Rumex dentatus L. phenolics ameliorate hyperglycemia by modulating hepatic key enzymes of carbohydrate metabolism, oxidative stress and PPARγ in diabetic rats

Article abstract of DOI:10.1016/j.fct.2020.111202

Rumex dentatus L. is a flowering plant with promising therapeutic effects. This study investigated the antioxidant efficacy of phenolic compounds isolated from R. dentatus L. in vitro and by conducting density function theory (DFT) studies to explore the mechanisms of action. The antioxidant, anti-inflammatory and antidiabetic effects of polyphenols-rich R. dentatus extract (RDE) were investigated in type 2 diabetic rats. Phytochemical investigation of the aerial parts of R. dentatus resulted in the isolation of one new and seven known compounds isolated for the first time from this species. All isolated phenolics showed in vitro radical scavenging activity. The antioxidant activity of the compounds could be oriented by the hydrogen atom transfer and sequential proton loss electron transfer mechanisms in gas and water phases, respectively. In diabetic rats, RDE attenuated hyperglycemia, insulin resistance and liver injury and improved carbohydrate metabolism. RDE suppressed oxidative stress and inflammation and upregulated PPARγ. In silico molecular docking analysis revealed the binding affinity of the isolated compounds toward PPARγ. In conclusion, the computational calculations were correlated with the in vitro antioxidant activity of R. dentatus derived phenolics. R. dentatus attenuated hyperglycemia, liver injury, inflammation and oxidative stress, improved carbohydrate metabolism and upregulated PPARγ in diabetic rats.

Full text of DOI:10.1016/j.fct.2020.111202

Journal Pre-proof
Rumex dentatus L. phenolics ameliorate hyperglycemia by modulating hepatic key
enzymes of carbohydrate metabolism, oxidative stress and PPARγ in diabetic rats
Rasha H. Elsayed, Emadeldin M. Kamel, Ayman M. Mahmoud, Ashraf A. El-
Bassuony, May Bin-Jumah, Al Mokhtar Lamsabhi, Sayed A. Ahmed
PII:
S0278-6915(20)30090-9
https://doi.org/10.1016/j.fct.2020.111202
FCT 111202
DOI:
Reference:
To appear in: Food and Chemical Toxicology
Received Date: 7 January 2020
Revised Date: 12 February 2020
Accepted Date: 13 February 2020
Please cite this article as: Elsayed, R.H., Kamel, E.M., Mahmoud, A.M., El-Bassuony, A.A., Bin-
Jumah, M., Lamsabhi, A.M., Ahmed, S.A., Rumex dentatus L. phenolics ameliorate hyperglycemia by
modulating hepatic key enzymes of carbohydrate metabolism, oxidative stress and PPARγ in diabetic
rats, Food and Chemical Toxicology (2020), doi: https://doi.org/10.1016/j.fct.2020.111202.
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Credit Author Statement:
A.M.M. conceived the study, designed and performed animal experiments, carried out the
biochemical, molecular and histologic studies, analyzed the data and prepared the figures.
A.M.M. and E.M.K wrote the manuscript. R.H.E.; E.M.K. and A.A.E. carried out the
phytochemical analysis. E.M.K. and A.L. performed the DFT studies. E.M.K. performed the
molecular docking studies. M.B-J. provided resources, participated in the assays and acquired
funding. A.M.M. and S.A.A. revised the manuscript. All authors read and approved the
manuscript for publication.

Rumex dentatus L. phenolics ameliorate hyperglycemia by modulating hepatic key enzymes
of carbohydrate metabolism, oxidative stress and PPARγ in diabetic rats
a#
a#
b#
Rasha H. Elsayed , Emadeldin M. Kamel , Ayman M. Mahmoud *, Ashraf A. El-
a
c
d,e
a
Bassuony , May Bin-Jumah , Al Mokhtar Lamsabhi , Sayed A. Ahmed
a
Chemistry Department, Faculty of Science, Beni-Suef University, Beni-Suef 62514, Egypt.
b
Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, Beni-
Suef 62514, Egypt.
c
Biology Department, College of Science, Princess Nourah bint Abdulrahman University,
Riyadh 84428, Saudi Arabia.
d
Departamento de Química, Módulo 13, Universidad Autónoma de Madrid, Campus de
Excelencia UAM-CSIC Cantoblanco, Madrid 28049, Spain.
e
Institute for Advanced Research in Chemical Sciences (IAdChem), Universidad Autónoma de
Madrid, Madrid 28049, Spain.
#R.H.E., E.M.K. and A.M.M. participated as first author.
Corresponding author:
Ayman M. Mahmoud, PhD
Physiology Division, Zoology Department, Faculty of Science, Beni-Suef University, Salah
Salim St., 62514, Beni-Suef, Egypt.
E-mail: ayman.mahmoud@science.bsu.edu.eg
1

Abstract
Rumex dentatus L. is a flowering plant with promising therapeutic effects. This study
investigated the antioxidant efficacy of phenolic compounds isolated from R. dentatus L. in
vitro and by conducting density function theory (DFT) studies to explore the mechanisms of
action. The antioxidant, anti-inflammatory and antidiabetic effects of polyphenols-rich R.
dentatus extract (RDE) were investigated in type 2 diabetic rats. Phytochemical investigation of
the aerial parts of R. dentatus resulted in the isolation of one new and seven known compounds
isolated for the first time from this species. All isolated phenolics showed in vitro radical
scavenging activity. The antioxidant activity of the compounds could be oriented by the
hydrogen atom transfer and sequential proton loss electron transfer mechanisms in gas and
water phases, respectively. In diabetic rats, RDE attenuated hyperglycemia, insulin resistance
and liver injury and improved carbohydrate metabolism. RDE suppressed oxidative stress and
inflammation and upregulated PPARγ. In silico molecular docking analysis revealed the
binding affinity of the isolated compounds toward PPARγ. In conclusion, the computational
calculations were correlated with the in vitro antioxidant activity of R. dentatus derived
phenolics. R. dentatus attenuated hyperglycemia, liver injury, inflammation and oxidative
stress, improved carbohydrate metabolism and upregulated PPARγ in diabetic rats.
Keywords: Oxidative stress; Diabetes; Phenolics; Cytokines; PPARγ; DFT.
2

1. Introduction
Plants of the genus Rumex (sorrel, Polygonaceae) are distributed worldwide and have been used
as vegetables. There are approximately 200 species belong to this genus and some possess
beneficial pharmacologic properties (Vasas et al., 2015). Rumex dentatus L. is a flowering plant
occurs in North Africa and Eurasia and commonly known as toothed dock (A Abou Elfotoh et
al., 2013). R. dentatus is rich in bioactive constituents with well-acknowledged antioxidant
effects (Elzaawely and Tawata, 2012; Humeera et al., 2013), and has been reported to
suppress proliferation and induce apoptosis in breast cancer MDA-MB-231 cell line in vitro
(
Batool et al., 2017). In addition, Zhang et al have reported the anti-proliferation activity of
an anthraquinone and naphthalene glucosides isolated from R. dentatus against breast cancer,
melanoma, gastric cancer and oophoroma cell lines (Zhang et al., 2012). The alcoholic
extracts of R. dentatus have shown potent antimicrobial activities against different pathogenic
bacterial strains (Humeera et al., 2013), and ameliorated paracetamol-induced liver injury in
mice (Saleem et al., 2014). Therefore, R. dentatus represents a promising source of bioactive
compounds with beneficial therapeutic activities. Although R. dentatus showed a radical
scavenging activity which was positively correlated with its phenolic content (Elzaawely and
Tawata, 2012), little information is available on the isolation and characterization of phenolics
from this plant. The leaves and roots of R. dentatus contain high levels of phenolic compounds
as previously demonstrated by GC-MS and HPLC analyses (A Abou Elfotoh et al., 2013;
Elzaawely and Tawata, 2012). The antioxidant activity of phenolics has been attributed to their
radical-scavenging properties (Kamel et al., 2016). In silico investigation of the antioxidant
potential of phenolic compounds can help disclosing the relation between the antioxidant
activity and structure of phenolics (Kamel et al., 2016; Payán-Gómez et al., 2010). Besides the
3

commonly known routes for phenolics mechanisms of action as antioxidants, hydrogen atom
transfer (HAT) and single-electron transfer followed by proton transfer (SET-PT), the
sequential proton loss electron transfer (SPLET) has been proposed (Litwinienko and Ingold,
2005; Musialik and Litwinienko, 2005).
Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia and
impaired insulin action and/or release. Type 2 DM (T2DM) is the most prevalent form of DM
and affects more than 380 million people worldwide (Cho et al., 2018). Chronic hyperglycemia
in DM can lead to serious complications and increase the risk of developing liver diseases if not
tightly controlled (Jellinger, 2007; Levinthal and Tavill, 1999). Besides hyperglycemia,
impaired insulin action in peripheral tissues can increase the demand to insulin and lead to
gradual destruction of β-cells (Halban et al., 2014). Hyperglycemia has also been associated
with excessive generation of reactive oxygen species (ROS), oxidative stress and inflammation
(Mahmoud et al., 2012). Excess ROS alter cell function, provoke lipid peroxidation (LPO),
inflammation and cell death (Tiwari et al., 2013), reduce insulin release and impair insulin
signaling, resulting in the development of serious diabetes complications (Newsholme et al.,
2016). Therefore, counteracting hyperglycemia and its associated oxidative stress represents an
effective strategy for the management of DM and its complications. In this context, the
consumption of phenolic compounds and plant extracts rich in polyphenols ameliorated
oxidative stress, inflammation and the metabolic derangements in DM (Ahmed et al., 2012;
Ahmed et al., 2010; Mahmoud et al., 2017a; Mahmoud et al., 2012). Given its rich content of
phenolics, R. dentatus can attenuate hyperglycemia and oxidative stress and improve insulin
sensitivity in T2DM.
4

In this study, the antioxidant potential of phenolic compounds isolated from R. dentatus has
been investigated and a density function theory (DFT) study on the structure-antioxidant
activity relationship (SAR) was conducted to correlate the experimental findings with the
theoretical predictions. In addition to the in vitro and in silico investigations, the antidiabetic
potential of a phenolic-rich R. dentatus extract has been evaluated, pointing to its modulatory
effect on peroxisome proliferator-activated receptor gamma (PPARγ), oxidative stress and
inflammation. PPARγ is a ligand-inducible transcription factor that regulate the expression of
multiple genes involved in metabolism and inflammation (Han et al., 2017). PPARγ activation
increases insulin sensitivity, decreases blood glucose and lipids, and suppresses ROS generation
and inflammation (Tontonoz and Spiegelman, 2008).
2. Materials and Methods
2.1. Phytochemical investigation
2.1.1. General
1
13
H-NMR and C-NMR spectra of the investigated compounds were measured on Bruker AV-
1
13
4
00 spectrometer operating at 400 MHz for H-NMR and 100 MHz for C-NMR. TMS was
used as the internal standard. Chemical shift (δ) values were reported in (ppm) and coupling
constants were calculated and expressed in Hz. HREI-MS mass data obtained by using
Micromass Autospec (70 eV) spectrometer. UV data was recorded by Shimadzu UV–Vis 160i
spectrophotometer. Optical rotation was measured by using (model: SR6) - USA polarimeter.
FT-IR spectroscopic data were obtained from Nexus 670 FT-IR FT-Ramen spectrometer on
KBr pellets.
5

2.1.2. Plant material
The aerial parts of R. dentatus L. were collected from Beni-Suef governorate between February
and April 2017. The species was authenticated by the Botanists from the Botany Department
and a voucher specimen was kept at the Herbarium of the Faculty of Science (Beni-Suef
University, Egypt).
2.1.3. Extraction and isolation
The air-dried aerial parts of R. dentatus L. (1 kg) were powdered and extracted at room
temperature with ethanol/water (7:3, 6 L x 4 times). The extracts were combined and
evaporated in vacuo at 50 °C till dryness to give brownish sticky mass (350 g). The extract was
dissolved in water and successively partitioned with dichloromethane (1 L x 4), ethyl acetate
(EtOAc) (1 L x 4) and n-butanol (1 L x 4) to afford dichloromethane (9 g), EtOAc (8 g), n-butyl
alcohol (39 g) and water-soluble extract (80 g), respectively. 7 g of the EtOAc extract was
fractionated on silica gel column (100 x 3 cm, 200 g) using gradient elution started with EtOAc
followed by EtOAc/methanol (MeOH) mixture of increasing polarity (9:1→ 0:10). 80 fractions
were collected and then combined into 6 fractions (A→F) where the fractions were monitored
by thin layer chromatography (TLC) and paper chromatography (PC). Fraction B (1.2 g) was
subjected to silica gel column (60 x 3 cm, 70 g) and eluted with a gradient of n-hexane/EtOAc
mixture to give 9 sub-fractions. Sub-fraction 6 (100 mg) was purified on silica column by n-
hexane/EtOAc (6:4) to afford compound 2 (28 mg). Fraction D (1.5 g) was chromatographed on
sephadex LH-20 column (60 x 3 cm) and eluted with MeOH-H O (2:8, 3:7, 4:6, 5:5, 6:4, 7:3,
2
8:2, 9:1 and 10:0) to yield 9 sub-fractions. Sub-fraction 3 (61 mg) was applied on sephadex LH-
20 column using 30% MeOH to give compound 7 (15 mg). Sub-fraction 4 (165 mg) was
6

chromatographed on sephadex LH-20 column and eluted with saturated butanol to afford
compound 3 (12 mg) and compound 6 (20 mg). Sub-fraction 5 (65 mg) was applied to sephadex
LH-20 column and eluted with gradient of MeOH-H O (3:7, 4:6, 5:5, 6:4, 7:3) to afford
2
compound 8 (5 mg). Fraction E (1.1 g) was subjected to sephadex LH-20 column (40 x 3.5 cm)
and eluted with saturated butanol to give 4 sub-fractions. Sub-fraction 3 (43.5 mg) was purified
on sephadex LH-20 column with 50% MeOH to afford compound 4 (11 mg) and sub-fraction 4
was also purified on sephadex LH-20 column with 25% MeOH to afford compound 5 (35 mg).
Fraction F (40 mg) was purified over sephadex LH-20 column (50 x 1 cm) using 25% MeOH to
give compound 1 (6 mg).
(S)- 4'-methylnonyl benzoate (1)
2
5
Yellow amorphous powder; [α]D - 36.4 (c 0.09, MeOH); UV (MeOH) λmax (log ε): 209.7
−
1
(
4.98.16), 232 (5.11), 304.7 (4.87) and 368.8 (4.91) nm; IR νmax cm (KBr): 2984, 2817, 1741,
−
1
1
13
1
631, 1561, 1473, 1085, 922 and 753 cm ; H, C NMR and selected HMBC data (Table 2);
+
HR-ESI-MS (positive ion mode): m/z 262.2500 [M+H] (calcd for C17
H O
26 2
, 262.1933).
2.2. Computational methods
The quantum mechanical calculations were performed using the Gaussian 09 package and DFT
was used for all calculations (Pv et al., 2017). The structure geometry of neutral compounds,
cations, anions and radicals was optimized using B3LYP functional level and 6-311G + (d,p)
was used as a basis set for these calculations. Frequency calculations were also performed at the
same level to depict the stationary points, obtain zero-point energy (ZPE) and confirm that the
ground states have no imaginary frequency. In addition, single point calculations were carried
out on the optimized structures at 6-311G ++ (d,p) basis set in gas and water phase (ε = 1 and
7

78.4). Self-consistent reaction field (SCRF) calculations were employed for the solvation effect
by using the polarizable continuum model (PCM) solvation method (Tomasi and Mennucci,
+
•
-
2
002). The enthalpy of proton (H ), hydrogen atom (H ) and electron (e ) in gas phase and water
phase were taken from the study of Rimarčík et al (Rimarčík et al., 2010).
2.3. Radical scavenging activity (RSA) assay
RSA of the isolated compounds against 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals was
evaluated as previously reported (Brand-Williams et al., 1995) using rutin as a reference
antioxidant.
2.4. In vivo antidiabetic effect of R. dentatus extract (RDE)
2.4.1. Experimental animals and induction of T2DM
Male Wistar rats, weighing 170-190 g, obtained from Vacsera (Giza, Egypt) were acclimatized
for one week before the onset of the experiment. The rats were housed under standard
conditions (23 ± 2°C and 50-60% humidity) and were supplied food and water ad libitum. The
study protocol and procedures have been implemented in compliance with the National
Institutes of Health guidelines (NIH publication No. 85–23, revised 2011) and were approved
by the local Animal Care and Use Committee of Beni-Suef University. To induce T2DM, the
rats were fed high fat diet (HFD; 58% fat, 25% protein and 17% carbohydrate) for 4 weeks
followed by a single intraperitoneal (i.p.) injection of 35 mg/kg streptozotocin (STZ; Sigma,
USA) dissolved in freshly prepared citrate buffer (pH 4.5). After 7 days, blood glucose was
determined in overnight fasted rats 2 h following the oral administration of 3 g/kg glucose and
rats having blood glucose levels ≥ 250ꢀmg/dl were selected.
8

2.4.2. Experimental groups and treatments
To investigate the antidiabetic effect of RDE, the rats were divided into 7 groups (n=6) as
following:
Group I (Control).
Group II (200 mg/kg RDE): received 200 mg/kg RDE orally for 4 weeks.
Group III (Diabetic).
Group IV (Diabetic + 50 mg/kg RDE): received 50 mg/kg RDE orally for 4 weeks.
Group V (Diabetic + 100 mg/kg RDE): received 100 mg/kg RDE orally for 4 weeks.
Group VI (Diabetic + 200 mg/kg RDE): received 200 mg/kg RDE orally for 4 weeks.
Group VII (Diabetic + PIO): received 10 mg/kg pioglitazone (PIO) (Abd El-Twab et al., 2016)
orally for 4 weeks.
Groups I and II were fed normal chow diet and received a single injection of citrate buffer (pH
4.5). RDE and PIO were dissolved in 0.5% carboxymethyl cellulose (CMC). Groups I and III
received 0.5% CMC orally for 4 weeks. The doses of RDE were selected based on previous
studies employed R. dentatus methanolic extract at 250 and 500 mg/kg body weight to
investigate its hepatoprotective effect in mice (Saleem et al., 2014). In another study, the in vivo
antioxidant activity of the ethyl acetate fraction of another species, R. tingitanus, was studied in
Wistar rats at a dose of 250 mg/kg body weight (Mhalla et al., 2018). Herein, we investigated
the effect of 50, 100 and 200 mg/kg ethyl acetate fraction of R. dentatus in diabetic rats.
9

Twenty-four h after the last treatment, overnight fasted rats were sacrificed, and blood samples
were collected on EDTA for glycated hemoglobin (HBA1c) estimation. Other blood samples
were left to coagulate for serum separation. The rats were dissected, and the liver was removed
to prepare 10% w/v homogenate in cold phosphate buffered saline (PBS). The homogenate was
centrifuged, and the clear supernatant was collected for the determination of ROS, LPO, nitric
oxide (NO), and the antioxidants reduced glutathione (GSH), superoxide dismutase (SOD) and
catalase (CAT). Other samples were fixed in 10% neutral buffered formalin and others were
kept frozen at -80°C for RNA isolation.
2.4.3. Oral glucose tolerance test (OGTT)
Blood samples were collected from the lateral tail vein of overnight fasted rats. The rats
received 3 g/kg glucose solution and blood samples were collected after 30, 60, 90 and 120 min
and glucose was measured according to the method of Trinder (Trinder, 1969) using
commercially available kit (Spinreact, Spain).
2.4.4. Assay of HBA1c, insulin and HOMA-IR
Blood HbA1c% and serum insulin were determined using Biosystems (Spain) and RayBiotech
USA) assay kits, respectively. To evaluate the effect of RDE on insulin sensitivity,
(
homeostasis model of insulin resistance (HOMA-IR) (Haffner, 2000) was calculated as
following:
2.4.5. Assay of liver glycogen and glucose-metabolizing enzymes
1
0

Liver glycogen content was determined in the liver of control and diabetic rats according to the
method of Seifter et al (Seifter et al., 1950). Hexokinase (Brandstrup et al., 1957), glucose-6-
phosphatase (G-6-Pase) (Koide and Oda, 1959), fructose-1,6-biphosphatase (F-1,6-BPase)
(
Freedland and Harper, 1959) and glycogen phosphorylase (Stalmans and Hers, 1975) activities
were assayed following previously described methods. Inorganic phosphorus (Pi) released
during these assays was determined using Spinreact (Spain) reagent kit according to the method
of Fiske and Subbarow (Fiske and Subbarow, 1925).
2.4.6. Assay of liver function and oxidative stress markers and cytokines
Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase
ALP) and lactate dehydrogenase (LDH) were assayed using Spinreact (Spain) reagent kits. The
(
levels of ROS were determined using 2′,7′-dichlorodihydrofluorescein diacetate (H DCF-DA)
2
as previously described (Hozayen et al., 2019). Malondialdehyde (MDA), a marker of LPO, and
NO were assayed in the liver homogenate following the methods of Preuss et al (Preuss et al.,
1998) and Grisham et al (Grisham et al., 1996), respectively. Hepatic GSH content and activity
of SOD and CAT were determined according to the methods of Beutler et al (Beutler et al.,
1963), Marklund and Marklund (Marklund and Marklund, 1974) and Cohen et al (Cohen et al.,
1970), respectively. TNF-α, IL-6 and IL-1β were determined using specific ELISA kits (R&D
Systems, USA).
2.4.7. Histology
Immediately after dissection of the animals, samples from the liver were fixed in 10% neutral
buffered formalin for 24 h. The fixed samples were processed for dehydration and embedding
1
1

in paraffin wax. 5-µm sections were cut using a rotary microtome, deparaffinized, rehydrated
and stained with hematoxylin and eosin (H&E) for microscopic examination.
2.4.8. Gene expression
The effect of RDE on phosphoenolpyruvate carboxykinase (PEPCK), TNF-α, IL-6, IL-1β,
iNOS, COX-2 and PPARγ in the liver of control and diabetic rats was determined by qRT-PCR
(Mahmoud, 2014; Mahmoud et al., 2017b). Briefly, TRIzol reagent (Invitrogen, USA) was used
to isolate RNA which was treated with RNase-free DNase (Qiagen, Germany) and then
quantified on a nanodrop. RNA samples with A260/A280 nm > 1.7 were selected for cDNA
synthesis that was amplified using SYBR Green master mix and the primers set listed in Table
−
∆∆Ct
1. The obtained data were analyzed by the 2
method (Livak and Schmittgen, 2001) and
normalized to β-actin.
2.5. Molecular docking
Molecular docking analysis was conducted by Autodock Tools (ADT) v1.5.6 and AutoDock
Vina software, and the obtained results were analyzed and visualized by using PyMOL v2.3.2.
The 3D crystal structure of PPARγ was obtained from Protein Data Bank,
(http://www.rcsb.org/pdb) (PDB, ID: 2PRG). The structure of the isolated compounds (1-8) was
built using ChemDraw Ultra version 13.0 (ChemBioOffice modeling software, CambridgeSoft,
UK) and then converted to .pdb 3D structure using UCSF Chimera (Pettersen et al., 2004). The
protein receptor was prepared for docking by optimization which includes the addition of polar
hydrogens and setting the grid box according to the best configuration of the active site amino
acid residues (Nolte et al., 1998). The size of grid box was set at 38 x 38 x 46 (x,y,z). The
center of the grid box was located at 52.893 x -34.142 x 12.877 (x,y,z).
1
2

2.6. Statistical analysis
All statistical comparisons were made by one-way ANOVA followed by Tukey’s test and the
differences were considered statistically significant at P<0.05. The results were presented as
mean ± SEM (standard error of mean). The statistical analysis was carried out using GraphPad
Prism 7 (La Jolla, CA, USA).
3. Results
3.1. Phytochemical study
Investigation of the EtOAc-soluble fraction revealed the isolation of one new compound (1)
along with seven known compounds (2-8) (Fig. 1). The structure of the compounds was
1
13
confirmed by H-, C- and 2D-NMR and spectroscopic tools (UV, MS and IR). The isolated
compounds were identified as quercetin (2) (Renda et al., 2017), emodin-1-O-β-D-
glucopyranoside (3) (El-Toumy et al., 2012), kaempferol-3-O-β-D-glucuronide (4) (Yang et al.,
2
010), querectin-3-O-β-D-glucuronide (5) (M. Soliman et al., 2002), querectin-3-O-β-D-
glucopyranoside (6) (Islam et al., 2012), kaempferol (7) and resveratrol 3-glucoside (8)
Vastano et al., 2000).
(
Compound 1 is yellow amorphous, and its IR spectrum displayed strong peaks at 1741, 1631
and 1561, demonstrating the presence of an ester group. The molecular formula C H O was
1
7
26
2
+
1
13
obtained from the HR-ESI-MS data (m/z 262.2500, [M+H] , calcd 262.1933). H and C NMR
and HMBC spectral analysis depicted in Table 2 indicated the presence of a monosubstituted
1
3
benzene ring (δ
H
7.69, H-2 and H-6; 7.70-7.74, H-3, H-4 and H-5) confirmed by aromatic C
NMR signals at δ
c
129.13 (C-1), 127.08 (C-2, C-6), 125.96 (C-3, C-5) and 132.07 (C-4). The
1
3

presence of oxygenated methylene protons (H-1') was detected by a triplet PMR signal at δ
H
1
3
4
.14 and a C NMR at δ 67.88. The HR-ESI-MS peak at m/z = 105 diagnostic for the loss of
c
alkoxy chain C H O. The characteristic pattern of methylnonyl benzoate group was deduced
1
0
21
1
3
from the appearance of fifteen C NMR signals that was detailed by DEPT experiment into two
1
3
quaternary carbons, two methyl, seven methylene, three methine and one CH group. The C
NMR displayed most down field signal at δ 167.46 indicative for the presence of the carbonyl
c
1
ester. The terminal methyl group was inferred by a H NMR signal at δ
H
= 0.88 (t, J = 6.2 Hz,
was elucidated by
1.26-
1
3
C-10
signals at δ
.36 were attributed to methylene protons of the methylnonyl group and confirmed by the C
NMR signals in the range 22.55-31.75. From the HMBC correlation spectral analysis (Fig. 2),
the location of methyl at position 4 of the nonyl group was estimated from the correlation of
the methylene protons at δ 1.36 (H-2 ) to δ 14.26 (C-5 ). As well, H-1 (δ 4.14) showed
) and weak correlation with δ 31.75
՝
) and a C NMR signal δ
C
11.36 (C-10
՝
). Also, the methyl at C-4
՝
H
= 1.24 (m, C-5 ) and δ
՝
C
14.26 (C-5
՝
). Seven two-protons multiplets at δ
H
1
3
1
՝
՝
՝
՝
H
C
H
strong correlation with δ 167.46 (CO) and δ 22.85 (C-3
՝
C
C
C
(
C-2
՝
). Furthermore, the terminal methyl resonance (δ
H
=0.88, C-10
՝ ) displayed a strong
2
D
5
HMBC correlation with C-8
՝
C
(δ 23.7).The optical rotation of compound 1 ([α]
- 36.4) was
levorotatory that established the configuration of C-4’ as 4’S. Alkaline hydrolysis of compound
o
1
afforded benzoic acid, mp 121-122 C (compared by Co TLC). Based on the described
spectral data and conventional chemical reactions (alkaline hydrolysis), the structure of
compound 1 was elucidated as (S)-4'-methylnonyl benzoate.
3.2. Computational studies and RSA
The isolated phenolics 2 and 4-7 contain the rings A, B and C of the flavone moiety (Fig. 1.).
The OH groups in positions 5, 7 and 4′ are common in these compounds. Meanwhile,
1
4

compounds 2, 5, 6 and 7 show extra OH group at position 3′ for 2, 5, and 6 and position 3 for
compound 7. In addition, a fifth OH group appeared in the structure of compound 2.
Compounds 2 and 7 are flavonoids aglycones, while compounds 4-6 are flavonoids glycosides
with a common position of the O-sugar moiety at position 3. Compound 3 is an anthraquinone
glucoside with two hydroxyl substituents at positions 6 and 8, while compound 8 is a
dihydroxystilbene glucoside with two OH groups at positions 5 and 4′. Whereas the primary
elucidation of the optimized structures of the isolated phenolics was relied on the
stereochemistry assessment provided through 1D and 2D NMR evaluation, many conformers
for each compound were screened to find out the most stable conformer for each compound by
altering the dihedral angle C2′–C1′–C2–C3 in compounds 2 and 4-7 and the angle C1′–C7–C8–
C1 for compound 8. With its structural features of stilbene, resveratrol (compound 8) adopts (E)
and (Z) isomeric conformers. Although the (E) isomer is the most biologically active and
abundant form of resveratrol in nature, the optimized geometrical structures of (E) and (Z)
isomers of this compound have been compared. Quantitative and qualitative analyses suggested
that E-resveratrol exhibited stronger effects than Z-resveratrol in all experimental and
computational assays. This fact is in agreement with the previously reported data (Lin et al.,
2018).
The suggested mechanisms for the radical scavenging activity routes are represented in Figure
−
1
3
. The Results of DFT calculations of BDE (kcal mol at 298.15 K) of various hydroxyl groups
in gas phase and aqueous medium of isolated compounds (2–8) are represented in Table 3.
Homolytic cleavage of each OH of the phenolics (2-8) afforded five radicals in compound 2,
four radicals in compounds 5-7, three radicals in compound 4 and two radicals in compounds 3
and 8. Among all radicals, those obtained from the OH bond cleavage at position 4' are the most
1
5

stable for compounds 2, 5, 6 and 8, while Flav-O radicals at positions 6, 7 and 3 are the most
stable in compounds 3, 4 and 7, respectively. Phenoxy radicals at position 7 are the active site
(lowest BDE) for compounds 4-6 in gas phase. This is might be associated with the linkage of
sugar moiety to position 3 in these phenolics.
The calculated reaction enthalpies for the active sites in each compound (lowest BDE) of
compounds under investigation in gas and water phases are represented in Table 4, respectively.
Results assessment indicates that the homolytic dissociation of the OH is the most favorable
mechanism in the gas phase. In contrast, the SPLET pathway is thermodynamically the most
favorable mechanism in water phase because the first step energy demand of this mechanism is
less than that required for homolytic OH bond cleavage. This indicates that phenolics in
aqueous phase are guided to stimulate radical scavenging by the proton affinity of these entities.
Interestingly, the highest antioxidant potential is compounds 2 which possess the lowest BDE
active site. This is confirmed by the lowest IC value of this compound in the DPPH assay. In
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addition, compound 2 can afford five radical species because of the five hydroxyl substituents.
3.3. RDE attenuates hyperglycemia and improves insulin sensitivity in diabetic rats
OGTT and HbA1c% were determined to evaluate the anti-hyperglycemic potential of RDE in
diabetic rats. Diabetic rats exhibited significant blood glucose elevation at all points of the
OGTT (Fig. 4A) as revealed by the area under curve (AUC) analysis (Fig. 4B). Hyperglycemia
was further confirmed by the significant elevation in HbA1c% (Fig. 4C). Treatment of the
diabetic rats with RDE decreased blood glucose (Fig. 4A,B) and HbA1c% (Fig. 4C)
significantly in a dose-dependent manner. In addition, PIO ameliorated hyperglycemia and
HbA1c% in diabetic rats. Besides hyperglycemia, serum insulin was decreased (Fig. 4D) and
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HOMA-IR value was increased significantly (Fig. 4E), effects that were reversed following
treatment with 50, 100 and 200 mg/kg RDE or PIO.
3.4. RDE improves glucose metabolizing enzymes and increases glycogen in liver of diabetic
rats
Hexokinase was significantly declined in HFD/STZ-induced rats when compared with the
control rats (P<0.001; Fig. 5A). In contrast, the activities of G-6-Pase (Fig. 5B), F-1,6-BPase
(
Fig. 5C) and glycogen phosphorylase (Fig. 5E) and mRNA abundance of PEPCK (Fig. 5D)
were increased in the liver of diabetic rats (P<0.001). Consequently, liver glycogen content was
significantly (P<0.001) decreased in diabetic rats as depicted in Figure 5F. Treatment with RDE
(50, 100 and 200 mg/kg) or PIO enhanced hexokinase and glycogen, and suppressed G-6-Pase,
F-1,6-BPase, PEPCK and glycogen phosphorylase in liver of diabetic rats. Oral administration
of 200 mg/kg RDE didn’t affect the activity of the assayed enzymes or glycogen content in
normal rats.
3.5. RDE prevents liver injury in diabetic rats
To evaluate the potential of RDE to prevent liver injury in diabetic rats, liver function markers
were assayed, and a histopathological investigation was carried out. Serum transaminases, ALP
and LDH were elevated in diabetic rats when compared with the control group (P<0.001; Fig.
6A-D). Treatment of diabetic rats with 50, 100 and 200 mg/kg RDE or PIO reduced serum
ALT, AST, ALP and LDH.
Histopathological investigation supported the hepatoprotective efficacy of RDE. Examination
of liver sections from diabetic rats revealed centrilobular hepatic vacuolation and fatty changes
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(
Fig. 7). Oral supplementation of 50 and 100 mg/kg RDE improved the histological architecture
of the liver of diabetic rats which showed less vacuolations. The 200 mg/kg RDE as well as PIO
remarkably prevented the histopathological alterations induced by diabetes in rat liver (Fig. 7).
3.6. RDE suppresses oxidative stress and enhances antioxidants in diabetic rats
ROS, MDA, NO and cellular antioxidants were determined to assess the protective effect of
RDE against oxidative stress in diabetic rats (Fig. 8). Hepatic ROS, LPO and NO were
increased in diabetic rats (P<0.001; Fig. 7A-C), whereas GSH content, and activities of SOD
and CAT were markedly decreased (Fig. 8D-F). All doses of RDE as well as PIO were able to
reduce hepatic ROS, LPO and NO, and enhance GSH, SOD and CAT. The effect of RDE on
hepatic ROS, NO, SOD and CAT in diabetic rats was dose dependent. In normal rats, RDE
didn’t affect hepatic ROS, LPO, NO and cellular antioxidants (Fig. 8).
3.7. RDE attenuates inflammation in diabetic rats
Diabetic rats exhibited significantly elevated serum TNF-α (Fig. 9A), IL-1β (Fig. 9B) and IL-6
Fig. 9C) when compared with the control rats (P<0.001). RDE and PIO decreased pro-
(
inflammatory cytokines in serum of diabetic rats. RDE exerted a dose-dependent effect on
serum TNF-α in diabetic rats.
The expression of pro-inflammatory cytokines, iNOS and COX-2 was assessed in the liver of
control and treated rats. TNF-α, IL-1β, IL-6, iNOS and COX-2 mRNA abundance was
significantly increased in the liver of HFD/STZ -induced rats (P<0.001; Fig. 9D-H). Oral
supplementation of RDE or PIO downregulated the expression levels of all assayed
inflammatory mediators. The effect of RDE on mRNA abundance of these mediators in the
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liver of diabetic rats was dose dependent. Of note, oral supplementation of 200 mg/kg RDE
didn’t affect serum levels and mRNA abundance of the determined inflammatory mediators in
normal rats.
3.8. RDE upregulates PPARγ expression in liver of diabetic rats
PPARγ gene expression was decreased in the liver of HFD/STZ diabetic rats when compared
with the control group (Fig. 10A). All doses of RDE and PIO increased hepatic PPARγ mRNA
abundance in diabetic rats.
Docking of R. dentatus isolated phytochemicals (1-8) onto PPARγ was carried out and the
binding affinities and hydrogen bond interactions were analyzed (Table 5). The Isolated
compounds showed binding affinities in the range of -7.2 kcal/mol to -8.7 kcal/mol with
compound 8 formed the most stable complex with PPARγ. All compounds fitted the same
pocket in PPARγ and showed binding toward the amino acids Phe226, Glu259, Arg288,
Cys285, Ser289, Glu291, Ala292, Met329, Leu330, Glu295, Ile296, His323, Ile326, Tyr327,
Leu333, SER342, Glu343, Thr268, Arg280, Phe363, Met364, His449 and Tyr473 (Fig. 10B).
The compounds exhibited variable number of hydrogen bonding interactions (ranging from two
to five) between their polar moieties and the amino acid residues (Table 5). The amino acids
involved in the polar interactions of all isolated phytochemicals are Glu259, Arg288, Glu291,
Thr 268, Ser342 and Glu343.
4. Discussion
This study investigated the antioxidant potential of compounds isolated from R. dentatus using
in vitro and computational approaches. In addition, the anti-hyperglycemic, antioxidant and
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anti-inflammatory effects of R. dentatus were evaluated in HFD/STZ-induced type 2 diabetic
rats, pointing to the possible role of PPARγ.
Phytochemical analysis of R. dentatus resulted in the isolation of one new and seven known
phenolic compounds. All isolated phenolic compounds exhibited a radical scavenging activity
evidenced by the in vitro DPPH assay. The antioxidant properties of natural phenolics have
been attributed to their potent radical scavenging activity which is associated with the existence
of OH at specific position on the phenolic nucleus (Kamel et al., 2016). The flavonoid
glycosides 4, 5 and 6 showed nearly similar active site BDE values but different IC . The main
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reason for this inconsistency is the different number of OH substituents. A second major reason
is linked to the medium that can play a significant role in the antioxidant activity. For various
OH groups in water, the BDE values order is nearly the same as that resulted for the gas phase.
At the active site on the phenolic compound core, homolytic or heterolytic OH bond cleavage
may occur depending mainly on the reaction medium. The H-abstraction process is believed to
be the main process underlying the antioxidant activity of these compounds (Barzegar, 2012).
This study showed that the radicals obtained from the hydrogen abstraction at position 4' and 3'
are commonly stable in the majority of phenolics and flavonoids (Kamel et al., 2016). This is
attributed to the hydrogen bonding between one oxygen radical and one OH after H atom
abstraction from one of the two OH substituents in positions C-3′ and C-4′ alternately.
Furthermore, the existence of sugar causes planarity deviations and affects the stability of
phenolics radicals (Alov et al., 2015). Consequently, aglycones could be more potent
antioxidants than their corresponding glycosides. The main reason for this discrimination might
be attributed to the electron withdrawing properties of the sugar moiety (Klein et al., 2009).
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HAT, SET-PT and SPLET are the mechanisms suggested to understand the antioxidant activity
of the phenolics (Nakanishi et al., 2005). The BDE values represent a tool for estimating the
homolytic OH bond dissociation energy in HAT mechanism. The SPLET pathway includes
heterolytic breakage of the OH bond followed by anion ionization (proton affinity (PA) +
electron transfer enthalpy (ETE)), whereas SET-PT is an ionization of the phenolic compound
followed by a proton transfer (ionization potential (IP) + proton dissociation enthalpy (PDE)).
The overall energy demand of SET-PT or SPLET pathways is similar to HAT. Owing to the
charge separation, SET-PT and SPLET are thermodynamically more favorable in polar media
and are preferred destination for radicals with an elevated electron affinity (Amić et al., 2013;
Zhang and Ji, 2006). Thus, the assessment of SAR of the isolated phenolics can be analyzed by
evaluating the PA, ETE, IP, PDE and BDE. The findings of this study demonstrate that SPLET
in aqueous medium and HAT in gas phase are the mechanisms behind the antioxidant activity
of phenolics. Compounds 2 and 6-8 are the most potent antioxidant phenolics where the
comparison between their PA and IP of the produced anion might prefer radical scavenging. On
the other hand, the SET-PT pathway is the disfavored mechanism in all isolated phenolics.
Moreover, the thermodynamic characteristics of these phenolics are affected by the nature of
the H-abstraction site and the neighboring substituents.
Next, we investigated the potential of RDE to attenuate hyperglycemia, IR and oxidative stress,
and to modulate carbohydrate metabolizing enzymes and PPARγ in type 2 diabetic rats.
Hyperglycemia resulting from impaired insulin secretion and/or action is the characteristic
feature of DM and can lead to serious complications if not tightly controlled (Jellinger, 2007).
In this study, HFD/STZ-induced rats exhibited glucose intolerance and elevated blood
HbA1c%, demonstrating hyperglycemia. In addition, these rats showed IR evidenced by the
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1

significantly increased HOMA-IR. Elevated blood glucose and HbA1c%, and impaired insulin
secretion and sensitivity have been previously demonstrated in HFD/STZ-induced rats (Guex et
al., 2019; Mahmoud et al., 2012). This model has been acknowledged to mimic human T2DM
(
Lee et al., 2011) and is therefore widely accepted to evaluate the therapeutic effects of new
agents. Interestingly, treatment of the diabetic rats with RDE improved glucose tolerance and
decreased HbA1c%, indicating its effective anti-hyperglycemic effect. Assessment of HbA1c is
a valuable test for the diagnosis and management of high blood glucose in diabetes and levels
lower than 7% reveal good glycemic control (Association, 2014). In addition, RDE alleviated
serum insulin levels and reduced HOMA-IR. Therefore, it is noteworthy assuming that
increased insulin sensitivity following treatment with RDE attenuated hyperglycemia in
diabetic rats.
Liver plays a central role in glucose homeostasis and the enzymes regulating hepatic glucose
production represent potential targets for the control of blood glucose levels. In this context,
uncontrolled hepatic glycogenolysis and gluconeogenesis and decreased peripheral glucose
utilization resulting from impaired insulin secretion and/or action provoke hyperglycemia in
diabetes (Nordlie et al., 1999). Thus, to explore the mechanism underlying the anti-
hyperglycemic effect of RDE, we determined the activity of hepatic glucose-metabolizing
enzymes and glycogen content in HFD/STZ-induced rats. The activity of hexokinase was
significantly decreased, whereas G-6-Pase, F-1,6-BPase and glycogen phosphorylase were
activated in diabetic rats. In accordance, previous studies have demonstrated a decrease in
hepatic hexokinase activity and increased G-6-Pase and F-1,6-BPase in HFD/STZ-induced
diabetic (Gothandam et al., 2019; Mishra et al., 2019). Decreased activity of hexokinase has been
associated with impaired insulin secretion and IR, resulting in diminished glucose oxidation via
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glycolysis and consequently hyperglycemia (Ahmed et al., 2010; Gupta et al., 1999). In contrast,
G-6-Pase and F-1,6-BPase promote gluconeogenesis where F-1,6-BPase dephosphorylates
fructose-1,6-bisphosphate into fructose-6-phosphate (Pilkis and Claus, 1991) and G-6-Pase
catalyzes glucose-6-phosphate dephosphorylation to glucose (Roden and Bernroider, 2003).
Consequently, glycogenolysis has also been provoked in the liver of HFD/STZ-induced rats as
evidenced by the increased glycogen phosphorylase activity and decreased glycogen content.
Exaggerated glycogenolysis and gluconeogenesis in diabetic rats indicate insulin deficiency and
IR because insulin stimulates glycogen synthase and suppresses glycogen phosphorylase and G-
6
-Pase activities (Golden et al., 1979). Therefore, increased insulin release and sensitivity
following treatment with RDE alleviated hexokinase, decreased F-1,6-BPase, G-6-Pase and
glycogen phosphorylase, and restored hepatic glycogen content in diabetic rats. To investigate
the anti-hyperglycemic effect of RDE separately from increased insulin sensitivity, PEPCK gene
expression was investigated. PEPCK catalyzes the initial step in hepatic gluconeogenesis (Quinn
and Yeagley, 2005) and its hepatic expression is upregulated in HFD/STZ-induced diabetes
(Song et al., 2019). Accordingly, PEPCK mRNA abundance was increased in the liver of
HFD/STZ diabetic rats in this study and this contributed to gluconeogenesis and hyperglycemia.
Given that PEPCK is insulin-independent (Scott et al., 1998), its downregulation following
treatment added support to the anti-hyperglycemic effect of RDE. Chronic hyperglycemia can
lead to serious complications, including neuropathy, retinopathy and nephropathy and accelerate
the progression of liver injury (Jellinger, 2007; Levinthal and Tavill, 1999). Accordingly, this
study showed increased serum transaminases, ALP and LDH accompanied with
histopathological alterations, particularly fatty changes, in the liver of diabetic rats. Hepatocyte
injury in diabetes occurs as a result of excess lipid accumulation, hyperglycemia and hepatic IR
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(
Levinthal and Tavill, 1999). Liver function markers have been reported to increase in serum of
HFD and/or STZ-induced rats (Sahin et al., 2019), and hepatic lipid infiltration occurs as a result
of fatty acids accumulation, increased lipogenesis and impaired mitochondrial β-oxidation
(Mohamed et al., 2016). Hyperglycemia can provoke liver injury by eliciting oxidative stress and
inflammation (Mahmoud et al., 2012). Excess ROS can damage lipids, DNA and other cellular
components, resulting in cell death (Tiwari et al., 2013). Additionally, ROS activate the redox-
sensitive transcription factor NF-κB which elicits the expression of TNF-α, IL-1β, IL-6, iNOS,
COX-2 and other inflammatory mediators. Therefore, hyperglycemia-mediated surplus ROS
generation contributes significantly to liver injury in diabetes. The potential of ROS to induce
hepatocyte dysfunction and damage is supported by studies showing attenuation of liver injury
following ROS suppression (Aladaileh et al., 2019; Ranneh et al., 2019). Here, ROS, LPO and
NO were increased, and the antioxidant defenses were diminished significantly in the liver of
diabetic rats, demonstrating oxidative stress. Reduced cellular antioxidant defenses in the liver of
diabetic rats has been previously demonstrated (Mahmoud et al., 2012; Sahin et al., 2019). In
addition, TNF-α, IL-1β and IL-6, both serum levels and hepatic mRNA abundance, were
increased in diabetic rats. Diabetic rats showed upregulation of hepatic COX-2 and iNOS gene
expression which explained the increased NO levels. These findings demonstrate oxidative stress
and inflammation in the liver of diabetic rats. In accordance, serum TNF-α and IL-6, and hepatic
NF-κB expression were increased in diabetes induced by HFD and STZ in rats (Mahmoud et al.,
2
012; Sahin et al., 2019). Moreover, the circulating levels of TNF-α and IL-6 are known to
increase in T2DM patients (Pickup et al., 2000). Increased pro-inflammatory cytokines has been
associated with IR and impaired glucose tolerance. TNF-α suppresses insulin-stimulated
peripheral glucose uptake, provokes hepatic glucose production (Lang et al., 1992), impairs
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insulin action in muscles (Del Aguila et al., 1999) and increases lipolysis in adipocytes (Green et
al., 1994). IL-6 impairs insulin signaling by reducing insulin receptor substrate (IRS)-1 tyrosine
phosphorylation and protein kinase B/Akt phosphorylation in hepatocytes (Senn et al., 2002) and
induces glucose intolerance (Müller et al., 2002). IL-1β has also been associated with the loss of
β cell mass in T2DM (Dinarello et al., 2010) and directly induce IR in hepatocytes in vitro (Nov
et al., 2010). Therefore, attenuation of oxidative stress and inflammation can ameliorate
hyperglycemia and IR in T2DM. Treatment of the diabetic rats with different doses of RDE
significantly reduced hepatic ROS, LPO and NO and boosted antioxidant defenses. In addition,
RDE suppressed inflammation as evidenced by the decreased serum pro-inflammatory cytokines
and hepatic mRNA abundance of TNF-α, IL-1β, IL-6, iNOS and COX-2. These findings
demonstrate the potent antioxidant and anti-inflammatory efficacies of RDE. The improved
insulin sensitivity and suppression of liver injury and lipid infiltration could be directly attributed
to attenuation of oxidative stress and inflammation following RDE supplementation.
Previous studies have demonstrated the in vitro antioxidant activity of R. dentatus (Elzaawely
and Tawata, 2012; Humeera et al., 2013). For instance, the EtOAc fractions of R. dentatus
leaves and roots showed strong DPPH radical scavenging activity which was positively
correlated with the high content of phenolic compounds (Elzaawely and Tawata, 2012).
However, the studies investigated the antidiabetic and hepatoprotective efficacies of R.
dentatus are scarce. In paracetamol-induced mice, R. dentatus methanolic extract at doses of
250 and 500 mg/kg reduced serum ALT, AST and ALP and prevented histological alterations
(Saleem et al., 2014). Our findings present the first evidence that RDE ameliorated
hyperglycemia, IR, oxidative stress, inflammation and liver injury, and modulated the activity
of carbohydrate metabolizing enzymes in HFD/STZ-induced rats. These beneficial effects are
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directly related to the active constituents of RDE, including emodin glucoside, quercetin, and
kaempferol. Emodin and its glucosides have shown anti-inflammatory and antidiabetic effects
(
Lee et al., 2014; Xue et al., 2010). In HFD/STZ-induced mice, emodin improved glucose
tolerance and insulin sensitivity (Xue et al., 2010) and emodin-6-O-β-D-glucoside suppressed
high glucose-induced ROS generation and inflammation in endothelial cells in vitro (Lee et al.,
2014). The anti-diabetic effect of quercetin has been well-demonstrated in different studies
(reviewed in (Shi et al., 2019)). In HFD/STZ-induced rats (Gaballah et al., 2017) and type 2
diabetic db/db mice (Jeong et al., 2012), quercetin ameliorated hyperglycemia, IR and oxidative
stress. Other putative antidiabetic mechanisms of quercetin include enhancement of glucose
uptake, increase in insulin release and inhibition of α-glucosidase (reviewed in (Shi et al.,
2019)). Moreover, kaempferol reduced blood glucose, increased insulin and attenuated
oxidative stress in the liver of STZ-induced diabetic rats (Al-Numair et al., 2015). Recently,
kaempferol has been reported to improve glucose tolerance and insulin sensitivity in HFD/STZ-
induced diabetic mice and increase glucose uptake in adipocytes in vitro (Varshney et al.,
2019).
To further explore the mechanisms underlying the antidiabetic effects of RDE, we investigated
its effect on PPARγ gene expression in diabetic rats. In addition, we performed a molecular
docking study to investigate the binding affinity of the isolated compounds with PPARγ. RDE
increased hepatic PPARγ gene expression in diabetic rats. In silico analysis showed that all
isolated compounds possess a binding affinity toward PPARγ. The amino acids involved in the
polar interactions with all isolated phytochemicals are Glu259, Arg288, Glu291, Thr268,
Ser342 and Glu343, most of which are crucial residues for ligand interactions and PPARγ
activation (Álvarez-Almazán et al., 2017; Irudayaraj et al., 2016). Given that PPARγ agonists
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6

such as PIO increases insulin sensitivity, ameliorate hyperglycemia and dyslipidemia in T2DM,
prevent excessive lipid accumulation in peripheral tissues, and suppress pro-inflammatory
cytokines (Tontonoz and Spiegelman, 2008), the antidiabetic effect of RDE is mediated, at least
in part, via PPARγ activation. Besides these metabolic effects, activation of PPARγ can
suppress inflammation, boost antioxidant enzymes and protect the liver (Hwang et al., 2005;
Kersten et al., 2000; Okuno et al., 2010; Remels et al., 2009). PPARγ suppresses ROS
production mediated via NADPH (Hwang et al., 2005), stimulates the gene expression of
antioxidant enzymes (Okuno et al., 2010), diminish p65 nuclear translocation and NF-κB-
dependent inflammatory genes transcription (Kersten et al., 2000; Remels et al., 2009).
5. Conclusions
Overall, 8 compounds including a new one, were detected in the EtOAc fraction of R. dentatus.
The order of antioxidant activity of the isolated compounds estimated by computational
analysis is dependent on the BDE values, nature and position of OH substituents. This
arrangement is in a good agreement with the experimentally determined IC50 in the DPPH
radical scavenging assay. The antioxidant mechanism of action of these phenolics is oriented by
SPLET in the aqueous phase and by HAT in gas phase. However, the total energy demand of
SPLET and SET-PT mechanisms is the same as that for the HAT pathway. Thus, we can rank
the antioxidant efficacies of the isolated phenolics based on their BDE values. In addition, the
current findings show for the first time that R. dentatus exhibited a potent antidiabetic activity
in T2DM. Treatment of the diabetic rats with RDE ameliorated hyperglycemia, improved
glucose tolerance and insulin sensitivity, increased liver glycogen and alleviated the activity of
carbohydrate metabolizing enzymes. RDE suppressed excess ROS generation, oxidative stress
and inflammation, prevented liver injury and enhanced antioxidant defenses in liver of diabetic
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rats. Increased expression of hepatic PPARγ mediated, at least in part, the beneficial effects of
RDE in diabetic rats. In silico study showed the binding affinity of R. dentatus phenolics toward
PPARγ. Therefore, R. dentatus represent a promising lead for the management of T2DM,
pending further investigations to explore the underlying mechanisms.
Supplementary Materials: Supplementary Figures 1-18: Spectral data of the isolated
compounds, and Supplementary Figure 19: E and Z isomers of resveratrol.
Author Contributions: A.M.M. conceived the study, designed and performed animal
experiments, carried out the biochemical, molecular and histologic studies, analyzed the data
and prepared the figures. A.M.M. and E.M.K wrote the manuscript. R.H.E.; E.M.K. and A.A.E.
carried out the phytochemical analysis. E.M.K. and A.L. performed the DFT studies. E.M.K.
performed the molecular docking studies. M.B-J. provided resources, participated in the assays
and acquired funding. A.M.M. and S.A.A. revised the manuscript. All authors read and
approved the manuscript for publication.
Acknowledgments
The authors thank the Deanship of Scientific Research at Princess Nourah bint Abdulrahman
University for supporting this research through the Fast-track Research Funding Program. This
work has DGI Project no. CTQ2015-63997-C2, a generous allocation of computing time at the
Centro de Computación Científica of the UAM is also acknowledged.
Conflicts of Interest
The authors declare no conflict of interest.
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