iodine complex (␣-amylase inhibition) and 550 nm for reactive oxygen species. However, complexation of pro-oxidative
stigmasterol. All developed plates were photographed both before metal ions in living systems with polyphenols can inhibit their
and after derivatization. catalytic activity. Therefore, in the present study two different
methods were used to determine and compare the antioxidant
properties of selected algal samples. The DPPH• assay was used
to evaluate the free radical scavenging effectiveness [26,33] while
2.3.2. Method validation derivatization with ferric chloride was used to evaluate polyphe-
The methods for gallic acid, fucoxanthin and stigmasterol nolic content. DPPH• is a stable radical, due to delocalisation of
determinations were validated by following the International Con- its spare electron over the whole molecule. The electron delo-
ference on Harmonization (ICH) guidelines [30]. The working range calisation causes a deep violet color with maximum absorbance
was assessed by plotting chromatographic peak areas against con- (␭max ) at around 520 nm. When DPPH• is reduced to its stable non-
centration of standards in ␮g/band. Linear ranges were established radical form, the purple color changes to pale yellow [34]. Thus,
using the least squared regression analysis in Excel. Specificity was after derivatization with DPPH• , compounds possessing radical-
assessed by the ability of the optimized mobile phase to separate scavenging activity are detected as bright yellow bands against a
samples. Repeatability was assessed by applying three repetitions purple background (Fig. 1c).
of each standard at three concentrations (low, medium and high) For visualisation of phenolics in visible light, plates were sprayed
within the calibration curve. Variance between repetitions was with a 2% ethanolic solution of ferric chloride. Ferric chloride in
expressed as a relative standard deviation (%RSD). solution gives a colored reaction with a number of organic deriva-
The sensitivity of measurements was estimated in terms of the tives, i.e. phenols, enols, oximes, hydroxamic acids, and some
limit of quantitation (LOQ) and limit of detection (LOD). LOQ and carboxylic acids. Fucoxanthin for example, seen as a yellow band
LOD were calculated by the use of equation LOD = 3 × Sd/B and under white light at Rf = 0.37, has produced blue color after deriva-
LOQ = 10 × Sd/B, where Sd is the standard deviation of the peak areas tization with ferric chloride (Fig. 1a). Colour formation on reaction
of the standards (n = 3), taken as a measure of noise, and B is the with alcoholic ferric chloride solution is an identification test
slope of the corresponding calibration curve. commonly used to determine the presence of simple phenolic com-
pounds (Fig. 1d) [35]. Triterpenes (C30) and phytosterols (C27)
were identified on the plate after derivatization with anisalde-
hyde/sulfuric acid [36]. Stigmasterol was visualized as blue-violet
bands under visible light (Fig. 1e) at Rf values of 0.79.
2.3.3. ˛-Amylase inhibitory activity Gallic acid was used as a standard compound (i.e. model ana-
A 1% (w/v) ␣-amylase solution was prepared by diluting approx- lyte), for the quantification of free radical scavenging activity and
imately 1.25 mL (1 g) of ␣-amylase from Bacillus licheniformis liquid total phenolic content. Free radical scavenging activity and pheno-
(Cat. No. A4862, Sigma-Aldrich, Denmark) with distilled water to lic content were expressed as mg equivalents of gallic acid/100 g
a volume of 100 mL [31]. The enzyme stock solution was refrig- by using the standard curve of gallic acid (calibration graph). Cal-
erated at 4 ◦ C until required. Developed plates were saturated in ibration curves were constructed by plotting the range of applied
␣-amylase solution, placed in a plastic container with plugs to lay volumes versus corresponding band areas in pixels (Table 2). High
the plate on. The container was filled with a little water, such that correlation of determination (R-squared or R2 ) values indicate high
the plate does not come into contact with the water, but enough to correlation of the fitted regression lines, both linear and non-linear,
keep humidity inside the container. Incubation was performed for to the data (Table 2). The linear range of the methods was estab-
30 min at 37 ◦ C for the primary reaction between the enzyme and lished as 0.5–10.0 ␮g for fucoxanthin under white light; 0.3–8.0 ␮g
enzyme inhibitors separated in the extracts. After incubation, the for gallic acid with DPPH• ; 0.3–10.0 ␮g for gallic acid with FeCl3
plate was dipped in the 1% starch solution, incubated for another and 0.4–10.0 ␮g for stigmasterol with anisaldehyde/sulfuric acid.
10–20 min for enzyme substrate reaction, and then washed with A good repeatability of the methods was confirmed by calculat-
Gram’s Iodine solution (detection solution) [32]. The image of the ing the coefficient of variation for five repeated measurements at
plate was then captured.
200 S. Agatonovic-Kustrin, D.W. Morton / J. Chromatogr. A 1530 (2017) 197–203

Fig. 1. HPTLC chromatograms of extracts under white light (a), under 366 nm (b), after derivatization with ferric chloride (c), after derivatization with DPPH• (d), after
derivatization with anisaldehyde/sulfuric acid (e), and after ␣-amylase assay (f). Mobile phase; n-hexane: ethyl acetate: acetic acid in a ratio of 20:9:1 (v/v/v). Tracks 1–10,
ethanol extracts; Tracks 11–20, ethyl acetate extracts. Applied 20 ␮L per band.

Table 2
Calibration, linearity, accuracy, and precision for gallic acid, fucoxanthin, and stigmasterol determination.

Standard Method Equations R Linear range (␮g) LOD (␮g) LOQ (␮g)

Gallic acid DPPH y = 329846x − 142959 0.987 0.3–8.0 0.3 1.1
y = −868.3 x2 + 397750x − 240247 0.990
Gallic acid. Ferric chloride y = 72300x + 62844 0.967 0.3–10.0 0.3 1.1
y = −4285.4 x2 −111234x + 7560 0.999
Fucoxanthin White light y = 10877x − 6616.5 0.953 0.5–10.0 0.5 0.6
y = −687.96 x2 + 18363x − 21994 0.980
Stigmasterol Anisaldehyde/ sulfuric y = 28706x − 6477 0.983 0.4–10.0 0.4 1.5
acid y = 415.33x2 + 23820x − 7615.5 0.985

three different concentrations at low, medium and high concentra- color on the TLC plate in the presence of iodine, with a blue zone
tion of standards in the calibration range. Averaged coefficients of around the bands indicating reduced ␣-amylase activity in the sam-
variations were 7.3%, 6.7%, 4.1% and 7.6% for fucoxanthin, gallic acid ple. The blue color comes from the starch-iodine complex formed
(ferric chloride), gallic acid (DPPH• ) and stigmasterol respectively. with starch that was not hydrolyzed by the ␣-amylase due to
The relatively low %RSD observed indicates good precision of the enzyme inhibition by the compounds in the sample.
method allowing quantification of phenolics, antioxidants, fucox- Starch is a heteropolysaccharide, composed of amylose
anthin and stigmasterol. Sensitivity of the method was determined (20–30%) and amylopectin (70–80%) polysaccharides, which are
by calculating LOD and LOQ. LODs of 0.5, 0.3, 0.3 and 0.4 and LOQs of assembled in a cluster structure [37]. Amylose has the ability
0.6, 1.1, 1.1 and 1.5 for fucoxanthin, gallic acid with ferric chloride, to form inclusion complexes with iodine. Although starch occurs
gallic acid with DPPH• and stigmasterol were determined. naturally as water-insoluble granules, amylose can be molecu-
␣-Amylase inhibitory activity was evaluated with a starch test larly dispersed in water once the granular structure is destroyed
using iodine solution as an indicator. Starch produces a dark-blue by heating, opening up the possibility to form complexes with
S. Agatonovic-Kustrin, D.W. Morton / J. Chromatogr. A 1530 (2017) 197–203 201

Table 3
Antioxidant activity, phenolic content, fucoxanthin content, stigmasterol content and ␣-amylase inhibitory activity in ethanol extracts.

Sample Antioxidant activity Phenolic content GAE Fucoxanthin Stigmasterol ␣-amylase inhibition
GAE (mg/100 g) (mg/100 g) (mg/100 g) (mg/100 g) (AU (area))
*
1 27.3 143.9 2.13 1078
2 27.8 259.7 413.0 3.47 1624
3 32.5 61.5 93.5 1.37 13027
4 34.2 349.9 427.9 2.90 3992
5 45.8 564.9 433.7 5.55 2832
6 39.4 672.9 500.0 3.61 3342
7 40.2 514.0 0.0 5.37 8676
8 42.2 631.5 0.0 9.40 7821
9 32.5 67.4 0.0 0.57 7173
10 27.9 39.0 0.0 1.44 750
average 35.0 330.5 207.6 3.58 5032

Correlation
Antioxidant activity 0.84 0.36 0.14 0.29
Phenolic content 0.84 −0.17 −0.01
Fucoxanthin −0.85 −0.52
Stigmasterol 0.11

Table 4
Antioxidant activity, phenolic content, fucoxanthin content, stigmasterol content and ␣-amylase inhibitory activity in ethyl acetate extracts.

Sample Antioxidant GAE Phenolics GAE Fucoxanthin Stigmasterol ␣-amylase inhibition


(mg/100 g) (mg/100 g) (mg/100 g) (mg/100 g) (AU (area))

1 22.8 47.3 * 2.04 750


2 48.2 126.4 327.6 5.25 2655
3 26.4 11.3 40.9 5.01 2114
4 74.2 254.7 401.8 5.53 3470
5 95.9 693.8 620.5 10.84 15752
6 76.6 522.4 691.1 8.26 21493
7 55.8 428.2 * 8.73 11908
8 81.3 745.3 * 11.00 22741
9 44.5 2.42 * 3.00 8912
10 24.8 63.2 * 1.75 2280
Average 55.1 289.5 416.4 6.14 9208

Correlation
Antioxidant 0.89 0.84 0.86 0.77
Phenolic content 0.91 0.94 0.87
Fucoxanthin 0.90 0.87
Stigmasterol 0.83

iodine [25]. Long chains of amylose act as a flexible coil and 470 nm that corresponds to observed blue bands at Rf = 0.85; on,
form a complex with iodine. The iodine molecule is held within before, and after the ␣-amylase assay.
a cage structure, one iodine molecule per helix formed with six The results show that the extracts from freeze dried fresh
glucose units [38]. The starch-iodine dark blue complex shows a samples (samples 1–8) have relatively higher antioxidant and ␣-
broad absorption peak with a ␭max at approximately 600–620 nm amylase inhibitory activity when compared to the commercial sun
and a shoulder peak with a ␭max at 460–470 nm. A critical chain dried samples (samples 9 and 10). Higher activity may be due to
length of 100 glycosyl residue is needed to make a stable and the presence of higher carotenoids and triterpenes contained in
ordered helix of glycosil residues that form a dark blue iodine fresh algae material. On average, higher antioxidant activity was
complex with a ␭max at 620 nm [39–41]. The shoulder peak in observed in the ethyl acetate extracts (55.1 mg/100 g GAE com-
the absorption spectrum is formed between Schardinger dextrins pared to 35.0 mg/100 g GAE) while, as expected, phenolic content
(water-soluble, low-molecular-weight polysaccharides) and iodine was higher in ethanol extracts (330.5 mg/100 g GAE compared to
[42]. The Schardinger dextrins or cyclodextrins are cyclic rings of 289.5 mg/100 g GAE) (Tables 3 and 4). Amounts of fucoxanthin,
glucose monomers linked by ␣-1,4 glycosidic linkage, obtained stigmasterol and ␣-amylase inhibitory activities were generally
with the further breakdown of starch [43]. higher in ethyl acetate extracts. Since ethyl acetate is less polar
The inhibition of ␣-amylase, seen as pale blue bands on the than ethanol, it is more effective in extracting the moderately polar
TLC plate, was observed at an Rf value of 0.85 (Fig. 1f). The sterols and triterpenes.
best resolution of the peaks in the chromatograms was achieved The highest ␣-amylase inhibitory activity was observed in green
at 470 nm, suggesting the formation of a complex between low algae samples 8 and 7 and brown algae samples 6 and 5, while the
molecular weight Schardinger dextrins and iodine (higher level of lowest activity was seen in red algae samples 1, 3, and 10. It is
starch decomposition). There was no bleaching of the DPPH• at accepted that different classes of algae possess characteristic sterol
the same position on the plate (Rf 0.85) to indicate antioxidant compositions. Green algae contain a complex mixture of sterols
activity. However, there was a characteristic blue-violet color reac- that is similar to the higher plants, while the sterol composition in
tion after derivatization with anisaldehyde, indicating the presence both brown and red algae is relatively simple and usually limited
of a terpenoid, probably a phytosterol and a pale blue color with to one primary sterol [44]. Furthermore, compared to brown algae,
ferric chloride indicating the presence of a phenolic compound. red algae is considered less advanced [45]. Therefore, the higher
The ␣-amylase inhibitory activity in the samples was analysed by enzyme inhibition of ethyl acetate extracts can be explained with
comparing the peak areas in TLC densitometric chromatograms at higher content of triterpenes and phytosterols. Tetracyclic triter-
202 S. Agatonovic-Kustrin, D.W. Morton / J. Chromatogr. A 1530 (2017) 197–203

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