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Original article
2021
:14;
202103
doi:
10.1016/j.arabjc.2020.102970

UHPLC-QToF-MS characterization of bioactive metabolites from Quercus robur L. grown in South Africa for antioxidant and antidiabetic properties

,
 Department of Life and Consumer Sciences, University of South Africa, Cnr Christiaan de Wet and Pioneer Ave, Private Bag X6, Florida 1710, South Africa

⁎Corresponding author. Unuofjo@unisa.ac.za (Jeremiah Oshiomame Unuofin)

Received: , Accepted: ,
© The Author(s) 2021
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Abstract

The leaves and stem bark extracts of Quercus robur L. grown in South Africa have been investigated for their bioactive metabolites using UHPLC-QToF-MS. The UHPLC-QToF-MS profiling of the various extracts revealed the presence of twenty-seven compounds. These compounds were majorly grouped into flavonoids, phenolic acid and triterpenoids such as alnusiin, (+)-catechin, cis-3,4-leucopelargonidin, (-)-gallocatechin, ellagic acids, gallic acid, luteoforol, kaempferol-3-o-(6-o-cis-coumaryl)glucoside, myricitrin, neohesperidin, neriifolin, pheophorbide a, pheophytin a, procyanidin B5, pedunculagin, quercetin 3-O-[beta-D-xylosyl-(1- > 2)-beta-D-glucoside and quercitrin. The aqueous bark extract had the highest total phenol content (TPC) (320 mg GAE/g DW) and total flavonoids content (TFC) (505 mg QE/g DW), whereas the 80% aqueous acetone leaves extract had the highest proanthocyanidins levels (Pro) (433 CE/g DW). The 80% aqueous acetone extract had the best 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging capability with an IC50 value of 8.16 µg/mL. The 80% aqueous ethanol extract had the highest 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) scavenging ability with an IC50 value of 7.73 µg/mL. The aqueous leaves extract had the best ferric reducing antioxidant power assay (FRAP) ability with values of 558 µg/mL GAE. The most significant inhibitory effects on digestive enzyme activity were obtained with the 80% aqueous methanol bark and aqueous bark extracts at the concentration of 7.81 µg/mL, yielding inhibition values of 62 and 97% on α-amylase and α-glucosidase, respectively higher than the positive control used (acarbose). These findings calls attention to the phytochemical content, antioxidant and inherent antidiabetic activity of Q. robur L. grown in South Africa and therefore potentiate its potential application in the food and pharmaceutical industries, with a focus in the prevention of diabetes.

Keywords

Diabetes
α-amylase
α-glucosidase
Bioactive compound
Quercus robur L.
UHPLC-QToF-MS
1

1 Introduction

Diabetes is one of the rising chronic diseases, it is stimulated by some conditions such as faulty insulin secretion, insulin inaction, and occasionally both (Elberry et al., 2015; Thirupathy Kumaresan et al., 2014; Unuofin and Lebelo, 2020) thus bringing about the onset of hyperglycemia and hyperlipidemia (Pereira et al., 2019). Reports from the International Diabetes Federation (IDF) in 2017 revealed that 451 million people most especially adults in the globe are suffering from diabetes, with a forecasted 693 million cases come 2045 (International Diabetes Federation (IDF), 2017). In recent times, diabetes has been shown to target numerous organs such as the pancreas, liver, muscles, kidney, and central nervous system, and also it has been implicated in several complications such as hypertension, stroke, blindness, and kidney disease (Gourgari et al., 2017; Pereira et al., 2019). The most prominent technique employed in the treatment of diabetes and its close associate hyperglycemia is by way of controlling the production of insulin thus abating and safeguarding blood glucose levels (Pereira et al., 2019; Unuofin and Lebelo, 2020). In recent times, there has been an upsurge in the number of drugs developed for specific enzymes and inhibitors (alpha-amylase, alpha-glucosidase, dipeptidyl peptidase-4 (DPP4) and glucagon-like peptide-1 (GLP-1) receptor) linked with diabetes. For instance, insulin and varied synthetic medications such as acarbose, biguanides, metformin, sulfonylureas, thiazolidinediones, and voglibose are some of the drugs used for the efficient management of hyperglycemia along with type 2 diabetes (T2D). Nevertheless, these medications have been reported to have myriads of side effects, as well as the high cost of purchasing them (Safavi et al., 2013; Unuofin and Lebelo, 2020; Yin et al., 2018). For these reasons, there is an urgent search for an alternative cure for diabetes in the form of natural products and medicinal plants. Natural products and medicinal plants have received huge attention because of their richness in polyphenolics such as flavonoids, tannins, and proanthocyanidins (Santos et al., 2013a, 2013b; Unuofin et al., 2017, 2018a; Jimoh et al., 2019). In recent times, different studies have proven the therapeutic potentials of polyphenolics compounds in several pathological conditions such as atherosclerosis, cancer, diabetes and other disorders (Tomé-Carneiro and Visioli, 2016; Unuofin et al., 2019; Unuofin and Lebelo, 2020). The abundance of these polyphenolics in medicinal plants is majorly swayed by the interplay between environmental conditions and plant (Gutbrodt et al., 2012; Oh et al., 2009; Pavarini et al., 2012; Unuofin et al., 2018a). The biosynthesis of secondary metabolites such as polyphenolics, that elicit pharmacological potentials is mainly hinged on the influence of the environment on the plant as it adapts to harsh conditions (Szakiel et al., 2011; Treutter, 2005; Winkel-Shirley, 2002).

The English oak (Quercus robur L.) is a large deciduous tree that belongs to the family Fagaceae. Its aboriginal distribution spans all of Europe and in recent times has spread into the Caucasus, Iran, Kazakhstan, and Russia (Haneca et al., 2009). Q. robur can thrive on harsh soil conditions and different continental weather but it favors fertile and well-watered soils (Ducousso and Bordacs, 2004). Q. robur is among the notable exotic species in South Africa. It is one of the first trees planted by early settlers (Dutch East India Trading Company) in the 350 years ago in Cape Town, it is also found in Johannesburg and other parts of South Africa (Ayayee and Chivandi, 2018; Campbell and Moll, 1977; Kemp, 2002).

The acorns of Q. robur gotten from Serbia has several potent phytoconstituent with antioxidant activity (tannins, gallic acid, ellagic acid, different galloyl and hexahydroxydiphenoyl derivatives) (Rakić et al., 2007, 2006). Kuliev et al., (1997) showed that more than twenty compounds (catechins and oligomeric and polymeric proanthocyanidins) were isolated from the bark of Q. robur.

According to Cadahía et al., (2001), an aqueous wood extract of Q. robur from Spain contained gallic acid, 5-(hydroxymethyl)furfural, 2-furanoic acid, aesculetin, vanillic acid, vanillic aldehyde, syringic acid, syringaldehyde, scopoletin, ferulic acid, coniferaldehyde, sinapaldehyde and ellagic acid using HPLC. Quinic acid and malic acid had higher quantities among 27 different phenolic acids were detected from the oak molasses by UHPLC-ESI-MS/MS method (Bursal and Boǧa, 2018).

Literature is replete with information about the biological activities of extracts and secondary metabolites isolated from acorns, stem barks, oak molasses and leaves of Q. robur L. These biological activities include anti-inflammatory, antimicrobial, hepatoprotective, gastro-protective, anti-oxidative and astringent activity (Andrenšek et al., 2004; Moharram et al., 2015; Popović et al., 2013; Zdravkovic et al., 2015).

Owing to climatic variation and strong adaptative potential of Q. robur to disparate environmental changes/settings, and its ability to give rise to natural products with curative potentials (Annighöfer et al., 2015; Bourtsoukidis et al., 2014; Kremer, 2016). Bearing in mind pharmacological potential of English oak (Quercus robur L.) grown in other parts of the world,

The objective of this study was to investigate Q. robur L. a renowned industrial crop worldwide; Q. robur used in this study was grown in South Africa. UHPLC-QToF-MS was employed in characterization of bioactive metabolites. The antioxidant property and probable antidiabetic activity were estimated. The outcome of our research can provide important information for commercial utilization of these plant parts in the areas of nutraceuticals, and pharmaceutical development.

2

2 Materials and methods

2.1

2.1 Chemicals

Folin and Ciocalteu’s phenol reagent, gallic acid, quercetin, 2,4,6-tripyridyl-s-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH), L-ascorbic acid, vanillin, hexahydrate aluminium chloride, ferric chloride, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were purchased from Sigma-Aldrich Chemical Co. Ltd. (Irvine, United Kingdom), were analytical grade.

2.2

2.2 Plant materials

Leaves and stem bark of English oaks Quercus robur L. were collected from Alice in the Eastern Cape of South Africa, in November 2017. Both plant parts were then washed in distilled water and oven-dried (40 °C) to constant mass, then blended to a fine powder using a mechanical blender and stored in air-tight containers.

2.3

2.3 Extraction of crude extracts

The oven-dried powdered plant samples (75 g for each sample) from each plant part was extracted with 750 mL (1/10: w/v) of following solvents – distilled water (aqueous), aqueous acetone 80% (acetone: water, 80:20 v/v), aqueous ethanol 80% (ethanol:water, 80:20 v/v) and aqueous methanol 80% (methanol:water, 80:20 v/v) for 48 h at room temperature in an orbital shaker (Gallenkamp, UK). The extracts were by filtering through Whatman No. 1 filter paper. The residues were extracted again with fresh solvent to achieve exhaustive extraction from the plant materials and thereafter the extracts combined. The combined extracts (80%- acetone, ethanol, and methanol) was concentrated in vacuo rotary evaporator before further dried with a freeze drier. The aqueous extract was lyophilized by the aid of a freeze dryer. The dried crude concentrated extracts were weighed to calculate the yield and stored in a refrigerator (−4 °C) until used for analyses.

2.4

2.4 Ultra-high-performance liquid chromatography-mass spectrometry analysis

To determine the phytochemical profiles, aqueous and 80% (acetone, ethanol, and methanol) of the leaves and bark extracts of Q. robur were subjected to UHPLC-QToF-MS. analysis. An Agilent ultra-high-performance liquid chromatography-mass spectrophotometer (Compass QToF Series 1.9, Bruker Instrument: Impact II) system was used for the UHPLC-QTOF-MS analysis. The chromatographic separation was carried out using an Acquity UPLC BEH C18 column 1.7 um, diameter 2.1 × 100 mm (Miscrosep Waters, Johannesburg, South Africa). The mobile phase consisted of formic acid (FA) in water and acetonitrile. The column flow was set at 0.3 mL/min, column oven temp at 35 °C, and draw speed at 3 µL/s with a total injection volume of 2 µL. The parameters for the mass spectrometer (MS) were as follows: capillary voltage 4500 V, drying gas 8 l/m, gas temperature 200 °C, ionization energy 4.0 eV, collision energy 7.0 eV, and cycle time 0.5 s. Data analysis was done using the Bruker Software (Bruker Compass Data Analysis 4.3, Bruker Daltonik GmbH, Bremen, Germany, 2014). The results were compared by using the online National Institute of Standards and Technology (NIST) library.

2.5

2.5 Measurement of phenol, flavonoid, and proanthocyanidins contents

To determine the phenol content of Quercus robur leaves and bark extracts, the method reported by Unuofin et al. (2017) was used. The flavonoid content of the leaves and stem bark extracts was estimated as a method described by Unuofin et al. (2018a). To measure the proanthocyanidin contents, the methods of Unuofin et al. (2018a) was used. Extracts were tested at a concentration of 1 mg/mL and the phenol, flavonoid, and proanthocyanidins contents of the sample were determined from standard curves (20–100 μg/mL) of gallic acid for phenol content (mg/g GAE) and standard curves of (200–1000 μg/mL) of quercetin for flavonoid content (mg/g QE) or catechin for proanthocyanidins content (mg/g CE) using the following formula: Phenol content (mg∕g GAE) = C × EV/M where C is the concentration (mg/mL) extrapolated from a standard curve, EV is the extract/standard volume used in the assay (mL), and M is the mass (g) of the extract/standard in EV (mL) of the sample solution.

2.6

2.6 Measurement of in vitro antioxidant capacity

2.6.1

2.6.1 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay

The scavenging activities of the leaves and bark extracts of Q. robur were determined following a proposed by Jimoh et al. (2019). Initially, 100 µL of 0.135 mM dissolved in methanol was reacted with 100 µL of each extract/standard with concentration ranging from 5 to 80 µg/mL. The absorbance of the mixture was read at 517 nm after 30 mins incubation in the dark. All experimental determinations were carried out in triplicate. The DPPH radical-scavenging potential of the extracts/standard was determined using the following equation:

2.6.2

2.6.2 2,2́-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging assay

ABTS radical scavenging abilities of the leaves and bark extracts of Q. robur followed a method previously described by Unuofin et al. (2017). Briefly, the ABTS radical was formed by reacting solutions of 7 mM ABTS and 2.45 mM potassium persulfate for a period of 16 h at room temperature in the dark. One millilitre of formed ABTS radical is mixed with 60 mL of methanol and the absorbance adjusted to 0.700 ± 0.008 at 734 nm to form ABTS solution. One hundred microlitres of the ABTS solution is then mixed with 100 µL of each extract/standard (5 to –80 µg/mL). The absorbance of the resulting mixture is read at 734 nm after 7 min incubation in the dark. All samples were run in three replicates. The ABTS radical-scavenging potential of the extracts/standard was determined using the following equation.

2.6.3

2.6.3 Ferric reducing antioxidant power assay (FRAP)

The ability of leaves and bark extracts of Q. robur to reduce ferric ions was carried out using the method described by Ahmed et al. (2015). The working FRAP reagent used for this study was freshly prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (tripyridyl triazine) solution and 20 mM FeCl3·6H2O solution in the ratio of 10:1:1 respectively. Fifty microlitre aliquot of extracts at 25–400 µg/mL and 50 µL of the standard solutions of gallic acid (20–100 µg/mL) was added to 950 µL of FRAP working reagent. Absorbance was read at 593 nm after 15 min of adding the working reagent. The blank sample consisted of 950 µL of FRAP working reagent and 50 µL of methanol. The reducing power capability of the various extracts was expressed as an equivalent concentration of antioxidants that gave a ferric reducing ability equivalent when compared to gallic acid standard (GAE).

  • GAE = CXV/M Where, GAE = Gallic acid equivalent of sample extract (mg GAE/g of sample);

  • C = concentration of gallic acid extrapolated from the standard curve in mg/mL

  • V = Volume of the sample extract in mL;

  • M = Weight of the sample extract in g.

2.7

2.7 Carbohydrates-hydrolysing enzymes inhibition

The ability of leaves and bark extracts of Q. robur to inhibit carbohydrate-hydrolyzing enzymes was carried out. The alpha-amylase inhibitory assay was performed as previously described by Unuofin et al. (2019) using extracts at the concentration of 7.8 and 1000 µg/mL. Absorbance was measured at 580 nm. The α-glucosidase inhibition test was performed following the published method of Unuofin et al. (2018a,b) using extracts at the concentration of 7.8 and 1000 µg/mL. Absorbance was measured at 405 nm. Acarbose used as a positive control in both assays. Inhibition activity (%) = [1 − (Abs of test sample 580/405nm/Abs of enzyme control 580/405nm)] × 100.

2.8

2.8 Data evaluation

All assays were conducted in three replicates, and the obtained results (mean ± SD) were statistically analyzed using MINITAB 17. Normality test data were subjected to Anderson-Darling analysis; One-way analysis of variance were performed to check the significant differences among samples. Means of samples were compared using Fischer’s least significant difference (LSD) at the 5% confidence level, when significant differences were found.

Then partial least squares discriminant analysis (PLS-DA) and Heatmap were achieved to identify the most discriminant biological activities and to characterize the clusters obtained from PLS-DA analysis. All analysis were performed under R v 3.6.1.

3

3 Results and discussion

3.1

3.1 Phytochemical content of the various extracts

3.1.1

3.1.1 Total phenolic content (TPC)

The total phenolic content of the extracts was measured using the Folin Ciocaltau method. TPC values were extrapolated from a standard curve y = 8.3188x + 0.0087 with R2 = 0.9606, where y is the absorbance and x is the concentration of the gallic acid solution (µg/mL) expressed as mg GAE/g DW of the sample. The TPC values of both the leaves and stem bark extracts of Q. robur ranged from 114.90 mg GAE/g DW for 80% aqueous acetone bark extract to 320.71 mg GAE/g dw for aqueous bark extract (Table 1). The decrease in TPC was in this order: aqueous bark extract > 80% aqueous acetone leaves extract > 80% aqueous ethanol bark extract > 80% aqueous methanol bark extract > aqueous leaves extract > 80% aqueous ethanol leaves extract > 80% aqueous methanol leaves extract > 80% aqueous acetone bark extract. The TPC of the 80% aqueous methanol leaves extract is not significantly higher than that of the 80% aqueous acetone bark extract (p < 0.05). This value is substantially greater than that found oak grown in Belgium (5.0–62.7 mg GAE/g DW) (Dedrie et al., 2015), Poland (55.4–79.3 mg GAE/g DW) (Dróżdż and Pyrzynska, 2018) and Serbia (12.86–32.37 mg CE/g DW) (Popović et al., 2013). These differences in value could be ascribed to changes in botanical and geographical origin.

Table 1 Percent yield, total flavonoid and total phenolic and proanthocyanidin contents of leaves and stem bark extracts of Quercus robur.
Plant part Solvent system Yield (%) TPC (mg GAE/g DW) TFC (mg QE/g DW) Pro (mg CE/g DW)
Leaves Aqueous 22.40% 178.16 ± 0.13e 212.65 ± 0.90e 135.80 ± 10.36c
80% aqueous acetone 22.53% 284.27 ± 0.73b 409.29 ± 35.91b 433.24 ± 12.82a
80% aqueous ethanol 22.40% 136.94 ± 0.43f 181.98 ± 7.57f 170.77 ± 12.60b
80% aqueous methanol 22.57% 115.44 ± 0.16 g 105.53 ± 4.36 g 71.51 ± 3.83d
Bark Aqueous 6.27% 320.71 ± 0.22a 505.62 ± 8.44a 83.46 ± 4.82d
80% aqueous acetone 8.27% 114.90 ± 0.40 g 134.31 ± 8.77 g NA
80% aqueous ethanol 6.27% 256.41 ± 0.68c 352.01 ± 9.59c 84.48 ± 1.32d
80% aqueous methanol 6.00% 228.23 ± 0.31d 294.59 ± 7.45d 34.13 ± 6.65e

Values in the same column not having common superscripts letter (a-g) differ significantly at p > 0.05. Mean ± SD (n = 3).

GAE = gallic acid equivalent; QE = quercetin equivalent; CE = cathechin equivalent; TFC = total flavonoid content; TPC = total phenol content. Pro = Proanthocyanidin. Expressed as 100 × (g dry extract/g dry leaves).

3.1.2

3.1.2 Total flavonoid content (TFC)

The TFC of the extracts is revealed in Table 1. The TFC values of both the leaves and stem bark extracts of Q. robur range from 105.53 mg QE/g DW for 80% aqueous methanol bark extract to 505.62 mg QE/g DW for aqueous bark extract (Table 1). The decrease in TFC was in this order: aqueous bark extract > 80% aqueous acetone leaves extract > 80% aqueous ethanol bark extract > acetone > 80% aqueous methanol bark extract > aqueous leaves extract > 80% aqueous ethanol leaves extract > 80% aqueous acetone bark extract > 80% aqueous methanol leaves extract. The TFC of the 80% aqueous methanol leaves extract is not significantly higher than that of the 80% aqueous acetone bark extract (p < 0.05). A similar trend of the result was obtained in the amount of TPC. Our study revealed higher total flavonoid content when compared with values gotten from Q. robur grown in Poland (33.1–78.4 mg GAE/g DW) (Dróżdż and Pyrzynska, 2018) and Serbia (0.23–2.09 mg rutin/g DW) (Popović et al., 2013). These differences in value could be ascribed to changes in botanical and geographical origin.

3.1.3

3.1.3 Proanthocyanidins contents (Pro)

The proanthocyanidins contents of both leaves and stem bark extracts of Q. robur are revealed in Table 1. The Pro values range from 34.13 mg CE/g DW for 80% aqueous methanol bark extract to 433.24 mg CE/g dw for 80% aqueous acetone leaves extract (Table 1). The decrease in Pro was in these orders: 80% aqueous acetone leaves extract > 80% aqueous ethanol leaves extract > aqueous leaves extract > 80% aqueous ethanol bark extract > aqueous bark extract > 80% aqueous methanol leaves extract > 80% aqueous methanol leaves extract. There was no significant difference in the Pro contents of 80% aqueous ethanol bark extract, aqueous bark extract and 80% aqueous methanol leaves extract (p < 0.05). Our study revealed higher proanthocyanidin content when compared with values gotten from Q. robur grown in Serbia (1.49–11.02 mg leukocyanidine/g DW) (Popović et al., 2013). Finally, our study corroborates the influence of environmental conditions on the composition of polyphenolic contents in different plant organs as it was revealed in Ruiz-Rodríguez and co-worker's study on the strawberry tree (Ruiz-Rodríguez et al., 2011). These polyphenolic contents such as phenolic acids, flavonoids, and proanthocyanidins (condensed tannins) are widely distributed into different plant part and they are produced as a result of adaptation to harsh climate condition and defense against herbivory animals. Polyphenolic content present in plant materials such as stem bark, leaves, rhizomes, corms, algae, and mushroom is of commercial and scientific interest because of their therapeutic potential in the management and treatment of oxidative stress-induced diseases such as diabetes, cancer, and obesity (Custódio et al., 2015; Soobrattee et al., 2005; Unuofin et al., 2018b).

3.2

3.2 Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-QToF-MS) analysis of leaves and stem bark extracts of Quercus robur

The UHPLC-MS analysis identified a total of twenty-seven secondary metabolites majorly flavonoids and phenolic acids which varied among Q. robur leaves and stem bark extracts (Table 2). The information gathered from other Quercus species revealed a number of phenolic acid such as In previous reports, several phenolic acids were identified in other Quercus species such as (+) catechin, chlorogenic, cinnamic, ellagic acid, esculetin, gallic, gentisic acids, protocatechuic, quercetin were the major compounds in corks, leaves and stem from Spain and Portugal (Kim et al., 2008; Santos et al., 2010; Touati et al., 2015). Our study is in agreement with Galiñanes et al. (2015) where reverse-phase high-performance liquid chromatography-electrospray ionization time-of-flight (RP-HPLC-ESI-TOF) mass spectrometry analysis of 2% Na2SO3 extract of Q. robur identified certain flavonoids and phenolic acids such as (+)-catechin, (-)- gallocatechin, ellagic acids and gallic acid. However, other flavonoids, polyphenolic acids and triterpenoid such as neriifolin, alnusiin, kaempferol-3-o-(6-o-cis-coumaryl)glucoside, procyanidin B5, cis-3,4-leucopelargonidin, pheophytin a, luteoforol, quercetin 3-O-[beta-D-xylosyl-(1->2)-beta-D-glucoside, myricitrin neohesperidin, quercitrin, camelliol C, cucurbitacin O and cucurbitacin F were the different leaves and stem bark extracts of Q. robur grown in South Africa. The difference in the bioactive of compounds found in our study could be attributed to changes in botanical and geographical origin of Q. robur.

Table 2 Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-QToF-MS) Analysis of leaves and stem bark extracts of Quercus robur.
No Name Molecular Formula Exact Mass RT (min) M+H M−H Extracts
HL AL EL ML HB AB EB MB
1 (-)-quinic acid C7H12O6 192.063 1.00 193.070 191.06 + +
2 Pedunculagin C34H24O22 784.076 1.78 783.079 +
3 Alnusiin C41H26O26 934.071 3.33 935.271 + + + +
4 Tribuloside (kaempferol-3-o-(6-o-cis-coumaryl)glucoside) C30H26O13 594.137 3.99 595.470 +
5 Procyanidin B5 C30H26O12 578.142 4.18 577.132 + + +
6 (+)-Gallocatechin C15H14O7 306.074 4.96 305.067 +
7 cis-3,4-Leucopelargonidin C15H14O6 290.079 4.54 291.084 + + + +
8 Pheophytin a C55H74N4O5 870.566 12.74 871.500 + +
9 Stearidonic acid C18H28O2 276.209 10.08 277.216 275.309 + +
10 (+)-catechin C15H14O6 290.079 4.6 291.200 +
11 Luteoforol C15H14O6 290.079 4.73 289.073 + + +
12 Quercetin 3-O-[beta-D-xylosyl-(1- > 2)-beta-D-glucoside C26H28O16 596.138 6.05 597.521 + +
13 Myricitrin C21H20O12 464.095 6.57 465.103 463.088 + +
14 Neohesperidin C28H34O15 0.76 609.010 +
15 Quercitrin C21H20O11 448.101 6.96 449.106 447.093 + +
16 Pteryxin C21H22O7 386.137 7.63 387.145 +
17 Cucurbitacin O C30H46O7 518.324 8.21 518.324 + +
18 Kaempferol 3-(3″,6″-diacetyl-2″,4″-di-p-coumaroylrhamnoside) C43H36O17 824.195 9.69 825.195 + +
19 3 beta-Hydroxy-4beta-methyl-5alpha-cholest-7-ene-4alpha-carbaldehyde C29H48O2 428.365 10.96 428.365 +
20 Crucigasterin E C18H33NO 279.256 11.08 277.368 + + +
21 Oleamide C18H35NO 281.272 11.46 282.279 + + +
22 Pheophorbide a C35H36N4O5 592.269 12.42 593.276 + + + +
23 Thermozeaxanthin-13 C59H90O8 926.664 12.69 926.664 +
24 Cucurbitacin F C30H46O7 518.324 8.07 517.319 + + + + + + +
25 Camelliol C C30H50O 426.386 11.63 426.387 +
26 Ellagic acid C14H6O8 302.006 6.94 303.014 300.999 +
27 Neriifolin C30H46O8 534.319 6.74 535.327 +

NB: HL = aqueous leaves extracts; AL = 80% acetone leaves extract; EL = 80% ethanol leaves extract; ML = 80% methanol leaves extract; HL = aqueous bark extracts; AL = 80% acetone bark extract; EL = 80% ethanol bark extract; ML = 80% methanol bark extract; RT = retention time.

3.3

3.3 Antioxidant assay

Free radicals in the form of reactive oxygen species (ROS) have been implicated to play a dual role (beneficial and deleterious) in the body. They play a beneficial role when present at a very low concentration because they act as a secondary messenger in the signal transduction of certain pathways (Custódio et al., 2015; Valko et al., 2007). Their deleterious effect is prompted by their overproduction in the body thus causing oxidative damage to biological molecules such as lipid, nucleic acid, and protein and in turn influence the onset of disease conditions such as diabetes, cancer and neurodegenerative diseases (Unuofin and Lebelo, 2020; Uttara et al., 2009). Over the years, synthetic antioxidants such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) have been used to suppress and mop up ROS but their harmful effect on the liver and ability to promote carcinogenic effects in human has promoted the exploration of natural antioxidants as a possible alternative owing to their disease prevention potential and less toxic effect (Boulekbache-Makhlouf et al., 2013; Morais et al., 2011; Touati et al., 2015; Unuofin and Lebelo, 2020). Three different antioxidant assays (2,2́-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays and ferric reducing antioxidant power (FRAP) method were used to estimate the antioxidant capacity of leaves and stem bark extracts of Q. robur.

3.3.1

3.3.1 ABTS (2,2́-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity

There was significant dose-dependent scavenging of ABTS radical by leaves and stem bark extracts of Q. robur. At the highest concentration of the various extracts, greater percentage scavenging potential for ABTS radical was recorded (Fig. 1). The ability of the various extracts to scavenge 50% of the radical (IC50) is expressed in this order (Table 3): 80% aqueous ethanol bark extract (7.73 µg/mL) < 80% aqueous acetone leaves extract (8.94 µg/mL) < 80% aqueous methanol bark extract (13.70 µg/mL) < 80% aqueous ethanol leaves extract (16.19 µg/mL) < 80% aqueous acetone bark extract (17.34 µg/mL) < 80% aqueous methanol leaves extract (19.04 µg/mL) < aqueous bark extract (33.95 µg/mL) < aqueous leaves extract (131.58 µg/mL). The IC50 value of ascorbic acid which served as antioxidant standard (96.73 µg/mL). From our study, the solvent extracts (leaves and stem bark) of Q. robur grown in South Africa exhibited higher ABTS radical quenching than the ascorbic acid solution. Also, our study exhibited far superior ABTS radical scavenging activity than Q. robur grown in Spain whose extractant were water (IC50 = 493 µg/mL), 1% NaOH (IC50 = 480 µg/mL) and 2% Na2SO3 (IC50 = 489 µg/mL).

ABTS (2,2́-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity of Quercus robur leaves and stem bark extracts in different solvents (concentration ranged between 5 and 80 µg/mL). The percentage inhibition values in the bar chart are expressed as mean ± SD (n = 3). Mean separation was achieved by LSD (p < 0.05). Sets of bars (the same concentration) with different alphabets are significantly different; p < 0.05.
Fig. 1 ABTS (2,2́-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) radical scavenging activity of Quercus robur leaves and stem bark extracts in different solvents (concentration ranged between 5 and 80 µg/mL). The percentage inhibition values in the bar chart are expressed as mean ± SD (n = 3). Mean separation was achieved by LSD (p < 0.05). Sets of bars (the same concentration) with different alphabets are significantly different; p < 0.05.
Table 3 IC50 values (µg/mL) of leaves and stem bark extracts of Quercus robur on ABTS and DPPH radical scavenging activity.
Plant part Extract/Standard ABTS (µg/mL) DPPH (µg/mL)
Leaves Aqueous 131.58 ± 0.83 h 86.455 ± 3.51 g
80% aqueous acetone 8.94 ± 0.67b 8.16 ± 0.44a
80% aqueous ethanol 16.19 ± 1.33d 18.93 ± 0.79d
80% aqueous methanol 19.04 ± 0.08e 22.46 ± 0.71e
Bark Aqueous 33.95 ± 2.76f 62.76 ± 2.53f
80% aqueous acetone 17.34 ± 1.53d 22.46 ± 0.13e
80% aqueous ethanol 7.73 ± 0.62a 8.85 ± 0.53b
80% aqueous methanol 13.70 ± 1.36c 13.88 ± 0.61c
Ascorbic acid 96.73 ± 1.00 g 19.05 ± 1.02d

Values in the same column not having common superscripts letter (a-g) differ significantly at p > 0.05. Mean ± SD (n = 3).

ABTS = 2,2́-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid); DPPH = 2,2-diphenyl-1-picrylhydrazyl; IC50 value = effective concentration of antioxidants necessary to decrease the radical concentration by 50%.

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of Quercus robur leaves and stem bark extracts in different solvents (concentration ranged between 5 and 80 µg/mL). The percentage inhibition values in the bar chart are expressed as mean ± SD (n = 3). Mean separation was achieved by LSD (p < 0.05). Sets of bars (the same concentration) with different alphabets are significantly different; p < 0.05.
Fig. 2 DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of Quercus robur leaves and stem bark extracts in different solvents (concentration ranged between 5 and 80 µg/mL). The percentage inhibition values in the bar chart are expressed as mean ± SD (n = 3). Mean separation was achieved by LSD (p < 0.05). Sets of bars (the same concentration) with different alphabets are significantly different; p < 0.05.

3.3.2

3.3.2 DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity

The scavenging effects of the various extracts on 2,2-diphenyl-1-picrylhydrazyl radicals were determined using varying concentrations (5–80 µg/mL) in comparison with ascorbic acid (Fig. 2 and Table 3). The 80% aqueous acetone leaves extract showed the most potent DPPH scavenging capacity and lowest IC50 (8.16 µg/mL), followed by 80% aqueous ethanol bark extract (IC50 = 8.85 µg/mL), 80% aqueous methanol bark extract (IC50 = 13.88 µg/mL), 80% aqueous ethanol leaves extract (IC50 = 18.93 µg/mL), 80% aqueous acetone bark extract (IC50 = 22.46 µg/mL), 80% aqueous methanol leaves extract (IC50 = 22.46 µg/mL), aqueous bark extract (IC50 = 62.76 µg/mL), aqueous leaves extract (IC50 = 86.46 µg/mL), with comparison to ascorbic acid used as antioxidant standard. The IC50 value of ascorbic acid which served as positive standard (IC50 = 19.05 µg/mL). Results obtained from a study carried out by Galiňanes et al. (2015) on DPPH radical scavenging potential of Q. robur bark grown in Spain revealed that water extract (IC50 = 493 µg/mL), 1% NaOH extract (IC50 = 480 µg/mL) and 2% Na2SO3 extract (IC50 = 489 µg/mL) but these scavenging capacity is less than obtained in our study (Table 3).

3.3.3

3.3.3 Ferric reducing antioxidant potential (FRAP)

For the FRAP assay, the absorbance at 593 nm of eight extracts of Q. robur stem bark and leaves grown in South Africa (Fig. 3). There was increased antioxidant potential with increasing concentration of the various extracts. The aqueous leaves extract showed the highest and best antioxidant potential to reduce ferric ion when compared to other extracts of Q. robur examined (p < 0.05), followed by 80% aqueous acetone leaves extract, 80% aqueous ethanol bark extract, aqueous bark extract, aqueous leaves extract, 80% aqueous methanol bark extract, 80% aqueous acetone bark extract, 80% aqueous methanol leaves extract and 80% aqueous ethanol leaves extract.

Ferric reducing antioxidant potential (FRAP) values of different extracts of Q. robur stem bark and leaves in terms of gallic acid equivalents (GAE) (n = 3) The gallic acid equivalent (GAE) values in the bar chart are expressed as mean ± SD (n = 3). Mean separation was achieved by LSD (p < 0.05). Sets of bars (the same concentration) with different alphabets are significantly different; p < 0.05.
Fig. 3 Ferric reducing antioxidant potential (FRAP) values of different extracts of Q. robur stem bark and leaves in terms of gallic acid equivalents (GAE) (n = 3) The gallic acid equivalent (GAE) values in the bar chart are expressed as mean ± SD (n = 3). Mean separation was achieved by LSD (p < 0.05). Sets of bars (the same concentration) with different alphabets are significantly different; p < 0.05.

3.4

3.4 Antidiabetic activity

Diabetes is a metabolic condition resulting in elevated levels of blood sugar, it promotes the onset of other severe conditions (Unuofin and Lebelo, 2020). The process of management or treatment patients with this condition is aimed at regulating their glycemic levels. Most allopathic medications used for the treatment and management of diabetes possess myriads of adverse effects. These adverse effects associated with allopathic medications have promoted the exploration of medicinal plants with antidiabetic properties as they could serve as useful sources for cheaper and safer antidiabetic drugs. The antidiabetic potential (α-amylase and α-glucosidase) of the different solvent extracts of leaves and stem bark of Q. robur was evaluated. The extracts had varying degrees of inhibition against α-amylase at 7.8 and 1000 µg/mL, while higher inhibitory potential against α-glucosidase was observed at 7.8 and 1000 µg/mL respectively (Table 4). Our findings are in support with previous studies where natural products and medicinal plant materials displayed a stronger inhibitory potential against α-glucosidase when measured up to α-amylase (Custódio et al., 2015; Kwon et al., 2008; Unuofin et al., 2019, 2018b). The results obtained showed that extracts of Q. robur grown in South Africa exhibits better α-amylase and α-glucosidase inhibition at 7.8 and 1000 µg/mL respectively. These values are substantially higher than that found in the methanol extracts from the acorn, acorn shell and cupule of Q. robur grown in Turkey which indicated 94.28, 37.14 and 26.86% α-glucosidase inhibitory activity respectively at 20 µg/mL and no inhibition of α-amylase at that same concentration (Güvenalp et al., 2016). These differences can be attributed to the disparity in botanical and geographical origin. The aqueous extract of stem bark at 7.8 µg/mL and leaves at 1000 µg/mL potentiated the highest reduction of α-glucosidase activity. The higher activity observed most especially in the aqueous extract of the stem bark could be due to its high phenolic contents. Our results are in support with findings of Custódio et al. (2015) and Unuofin et al. (2019), who revealed that there is a strong association between high phenolic contents and high α-glucosidase inhibitory activity of plant extracts. This further justifies the modulatory effect of phenolic compounds in glucose metabolism and their role in the management of T2DM (Unuofin and Lebelo, 2020). The presence of flavonoids, phenolic acid and triterpenoids such as cucurbitacins, pedunculagin, kaempferol-3-O-β-D-(6″-O-coumaryl) glycoside, neohesperidin, quercitrin, and pheophorbide a (Table 2) have been shown to inhibit these digestive enzymes through a different mechanism of action both in vitro and in vivo experimental models (Babujanarthanam et al., 2011; Chauhan et al., 2010; Jia et al., 2015; Kim et al., 2011; Lee et al., 2013; Tan et al., 2008).

Table 4 Inhibitory activity (%) of leaves and stem bark extracts of Quercus robur on α-amylase and α-glucosidase.
Plant part Extract/Standard α-amylase α-glucosidase
7.8 µg/mL 1000 µg/mL IC50 (µg/mL) 7.8 µg/mL 1000 µg/mL IC50 (µg/mL)
Leaves Aqueous 16.37 ± 0.03c 38.18 ± 0.40f 1467.90 52.32 ± 2.41b 92.70 ± 0.00d 7.60
80% aqueous acetone 38.74 ± 0.04 g 3.57 ± 0.04a 1497.09 92.79 ± 1.59f 81.47 ± 0.24a 4.35
80% aqueous ethanol 32.75 ± 0.04e 28.07 ± 0.13d 1701.01 95.44 ± 0.17 g 85.71 ± 0.01b 3.21
80% aqueous methanol 30.11 ± 0.04d 34.69 ± 0.08e 1539.18 77.98 ± 3.42d 91.21 ± 0.00d 4.93
Bark Aqueous 7.79 ± 0.18 a 14.73 ± 0.20b 3171.20 96.50 ± 0.32 h 85.46 ± 0.05b 3.18
80% aqueous acetone 34.42 ± 0.03f 23.00 ± 0.10c 1576.46 75.02 ± 2.11c 89.06 ± 0.05c 5.03
80% aqueous ethanol 41.51 ± 0.14 h 53.10 ± 0.07 h 869.10 94.28 ± 1.03 g 85.77 ± 0.00b 3.25
80% aqueous methanol 62.43 ± 0.01i 49.30 ± 0.05 g 84.55 82.49 ± 0.08e 88.60 ± 0.02c 4.23
Acarbose 13.12 ± 0.01b 63.50 ± 0.20i 635.04 12.89 ± 1.02a 62.05 ± 0.20d 661.20

Values in the same column not having common superscripts letter (a-g) differ significantly at p > 0.05. Mean ± SD (n = 3).

3.5

3.5 Multivariate analysis

As a preliminary step, whole pharmacological activities expressed by both leaves and bark of Q. robur and their respective solvent of extraction was analyzed by Partial least squares discriminant analysis (PLS-DA) was achieved using ‘‘organs” as class membership.

PLS-DA is a supervised algorithm that refines partition between different clusters of samples, by way of connecting two data matrices, e.g. a raw data (X) and an equivalent dependent class membership (Y). Fig. 4A revealed that both organs were closely related, suggesting that certain pharmacological activities of leaves and bark were not significantly different. Using Student’s t-test or Wilcoxon, it appears clearly that leaves were most active against DPPH, ABTS FRAP, and α-glucosidase than bark whereas α-amylase was most active in the plant bark than the leaves (Fig. 4B).

Supervised Partial Least Squares Discriminant Analysis. A: Samples plot. B: Characterization of bark and leaves samples taking account of the identified most discriminant biological activities. C: Correlation coefficients between total bioactive compounds and biological activities (Pearson Correlation Coefficient (R), p < 0.05). D: Heatmap showing variation of biological activities between pulp and peel extracts. red color indicates high activity. Green color indicates low activity.
Fig. 4 Supervised Partial Least Squares Discriminant Analysis. A: Samples plot. B: Characterization of bark and leaves samples taking account of the identified most discriminant biological activities. C: Correlation coefficients between total bioactive compounds and biological activities (Pearson Correlation Coefficient (R), p < 0.05). D: Heatmap showing variation of biological activities between pulp and peel extracts. red color indicates high activity. Green color indicates low activity.

Thereafter the PLS-DA analysis, it was important to assess the impact of the second factor e.g. extraction of solvents on the biological activities of each organ. Consequently, correlation coefficients between total bioactive compounds and biological activities using Pearson Correlation Coefficient and heatmap were generated, by visual examination of the corresponding results reported in Fig. 4C and D; The effect of leaves was remarkable in ABTS, DPPH, FRAP, and α-glucosidase inhibition assays particularly the aqueous extract. The 80% aqueous acetone had more contents of TFC and TPC compared to other leaves extract solvents. Regarding the bark, the aqueous extract had more contents of TFC and TPC content in this study. In addition, its effect was observed to be most active in α-amylase inhibition assays. The 80% aqueous ethanol extract had the highest contents of Pro in this study. According to these observations, the choice of the solvent of extraction for phytochemical compounds extraction must be based upon their respective pharmacological activity evaluation owing to fact that better biological activity was observed in leaves than in the bark of Q. robur. In fact, although the leaves extract portrayed better biological activity, both plant parts are showed substantive biological activities across board.

4

4 Conclusion

The present study revealed a detailed chemical characterization of secondary metabolites of leaves and stem bark from Quercus robur L. grown in South Africa by UHPLC-QToF-MS as well as evaluating the antioxidant and anti-diabetes activities of the different extracts. Our results indicated that Q. robur L. grown in South Africa may constitute a potential source of antioxidant with application in the food and pharmaceutical industries, with a focus in the prevention of diabetes. In all, our results suggest possible applications of both leaves and stem bark to the nutraceutical industry.

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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