Antidiabetic and antioxidant profiling of 67 African trifoliate yam 
accessions by planar on-surface assays versus in vitro assays

Priscilla O. Aiyedun a,b,c,1, Mubo A. Sonibare a, Badara Gueye b, Dirk C. Albach c, Julia Heil d,  
Gertrud E. Morlock d,e,*,1

a Department of Pharmacognosy, Faculty of Pharmacy, University of Ibadan, Oduduwa Road, 200132 Ibadan, Oyo, Nigeria
b Genetic Resources Centre, International Institute of Tropical Agriculture, Oyo Road, 200001 Ibadan, Oyo, Nigeria
c Institute of Biology and Environmental Sciences, Carl von Ossietzky University Oldenburg, Carl-von-Ossietzky-Str. 9-11, 26129 Oldenburg, Germany
d Institute of Nutritional Science, Chair of Food Science, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, Germany
e Center for Sustainable Food Systems, Justus Liebig University Giessen, Senckenbergstr. 3, 35390 Giessen, Germany

A R T I C L E  I N F O

Keywords:
Dioscorea dumetorum
Bioactivity
Effect-directed analysis or assays
High-performance thin-layer chromatography
HPTLC–EDA
HPTLC–HRMS

A B S T R A C T

Trifoliate yam (Dioscorea dumetorum) is traditionally used to treat diabetics in Nigeria. However, almost no in
formation is available on its antidiabetic constituents and their natural variance. Hence, the activity of meth
anolic tuber extracts of 67 trifoliate yam accessions from the largest collection in Africa was proven by four 
colorimetric antidiabetic and antioxidant in vitro assays, as diabetes is also linked with oxidative stress. For the 
first time, selected accessions were also analyzed by planar bioactivity profiling. It has a comparatively higher, 
more differentiated information content, is more sustainable in terms of material consumption, and enables 
straightforward compound prioritization and characterization. Up to a dozen individual antioxidant zones were 
revealed as well as one prominent zone inhibiting α-glucosidase and α-amylase. The latter inhibition zone was 
tentatively assigned to palmitic, linoleic, oleic, linolenic, oxo-nonanoic fatty acids by direct elution to heated 
electrospray ionization high-resolution mass spectrometry.

1. Introduction

Diabetes is classified as a civilization disease and a major health 
burden worldwide [1]. According to the Diabetes Atlas, there has been a 
continued increase in diabetes prevalence globally with approximately 
537 million adults (20–79 years) living with diabetes and 6.7 million 
deaths caused by diabetes in 2021. An increase to 643 million by 2030 
and 783 million by 2045 was also projected [2]. Diabetes is considered 
to be the leading cause of cardiovascular diseases, blindness, kidney 
failure and lower limb amputation in most high-income countries, and 3 
out of every 4 adults live with diabetes in low- and middle-income 
countries. Different synthetic medications used to treat diabetes, such 
as biguanides (e.g., metformin), sulfonylureas (e.g., glibenclamide), 
thiazolidinediones (e.g., rosiglitazone and pioglitazone), α-glucosidase 
inhibitors (e.g., acarbose, miglitol and voglibose) are frequently ineffi
cient and have side effects. Such undesirable impacts include weight 
gain, lactic acidosis, hepatoxicity, kidney toxicity, pancreatitis, 

cardiovascular diseases, hypoglycemia, and gastrointestinal symptoms 
such as nausea, vomiting, indigestion, diarrhea, belching, and flatulence 
[3–5].

Searching for more effective and safer antidiabetic agents, natural 
plant-based products as ethnomedicinal functional food have always 
been more sought after as the basis for the treatment of human diseases 
[6]. Medicinal plants have comparatively few side effects, contain fewer 
heavy metals, and show a broader distribution of molecular properties, 
partition coefficient, and structural diversity [7,8]. They also have more 
interactions with proteins, enzymes and other biological molecules and 
have greater molecular rigidity when compared with synthetic com
pounds [9]. One severely understudied plant is the trifoliate yam Dio
scorea dumetorum (Kunth) Pax belonging to the family Dioscoreaceae. It 
is one of the most important plant families in the economy of many sub- 
Saharan African countries. With over 600 species worldwide, the tubers 
of many Dioscorea species (yams) including D. dumetorum are highly 
important for subsistence as proven by their continued cultivation for 

* Corresponding author at: Institute of Nutritional Science, Chair of Food Science, Justus Liebig University Giessen, Heinrich-Buff-Ring 26-32, 35392 Giessen, 
Germany.

E-mail address: Gertrud.Morlock@uni-giessen.de (G.E. Morlock). 
1 Both authors contributed equally.

Contents lists available at ScienceDirect

Fitoterapia

journal homepage: www.elsevier.com/locate/fitote

https://doi.org/10.1016/j.fitote.2024.106299
Received 30 June 2024; Received in revised form 7 November 2024; Accepted 10 November 2024  

Fitoterapia 180 (2025) 106299 

Available online 14 November 2024 
0367-326X/© 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). 

mailto:Gertrud.Morlock@uni-giessen.de
www.sciencedirect.com/science/journal/0367326X
https://www.elsevier.com/locate/fitote
https://doi.org/10.1016/j.fitote.2024.106299
https://doi.org/10.1016/j.fitote.2024.106299
http://creativecommons.org/licenses/by/4.0/


Table 1 
Antioxidant and antidiabetic screening of the 67 Dioscorea dumetorum accessions by colorimetric in vitro assays (in bold: twelve samples selected for reproducibility 
study as in Table 2 and for comparison with the HPTLC− UV/Vis/FLD − EDA profiling as in Figs. 1–3).

Landraces of Dioscorea dumetorum Colorimetric in vitro assays <LOD: below detection limit.

No. Accession 
TDd-xxxx

Origin Cultivar name TPC (mg GAE/g) DPPH• SC50 (mg/mL) α-Glucosidase IC50 (mg/mL) α-Amylase IC50 (mg/mL)

1 2788 Nigeria Dumetorum 53 ± 0.42 1.2 ± 0.03 155 ± 0.36 4 ± 0.05
2 3093 Togo Vote 333 165 ± 0.33 0.7 ± 0.01 150 ± 0.25 <LOD
3 3094 Togo Ic-35 157 ± 0.46 1.6 ± 0.02 125 ± 0.23 <LOD
4 3095 Togo Koute 281 <LOD 0.7 ± 0.01 148.3 ± 0.31 <LOD
5 3096 Togo D. dumentorum 101 ± 0.34 1.4 ± 0.01 150 ± 0.23 <LOD
6 3097 Togo Votedre 340 224 ± 0.22 0.9 ± 0.01 201 ± 0.27 <LOD
7 3098 Togo Fa 26 174 ± 0.51 3.4 ± 0.01 129 ± 0.26 <LOD
8 3100 Togo Dokoute 33 75 ± 0.37 <LOD 118 ± 0.24 <LOD
9 3102 Togo Votehe 341 115 ± 0.32 1.0 ± 0.01 148 ± 0.25 <LOD
10 3103 Ghana Kamfo 101 ± 0.27 1.4 ± 0.01 153 ± 0.24 11 ± 0.04
11 3104 Togo Agbote 93 357 ± 0.43 0.2 ± 0.00 85 ± 0.23 13 ± 0.03
12 3105 Nigeria D. domentorum (44) 99 ± 0.40 <LOD 210 ± 0.35 <LOD
13 3106 Togo Agbote 19 206 ± 0.39 1.0 ± 0.01 127 ± 0.24 14 ± 0.06
14 3107 Togo Agbote 104 182 ± 0.47 0.5 ± 0.01 111 ± 0.22 <LOD
15 3108 Ghana Fa/89/007 163 ± 0.24 0.7 ± 0.01 163 ± 0.25 <LOD
16 3109 Ghana Nya 160 ± 0.44 1.4 ± 0.01 190 ± 0.21 <LOD
17 3110 Togo Agbota 457 38 ± 0.16 2.2 ± 0.01 243 ± 0.23 <LOD
18 3111 Togo Vote 293 98 ± 0.38 2.2 ± 0.01 <LOD <LOD
19 3112 Nigeria D. domentorum 29 ± 0.20 2.4 ± 0.01 93 ± 0.23 <LOD
20 3113 Gabon – 138 ± 0.27 1.2 ± 0.01 126.6 ± 0.22 <LOD
21 3114 Gabon – 104 ± 0.25 11 ± 0.01 103 ± 0.21 <LOD
22 3686 Nigeria Ighu(wild) 114 ± 0.36 0.8 ± 0.01 118 ± 0.21 11 ± 0.04
23 3687 Nigeria Wild(ighu) 83 ± 0.27 1.2 ± 0.01 131 ± 0.23 <LOD
24 3710 Congo Isciuli 321 ± 0.46 0.1 ± 0.00 114 ± 0.20 <LOD
25 3717 Congo Assoulu cui 77 ± 0.19 2.3 ± 0.01 154 ± 0.24 <LOD
26 3770 Nigeria – 121 ± 0.41 1.9 ± 0.01 162 ± 0.24 <LOD
27 3771 Nigeria – 279 ± 0.40 0.6 ± 0.01 73 ± 0.21 6 ± 0.04
28 3775 Nigeria Unu-oji 602 ± 0.51 0.4 ± 0.00 129 ± 0.26 9 ± 0.05
29 3776 Nigeria Obiauturugo 229 ± 0.39 0.9 ± 0.01 83 ± 0.23 <LOD
30 3777 Nigeria Oyoyo 132 ± 0.33 0.9 ± 0.01 113 ± 0.22 <LOD
31 3778 Nigeria Ameh 63 ± 0.39 <LOD 129 ± 0.23 <LOD
32 3779 Nigeria Amola 126 ± 0.37 0.4 ± 0.01 36 ± 0.21 5 ± 0.05
33 3790 Nigeria Peeba 154 ± 0.33 2.1 ± 0.01 104 ± 0.25 <LOD
34 3804 Benin – 310 ± 0.34 0.5 ± 0.01 90 ± 0.23 <LOD
35 3810 Benin Assan-te 91 ± 0.28 1.5 ± 0.01 144 ± 0.27 <LOD
36 3829 Benin Klipm-36 252 ± 0.30 0.5 ± 0.01 150 ± 0.21 <LOD
37 3848 Benin Leife 413 ± 0.46 0.4 ± 0.01 74 ± 0.21 <LOD
38 3906 Nigeria – 87 ± 0.37 1.5 ± 0.01 209 ± 0.37 21 ± 0.05
39 3910 Nigeria – 456 ± 0.38 0.5 ± 0.01 126 ± 0.22 <LOD
40 3935 Benin – 48 ± 0.18 1.2 ± 0.01 110 ± 0.22 4 ± 0.04
41 3946 Nigeria Esuru-funfun 740 ± 0.43 0.2 ± 0.00 27 ± 0.21 <LOD
42 3947 Nigeria – 100 ± 0.35 2.5 ± 0.01 115 ± 0.25 <LOD
43 4117 Togo Ikafugaga 481 ± 0.44 0.6 ± 0.01 182 ± 0.24 <LOD
44 4118 Togo N’kafo 144 ± 0.27 0.8 ± 0.01 114 ± 0.21 <LOD
Cultivated
45 4119 Nigeria Esuru (white) 225 ± 0.44 0.8 ± 0.01 152 ± 0.23 <LOD
46 4120 Nigeria Esuru (white) 165 ± 0.27 1.0 ± 0.01 153 ± 0.22 <LOD
47 4122 Nigeria Esuru 133 ± 0.31 2.7 ± 0.01 123 ± 0.23 <LOD
48 4123 Nigeria Esuru 76 ± 0.18 1.5 ± 0.01 130 ± 0.36 <LOD
49 4124 Nigeria Esuru 121 ± 0.39 1.5 ± 0.01 105 ± 0.22 <LOD
50 4125 Nigeria Esuru 120 ± 0.24 1.3 ± 0.01 126 ± 0.23 <LOD
51 4126 Nigeria Esuru 253 ± 0.35 0.8 ± 0.01 105 ± 0.22 <LOD
52 4127 Nigeria Esuru 220 ± 0.46 0.7 ± 0.01 198 ± 0.22 <LOD
53 4128 Nigeria Esuru 156 ± 0.25 1.0 ± 0.01 35 ± 0.21 3 ± 0.03
54 4129 Nigeria Esuru 163 ± 0.38 1.4 ± 0.01 151.3 ± 0.27 <LOD
55 4130 Nigeria Esuru 63 ± 0.24 0.9 ± 0.01 232 ± 0.26 <LOD
56 4131 Nigeria Esuru 120 ± 0.34 0.9 ± 0.01 208 ± 0.25 <LOD
57 4132 Nigeria Esuru 157 ± 0.38 3.2 ± 0.01 200 ± 0.26 <LOD
58 4133 Nigeria Esuru 155 ± 0.49 1.5 ± 0.01 189 ± 0.26 <LOD
59 4134 Nigeria Gududu (poison) 116 ± 0.32 2.3 ± 0.01 144 ± 0.27 <LOD
60 4135 Nigeria Esuru 76 ± 0.26 2.0 ± 0.01 208 ± 0.27 <LOD
61 4136 Nigeria Esuru 82 ± 0.36 1.7 ± 0.01 108 ± 0.22 <LOD
62 4137 Nigeria Esuru 53 ± 0.27 2.7 ± 0.01 173 ± 0.28 <LOD
63 4138 Nigeria Esuru 144 ± 0.36 0.8 ± 0.01 131 ± 0.25 <LOD
64 4139 Nigeria Esuru 257 ± 0.40 0.9 ± 0.01 102 ± 0.22 <LOD
65 4140 Nigeria Esuru 81 ± 0.43 1.2 ± 0.01 173 ± 0.25 <LOD
66 4141 Nigeria Esuru 24 ± 0.22 4.7 ± 0.01 237 ± 0.22 <LOD
67 4142 Nigeria Esuru 42 ± 0.29 2.4 ± 0.01 216 ± 0.25 19 ± 0.04

P.O. Aiyedun et al.                                                                                                                                                                                                                             Fitoterapia 180 (2025) 106299 

2 



food and medicinal purposes in Africa [10]. In West African folk medi
cine practices, D. dumetorum is used in the treatment of several free 
radical-mediated diseases [11] such as hemorrhoid, measles, jaundice, 
fever, malaria, ulcers, scorpion bites, sexual weakness [12–14]. The 
alcohol and water extracts of tubers are also used to treat confirmed 
cases of diabetes [15,16]. Several studies have reported in vitro and/or in 
vivo hypoglycemic [13], antioxidant, anti-nociceptive [17], antifungal 
[18], anticholinesterase [19], and analgesic [20] activities of 
D. dumetorum most likely due to the presence of phenols, flavonoids, 
alkaloids, tannins, phytates, and oxalates. The ability of a plant extract 
to inhibit digestive starch-hydrolyzing enzymes (salivary and pancreatic 
α-amylase, or intestinal α-glucosidase) is indicative of the antidiabetic 
effect. Researchers have reported the general antidiabetic effects of 
D. dumetorum without narrowing it down to the specific compounds 
responsible [13,21–25]. So far, only one study has proposed an active 
compound, i.e. dioscoretine, as hypoglycemic agent [26].

Difficulties were faced in discovering drug compounds from plants 
[6,27]. Apart from tedious workflows, in vitro assay protocols may 
discriminate lipophilic compounds by removal in a defattening step or 
by insolubility in the polar assay medium or by adsorption to plastic 
material. Mixed effect mechanisms can mislead the decision taken from 
results of in vitro assays, which only provide a sum value for a complex 
mixture. Hence, comparing in vitro assays with on-surface assays is 
essential for sound information on bioactivity. The combination of a 
planar separation technique and a planar biological or enzymatic assay 
detection has unique benefits [28]. Planar multiplex assays allow dif
ferentiation of the various effects caused by agonists, antagonists, false- 
positives, and synergists [29,30]. As a straightforward hyphenation, 
high-performance thin-layer chromatography–effect-directed analysis 
(HPTLC–EDA) including multi-imaging by ultraviolet (UV), visible (Vis, 
white light illumination), and fluorescence detection (FLD) directly 
points to bioactive compound zones. This is a great assistance in 
deciding which specific compounds should be further characterized out 
of the abundance of compounds present in natural samples [31,32].

In this study, 67 African D. dumetorum accessions were analyzed to 
provide an understanding of their variability regarding total phenolic 
content (TPC) as well as antioxidative and antidiabetic properties across 
the botanical diversity. It was hypothesized that ethnomedicinal uses of 
D. dumetorum will be confirmed and support its properties as a func
tional food, that in vitro assays will at best correlate with planar assays, 
that bioactivity profiles of D. dumetorum obtained for the first time are 
meaningful and can be used for compound prioritization and future 
authenticity verification, and that a straightforward hyphenation to 
heated electrospray ionization high-resolution mass spectrometry 
(HESI-HRMS) will be able to tentatively assign prominent bioactive 
compounds.

2. Materials and methods

2.1. Chemicals and reagents

All chemicals were of analytical grade. Acetic acid (100 %), ani
saldehyde (4-methoxy benzaldehyde) sulfuric acid (96 %), gallic acid 
(≥98 %), citric acid (p. a.), disodium hydrogen phosphate (≥99 %) were 
obtained from Carl Roth, Karlsruhe, Germany. Potassium sodium 
tartrate tetrahydrate was purchased from Molychem, Mumbai, India. 
Potato starch, sodium dihydrogen phosphate dihydrate (≥99 %), diso
dium hydrogen phosphate dihydrate (≥99 %), sodium carbonate, so
dium hydroxide pellets (≥98 %), methanol (MS quality) and formic acid 
(99 %) were from VWR, Darmstadt, Germany, whereas 3,5-dinitrosali
cylic acid was from Kemlight Spechem Labs, Mumbai, India (97 %). 
Gram's iodine solution, acarbose (for pharm.), α-glucosidase from 
Saccharomyces cerevisiae (1000 U/vial), sodium acetate, sodium chlo
ride, α-amylase from hog pancreas (50 U/mg), α-amylase from porcine 
pancreas (9 U/mg), p-nitrophenyl-α-D-glucopyranoside (≥99 %), L- 
ascorbic acid, dimethyl sulfoxide, methanol (99.6 %) and ethanol (99 %) 

were delivered by Sigma-Aldrich, Steinheim, Germany. 2-Naphthyl-α-D- 
glucopyranoside (Fluorochem, Hadfield Derbyshire, UK), and Fast Blue 
B salt (95 %) was purchased from MP Biomedicals, Eschwege, Germany. 
2,2-Diphenyl-1-picrylhydrazyl (DPPH•, 95 %) was delivered by Alfa 
Aesar, Schwerte, Germany. Acetonitrile (Honeywell) was from Fluka, 
Seelze, Germany. Ethyl acetate (≥99.8 %) was purchased from Th. 
Geyer, Renningen, Germany. HPTLC plates silica gel 60 F254 MS-grade 
and silica gel 60 (both 20 cm × 10 cm) were provided by Merck, 
Darmstadt, Germany. Folin–Ciocalteu reagent was delivered by LOBA
Chemie, Tarapur, India. Bidistilled water was produced by Purelab 
Chorus (ElgaVEOLIA, Woodridge, IL, USA).

2.2. Plant materials and preparation

Field-grown tubers of 67 yam accessions were harvested in 
December 2018, at the International Institute of Tropical Agriculture, 
Ibadan, Nigeria. These originate from the six African countries Nigeria, 
Benin, Ghana, Gabon, Togo, and Congo (Table 1), representing the ge
netic diversity of Dioscorea dumetorum in the field bank at the Genetic 
Resource Center of the Institute. The tubers were washed thoroughly 
with running tap water and peeled with a sharp knife. The flesh was 
sliced into thin strips, freeze-dried for 120 h (freeze dryer FreeZone, 
Labconco, Kansas City, MO, USA), and pulverized (50–300 mesh, speed 
1, grinder HYDDNice 2500 W, Hangzhou, China). Each powder (50 g) 
was macerated in methanol (500 mL) at room temperature with regular 
stirring for 72 h. The extracts were filtered using a Buchner funnel fitted 
with a filter paper (185 mm diameter, Whatman, GE Healthcare, 
Buckinghamshire, UK) and connected to a vacuum pump. The filtrates 
were concentrated in vacuo at 40 ◦C and allowed to dry in the open air 
for 48 h. The dry extracts were stored in a refrigerator (4 ◦C) and dis
solved in solvent or buffer as described for the respective in vitro assay 
analysis. For HPTLC, extract solutions (50 mg/mL) were prepared in 
ethyl acetate – ethanol – water, 1:1:1 (V/V/V), ultra-sonicated (10 min), 
and centrifuged (17,000 xg, 5 min).

2.3. TPC cuvette assay

The TPC of the extracts was determined by the colorimetric 
Folin–Ciocalteu assay [33]. Briefly, 2.5 mL Folin–Ciocalteu reagent – 
bidistilled water (1,10, V/V) was mixed with 0.5 mL extract solution (1 
mg/mL in methanol) and 2 mL of sodium carbonate solution (7.5 g/L in 
bidistilled water). The resultant mixture was incubated at 25 ◦C for 30 
min and the absorbance of the resulting blue-colored complex was 
measured at 765 nm in a cuvette spectrophotometer (UV-3100PC, VWR 
International, Radnor, PA, USA). The higher the amount of reducing 
phenolic compounds, the deeper the intensity of the blue-colored com
plex formed [34]. A calibration curve (6.25–200 μg/mL, y = 9.766× +

0.0538, R2 = 0.9978) was constructed for gallic acid (6.25, 12.5, 25, 50, 
100, and 200 μg/mL in methanol). TPC of the plant extracts was 
expressed as milligrams gallic acid equivalent (GAE) per g of extract.

2.4. DPPH• scavenging cuvette assay

In the colorimetric assay, the antioxidant activity of the extracts was 
determined by their ability to scavenge the stable DPPH• [35,36]. 
Briefly, six 2-mL extract solutions (ranged 15, 30, 60, 125, 250, and 500 
μg/mL in methanol) were mixed each with 3 mL DPPH• solution (freshly 
prepared, 0.1 mM in methanol) and incubated at 25 ◦C for 30 min in the 
dark. The deep purple color of the DPPH• changes to yellow in the 
presence of antioxidant compounds [37]. The absorbance of the purple 
color was measured at 517 nm in the UV-3100PC cuvette spectropho
tometer. The negative control was methanol in equal volume instead of 
extract.

P.O. Aiyedun et al.                                                                                                                                                                                                                             Fitoterapia 180 (2025) 106299 

3 



2.5. α-glucosidase inhibition microtiter plate assay

The colorimetric assay was carried out as described [38]. Briefly, five 
20-μL extract solutions (ranged 50, 40, 30, 20 and 10 mg/mL in 
dimethyl sulfoxide – distilled water, 1:1, V/V) were placed in a 96 well 
plate and 50 μL α-glucosidase (1 U/mL) in 100 mM phosphate buffer 
(pH 6.9) were added. Each mixture was incubated 37 ◦C for 10 min, after 
which 30 μL substrate solution (5 mM p-nitrophenyl-α-D-glucopyrano
side in 0.1 M sodium phosphate buffer of pH 6.9) were added and 
incubated at 37 ◦C for 30 min. The absorbance of the yellow color 
(formed nitrophenol) was measured at 405 nm at 37 ◦C in a microplate 
reader (Infinite 200 PRO, Tecan, Switzerland). Sample blanks were 
prepared as described [39] to quantify the absorbance contributed due 
to colored and/or turbidity. The positive control was acarbose (ranged 
50, 40, 30, 20 and 10 mg/mL in dimethyl sulfoxide – distilled water, 1:1, 
V/V). The negative control contained dimethyl sulfoxide and distilled 
water in equal volume instead of extract.

2.6. α-amylase inhibition microtiter plate assay

The colorimetric assay was carried out as described [40,41]. Briefly, 
five 50-μL extract solutions (ranged 50, 25, 12.5, 6.25, 3.125 mg/mL in a 
4:1 mixture, V/V, of methanol and 0.02 M sodium phosphate buffer of 
pH 6.9 containing 0.006 M sodium chloride) were placed in a 96-well 
plate and 50 μL α-amylase solution (0.5 mg/mL in the mentioned 
buffer) were added. Each mixture was incubated at 25 ◦C for 10 min, 
after which 50 μL starch solution (1 % in the mentioned buffer) were 
added and again incubated at 25 ◦C for 10 min. The reaction was 
terminated by adding 100 μL of dinitrosalicylic acid reagent (96 mM 3,5- 
dinitrosalicylic acid, 5.31 M sodium potassium tartrate in 2 N sodium 
hydroxide) to each mixture, which was then heated at 100 ◦C (boiling 
water vapor) for 10 min, cooled down to room temperature, and diluted 
with 100 μL distilled water. The absorbance of the red color (formed 3- 
amino-5-nitrosalicylic acid) was measured at 540 nm in a microplate 
reader (LT-4500, Labtech, Uckfield, UK). The positive control was 
acarbose (1 mg/mL in methanol – mentioned buffer, 4,1, V/V). The 
negative control was 80 % methanol in equal volume instead of extract.

2.7. Calculations and statistical analysis

The in vitro assay measurements were carried out in triplicates and 
the results are expressed as the mean including its standard error. The 
percentage inhibition was calculated as absorbance difference (negative 
control minus sample) times 100 divided by the absorbance of the 
negative control. The 50 % scavenging (SC50) or 50 % inhibition con
centrations (IC50) were determined graphically from dose-response 
curves. The individual sample results were compared by one-way 
analysis of variance (ANOVA) using the R Statistical Package (Version 
4.1.2, R Foundation, Vienna, Austria), whereby significant differences 
were compared by Duncan's multiple range test in R. Pearson's corre
lation analysis was performed using SAS (Version 9.4, SAS Institute, 
Cary, NC, USA) to assess the correlation between assays considered 
significant for p < 0.05.

2.8. HPTLC-UV/Vis/FLD method

The extracts (3 μL/band, but 1.5 μL/band for enzyme inhibition as
says) were applied as bands on a prewashed (with methanol-water, 4:1 
(V/V), and dried at 100 ◦C for 20 min) HPTLC plate silica gel 60 F254 
(Automatic TLC Sampler 4, CAMAG, Muttenz, Switzerland). The HPTLC 
separation was performed in a twin trough chamber (20 × 10 cm, 
CAMAG) using 6 mL mobile phase up to a migration distance of 70 mm. 
Acetonitrile – water 4:1 (V/V) was used as a polar mobile phase system 
(plates after derivatization and DPPH• assay), whereas ethyl acetate – n- 
hexane 7:5 (V/V) was applied as an apolar mobile phase system (used for 
both enzyme assays). The ambient air had 20–33 % relative humidity 

depending on the day. Each chromatogram was dried in a cold air stream 
(hair dryer) and detected at Vis, UV 254 nm, and FLD 366 nm (TLC 
Visualizer, CAMAG).

2.8.1. Detection via derivatization reagent
The chromatogram was derivatized with the anisaldehyde sulfuric 

acid reagent (1.5 mL 4-methoxy benzaldehyde in 210 mL methanol, 25 
mL acetic acid, and 13 mL concentrated sulfuric acid added dropwise) 
via piezoelectrical spraying (3 mL, blue nozzle, level 5), followed by 
heating at 110 ◦C and 170 ◦C each for 3 min (TLC Plate Heater, CAMAG), 
accompanied by detection at Vis and FLD 366 nm.

2.8.2. Detection via planar DPPH• assay
The positive control (0.125 mg/mL gallic acid in ethanol, 0.5, 1.3 

and 2.0 μL/band) was applied on the upper right plate edge, followed by 
drying for 0.5 min. The chromatogram was treated with the DPPH•

assay reagent (0.04 % DPPH• in methanol containing 10 % ethanol and 
10 mM sodium chloride, 9,1, V/V) via piezoelectrical spraying (3 mL, 
green or blue nozzle, level 5) followed by plate heating at 60 ◦C for 1 min 
and detection at Vis (repeated after one day as zones can partially be 
more intense).

2.8.3. Detection via planar α-glucosidase inhibition assay
The positive control (3 mg/mL acarbose in methanol; 1, 3, and 6 μL/ 

band) was applied on the upper right plate edge, followed by drying for 
0.5 min. The substrate solution (12 mg 2-naphthyl-α-D-glucopyranoside 
in 9 mL ethanol and 1 mL 10 mM aqueous sodium chloride solution) was 
sprayed piezoelectrically on the chromatogram (2 mL, yellow nozzle, 
level 6), and dried for 2 min. Then, α-glucosidase solution (10 U/mL in 
sodium acetate buffer pH 7.5) was sprayed onto the chromatogram (2 
mL, yellow nozzle, level 6), followed by incubation at 37 ◦C for 15 min. 
Finally, the chromogenic reagent solution (4 mg/mL Fast blue B salt in 
water) was sprayed piezoelectrically on the chromatogram (0.5 mL, red 
nozzle, level 6) followed by drying at 50 ◦C for 3 min and detection at 
Vis.

2.8.4. Detection via planar α-amylase inhibition assay
The positive control (0.1 mg/mL acarbose in methanol; 0.3, 0.6, and 

0.9 μL/band) was applied on the upper right plate edge, followed by 
drying for 0.5 min. The α-amylase solution (62.5 U/mL in sodium ace
tate buffer pH 7.5) was piezoelectrically sprayed onto the chromatogram 
(2 mL, red nozzle, level 5), followed by incubation at 37 ◦C for 30 min. 
Then, the substrate solution (2 % soluble starch solution in water) was 
piezoelectrically sprayed (1 mL, red nozzle, level 5) onto the chro
matogram, followed by incubation at 37 ◦C for 20 min. For visualization, 
Gram's iodine solution was piezoelectrically sprayed (500 μL, yellow 
nozzle, level 5), followed by plate heating and detection at Vis.

2.9. Zone characterization via HESI-HRMS

Zones of interest were eluted with methanol at a flow rate of 0.1 mL/ 
min. The TLC-MS Interface with an oval elution head (CAMAG) and 
inline C18 guard cartridge (SecurityGuard cartridge, Phenomenex, 
Torrance, CA, USA) was connected to the pump (Ultimate HPG-3200SD 
standard binary pump, Dionex Softron, Germering, Germany) and the 
heated electrospray ionization source (Ion-Max HESI-II probe Q) of the 
Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo 
Scientific, Dreieich, Germany) with the following ionization parameters 
set: sheath gas 20 AU, aux gas 10 AU, spray voltage ±3.5 kV, capillary 
temperature 270 ◦C, probe heater temperature 200 ◦C and S-lens RF 
level 50 AU. HRMS spectra were recorded as full scan (m/z 50–750) in 
the positive and negative ionization mode with a resolution of 280,000 
full widths at half-maximum (FWHM) at m/z 200 with the following 
settings: automatic gain control (AGC) target 3e6, maximum inject time 
100 ms, without lock mass injection. External mass calibration was 
performed weekly (using Pierce LTQ Velos ESI positive and negative ion 

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4 



calibration solution, Thermo Scientific). The instruments were operated 
by Xcalibur 4.2.47 with Foundation 3.1.261.0 and SII for Xcalibur 
1.5.0.10747, and spectra processing was performed using Qual Browser 
Excalibur 3.0.63 (all Thermo Scientific). Mass spectra of the plate 
background were recorded and subtracted from the analyte spectrum.

3. Results and discussion

The 67 African trifoliate yam accessions (Table 1) of the largest 
collection of D. dumetorum in Africa were grown and harvested in 
December 2018. Methanolic tuber extracts were prepared. Their TPC as 
well as the DPPH• scavenging, α-amylase, and α-glucosidase inhibition 
properties were analyzed using colorimetric in vitro assays. Based on 
these results of the colorimetric in vitro assays, twelve accessions 
(Table 2) were selected for HPTLC–EDA profiling. The method protocol 
of the latter had to be developed first. These effect-directed techniques 
were used to clarify the rationale for ethnomedicinal applications, how 
much the accessions differ in terms of antioxidant and antidiabetic ac
tivities, whether the results of the in vitro assays are consistent with the 
HPTLC-EDA profiles, whether the bioactivity profiles can be meaningful, 
and whether prominent bioactive compounds can be tentatively 
assigned.

3.1. TPC cuvette assay results

The TPC was analyzed using the Folin− Ciocalteu method (colori
metric assay), which measures the reducing activity of the sample. The 
Folin− Ciocalteu reagent reacts with all reducing molecules, considered 
to be mostly phenolic compounds, and thus is ought to resemble the 
TPC. The TPC varied considerably among the accessions ranging from 
24 to 740 mg GAE/g extract with accession 3946 having the highest 
phenolic content and accession 4141 the lowest (Table 1). There was no 
geographic bias in the TPC of the accessions, since those samples that 
showed the highest total phenolic content (TPC ≥ 300 mg GAE/g) 
originated from different countries. There was a significant difference in 
the TPC across the accessions (Table 1). Phenolic compounds, such as 
phenolic acids, flavonoids, tannins, and polyphenols that make up the 
phenolic content of the accessions, have been reported to be the most 
abundant antioxidants in medicinal plants. These can also express 
various bioactivities including antidiabetic, anti-inflammatory, and 
anti-cancer. Some plant-derived antidiabetic phenolic compounds 
include kaempferitin, isoscutellarein, vitexin, isovitexin, and iso
rhamnetin rutinoside [42–47]. The Folin–Ciocalteu assay could also be 
seen as a measure of total antioxidant capacity and not just phenolic 
content since the Folin–Ciocalteu reagent also reacts with other anti
oxidants other than phenolics [34,48]. These other non-phenolic anti
oxidants include proteins, vitamins, amines, aldehydes and ketones, 
carbohydrates, thiols, and unsaturated fatty acids among others.

3.2. DPPH• radical scavenging cuvette assay results

The samples showed a dose-dependent DPPH• activity. The SC50 (the 
value is inversely proportional to the extract activity and concentration 
that will neutralize 50 % of the oxidative potential of DPPH•) ranged 
from 0.1 mg/mL for accession 3710 to 2.5 mg/mL for accession 3947 
(Table 1) when compared with that of the standard ascorbic acid (0.02 
mg/mL). Accession 3946, which had the highest TPC, also had a rela
tively low SC50 of 0.2 mg/mL (third lowest), which indicates a high 
antioxidant activity. A similar SC50 (0.1 mg/mL) was recently reported 
for the DPPH• scavenging activity of the methanol extract of 
D. dumetorum tubers collected in Nigeria [49]. Consequently, this indi
cated that the reducing activity of phenolic compounds in the accessions 
studied seems to contribute to their corresponding strong radical scav
enging activities. The general DPPH• scavenging activity of Dioscorea 
species has been reported previously [50–56]. Specific phenolic com
pounds, such as chrysin, quercetin rhamnoside, quercitrin, catechin, 
epicatechin, hyperin, procyanidin B2, quercetin arabinofuranoside, and 
hydroxyphloretin were detected in several plants [57,58]. The anti
oxidative activity of polyphenolic compounds was also reported as a 
mechanism by which they exhibited antidiabetic effects [59].

3.3. α-glucosidase inhibition microtiter plate assay

The results revealed that all samples exhibited a dose-dependent 
inhibition of the α-glucosidase with IC50 ranging from 27 mg/mL to 
129 mg/mL (Table 1). The IC50 of the Nigerian accessions 3946 (27 mg/ 
mL) and 4128 (35 mg/mL) elicited the most potent effects, compared to 
extracts of other accessions and the positive control acarbose (63 mg/ 
mL). The high TPC (740 mg GAE/g) and α-glucosidase inhibition of 
accession 3946 are consistent with the trends in previous studies [60], in 
which the α-glucosidase inhibitory activity of medicinal plants was 
attributed to being a function of their phenolic content. Other studies 
have also established the presence of α-glucosidase inhibitors in tubers 
of D. dumetorum [21] and attributed this activity in extracts of Dioscorea 
species to their high phenolic content [61]. However, this correlation is 
not necessarily valid, suggesting that the phenolic compounds are not 
mainly responsible for their anti-diabetic effect as given in the two 
following examples. The extract from accession 4128 (IC50 35 mg/mL) 
showed the second-highest antidiabetic activity but had a low TPC (156 

Table 2 
Reproducibility of the antioxidant and antidiabetic activities of twelve selected 
Dioscorea dumetorum accessions (Table 1) by colorimetric in vitro assays.

Landraces Colorimetric in vitro assays

Antioxidant 
activity

Antidiabetic activity

No. Accession 
TDd-xxxx

Origin TPC 
(mg 

GAE/ 
g)

DPPH•

SC50 

(mg/ 
mL)

α-Glucosidase 
IC50 (mg/mL)

α-Amylase 
IC50 (mg/ 

mL)

11 3104 Togo 357 
±

0.43d

0.2 ±
0.00k

85 ± 0.23f 13 ± 0.03a

19 3112 Nigeria 29 ±
0.20l

2.4 ±
0.01b

93 ± 0.23d <LOD

24 3710 Congo 321 
±

0.46e

0.1 ±
0.00l

114 ± 0.19c <LOD

27 3771 Nigeria 279 
±

0.40g

0.6 ±
0.01e

73 ± 0.21i 6 ± 0.04c

28 3775 Nigeria 602 
±

0.51b

0.3 ±
0.00i

129 ± 0.26a 9 ± 0.05b

29 3776 Nigeria 229 
±

0.39h

0.8 ±
0.01d

83 ± 0.23g <LOD

32 3779 Nigeria 126 
±

0.37j

0.4 ±
0.01h

36 ± 0.21j 5 ± 0.05d

34 3804 Benin 310 
±

0.34f

0.4 ±
0.01f

90 ± 0.23e <LOD

37 3848 Benin 413 
±

0.46c

0.4 ±
0.01g

74 ± 0.21h <LOD

41 3946 Nigeria 740 
±

0.43a

0.2 ±
0.00j

27 ± 0.21l <LOD

42 3947 Nigeria 100 
±

0.35k

2.5 ±
0.01a

115 ± 0.25b <LOD

53 4128 Nigeria 156 
±

0.25i

1.0 ±
0.01c

35 ± 0.21k 3 ± 0.03e

Values with the same letter are not significantly different. < LOD: below 
detection limit.

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mg GAE/g). Accession 3775 with a high TPC (602 mg/mL) showed a low 
α-glucosidase inhibition.

The majority (75 %) of the selected accessions showed an inhibition 
of the α-glucosidase (IC50 ≤ 100 mg/mL). In literature, the IC50 of 
acarbose (71 mg/mL) at the same enzyme concentration is comparable 
[38]. However, with acarbose, severe gastrointestinal side effects were 
observed, such as flatulence, diarrhea, meteorism and abdominal 
distention [62–66]. Hence, the inhibition of the α-glucosidase via nat
ural multi-component mixtures such as D. dumetorum is of pharmaco
logical importance to substitute synthetic α-glucosidase inhibitors with 
side effects, such as acarbose, miglitol, and voglibose.

3.4. α-amylase inhibition microtiter plate assay

The majority (82 %) of the selected accessions showed no inhibition 
of porcine pancreatic α-amylase. The results revealed that 9 samples 
exhibited a dose-dependent inhibition of α-amylase with IC50 ranging 
from 3 mg/mL to 23 mg/mL while three accessions exhibited a dose- 
independent inhibition (Table 1). Eight samples that showed activity 

originate from Nigeria, two from Togo and one each from Benin and 
Ghana (Table 1). The positive control acarbose exhibited a dose- 
dependent inhibition of the pancreatic α-amylase with an IC50 of 0.05 
mg/mL and a percentage inhibition range between 75 % at 1 mg/mL and 
45 % at 0.03 mg/mL. The results supported previous studies that found 
acarbose at the same concentration of 1 mg/mL to show 57 % [67] and 
66 % [66] inhibition of pancreatic α-amylase. Accession 4128 which 
showed high inhibition of α-glucosidase (IC50 35 mg/mL) elicited the 
most potent α-amylase inhibition (IC50 3 mg/mL), followed by acces
sions 3935 and 2788 (both with IC50 4 mg/mL). This suggests that the 
antidiabetic compound present in accession 4128 inhibits both 
α-amylase and α-glucosidase and is not a phenolic compound since it had 
a low TPC (156 mg GAE/g). The accessions 3779, 3771, 3775, 3104, 
which showed high inhibition of α-glucosidase, also showed antidiabetic 
activity via inhibition of α-amylase. However, accession 3946, which 
showed the highest inhibition of α-glucosidase (IC50 27 mg/mL), did not 
inhibit α-amylase. Extracts from accessions 3935 (IC50 4 mg/mL), 2788 
(IC50 4 mg/mL), 3686 (IC50 11 mg/mL), 3103 (IC50 11 mg/mL), 3106 
(IC50 14 mg/mL), 4142 (IC50 19 mg/mL) and 3906 (IC50 21 mg/mL) 
showed inhibition of α-amylase despite having low TPC, DPPH• scav
enging activity and α-glucosidase inhibition. There has been little 
research into the inhibitory effects of D. dumetorum tuber in vitro 
[21,68], whereas several studies have established its antidiabetic effect 
in vivo [13,21,25,69]. Previous studies have also established a higher 
inhibitory effect on α-glucosidase than on α-amylase in D. dumetorum 
tuber [21] and other plants [64]. However, the inhibitory effects of 
other Dioscorea species on these two key diabetes-related enzymes have 
been established [50,61,70].

Table 3 
Pearson’s correlation coefficients (r; *significant correlation at p < 0.05) of the 
TPC and of enzyme inhibition activities, all analyzed by in vitro assays.

Parameters TPC DPPH•

scavenging
α-Glucosidase 

inhibition

TPC 1
DPPH• scavenging − 0.6516* 1
α-Glucosidase 
inhibition

− 0.0611 0.30915 1

Fig. 1. HPTLC− UV/Vis/FLD profiles of twelve selected Dioscorea dumetorum accessions (Table 2, 150 μg/band, 3 μL/band of 50 mg/mL extract solution, solvent 
blank for comparison) applied on the HPTLC plate silica gel 60 F254 with acetonitrile − water 4:1 (V/V) up to 70 mm, detected at (A) UV 254 nm, (B) FLD 366 nm, 
and (C/D) after derivatization with anisaldehyde sulfuric acid reagent, followed by plate heating at 110 ◦C and then (E/F) 170 ◦C at white light illumination (C/E, 
reflectance/transmission mode, white balance adjusted) and FLD 366 nm (D/F).

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3.5. Correlation analysis of the colorimetric in vitro assay results

A significantly negative (r = − 0.6516, p < 0.05) correlation was 
observed between TPC and DPPH• scavenging activity (Table 3) through 
Pearson's correlation matrix. This means that an increase in the TPC 
decreases the IC50 values, connoting high antioxidant activity. This 
corroborates the use of both assays as a measurement of antioxidant 
activity of plant extracts in previous reports since the magnitude of IC50 
of DPPH• scavenging activity is inversely proportional to the antioxi
dant activity of samples, while TPC values have a direct proportion to 
antioxidant capacity. The observed correlation confirms that all DPPH•

scavenging constituents in the phenolic compounds act in synergy to 
make up the overall antioxidant activity of the extracts. In contrast, the 
α-glucosidase inhibition assay had no significant associations with the 
TPC (r = − 0.0611) and the antioxidant activity expressed as DPPH•

scavenging activity (r = 0.3091) of the samples.

3.6. Development of the HPTLC method

Based on the in vitro results, twelve accessions (Table 2) were 
selected for HPTLC− EDA profiling, which was performed for the first 
time in this study. Different mobile phase systems were studied on 
HPTLC plates silica gel 60 F254 for the intended effect-directed profiling. 
Among the tested ones, acetonitrile − water 4:1 (V/V) allowed the best 
polar separation. This mobile phase system was used later for the 
derivatization experiment (Fig. 1) and DPPH• assay (Fig. 2). As an 
apolar mobile phase system, the mixture ethyl acetate – n-hexane 7:5 
(V/V) was selected and used later for both enzyme assays since a more 
apolar highly active compound zone was observed (Fig. 3). Both mobile 

phases took only about 23 min and allowed for fast sustainable profiling 
in the polar and apolar substance range. Only about 0.5 mL solvent per 
sample was consumed for each analysis. These developed chromato
graphic systems were found to be a good start for chemical profiling as 
well as non-target effect-directed profiling.

3.6.1. Chemical profiling at UV/Vis/FLD including derivatization
The UV/Vis/FLD multi-imaging of the polar HPTLC chromatograms 

revealed UV-absorbing (Fig. 1A) and mainly blue fluorescent (Fig. 1B) 
compound bands, which were spread along the migration distance. 
Accessions 3104, 3710, 3848, 3946, and 4128 with comparatively 
darker UV-absorbing bands (Fig. 1A) also expressed high TPCs. Previous 
studies have shown blue fluorescent bands (attributable to plant acids) 
to be indicative of phenolic compounds [71,72]. In previous studies, 
phenolic and flavonoids were said to be present in D. dumetorum but 
specific compounds were not identified [49,73–75]. Among the tested 
derivatization reagents, the anisaldehyde sulfuric acid reagent 
(Fig. 1C− F) detected most organic compounds. The previously 
mentioned dark UV-absorbing bands turned blue fluorescent (Fig. 1D) 
when the plate was heated at 110 ◦C. Heating the same chromatogram 
up to 170 ◦C turned them into a rusty color (Fig. 1F). Additional pink 
bands were revealed for accessions 3104, 3710, 3771, and 3779 under 
white light illumination (Fig. 1E), expressed as prominent orange- 
colored fluorescent bands when viewed at FLD 366 nm (Fig. 1F). All 
in all, the obtained chemical profiles of D. dumetorum are meaningful 
and can be considered for future authenticity proofs or as reference 
profiles. Especially the chemical derivatization proved that 
D. dumetorum is rich in phytochemicals, and it was wondered which are 
the important bioactive ones.

Fig. 2. HPTLC− UV/Vis/FLD − DPPH• profiles of twelve selected Dioscorea dumetorum accessions (Table 2, 150 μg/band, 3 μL/band of 50 mg/mL extract solution, 
solvent blank for comparison) applied on the HPTLC plate silica gel 60 F254 with acetonitrile − water 4:1 (V/V) up to 70 mm, and then 3:1 up to 30 mm to better 
separate the polar antioxidants, detected at (A) Vis, (B) UV 254 nm (enhanced), (C) FLD 366 nm (enhanced), and (D − F) after the DPPH• assay at white light 
illumination via (D) reflectance mode, (E) transmission mode and (F) both latter modes.

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3.6.2. HPTLC–DPPH• assay–Vis profiles
Characteristic antioxidant profiles of D. dumetorum were observed. 

The HPTLC− DPPH• assay–Vis autogram revealed DPPH• scavenging 
compounds as yellow bands on a purple background under white light 
illumination (Figs. 2D− F). The previously mentioned pink-colored and 
orange fluorescent bands in the HPTLC chromatogram derivatized with 
the anisaldehyde sulfuric acid reagent (Fig. 1E/F) showed no scavenging 
activity. However, many other natively blue fluorescent compounds 
(indicating plant acids among others) showed antioxidant activity. The 
number (up to a dozen compounds per sample) and intensity (from weak 
to very intense) of the antioxidant compounds substantially differed 
between the accessions. There was no profile correlation between the 
accessions and their countries of origin. The results align with what was 
obtained previously from the cuvette assay as the accessions with the 
highest SC50 (lowest activity) also showed the fewest and least intense 
bands (Fig. 2D− F; 3947, 4128, and 3112). Our findings are confirmed by 
several studies reporting the presence of DPPH• scavenging compounds 
in D. dumetorum via in vitro and in vivo assays [17,69,74].

3.6.3. HPTLC–α-glucosidase inhibition assay–Vis profiles
The polar mobile phase revealed α-glucosidase inhibitors only in the 

mobile phase front, and thus an apolar mobile phase system was 
developed and used for this assay. In contrast to the polar separation 
system, the compounds detected at UV/Vis/FLD (Fig. 3A–C) remained at 
the start zone due to the weak solvent strength of the apolar mobile 
phase system. Characteristic α-glucosidase inhibition profiles of 
D. dumetorum are revealed for the first time in this study. The autogram 
showed α-glucosidase inhibitors as colorless zones on a purple back
ground under white light illumination (Fig. 3D). One prominent intense 
α-glucosidase inhibitor was detected in all accessions. This suggests the 

presence of the same bioactive compound that can be a characteristic 
signature antidiabetic compound of D. dumetorum and could be 
responsible for the inhibitory activities obtained previously from the in 
vitro assay. Depending on the sample, up to two further but substantially 
weaker inhibitors were observed, apart from some compounds that also 
showed a weak response and remained in the start zone. There was also 
no relationship between the variation of enzyme inhibition across the 
samples and their origin. The accessions 3112, 3710, 3776, 3779, 3804, 
3947 and 4128 with the highest response correlated with high activities 
(ranged from 34.9 mg/mL for 4128 to 115 mg/mL for 3947) which was 
observed by the colorimetric microtiter plate assay. Our finding is in line 
with previous studies in which α-glucosidase inhibitors were reported in 
tuber of D. dumetorum, but without assignment to any particular com
pound [21].

3.6.4. HPTLC–α-amylase inhibition assay–Vis profiles
Analogously, the polar mobile phase did reveal inhibitors only in the 

mobile phase front, and the previous apolar mobile phase system was 
used also for this assay (Fig. 3E). Characteristic α-amylase inhibition 
profiles of D. dumetorum are revealed for the first time in this study. The 
autogram showed α-amylase inhibitors as dark purple zones on a bright 
background under white light illumination (Fig. 3F). Similar to the 
previous α-glucosidase inhibition profile, the same prominent intense 
α-glucosidase and now also α-amylase inhibitor was detected in all ac
cessions, since the horizontal intensity pattern of the compounds zones 
across all accessions was the same. Identical horizontal compound pat
terns are a clear indication that the same compound is present. Again, 
accessions 3112, 3710, 3776, 3779, 3804, 3947 and 4128 showed the 
highest response. This correlated with the inhibition activity observed 
by the colorimetric microtiter plate assay for accessions 3779 and 4128 

Fig. 3. HPTLC− UV/Vis/FLD − α-glucosidase/α-amylase inhibition profiles of twelve selected Dioscorea dumetorum accessions (Table 2, 75 μg/band, 1.5 μL/band of 
50 mg/mL extract solution, solvent blank for comparison) applied on the HPTLC plate silica gel 60 F254 with ethyl acetate – n-hexane 7:5 (V/V) up to 70 mm, detected 
at (A) Vis, (B) UV 254 nm, (C/E) FLD 366 nm, and after the respective (D) α-glucosidase and (F) α-amylase inhibition assays at white light illumination (reflectance 
mode; for α-amylase, white balance adjusted).

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but not for 3112, 3710, 3776, 3804, and 3947 which were not observ
able (below detection limit) by the colorimetric microtiter plate assay. 
This discrepancy was explained by solubility problems of this obviously 
very apolar compound in the highly polar bioassay medium or adsorp
tion to plastic material (polystyrene) of the in vitro assay since the true 
concentration of this very apolar extract part was not analytically 
determined. In contrast to the previous α-glucosidase inhibition assay 
containing dimethyl sulfoxide as solubilizer, the α-amylase inhibition 
assay did not contain such a solubilizer and discriminated the more 
lipophilic compounds present. This demonstrated that the planar assays 
were more robust regarding solubility issues than the corresponding in 
vitro assays. Although most of the samples did not exhibit α-amylase 
inhibition in the colorimetric microtiter plate assay, the observed 
HPTLC data clearly indicate the potential of D. dumetorum as a source of 
antidiabetic α-amylase inhibitors. There was also no relationship be
tween the variation of enzyme inhibition across the samples and their 
origin. Our results confirm the findings from previous studies 
[21,68,76], which already described the presence of α-amylase in
hibitors (phenolic compounds and phytates) in tubers of D. dumetorum 
but without assignment to any particular compound.

3.6.5. Characterization of the prominent antidiabetic compound zone
The prominent antidiabetic compound zone (Fig. 3D/F) was not 

visible (Fig. 3A), not UV-active (Fig. 3B), and not fluorescent (Fig. 3C), 
but got colored greyish-blue after derivatization with the anisaldehyde 

sulfuric acid reagent (Fig. 1C/E, hRF 93 in the polar mobile phase sys
tem). HPTLC− HESI-HRMS spectra (Fig. 4) were recorded from the 
prominent antidiabetic zone using the apolar separation due to the 
better zone resolution and less interference from other compounds (left 
at the start zone). The mass signals at m/z 559.4729 [2M1-H]− (Δ ppm 
0.5), m/z 279.2329 [M1-H]− (Δ ppm 0.2), m/z 627.4338 [2M1- 
2H+3Na]+ (Δ ppm − 1.3), m/z 605.4518 [2M1-H+2Na]+ (Δ ppm − 0.3), 
and m/z 325.2112 [M1-H+2Na]+ (Δ ppm 0.5) tentatively indicated 
linoleic acid (C18H32O2). Further mass signals revealed the coelution of 
further fatty acids in the same zone, which was expected in a normal 
phase separation system. Tentatively, palmitic acid (C16H32O2) at m/z 
511.4728 [2M2-H]− (Δ ppm 0.7) and m/z 255.2331 [M2-H]− (Δ ppm 
− 0.6), oleic acid (C18H34O2) at m/z 281.2483 [M3-H]− (Δ ppm 1.2), and 
linolenic acid (C18H30O2) at m/z 277.2176 [M4-H]− (Δ ppm − 0.9) and 
m/z 301.2134 [M4+Na]+ (Δ ppm 0.3) were assigned. This tentative 
assignment to fatty acids is consistent with the earlier hypothesis that 
the α-amylase and α-glucosidase inhibiting compound was not a 
phenolic compound because it did not correlate with the TPC. This 
HPTLC− HESI-HRMS results were in accordance with other studies 
showing the anti-diabetic effect for individual fatty acids [77–80]. 
Another oxidized structure evident at m/z 171.1027 [M5-H]− (Δ ppm 
− 0.4), m/z 217.0811 [M5-H+2Na]+ (Δ ppm 0.1) and m/z 195.0992 
[M5+Na]+ (Δ ppm − 0.2) was tentatively assigned to oxo-nonanoic acid 
(C9H16O3). Further unassigned oxidized structures, such as C12H20O3 at 
m/z 257.1123 [M6-H+2Na]+ (Δ ppm 0.6), m/z 235.1304 [M6+Na]+ (Δ 

Fig. 4. HPTLC− HESI-HRMS spectra in the (A) negative and (B) positive ionization mode of the prominent zone inhibiting the α-glucosidase and α-amylase, 
exemplarily shown for accession 3848 (75 μg/band, 1.5 μL/band of 50 mg/mL extract solution).

P.O. Aiyedun et al.                                                                                                                                                                                                                             Fitoterapia 180 (2025) 106299 

9 



ppm 0.3) and m/z 211.1340 [M6-H]− (Δ ppm − 0.4), and C10H13O6 at 
m/z 230.0787 [M7+H]+ (Δ ppm − 1.0), were detected. Oxidized struc
tures with comparatively low HRMS signal intensity can nevertheless 
have an impact on the bioactivity of samples [81] and are worth to 
mention.

4. Conclusions

This study provided new insights into the bioactivity of methanolic 
tuber extracts from 67 different African trifoliate yam accessions, which 
showed clear differences in the antioxidant and antidiabetic activities. 
For the first time, characteristic antidiabetic bioactivity profiles were 
obtained which are helpful as authentic reference profiles for further 
food processing. Up to a dozen individual antioxidants were revealed as 
well as one prominent α-glucosidase and α-amylase inhibiting com
pound zone. The HPTLC− HRMS exemplarily allowed the straightfor
ward characterization and tentative assignment of the latter zone to be 
coeluting fatty acids. This represents substantial progress in under
standing the importance of African trifoliate yam in ethnomedicinal 
diabetes treatment and could explain the mechanisms by which they 
lower blood sugar levels. It also suggests it as source of functional food.

The HPTLC− EDA analysis was performed miniaturized on the same 
planar surface for many samples in parallel, from screening to effect 
detection and tentative molecular formula assignment. It allowed for the 
prioritization of compounds, including lipophilic compounds, and pro
vided more differentiated information. The workflow was compara
tively faster, simpler, more sustainable and less laborious. The results of 
in vitro assays and planar on-surface assays correlated to a high degree. 
However, in the colorimetric microtiter plate assay, α-amylase inhibi
tion was comparatively very weak if not absent, which was explained by 
solubility problems of this obviously very apolar fatty acid structures in 
the highly polar bioassay medium, containing no dimethyl sulfoxide as 
solubilizer and thus discriminating the more lipophilic compounds 
present.

Funding

For the research stay of POA at University of Oldenburg, the project 
was supported by the Alexander von Humboldt-Stiftung, Research 
Group Linkage Programme – 1128007-NGA-IP, the Genetic Resource 
Center of the International Institute of Tropical Agriculture, and the 
German Academic Exchange Service (DAAD), Short-Term Research 
Grant – 57588367. At JLU Giessen, thank is owed to Annabel Mehl, Food 
Science group, for HRMS recording; instrumentation was partially fun
ded by the Deutsche Forschungsgemeinschaft (DFG, German Research 
Foundation) – INST 162/471–1 FUGG; INST 162/536–1 FUGG.

Compliance with ethical standards

The research did not involve human participants and/or animals.

Declaration of generative AI and AI-assisted technologies in the 
writing process

Generative AI and AI-assisted technologies were not used during the 
preparation of this work.

CRediT authorship contribution statement

Priscilla O. Aiyedun: Writing – original draft, Investigation, Formal 
analysis. Mubo A. Sonibare: Writing – review & editing, Supervision, 
Resources, Methodology, Conceptualization. Badara Gueye: Writing – 
review & editing, Supervision, Resources, Methodology. Dirk C. 
Albach: Writing – review & editing, Supervision, Resources, Method
ology. Julia Heil: Investigation. Gertrud E. Morlock: Writing – review 
& editing, Writing – original draft, Supervision, Resources, 

Methodology, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial 
interests or personal relationships that could have appeared to influence 
the work reported in this paper.

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	Antidiabetic and antioxidant profiling of 67 African trifoliate yam accessions by planar on-surface assays versus in vitro  ...
	1 Introduction
	2 Materials and methods
	2.1 Chemicals and reagents
	2.2 Plant materials and preparation
	2.3 TPC cuvette assay
	2.4 DPPH• scavenging cuvette assay
	2.5 α-glucosidase inhibition microtiter plate assay
	2.6 α-amylase inhibition microtiter plate assay
	2.7 Calculations and statistical analysis
	2.8 HPTLC-UV/Vis/FLD method
	2.8.1 Detection via derivatization reagent
	2.8.2 Detection via planar DPPH• assay
	2.8.3 Detection via planar α-glucosidase inhibition assay
	2.8.4 Detection via planar α-amylase inhibition assay

	2.9 Zone characterization via HESI-HRMS

	3 Results and discussion
	3.1 TPC cuvette assay results
	3.2 DPPH• radical scavenging cuvette assay results
	3.3 α-glucosidase inhibition microtiter plate assay
	3.4 α-amylase inhibition microtiter plate assay
	3.5 Correlation analysis of the colorimetric in vitro assay results
	3.6 Development of the HPTLC method
	3.6.1 Chemical profiling at UV/Vis/FLD including derivatization
	3.6.2 HPTLC–DPPH• assay–Vis profiles
	3.6.3 HPTLC–α-glucosidase inhibition assay–Vis profiles
	3.6.4 HPTLC–α-amylase inhibition assay–Vis profiles
	3.6.5 Characterization of the prominent antidiabetic compound zone


	4 Conclusions
	Funding
	Compliance with ethical standards
	Declaration of generative AI and AI-assisted technologies in the writing process
	CRediT authorship contribution statement
	Declaration of competing interest
	References