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Exploring the therapeutic potential of Terminalia ferdinandiana (Kakadu
Plum) in targeting obesity-induced Type 2 diabetes and chronic
inflammation: An in silico and experimental study
Md. Niaj Morshed
a,b,1
, Muhammad Awais
a,1
, Reshmi Akter
a
, Juha Park
b
, Li Ling
c
,
Byoung Man Kong
d
, Deok Chun Yang
a,d
, Dong Uk Yang
e
, Se Chan Kang
a
, Seok-Kyu Jung
f,
*
a
Graduate School of Biotechnology, College of Life Sciences, Kyung Hee University, Yongin-si 17104, Gyeonggi-do, Korea
b
Department of Biopharmaceutical Biotechnology, College of Life Science, Kyung Hee University, Yongin-si 17104, Gyeonggi-do, Republic of Korea
c
Jilin Ginseng Academy, Changchun University of Chinese Medicine, Changchun, 130117, China
d
Department of Oriental Medicinal Biotechnology, College of Life Sciences, Kyung Hee University, Yongin-si 17104, Gyeonggi-do, Korea
e
Hanbangbio Inc., Yongin-si 17104, Gyeonggi-do, Republic of Korea
f
Department of Horticulture, Kongju National University, Yesan 32439, Republic of Korea
ARTICLE INFO
Article History:
Received 8 January 2024
Revised 16 May 2024
Accepted 28 May 2024
Available online xxx
Edited by Dr S.C. Pendota
ABSTRACT
This study investigates the intricate relationship between excessive adipocyte production, leading to Type 2
Diabetes (T2D) and chronic inflammation characterized by elevated reactive oxygen species (ROS) and nitric
oxide (NO) levels. Leveraging in silico studies, computational analyses unravel the interaction between major
compounds of Kakadu Plum (KKD-NT) including ellagic acid (EA), gallic acid (GA), daidzein (DD), and ascorbic
acid (AA) with pivotal obesity-related biomolecules (PPARg, C/EBPa,b-catenin). Notably, EA and DD display
a superior binding affinity with active residues compared to GA, AA, and the control drug Resveratrol (RSV).
Experimental validation showcases the capacity of KKD-NT to diminish intracellular ROS in hypertrophied
adipocytes by amplifying antioxidant defense enzymes (SOD, Catalase, GPx). KKD-NT further mitigates obe-
sity-induced inflammation by reducing lipid accumulation, NO production, influencing adipogenesis factors
(PPARg, CEBPɑ, and FAS), Wnt signaling (b-catenin), and pro-inflammatory mediators (TNF-ɑ, IL-6, leptin) in
3T3-L1 cells. In vitro evaluations attest to anti-diabetic properties of KKD-NT as evidenced by enhancing glu-
cose uptake and inhibiting a-glucosidase activity. The up-regulation of GLUT4 and adiponectin mRNA
expression suggests potential benefits for obesity and diabetes. While acknowledging the need for further
research to assess safety and in vivo effectiveness, this study suggests that KKD-NT and its bioactive compo-
nents hold promise as innovative therapeutic interventions for obesity and related metabolic diseases, open-
ing avenues for future exploration in clinical applications.
© 2024 SAAB. Published by Elsevier B.V. All rights are reserved, including those for text and data mining, AI
training, and similar technologies.
Keywords:
Obesity
Antioxidant
Inflammation
Diabetes
Kakadu plum
1. Introduction
Obesity is a prominent global health problem that is becoming an
epidemic level around the world. Obesity has already been recog-
nized as the second most frequent cause of preventable death over
the past two decades (Awais et al., 2023). The World Health Organi-
zation (WHO) estimates that 650 million individuals were obese in
2016 and the frequency nearly tripled between 1975 and 2016. If the
current pace continues, it is estimated that over a billion people will
be obese by 2025 (Lobstein et al., 2022). Additionally, a comparative
wave is anticipated that approximately 417.3 million individuals are
expected to have either detected or undetected type 2 diabetes (T2D)
by 2030 (Karuranga et al., 2019). T2D also known as non-insulin-
dependent diabetes mellitus, is rooted in the dysregulation of glucose
and lipid metabolism (Milardi et al., 2021). This metabolic disorder is
characterized by insulin resistance (IR), a condition arising from fac-
tors such as obesity, genetic predisposition, suboptimal dietary prac-
tices, environmental influences, and eventual metabolic breakdown
at the cellular level (Zhang et al., 2020b). Obesity contributes to IR
and compromised b-cell functionality. These factors collectively fail
to adequately counterbalance the diminished insulin sensitivity
observed in liver tissue, adipose tissue, and skeletal muscle, thereby
culminating in the onset of T2D (Czech, 2017). Furthermore, the
growth of obesity and IR is significantly influenced by the dysfunction
of adipokines, known as bioactive substances released by adipocytes
(Lee et al., 2010). Among the adipokines, adiponectin is down-
* Corresponding author.
E-mail address: jungsk@kongju.ac.kr (S.-K. Jung).
1
These authors equally contributed to this work.
https://doi.org/10.1016/j.sajb.2024.05.056
0254-6299/© 2024 SAAB. Published by Elsevier B.V. All rights are reserved, including those for text and data mining, AI training, and similar technologies.
South African Journal of Botany 171 (2024) 3244
Contents lists available at ScienceDirect
South African Journal of Botany
journal homepage: www.elsevier.com/locate/sajb
regulated in the obese and diabetic state. Conversely, in adipose tis-
sue of the obese and IR states, inflammatory markers such as tumor
necrosis factor (TNF-a), and interleukin (IL-6) are increased (Yu et al.,
2017). Additionally, TNF-aand IL-6 inhibit insulin signaling through
the suppression of insulin receptor substrate-1 (IRS-1) and glucose
transporter-4 (GLUT-4) (Hotamisligil et al., 1993). Furthermore, TNF-
apromotes insulin resistance in adipose tissue by controlling the
expression of genes linked to adipogenesis, lipolysis, and adipokines
(Li et al., 2017). Despite the high incidence of obesity and T2D, the
number of FDA-approved therapies and the efficacy of medication
therapy for these diseases remain fairly low with undesirable side
effects (Dragano et al., 2020). Therefore, it is much desired to discover
novel drugs that promote muscle or adipose cell uptake of glucose
but do not cause weight gain or other side effects, in contrast to T2D
or insulin (Alonso-Castro et al., 2008).
However, plant-based Dietary antioxidants may offer a cost-effec-
tive way of treating oxidative damage and chronic inflammation to
improve obesity-related health (Xiao et al., 2022). Additionally,
herbal supplements and products have drawn the interest of natural
product researchers as a secure therapeutic approach for the man-
agement of several ailments including obesity, diabetes, inflamma-
tion, oxidative stress, etc.(Welz et al., 2018). It has been reported that
organic dietary phytochemicals, such as phytosterols, terpenoids,
organosulfur, and polyphenols, have demonstrated potential as anti-
obesity, anti-diabetic, and anti-inflammatory agents (Goktas et al.,
2020). Moreover, Garcia-Diaz et al. proposed that vitamin C (ascorbic
acid) may have the following positive effects on mechanisms relating
to obesity: (a) modulating adipocyte lipolysis; (b) controlling the
release of glucocorticoids from adrenal glands; (c) inhibiting glucose
metabolism and leptin secretion on isolated adipocytes; (d) improv-
ing hyperglycemia and lowering glycosylation in obese-diabetic
models, and (e) lowering the inflammatory response (Garcia-Diaz et
al., 2014).
The Kakadu plum (KKD), formerly known as Terminalia ferdinandi-
ana, has historically been the most widespread fruit among Austra-
lian Aboriginal tribes. The Aboriginal people of the Northern
Territory (NT) use the fruit of KKD as a significant commercial prod-
uct. Recently we reported that the fruit of KKD-NT is rich in ascorbic
acid, daidzin, a variety of phytochemicals such as phenolic com-
pounds and flavonoids, including proanthocyanidins, ellagic acid
(EA) and gallic acid, gallo-tannins, ellagitannins, and phenolic acids
after performing HPLC (Akter et al., 2022a). Additionally, KKD-NT has
shown pharmacological activity against cancer (Tan et al., 2011a),
rheumatoid arthritis (Sirdaarta et al., 2015), inflammation (Tan et al.,
2011b), bacteria (Wright et al., 2016), and other conditions. It can
serve as a potential antioxidant and thus can show protective effects
on the liver from alcohol-induced cytotoxicity (Akter et al., 2022a).
However, the activity of KKD-NT against obesity, obesity-induced
diabetes, and inflammation has not been reported yet. The goal of
this study was to establish an in silico and in vitro model that would
investigate the ability of the KKD-NT against obesity, diabetes, and
inflammation.
2. Materials and methods
2.1. Reagents and chemicals
3T3-L1, Preadipocyte cell lines from an embryo of the mouse were
purchased from ATCC. DMEM (Dulbecco’s Modified Eagle’s Medium),
FBS (Fetal Bovine Serum), and BCS (Bovine calf serum) were pur-
chased from Welgene, South Korea. Penicillinstreptomycin was
obtained from GeneDEPOT, human recombinant insulin, 3-isobutyl-
1-methylxanthine, and dexamethasone was obtained from Wako,
Japan. 2-NBDG 2-[N-(7nitrobenz-2-oxa-1, 3-diazol-4-yl) amino]2-
depxy-D-glucose, and P-NPG (para-Nitrophenyl Glucopyranoside)
were purchased from Thermo Fisher Scientific (USA). References
compound Ellagic acid (EA), Gallic acid (GA), Daidzin (DD), Ascorbic
acid (AA), Resveratrol (RSV), and a-glucosidase, acarbose were pur-
chased from Sigma, St. Louis, MO, USA.
2.2. Collection and extraction process of plant material
The dried fruit specimens of T. ferdinandiana from the Australian
Northern Territory (NT) were provided by KAKADU LIFE NT. These
fruits were crushed in a blender to perform the reflux method of
extraction. In brief, 2 g of ground material has been boiled in 40 mL
water for 2 h at 95 °Celsius, (3 repetitions). In each processing period,
the Whatman filter paper # 41 was utilized to filter the mixture, and
then the cooled filtrate was taken out. The solvent was then evapo-
rated using a rotary evaporator. Finally, extracts were obtained and
weighed to calculate the yield of extraction from the crude fruit.
2.3. In silico analysis
2.3.1. Physiochemical and ADMET properties analysis
The selected compounds have been subjected to in-silico physico-
chemical evaluations. The canonical SMILES of the metabolites were
obtained from the PubChem Database (www.pubchem.ncbi.nlm.nih.
gov) and used to predict ADMET properties using the normal param-
eters. The SwissADME (http://www.swissadme.ch/index.php)(Daina
et al., 2017), ADMETlab2.0 (https://admetmesh.scbdd.com/)(Xiong et
al., 2021), pkCSM (https://biosig.lab.uq.edu.au/pkcsm/)(Pires et al.,
2015) were used to investigate drug probability, absorption, distribu-
tion, metabolism, excretion that stated the compounds pharmacoki-
netics, physical and chemical parameters, and drug-likeliness
properties. Furthermore, the Protox-II webserver (https://tox-new.
charite.de/protox_II/)(Banerjee et al., 2018) was used to predict the
toxicity of oral consumption. This toxicity prediction serves to assess
the immunotoxicity, hepatotoxicity, carcinogenicity, and toxicologi-
cal pathways (Ghosh and Gemma, 2014).
2.3.2. Preparation of proteins and ligands
The crystal structure of PPAR-g(PDB: 2PRG) (Srinivasa et al.,
2023), CEBP-a(PDB: 1NWQ) (Mladenova et al., 2021), and b-catenin
(PDB:4DJS) (Gill et al., 2018), have been retrieved from the RCSB
(www.rcsb.org, accessed on 11 October 2023) protein data bank
(https://www.rcsb.org/)(Pallesen et al., 2017). Cofactors, water, and
metal ions from the complex structure were removed to prepare pro-
teins. Gasteiger charges for the protein were calculated after the non-
polar hydrogen atoms were merged and polar hydrogen atoms were
added (Opo et al., 2021). Utilizing AutoDock Tools, the non-polar
hydrogens were merged, aromatic carbons were found, and the ’tor-
sion tree’of the molecules was configured. These results were
obtained in PDBQT format for further screening. Additionally, the
PubChem Database was used to retrieve the 3D structures of the
active ingredients. (https://pubchem.ncbi.nlm.nih.gov/)(Zhang et al.,
2020a). The download format was an SDF file.
2.3.3. Identification of binding sites and generation of grid boxes
Analysis of comparable pockets from known protein-ligand inter-
actions can identify binding sites. The identified and unidentified
active sites of protein structures were obtained using PDB (https://
www.rcsb.org/) and CASTp (http://sts.bioe.uic.edu/castp/), respec-
tively, and the binding site of proteins has been analyzed through the
use of BIOVIA Discovery Studio Visualizer v19.1 (BIOVIA). The recep-
tor grid was built via molecular docking with the PyRx online screen-
ing tool, utilizing the binding sites obtained from the integrated
composition.
2.3.4. Molecular docking simulation
Using the PyRx tool, a molecular docking simulation has been car-
ried out to determine the best-fit candidates against target proteins.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
33
PyRx is a freely available computational screening program including
both AutoDock Vina and AutoDock 4 as a docking wizard that can
analyze a sizable dataset against a particular biological targeted mac-
romolecule. The AutoDock Vina Wizard was used through PyRx as
the default parameter to simulate molecular dockingOut of all the
compounds, the top compounds had the greatest binding affinities
(kcal/mol) to the required protein. Ultimately, the default configura-
tion was used to construct the receptor grids.
2.4. Cell viability assay
To cultivate the cells, 3T3-L1 fibroblast preadipocytes were cul-
tured in a complete medium (CM) that included DMEM with 10 %
BCS and 1 % P/S and were then placed in an incubator with 5 % CO2.
Afterward, 1 £10
4
cells/well were seeded into 96-well plates and left
to incubate for a full 24 h. Following the incubation times, cells were
subjected to a serum-free media that contained varying KKD-NT
doses (31.25500 mg/mL). After incubating the cells for 24 h, 20 ml
of MTT solution was added to each well, and the cells were kept in
the incubator for 3 h. Finally, 100 mL dimethyl sulfoxide (DMSO)
solution was added to the medium after three hours, and the absor-
bance at 570 nm was measured using an ELISA reader (Synergy-2,
Bio-Tek Instruments, Inc., 178 Vinooski, VT, USA) (Akter et al.,
2022b).
2.5. Differentiation induction in 3T3-L1 cells
The 3T3-L1 pre-adipocyte cell line was cultivated in a 75 cm2 cul-
ture flask containing basal media and incubated at 37 °C with 5 %
CO2 until 80 % confluence was reached. The differentiation assay for
cell culture was conducted as previously reported by Manzanarez et
al. (Manzanarez-Quin et al., 2023). After seeding cells into a 12-well
plate at a density of 1 £10
4
cells/well with basal media, cells were
incubated for 48 h, or until 70 % confluence was reached (Day 0).
After replacing the media with basal medium supplemented with
0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 mg/mL dexametha-
sone (DX), and 10 mg/mL insulin (differentiation medium I, MDI),
cells were then cultured for two days. After two days of incubation,
the medium was changed to a basal medium containing 10 mg/mL
insulin on the second and fourth days. In the end, the medium was
changed to the fundamental medium treated with KKD-NT, EA, GA,
DD, AA, and RSV at varying concentrations on the sixth day.
2.6. Lipid accumulation and triglyceride measurement assay
To observe the lipid droplets that demonstrate adipogenesis in
3T3-L1, an oil-red-O experiment was performed after differentiation
(Hallenborg et al., 2016). Then, the matured adipocytes were rinsed
with 1£PBS, followed by the fixation using 10 % formalin for 1 h or a
few days. After the fixation, cells were soaked in isopropanol (60 %)
and allowed to air dry. Matured adipocytes were then stained using
ORO solution for 15 min and washed with distilled water to remove
the excess stain. Phenotypic alterations in fully mature cells were
seen using an inverted light microscope (Nikon Instruments, Melville,
NJ, USA). In the end, the mature adipocytes were exposed to 100 %
isopropanol to quantify the triglyceride content, or lipid accumula-
tion, of the cells. Then the reactions were incubated at room tempera-
ture for 10 min, and the absorbance was read at 630 nm (Simu et al.,
2019).
2.7. Measurement of ROS level on 3T3-L1 cells
The H2DCFDA (20,7
0-dichlorofluorescein diacetate) fluorogenic
probe is often used to assess a certain level of reactive oxygen species
(ROS) production. The cellular ROS formation was evaluated by wash-
ing differentiated cell lines with PBS at room temperature. Following
cell washing, a nonfluorescent probe of H2DCFDA (10 mM) (Sigma,
St. Louis, MO, USA) was applied for 30 min and incubated at 37 C in
the dark. Finally, the fluorescence emission intensity was measured
between 485 and 495 nm using a Spectra Fluor multi-well fluores-
cence reader (Tecan, Maninder, Austria) by Reshmi et al. (Akter et al.,
2022b) with slight modifications.
2.8. Nitric oxide (NO) determination
Estimation of NO production in 3T3-L1 cells was conducted by
employing Greiss reagent following the previously described proto-
col (Lee et al., 2017) after slight modification. After differentiation,
each cell-free culture supernatant (100 mL) was mixed with an equal
Griess reagent (1 % naphthyl ethylenediamine dihydrochloride and
1 % sulfanilamide in 30 % acetic acid) for 15 min. The mixtures were
measured at 562 nm absorbance, using an EZRead 400 Microplate
reader (Biochrom, Cambridge, United Kingdom). The amount of inhi-
bition obtained using the following equation was used to display the
results: [{(Control
abs
-Sample
abs
)/Control
abs
} *100].
2.9. a-glucosidase inhibitory activity
A previously described approach (Li et al., 2023) was slightly mod-
ified to measure a-Glucosidase inhibitory activity. In short, 1 U/mL of
a-GLU working solution was created by adding a-GLU solid powder
to 0.1 M PBS. In a 96-well plate, 50 mL of samples or standards and
100 mLofa-GLU working solution were added, and the plate was
then incubated at 37 C for 10 min to initiate the reaction. Subse-
quently, 100 mL of a 5 mM p-NPG solution was incorporated and
incubated at 37 °C for 20 min. At 405 nm, the absorbance value was
measured after the reaction was finally stopped by adding 200 mLof
0.5 M sodium carbonate solution. Acarbose (1 mM) was used as a pos-
itive control. The inhibition rate (%) was calculated using the follow-
ing formula: [{(Control
abs
-Sample
abs
)/Control
abs
} *100].
2.10. Measurement of glucose uptake levels on 3T3-L1 cells
After some adjustments, the previously published methodology
(Lee et al., 2021) was used to measure the amount of glucose uptake
using a fluorescent glucose derivative, 2-NBDG. 10 % FBS-containing
glucose-free media was used to seed 3T3-L1 adipocytes in 96-well
plates. Following a 24-hour incubation period, KKD-NT extract in a
dose-dependent manner (31, and 250 mg/ml) its phytochemicals
(20 mM), and insulin (100 nM) (serving as a positive control) were
administered to the cells either with or without 50 mM 2NBDG. After
incubation for one hour, the cells were washed with PBS, and the cell
lysates were treated for ten minutes with 70 mL of 1 % Triton X-100
in PBS and 0.1 M potassium phosphate. The 2-NBDG fluorescence sig-
nal was measured at excitation/emission wavelengths of 460/535 nm
using a plate reader (Tecan Spark, Tecan, Switzerland) to quantify the
fluorescence.
2.11. RNA isolation and qRT-PCR
After the differentiation and treatment of 3T3-L1 cells with differ-
ent doses of KKD-NT, EA, GA, DD, AA, and RSV, qiazol reagent (Takara
Bio, China) was used to extract total RNA by manufacturer’s recom-
mendations. 2.5 mg of RNA was used in a 20 mL reaction volume to
synthesize the cDNA using the RevertAid First Strand cDNA Synthesis
Kit (TFS, USA). The expression levels of genes involved with adipo-
genesis were measured using quantitative real-time PCR on an
Applied Biosystems Inc. (Zebisch et al.) 7500 Real-Time PCR System
with a 20 mL SYBR Green PCR master mix (Invitrogen, Carlsbad, CA,
USA). The relative expression of gene-specific outputs was deter-
mined using the 2(-DDCt) technique, which was subsequently nor-
malized to matching b-actin levels. All findings were confirmed by
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
34
repeating the trials three times. The primers used are listed in sup-
porting information Table S1.
3. Results
3.1. Physiochemical and ADMET properties analysis of lead compounds
We observed that all compounds possessed several favorable
physiochemical features as per Lipinski rules of 5 (LO5). LO5 stated
that an orally administered drug must have hydrogen bond acceptor
(HBA) 10 & hydrogen bond donor (HBD) 5, molecular weight <
500 Daltons, polar surface area (PSA) <140 A
, rotatable bonds <10,
lipophilicity Log p<5(Lipinski et al., 2012). In the ADMET properties
analysis, we found all the reference compounds did not violate the
LO5 and maintained the standard ranges of all parameters. Moreover,
these compounds are depicted as non-toxic or less toxic in several
toxicity parameters (Supporting information Table S2). Along with
this, some major parameters of ADMET properties are shown in
(Fig. 1).
3.2. Molecular docking analysis
Molecular docking was used to investigate the molecular
interactions of the obesity-related targets PPARg,CEBPa,and
b-catenin with EA, GA, DD, and AA. Resveratrol was used as a
control drug. The formation of hydrogen bonds and the binding
energy to the key active residues and ligands confirmed the inter-
action results. Supporting information Table S3 featured informa-
tion on the ligands used on the selected targets, binding affinity,
active site residues, grid box size, and center characteristics. The
molecular docking demonstrated that EA and DD showed a strong
binding affinity with PPARg(7.9 k/mol, and 8.4k/mol), CEBPa
(7.2 k/mol, and 7.1 k/mol), and b-catenin (6.7 k/mol, and
6.9) compared to positive control RSV. AA displayed medium
binding energy with several hydrogen bonds. To inhibit PPARg,
EA formed a hydrogen bond with GLU343 residue, GA established
3 hydrogen bonds with GLN286, SER289, and HIS323 residues,
and AA assembled hydrogen bonds with SER289, and HIS323 resi-
dues, where positive control RSV did not show any hydrogen
bond (Fig. 2).
Moreover, the interaction of CEBPɑwith EA, and DD formed a
hydrogen bond at ARG288, ARG289, and GLU309 active sites respec-
tively. GA and AA formed hydrogen bonds with ASP320, ARG325
ARG388, ARG398, and TYR285 residues, where RSV showed no
hydrogen bond interaction with CABPɑreceptor (Fig. 3).
Furthermore, the interaction among the mentioned compounds
and b-catenin exposed two hydrogen bonds with EA at HIS219, and
THR257 residues, GA at GLN302, LEU263, ILE303 residues, DD at
LYS270, GLN302, and AA at HIS223, HIS224, SER179, and ASN220 res-
idues (Fig. 4).
Fig. 1. ADMET properties of (A) Ellagic Acid, (B) Gallic acid, and (C) Daidzin (D) Ascorbic acid, and (E) resveratrol. Abbreviations: MW: Molecular weight; nRig: number of rigid
bonds; fChar: formal charge; nHet: number of heteroatoms; MaxRing: number of atoms in the biggest ring; nRing number of rings; nRot: number of rotatable bonds; TPSA: topolog-
ical polar surface area; nHD: number of hydrogen bond donors; nHA: number of hydrogen bond acceptor; LogD: logP at physiological pH 7.4; logS: log of the aqueous solubility; and
LogP: log of the octanol/water partition coefficient.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
35
3.3. Effect of KKD-NT on cell viability of 3T3-L1 cells
In the initial investigation, 3T3-L1 preadipocyte cells were culti-
vated for 24 h with different concentrations of KKD-NT to study its
effects on the differentiation and proliferation of the cells. To get
more than 85 % cell viability, the cells were administered with 31.25,
62.50, 125, 250, and 500 g/mL of KKD-NT, as shown in Fig. 5. Based
on the results, the selected concentration did not disrupt the usual
structure of cells after 48 h indicating non-toxic effects. By the find-
ings from our previous investigation, high-performance liquid chro-
matography (HPLC) analysis revealed that our samples exhibit a
substantial concentration of vitamin C (ascorbic acid) (Akter et al.,
2021). Vitamin C is recognized for its capacity to safeguard cells from
oxidative damage through the reduction of reactive oxygen species
(ROS). It is noteworthy, however, that elevated levels of ascorbic acid
have been associated with cytotoxic effects, as they can lead to the
excessive generation of ROS, while concurrently disrupting the
energy homeostasis in cancerous cells.
In light of these observations, we have made the strategic decision
to limit the concentrations employed in all subsequent experiments
within the range of 31.25250 mg/mL. This carefully chosen concen-
tration range aims to strike a balance, ensuring the potential benefits
of vitamin C are harnessed for cellular protection, while mitigating
the adverse effects associated with higher concentrations, particu-
larly in the context of ROS generation and interference with energy
homeostasis in cancer cells. This approach will enable a more
nuanced and targeted exploration of the therapeutic potential of vita-
min C in our ongoing investigations.
3.4. KKD-NT inhibits lipid accumulation in 3T3-L1 cells
Pre-adipocytes were developed to mature adipocytes in MDI
media regardless of the samples to form lipid droplets. The micro-
scopic analysis demonstrated that KKD-NT treatment suppressed
intracellular triglyceride formation compared to the MDI treatment.
The active components of KKD-NT, such as EA, GA, DD, and AA, have
similarly significantly inhibited the formation of intracellular lipids
(Fig. 6). According to the quantitative analysis, the lipid level in the
MDI-treated group was roughly 250 %. However, KKD-NT doses of
31.25 mg/mL and 250 mg/mL reduced intracellular lipid content by
220 % and 150 %, respectively, as compared with MDI-treated 3T3-L1
cells. In addition, treatment with EA, GA, DD, and AA decreased the
lipid contents by 170 %, 200 %, 190 %, and 210 % respectively. Further-
more, RSV which is generally known to have anti-obesity properties,
was implemented as a control agent in our investigation. Moreover,
RSV has decreased lipid by 140 %.
3.5. Suppressive effect of KKD-NT on ROS generation in 3T3-L1 pre-
adipocyte cells
To investigate the inhibitory impact of intracellular ROS produc-
tion in 3T3-L1 adipocyte cells, ROS level was measured using a
DCFDA probe during adipogenesis. In comparison to cells without
treatment, intracellular ROS levels in MDI-differentiated cells were
much higher. Nonetheless, when DMI-induced cells were adminis-
tered with samples (KKD-NT, EA, GA, DD, AA, and RSV), the ROS pro-
duction was reduced in a dose-dependent manner (Fig. 7A). As a
result of their antioxidant characteristics, KKD-NT can reduce ROS
production in 3T3-L1 cells. It is exposed that, MDI treated cells pro-
duced 280 % ROS, whereas, KKD-NT and its compounds EA, GA, DD,
and AA decreased ROS generation by 150 %, 190 %, 200 %, 190 %, and
190 % respectively. RSV was used as a control drug that reduced ROS
production by 170 %.
3.6. Inhibitory effect of KKD-NT on NO generation in 3T3-L1 pre-
adipocyte cells
Previous research revealed that obesity-related IR can be caused
by an excess of the inflammatory cytokine nitric oxide (NO) (Kata-
shima et al., 2017). The study employed MDI-induced 3T3-L1 cells to
Fig. 2. 3D and 2D interactions of PPARg(2PRG) with (A)Ellagic Acid, (B) Gallic Acid, (C) Daidzin, (D) Ascorbic acid, and (E) Resveratrol.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
36
examine the impact of crude extract and isolated components on the
production of nitric oxide. As shown in (Fig. 7B), the bar chart demon-
strated that KKD-NT slightly inhibited the production of NO in MDI-
induced 3T3-L1 cells. EA, GA, DD, and AA also displayed an inhibi-
tory activity of NO generation in a dose-dependent manner. The
positive control, RSV, demonstrated strong anti-inflammatory
activity that was noticeably greater than that of all the samples.
Based on these findings, it was possible for the separated chemi-
cals to moderately suppress the generation of nitric oxide. Conse-
quently, it is conceivable that the minimal inhibitory effect of
these KKD-NT and its isolates on NO generation will help to
increase insulin sensitivity.
3.7. a-glucosidase inhibition by KKD-NT
a-glucosecosidase inhibition experiment was used to evaluate the
anti-diabetic potential of KKD-NT. To the standard positive control
acarbose (1 mM), different concentrations of KKD-NT (31.25, and
250 mg/ml), and its phytochemicals EA, GA, DD, and AA (20 mM)
were used. According to our findings, there is a positive relationship
between the inhibition levels of a-glucosidase and KKD-NT concen-
trations. The extract inhibited a-glucosidase activity by 14.88 % and
44.12 % at the concentrations of 31.25 and 250 mg/ml. Moreover, the
inhibitory activity of EA and DD were 64.37 % and 59.32 % respec-
tively which were stronger than the inhibitory activity of acarbose
(51 %) (Fig. 8A).
3.8. 2-NBDG uptake in 3T3-L1 cells
The effect of KKD-NT on the determination of glucose uptake of
3T3-L1 adipocyte cells has been shown in Fig. 8B. As the data shows
the untreated adipocytes have the lowest rate of glucose uptake.
Additionally, Insulin-treated cells absorbed substantially more glu-
cose than untreated cells. On the other hand, the cells treated with
KKD-NT extract with different concentrations (31.25, 250 mg/ml)
were reported to have gradually increased the glucose uptake than
the control. KKD-NT extract stimulated the 2-NBDG uptake into adi-
pocytes in a concentration-dependent manner. Furthermore, there
was a higher uptake of 2-NBDG at EA and DD compared to the con-
trol.
3.9. Effects of KKD-NT on adipogenic and pro-inflammatory gene
transcription in 3T3-L1 cells
Following that, qRT-PCR experiments were performed to find out
that KKD-NT affects the expression of significant transcription ele-
ments for adipogenesis. Excessive ROS production causes oxidative
stress in mature adipocytes by inhibiting antioxidant enzymes such
as SOD, CAT, and GPx (Taherkhani et al., 2021). However, KKD-NT
and its major constituents inhibited the production of ROS by
increasing the expression of these antioxidant enzymes (Fig. 9A). In
addition, Key adipogenic transcription factors such as PPARg,C/
EBPa, and b-catenin and lipogenic factor FAS, adipokines including
leptin and adiponectin are uncontrolled in MDI-induced mature
Fig. 3. 3D and 2D interactions of CEBPɑ(6DC0) with (A) Ellagic Acid, (B) Gallic Acid, (C) Daidzin, (D) Ascorbic acid, and (E) Resveratrol.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
37
Fig. 4. 3D and 2D interactions of b-Catenin (4DJS) alpha (6DC0) with (A) Ellagic Acid, (B) Gallic Acid, (C) Daidzin, (D) Ascorbic acid, and (E) Resveratrol.
Fig. 5. (A) Cell viability of treatment with KKD-NT on 3T3-L1 cell line for 24 h (B) Trypan blue cell viability images before and after treatment with KKD-NT on 3T3-L1 cells. Each set
of data represents the mean of the triplicate experiment §standard deviation. A significant difference between the groups was calculated using a two-tailed Student’st-test. ns rep-
resents the non-significant difference in cell viability of the sample compared with a non-treated control group.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
38
adipocytes. The insulin-regulated glucose transporter GLUT4 is
down-regulated after adipocyte differentiation. The mRNA expres-
sion of PPARg, C/EBPa, FAS,(Fig. 9B), and leptin was shown to be lower
after treatment with KKD-NT compared to the MDI-induced group.
Furthermore, EA, GA, DD, AA, and RSV treatment significantly
reduced the adipogenic markers. Moreover, GLUT4, adiponectin, and
b-catenin (Fig. 9C) were up-regulated after the treatments of KKD-NT
and its phytochemicals. On the other hand, the development of adi-
pose tissue in obese people causes a rise in pro-inflammatory cyto-
kines as well as a reduction in anti-inflammatory markers, which
results in regional and overall inflammation as well as issues with
glucose homeostasis (Akter et al., 2023a). To investigate the activity
Fig. 6. Inhibitory effect of the lipid accumulation for KKD-NT and its major phytochemicals on MDI-induced 3T3-L1 adipocytes. (A) Fat droplets were measured by oil red O staining
and observed using a microscope (at £20). (B) The absorbance of lipid accumulation, which was oil red O dye, was dissolved in isopropyl alcohol (520 nm). The data are mean values
of three experiments §SEM; ### <0.001 compared with control, * p<0.05, ** p<0.01, *** p<0.001 compared with the MDI.
Fig. 7. (A) Inhibition of reactive oxygen species (ROS) generation by KKD-NT and its major phytochemicals in MDI-induced 3T3-L1 adipocytes was determined by the DCFDA
method. KKD-NT treatment reduced intracellular ROS production dose-dependently. EA, GA, DD, AA, and RSV treatment decreased ROS levels significantly. (B) Nitric Oxide Inhibi-
tion Activity of KKD-NT, EA, GA, DD, AA, and RSV in MDI-induced 3T3L1 adipocytes was evaluated by using a Griess reagent. The intracellular NO production was reduced in a dose-
dependent manner by the treatment with these samples. Data are expressed as a percentage of control. ### p<0.001 MDI vs. control, while ** p<0.01 and *** p<0.001 treatment
vs. MDI.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
39
of KKD-NT on obesity-mediated inflammation, we observed that,
KKD-NT can down-regulate the mRNA levels of TNF-a, and IL-6. Our
findings showed that treating differentiated adipocytes with KKD-NT
considerably lowered the levels of these cytokines compared to MDI-
treated groups. EA, GA, DD, and AA have also significantly reduced
TNF-
ɑ
and IL-6 mRNA levels (Fig. 9C). A plethora of studies, spanning
from experiments using cell cultures to clinical trials on humans, has
provided solid evidence confirming the unique ability of RSV to con-
trol inflammation. This ability is especially important because inflam-
mation is involved in both metabolic syndrome and a variety of
chronic diseases. RSV was utilized as a favorable control in our inves-
tigation.
4. Discussion
A growing number of metabolic diseases associated with aging
pose a significant threat to the standard lifestyle of elderly individu-
als. Moreover, the prevalence of obesity, T2D, non-alcoholic fatty liver
disease, gout, and osteoporosis among young individuals emphasizes
the importance of addressing these global health issues (Clarke et al.,
2023). Obesity is induced by nutritional imbalances; that is, a high
consumption of calories combined with a lack of energy expenditure
can lead to obesity. Obesity can also lead to elevated T2D, blood pres-
sure, cancer, fatty liver disease, and cardiovascular disease (Pu et al.,
2022). However, one of the processes investigated in this study is
that excessive adipocyte production leads to T2D and chronic inflam-
mation, and increases the ROS and NO levels in cells. We attempted
to explore viable therapeutics that modulate obesity, diabetes, and
chronic inflammation during adipogenesis, as well as antioxidant lev-
els, using natural sources and bioactive components through in-silico
and in-vitro experiments. As a result, our investigation demonstrated
that KKD-NT significantly reduced obesity in MDI-induced obese cells
by decreasing fat formation and cytokines, enhancing antioxidant
activity by promoting antioxidant enzymes, and inhibiting the forma-
tion of ROS and NO. Additionally, KKD-NT gradually increased a-glu-
cosidase inhibition, and glucose uptake levels in dose-dependent
manner.
In silico studies were used to improve the general accuracy and
specificity of the experimental findings. Computational approaches
including physiological, ADMET properties and molecular docking
analysis provide a new insight into the interaction among
components of KKD-NT (EA, GA, DD, and AA) obesity-related target
biomolecules (PPARg, C/EBPa, and b-catenin). These tools also made
it easier to anticipate pharmacokinetic parameters and analyze
potential toxicological hazards. When compared to traditional
approaches, the in-silico projections for molecular descriptors, drug
similarity, and ADMET features aided in the finding of novel lead
compounds in a shorter timeframe. According to the protein-ligand
binding affinity analysis, EA, and DD had a greater binding interface
with the active residues of PPARg, C/EBPa, and b-catenin than GA,
AA, and control drug RSV. RSV is a phenolic compound found in
nature that possesses a wide range of biological and pharmacological
activities, notably anti-inflammatory and antioxidant abilities. RSV
supplementation increased glucose tolerance in pigs with obese syn-
drome by regulating the metabolism of glucose in skeletal muscle
and liver (Burgess et al., 2011). It also had a favorable effect on hepa-
tocellular ROS in an animal model of metabolic syndrome fed a high-
sugar diet (Aguirre et al., 2014). RSV has an anti-obesity impact by
lowering lipogenesis and enhancing lipolysis in fat cells (Bagul et al.,
2012). Furthermore, RSV stimulates brown adipose tissue activity,
which helps to restore thermogenesis (Fern
andezQuintela et al.,
2017).
Oil Red O (O-R-O)is an agent that stains adipocytes and neutral
lipids, used to visualize fat droplets (Zebisch et al., 2012). The anti-
obesity effect of KKD-NT was tested on 3T3-L1 adipocytes by O-R-O
staining with or without MDI media. Our findings suggest that KKD-
NT and its major components (EA, GA, DD, and AA) reduced lipid
accumulation in differentiated 3T3-L1 adipocytes. Based on the
results, we determined that KKD-NT showed significant efficacy in
decreasing lipid accumulation at 250 mg/ml without causing toxicity.
Obesity and diabetes have been linked to oxidative stress(Gilani et
al., 2021). Oxidative stress is defined as the imbalance of pro-oxidant
and the antioxidant protective capacity that promotes ROS or RNS
which might cause potential damage. Natural antioxidants have a
variety of biological effects, including the prevention of ROS, RNS for-
mation, and the inhibition of free radicals. The antioxidant system is
made up of enzymes (SOD, CAT, GPx, GST, and so on) that prevent the
overproduction of ROS and RNS (M€
oller et al., 2022). Furthermore,
research has shown that excessive levels of ROS and NO can develop
IR in obese people (Katashima et al., 2017). Previously we have
reported that KKD-NT is a strong antioxidant as it contains anti-oxi-
dative components such as phenolic acids (ellagic acid, gallic acid,
Fig. 8. (A) Percentage of a-glucosidase inhibition of different concentrations of KKD-NT extracts and its major phytochemicals. ACR (Acarbose) was used as a control. (B) 2-NBDG
uptake assay in 3T3L1 cells. The glucose uptake effects of KKD-NT on 3T3L1 cells using a fluorescent derivate of glucose 2-NBDG in the presence or absence of KKD-NT, and its phyto-
chemicals for 24 h. Phytochemicals of KK-NT were treated to differentiated cells at 20 mM. Insulin (100 nM) was used as a positive control Data are expressed as a percentage of con-
trol. ### p<0.001 MDI vs. control, while ** p<0.01 and *** p<0.001 treatment vs. MDI.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
40
ascorbic acid), and isoflavones (daidzin) (Akter et al., 2021). Our
results demonstrated that KKD-NT is a potent natural antioxidant
that can inhibit the production of intracellular ROS and NO in 3T3-L1
cells.
Adipocyte formation is an elaborate process involving several hor-
monal substances and transcription factors. Adipocyte the differenti-
ation process and TG production are regulated by the main
adipogenesis factors PPARgand C/EBPa. Additionally, b-catenin is an
important part of the Wnt signaling system that regulates cell prolif-
eration and differentiation in obesity (Chen and Wang, 2018). The
canonical Wnt signaling pathway begins with the binding of Wnt
ligands to their receptors, such as low-density lipoprotein receptor-
related protein 5 or 6 (LRP5/6) and the Frizzled receptor. These recep-
tors are situated on the cell membrane and play a key role in initiat-
ing the cascade. Activation of the pathway involves the inhibition of
GSK3bin the cytoplasm, a process facilitated by the interaction
between Wnt ligands and their receptors. This inhibition leads to the
stabilization and accumulation of b-catenin, a crucial transcriptional
mediator in canonical Wnt signaling. The release of b-catenin from
degradation allows it to enter the cell nucleus, where it regulates the
Fig. 9. Effects of KKD-NT and its major phytochemicals on mRNA expression levels of antioxidant, adipogenesis, and insulin-regulated genes in 3T3-L1 cells. ### <0.001 compared
with control, ** p<0.01, *** p<0.001 compared with the MDI.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
41
expression of genes involved in various cellular processes, including
those related to obesity. Given the significance of b-catenin in canon-
ical Wnt signaling, strategies aimed at enhancing its stability are con-
sidered promising for potential therapeutic interventions in
addressing obesity (Nijhawan et al., 2020). Moreover, during obesity,
cytokines that promote inflammatory factors such as TNF-aand IL-6
are secreted by adipose tissue. These cytokines have been linked to
chronic inflammation. Our experimental data has been revealed that
250 mg/ml concentration of KKD-NT has been shown potent antioxi-
dant as increased the expression of SOD, CAT, and GPx and also signifi-
cantly down-regulate the expression of the major adipogenic
markers (PPARg, C/EBPa), lipogenic marker (FAS) and up-regulate
b-catenin, indicating its potential as an anti-obesity supplementation.
In addition, KKD-NT (250 mg/ml) and the phyto-constituents
(20 mM) up-regulated the expression levels of GLUT4, and adiponec-
tine and down-regulated the expression of leptin which are co-
related with both obesity and diabetes. Also, the treatment of KKD-
NT down-regulates the mRNA level of TNF-aand IL-6. Furthermore,
20 mM concentrations of EA, GA, DD, AA, and RSV were applied to
observe the mRNA expression of the aforementioned markers. The
data compared the gene expression levels among the compounds
and the control drug.
It is essential to recognize that KKD-NT contains polyphenols and
isoflavones that can activate the wnt/b-catenin pathway. Thus, it is
possible to credit the combined actions of these phytochemicals for
the effects of KKD-NT that have been seen, including its previously
reported activity. However, our research concentrates on EA, GA, DD,
and AA as the key ingredients of KKD-NT, to evaluate their potential
anti-obesity effects (Akter et al., 2023b). The in silico and in vitro vali-
date the experimental results and offer a better comprehension of
the pharmacokinetic characteristics and molecular interactions of EA,
GA, DD, and AA. It is necessary to do additional research to evaluate
the safety and in vivo effectiveness of KKD-NT, opening the door to
its possible use as a cutting-edge therapeutic approach to treat obe-
sity.
5. Conclusions
In this research, the possible pharmacological activities of KKD-NT
as a natural source of antioxidant, anti-adipogenic, anti-inflamma-
tory, and anti-diabetic substances. KKD-NT is a potent antioxidant
agent, as it reduces oxidative stress by increasing the expression of
antioxidant enzymes. This research also demonstrated that KKD-NT
extract and its phytochemicals can regulate the expression of pro-
inflammatory cytokines, adipogenic markers, and adipokines. This
extract and its phytochemicals reduced the production of ROS, and
NO to diminish inflammatory activity and increased a-glucosidase
inhibition, and glucose uptake to inhibit T2D. The findings of this
study pave the way for further study into KKD-NT for the develop-
ment of herbal medications with antioxidant, anti-obesity, anti-
inflammatory, and anti-diabetic effects.
Institutional review board statement
Not applicable.
Fig. 9. Continued.
M.N. Morshed, M. Awais, R. Akter et al. South African Journal of Botany 171 (2024) 3244
42
Informed consent statement
Not applicable.
Ethical approval
Not applicable.
Availability of data and materials
All data generated or analyzed during this study are included in
this published article (and its Supplementary Information files).
Declaration of data authenticity
All figures submitted have been created by the authors, who con-
firm that the images are original with no duplication and have not
been previously published in whole or in part.
Declaration of competing interest
The authors declare no conflict of interest. The funders had no role
in the design of the study; in the collection, analyses, or interpreta-
tion of data; in the writing of the manuscript; or in the decision to
publish the results.
CRediT authorship contribution statement
Md. Niaj Morshed: Conceptualization, Data curation, Software,
Writing original draft. Muhammad Awais: Data curation, Formal
analysis, Methodology, Writing original draft. Reshmi Akter: Con-
ceptualization, Formal analysis, Methodology. Juha Park: Validation,
Visualization. Li Ling: Investigation, Visualization. Byoung Man
Kong: Investigation, Writing review & editing. Deok Chun Yang:
Formal analysis, Project administration, Resources. Dong Uk Yang:
Formal analysis, Validation. Se Chan Kang: Resources, Supervision,
Funding acquisition. Seok-Kyu Jung: Funding acquisition,
Supervision.
Data availability statement
Not applicable.
Acknowledgments
The authors acknowledge the World Class plus 300 Project (R&D)
(P0024386, Development of cosmetics and health functional foods
through standardization of smart farm cultivation) of the MOTIE,
MSS (Korea) for providing infrastructural support.
Funding
This work was supported by the World Class plus 300 Project
(R&D) (P0024386, Development of cosmetics and health functional
foods through standardization of smart farm cultivation) of the
MOTIE, MSS (Korea).
Supplementary materials
Supplementary material associated with this article can be found
in the online version at doi:10.1016/j.sajb.2024.05.056.
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