Article

Anti-Diabetic Potential of Sargassum horneri and Ulva australis

Extracts In Vitro and In Vivo
Young-Hyeon Lee 1,† , Hye-Ran Kim 2,† , Min-Ho Yeo 1 , Sung-Chun Kim 3 , Ho-Bong Hyun 3 ,
Young-Min Ham 3 , Yong-Hwan Jung 3 , Hye-Sook Kim 4 and Kyung-Soo Chang 1, *

1 Department of Clinical Laboratory Science, Catholic University of Pusan, Busan 46252, Republic of Korea;
[email protected] (Y.-H.L.); [email protected] (M.-H.Y.)
2 Department of Biomedical Laboratory Science, Dong-Eui Institute of Technology,
Busan 47230, Republic of Korea; [email protected]
3 Biodiversity Research Institute, Jeju Technopark, Jeju 63608, Republic of Korea; [email protected] (S.-C.K.);
[email protected] (H.-B.H.); [email protected] (Y.-M.H.); [email protected] (Y.-H.J.)
4 Division of International Infectious Diseases Control, Faculty of Pharmaceutical Sciences, Okayama
University, Tsushima-Naka, Kita-Ku, Okayama 700-8530, Japan; [email protected]
* Correspondence: [email protected]; Tel.: +82-51-510-0565
† These authors contributed equally to this work.

Abstract: Sargassum horneri (SH) and Ulva australis (UA) are marine waste resources that cause
environmental and economic problems when entering or multiplying the coastal waters of Jeju
Island. We analyzed their anti-diabetic efficacy to assess their reusability as functional additives. The
alpha-glucosidase inhibitory activity of SH and UA extracts was confirmed, and the effect of UA
extract was higher than that of SH. After the induction of insulin-resistant HepG2 cells, the effects of
the two marine extracts on oxidative stress, intracellular glucose uptake, and glycogen content were
compared to the positive control, metformin. Treatment of insulin-resistant HepG2 cells with SH and
UA resulted in a concentration-dependent decrease in oxidative stress and increased intracellular
glucose uptake and glycogen content. Moreover, SH and UA treatment upregulated the expression of
IRS-1, AKT, and GLUT4, which are suppressed in insulin resistance, to a similar degree to metformin,
Citation: Lee, Y.-H.; Kim, H.-R.; Yeo, and suppressed the expression of FoxO1, PEPCK involved in gluconeogenesis, and GSK-3β involved
M.-H.; Kim, S.-C.; Hyun, H.-B.; Ham, in glycogen metabolism. The oral administration of these extracts to rats with streptozotocin-induced
Y.-M.; Jung, Y.-H.; Kim, H.-S.; Chang, diabetes led to a higher weight gain than that in the diabetic group. Insulin resistance and oral
K.-S. Anti-Diabetic Potential of glucose tolerance are alleviated by the regulation of blood glucose. Thus, the SH and UA extracts
Sargassum horneri and Ulva australis may be used in the development of therapeutic agents or supplements to improve insulin resistance.
Extracts In Vitro and In Vivo. Curr.
Issues Mol. Biol. 2023, 45, 7492–7512.
Keywords: diabetes; Sargassum horneri; Ulva australis; extracts
https://doi.org/10.3390/
cimb45090473

Academic Editor: Myunggon Ko

1. Introduction
Received: 8 August 2023
Revised: 11 September 2023 Diabetes mellitus (DM) is a severe and chronic metabolic disease characterized by in-
Accepted: 13 September 2023 creased blood glucose levels due to an imbalance in glucose homeostasis caused by reduced
Published: 13 September 2023 insulin secretion, insulin resistance (IR), or a combination of these factors [1,2]. According
to the latest report from the International Diabetes Federation (IDF), over 460 million adults
worldwide have been diagnosed with diabetes, and this number is expected to increase to
approximately 700 million by 2045 [3]. Diabetes is classified into type 1 diabetes, which is
Copyright: © 2023 by the authors. caused by a decrease in insulin secretion due to a reduction in β cells in the pancreas, and
Licensee MDPI, Basel, Switzerland. type 2 diabetes, which is caused by a reduction in insulin secretion and IR [4]. More than
This article is an open access article 90% of the people worldwide with the illness have type 2 diabetes [5].
distributed under the terms and
Type 2 DM can develop at a younger age because of a high-fat diet, high-calorie diet,
conditions of the Creative Commons
a lifestyle with low physical activity, IR, and insufficient insulin metabolism [6]. IR is
Attribution (CC BY) license (https://
defined as impaired glucose uptake, metabolism, and storage [7]. Liver, skeletal muscle,
creativecommons.org/licenses/by/
and adipose tissues are representative insulin-sensitive tissues that cause hyperglycemia,
4.0/).

Curr. Issues Mol. Biol. 2023, 45, 7492–7512. https://doi.org/10.3390/cimb45090473 https://www.mdpi.com/journal/cimb

Curr. Issues Mol. Biol. 2023, 45 7493

hyperinsulinemia, and dyslipidemia. As the liver absorbs 34% of orally administered

glucose and supplies 90% of the glucose required by the body in a fasting state, there is
a growing need for research on glucose absorption and hypoglycemic mechanisms [8,9].
Accordingly, some studies have been conducted to evaluate the antidiabetic efficacy by
increasing enzyme activity or enzyme expression in hepatocytes whose activity is reduced
due to diabetes [10]. In addition, because the expression of genes, such as insulin receptor
substrate-1 (IRS1), protein kinase B (AKT), and GLUT4, tends to significantly reduce insulin
resistance, gene regulation related to insulin metabolism can be used as an index to evaluate
diabetes treatment [11]. Based on this evidence, drugs for treating diabetes are being devel-
oped and a combination of exercise and diet is being used to manage diabetes. However,
no fundamental treatment has yet been established for this condition. Biguanides, sulfony-
lureas/glucosidase inhibitors, and oral hypoglycemic agents such as thiazolidinedione and
insulin are used to treat diabetes; however, they cause gastrointestinal side effects such
as abdominal pain, diarrhea, and nausea [12,13]. To address these side effects, extensive
research has been conducted on natural substances that effectively prevent and treat type
2 diabetes.
Various foods with a low glycemic index, including nuts, vegetables, and seaweeds,
are known to prevent and improve DM [14–16]. Seaweeds are generally categorized into
red, brown, and green algae according to their color and are reported to be rich in proteins,
minerals, and vitamins, as well as in dietary fiber, a non-starch polysaccharide that digestive
enzymes cannot break down [17]. Seaweed is also effective in preventing and managing
diabetes because it is low in calories, has a low glycemic index, and is rich in antioxidant
nutrients [18]. Sargassum horneri (SH), used in the present study, is a delicacy in East Asia,
whereas Ulva australis (UA) is used as food in Europe. However, SH and UA have been
identified as the main causes of coastal pollution on Jeju Island [19,20].
SH farming is promoted in Chinese waters, and fragments of growing SH are scattered
all over the southern coast and in Japan, with an average of 1000–9000 tons introduced to
Jeju Island [21]. UA is found mainly along the coast, where algal circulation is inactive, and
its annual generation is estimated to be 10,000 tons [22]. SH and UA are introduced and
generated in coastal waters, causing loss of beach function and foul odors due to decay,
adding to the inconvenience of nearby residents and tourists. Climate change is expected to
exacerbate this situation [23]. Various studies have been conducted to determine the poten-
tial applications of these two seaweeds, which are recognized as organic waste resources.
Several studies have highlighted that SH has antitumor [24], anti-inflammatory [25], and
anti-allergy effects [26]. Studies of the antihyperlipidemic [27], antiviral [28], polysaccha-
ride structure, and physicochemical properties [29] of UA are currently in progress. Both
seaweeds are potentially valuable for blood sugar control; however, research to elucidate
their comprehensive efficacy against hepatic glucose metabolism disorders and the as-
sociated mechanisms remains scarce. In addition, according to the results of previous
studies, neophytadien, hexadecanoic acid, and ethyl ester were identified as major com-
pounds in SH extracts, and 2-Hexadecen-1-ol, 3,7,11,15-tetramethyl-acetate, [R-[R*,R*-(E)]]
in UA extracts [30]. There is a need for research on blood sugar control using SH and
UA extracts containing compounds with physiological activities such as antibacterial and
antioxidant effects.
Therefore, in the present study, the enzyme inhibitory potential of SH and UA extracts
was analyzed and their effects on the regulation of glucose metabolism, gluconeogenesis,
and IRS/AKT signaling pathways were compared in insulin-resistant HepG2 cells. In
addition, by analyzing whether insulin resistance could be improved by administering the
two extracts to STZ-induced diabetic mice, we sought to confirm the possibility of using
SH and UA to improve diabetes.
Curr. Issues Mol. Biol. 2023, 45 7494

2. Materials and Methods

2.1. Preparation of SH and UA Ethanol Extracts
SH and UA were collected from the coast of Jeju Island and washed, dried, and
powdered before use. The dried samples (50 g) were immersed in 1 L of 80% ethanol and
extracted by stirring at room temperature for 24 h. The stirred extracted material was
filtered using a 0.45 µm bottle-top vacuum filter (Corning Inc., Corning, NY, USA), and the
filtered material was evaporated from ethanol using a rotary concentrator (R100 + B100,
Lab Scitech, Corona, CA, USA) and then lyophilized to prepare an extract powder. Sample
extraction was performed twice using the same procedure. The prepared samples were
stored at −20 ◦ C, suspended in a cell culture medium or sterile distilled water at 10 mg/mL
concentration, and diluted before use.

2.2. α-Glucosidase Inhibitory Assay

α-glucosidase inhibitory activity was analyzed using an α-glucosidase inhibitor screen-
ing kit (K938-100, BioVision, Milpitas, CA, USA). After diluting the extract, 10 µL was
dispensed into a 96-well plate, and 10 µL of α-glucosidase enzyme solution was added.
The volume of each well was adjusted to 80 µL with assay buffer and incubated for 15 to
20 min at room temperature. Then, α-glucosidase assay buffer and α-glucosidase substrate
mix were mixed, and 20 µL of the solution was added to each well. The absorbance was
measured at 410 nm using a microplate reader (Varioskan LUX, Thermo Fisher Scientific,
Waltham, MA, USA).

2.3. Cell Culture and Induction of Insulin Resistance Model

The HepG2 human liver cancer cell line was purchased from Korean Cell Line Bank
(Seoul, Republic of Korea). HepG2 cells were cultured in Minimum Essential Medium
Eagle (MEM, Welgene, Seoul, Republic of Korea) medium supplemented with 10% fetal
bovine serum (FBS, Welgene) and 1% penicillin–streptomycin (PS, Welgene) at 37 ◦ C and
5% CO2 incubator. The cells were cultured to approximately 70–80% confluence, passaged
every 2–3 days, and used for the experiments. The glucose concentrations in the MEM
were 5 and 30 mM.
The IR model was established based on the results of a previous study [31]. HepG2
cells were seeded in a plate and then cultured in an incubator at 37 ◦ C and 5% CO2 for 24 h
using a culture medium. To induce IR-HepG2 cells, 1 µM insulin (Sigma-Aldrich, St. Louis,
MI, USA) was added to serum-free MEM, and D-glucose was added at a concentration
of 30 mM and cultured for 48 h. As a control, normal HepG2 cells were cultured in
insulin-free medium containing 5 mM D-glucose. Next, the cells were treated with different
concentrations of the extract or metformin (Sigma-Aldrich) at a concentration of 1 mM and
cultured for 24 h. Finally, insulin (100 nM) was added, incubated for 30 min, and used for
subsequent analysis.

2.4. Cell Viability Assay

The effect of the extract on the viability of HepG2 and IR-HepG2 cells was evaluated
using the WST-1 reagent (Cellvia, AbFrontier, Seoul, Republic of Korea). HepG2 cells were
seeded in 96-well culture plates at 1 × 105 cells/mL and cultured for 24 h. Extracts were
then prepared at different concentrations (125, 250, 500, and 1000 µg/mL) in a medium
without FBS, treated with cells, and cultured in a 37 ◦ C 5% CO2 incubator for 24 h. Next,
10 µL of WST-1 reagent was added to the medium and incubated for 30 min in a 37 ◦ C,
5% CO2 incubator. The absorbance was measured at 450 nm using a microplate reader
(Varioskan LUX, Thermo Fisher Scientific). Cell viability was compared to the absorbance
of the control group.

2.5. Reactive Oxygen Species (ROS) Detection Assay

Reactive oxygen species (ROS) were analyzed using 20 ,70 -dichlorofluorescein diacetate
(DCFH-DA, Sigma, USA). After the cells were dispensed at 1 × 105 cells/mL in a 96-
Curr. Issues Mol. Biol. 2023, 45 7495

well plate, insulin resistance was induced and the cells were treated with the extract or
metformin. After washing the cells twice with phosphate-buffered saline (PBS), 10 µM
DCFH-DA was added, and the solution was incubated for 40 min in a 37 ◦ C and 5% CO2
incubator. The fluorescence intensity of DCFH-DA was measured at 485 nm (excitation) and
525 nm (reflection) using a microplate reader (Varioskan LUX, Thermo Fisher Scientific).
HepG2 cells were seeded onto a Lab-Tek 4 well chamber slide and treated according
to a previously described protocol. The fluorescence intensity was confirmed using a
fluorescence microscope (BX51; Olympus Optical Co., Tokyo, Japan).

2.6. Cellular Glucose Uptake Assay

Intracellular glucose uptake was analyzed using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-
4-yl)amino]-D-glucose (2-NBDG; Invitrogen, Waltham, MA, USA), as previously described [32].
After preparing the insulin resistance model, the extract and metformin were administered
and incubated for 24 h. The cells were then treated with insulin (100 nM) for 30 min, and
2-NBDG (40 µM) was added and incubated for 30 min. Fluorescence was measured after
washing the cells three times with cold PBS. The fluorescence intensity of 2-NBDG was mea-
sured at 460 nm (excitation) and 528 nm (reflection) using a microplate reader (Varioskan LUX,
Thermo Fisher Scientific).
HepG2 cells were seeded onto Lab-Tek 4 well chamber slide and treated according
to the same protocol. To confirm the number of cells, the nucleic acid dye Hoechst 33342
(Invitrogen) was added and the cells were incubated for 30 min. After washing with cold
PBS, cells were examined under a fluorescence microscope (BX51; Olympus Optical Co.).

2.7. Glycogen Content Assay

After inducing insulin resistance in HepG2 cells, the extract and metformin were
administered as previously described. HepG2 cells were homogenized in 200 µL of dH2 O.
The homogenate was boiled for 10 min to inactivate the enzymes, centrifuged at 16,000× g,
and the supernatant was stored on ice. The glycogen content of the supernatant was
analyzed using a glycogen colorimetric assay kit (Biovision, Milpitas, CA, USA) according
to the manufacturer’s instructions. The glycogen content of the cells was calculated using
the total glucose level minus the free glucose background for each sample and normalized
to the protein level.

2.8. Quantitative RT-PCR Analysis

HepG2 cells were washed with 1 × PBS and total RNA was extracted using an
Accuprep® universal RNA extraction kit (Bioneer, Daejeon, Republic of Korea). Extracted
RNA was quantified using a NanoDrop 2000 (Thermo Fisher Scientific). Next, 1 µg of RNA
was subjected to qRT-PCR on a 7500 Fast Real-Time PCR system (Applied Biosystems,
Waltham, MA, USA) using a One-Step TB Green® PrimeScript™ RT-PCR Kit (Takara, Japan).
The conditions were 42 ◦ C for 50 s, 95 ◦ C for 10 s, followed by 40 cycles of 95 ◦ C for 5 s, 55
or 58 ◦ C for 30 s, and 72 ◦ C for 30 s. The primers used for qRT-PCR analysis are listed in
Table 1. The mRNA levels of the target gene in the same sample were normalized to that
of β-actin, a housekeeping gene. Data were expressed as relative fold change in transcript
levels relative to controls using the 2−∆∆CT equation.

2.9. Animal Experiments

The purchased six-week-old male ICR mice were raised under specific pathogen-free
conditions (KOSA BIO Inc., Sungnam, Republic of Korea). The mice were maintained in a
stable breeding environment with a light/dark cycle of 12 h a day at a temperature of 23 ±
3 ◦ C, relative humidity of 50 ± 10%, and 150 to 300 Lux. The experimental animals were
acclimated to the environment for one week before the experiment and allowed free access
to standard feed (Samtako Bio Korea, Seoul, Republic of Korea) and water. After the adapta-
tion period, just before administration, streptozotocin (STZ, Sigma-Aldrich) was dissolved
in cold 0.09 M citrate buffer (Sigma-Aldrich) and intraperitoneally administered to the ani-
Curr. Issues Mol. Biol. 2023, 45 7496

mals at 40 mg/kg for 5 days [33,34]. To confirm whether hyperglycemia was induced 72 h
after the injection, blood glucose levels were measured in the tail vein using a glucometer.
Animals with blood glucose levels of 250 mg/dL were selected for this study. The ICR mice
were divided into six groups: (1) normal, (2) diabetic, (3) metformin (100 mg/kg/day),
(4) SH (500 mg/kg/day), and (5) UA (500 mg/kg/day) (Scheme 1) [20,35]. The groups were
fed the same diet and divided into six sub-groups of five animals each. The diabetic group
received PBS by oral administration, and the experimental group received metformin or its
extract by oral administration for 6 weeks. This study was approved by the Institutional
Animal Care and Use Committee of Catholic University of Pusan (CUP 2022-003).

Table 1. Primers used in quantitative real-time PCR.

GenBank
Gene Forward Primer (50 to 30 ) Reverse Primer (50 to 30 ) Amplicon (bp)
Accession
IRS-1 1 NM_005544.3 GAG TCC CAG CAC CAA CAG AA CCT TGC CAC CCA TGC AGA TA 374
AKT 2 NM_005163.2 GGA CAA GGA CGG GCA CAT TA CGA CCG CAC ATC ATC TCG TA 192
GLUT4 3 NM_001042.3 GCT GAA GGA TGA GAA GCG GA TGT CTC GAA GAT GCT GGT CG 172
GSK-3β 4 NM_002093 CGA GAC ACA CCT GCA CTC TT TCT GTC CAC GGT CTC CAG TA 163
FoxO1 5 NM_002015 GTG GAT GGT CAA GAG CGT GC TGC CAC CCT CTG GAT TGA GC 170
PEPCK 6 NM-002591.3 AAG AGA CAC AGT GCC CAT CC ACG TAG GGT GAA TCC GTC AG 201
β-actin 7 NM_001101.5 ATG GAT GAT GAT ATC GCC GCG TCT CCA TGT CGT CCC AGT TG 250
1IRS-1, insulin receptor substrate 1; 2 AKT, protein kinase B; 3 GLUT4, Glucose Transporter Type 4; 4 GSK-3β,
glycogen synthase kinase-3 beta; 5 FoxO1, forkhead box O1; 6 PEPCK, phosphoenolpyruvate carboxykinase; 7
β-actin, homo sapiens actin beta.

Scheme 1. Experimental plan for amelioration of diabetes in streptozotocin-induced diabetic animals

by treatment with SH and UA extracts. After 5 days of IP dosing with Streptozotocin, confirm that
blood glucose reaches 250 mg/dl or higher. Administer the drug orally for the next 6 weeks. NC,
normal control; DC, diabetic control; Met, metformin 100 mg/kg; SH, Sargassum horneri extract
500 mg/kg; UA, Ulva australis extract 500 mg/kg (n = 5).

2.10. Weekly Body Weight and Blood Glucose Analysis

To analyze the changes in the weight of the experimental animals, they were weighed
using an electronic balance once a week at a fixed time (10:00 a.m.). Body weight gain was
expressed as a percentage by comparing the weight measured at the end of the experiment
with that measured at the beginning.
Changes in blood glucose levels were assessed every 10 days using a glucometer. The
diet was restricted for 6 h before blood glucose measurement, and tail vein blood was
collected for subsequent analyses.

2.11. Insulin Tolerance Test and Oral Glucose Tolerance Test

The insulin tolerance test (ITT) was performed on mice that fasted for 6 h, and
0.75 units/kg of insulin (Sigma-Aldrich) was intraperitoneally injected. Blood glucose
levels were measured before injection and 30, 60, 90, and 120 min after injection. The area
under the curve (AUC) was calculated to analyze insulin sensitivity.
Curr. Issues Mol. Biol. 2023, 45 7497

An oral glucose tolerance test (OGTT) was performed after all animals had fasted for
12 h. Blood was collected from the tail vein of the experimental animals at 0, 30, 60, 90, and
120 min after oral administration of 2 g/kg glucose solution, and changes in blood glucose
were analyzed.

2.12. Serum Biochemical Analysis

The experimental animals were fasted for 12 h before autopsy, and a mixture of
25 mg/kg alfaxalone (Alfaxan® ; Careside, Subiaco, WA, Australia) and 5 mg/kg xylazine
(Rompun® ; Bayer Korea, Seoul, Republic of Korea) was used as an anesthetic. Blood
was collected from the hearts of anesthetized experimental animals, coagulated at room
temperature, and analyzed using plasma obtained by centrifugation at 2500 rpm for 30 min.
The parameters analyzed were aspartate aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALP), total protein (TP), triglycerides (TG), total cholesterol
(CHO), high-density lipoprotein cholesterol (HDL-C), glucose (GLU), blood urea nitrogen
(BUN), and creatinine (CRE). Analysis was performed using a blood biochemistry analyzer
BT1500 (Biotecnica Instrument SpA, Rome, Italy).

2.13. Organ Weight and Histopathological Examination

The liver, kidney, pancreas, spleen, and heart of the experimental animals were re-
moved and weighed, and the absolute organ weight was compared to the body weight
of the experimental animals. For the histopathological examination, the liver and pan-
creas were partially resected and fixed in 10% neutral formalin, and paraffin blocks were
prepared. After that, slices 3 µm thick were cut to prepare serial sections, which were
then deparaffinized, rehydrated by gradually passing ethanol, washed, stained with hema-
toxylin and eosin, and finally evaluated using a microscope (Olympus BX51, Olympus
Optical Co., Rome, Italy).

2.14. Statistical Analysis

GraphPad Prism program 5.0 software (GraphPad Software Inc., SanDiego, CA, USA)
was used for data analysis. Values were expressed as mean ± standard deviation and tested
with one-way ANOVA for significant differences. The significance level was set at p < 0.05.

3. Results
3.1. α-Glucosidase Inhibitory Activity of SH and UA
To analyze the anti-diabetic effects of SH and UA ethanol extracts, their glucosidase
inhibitory abilities were evaluated. The SH extract showed α-glucosidase inhibitory activity
of 77.1 ± 1.9, 85.7 ± 0.8, and 89.6 ± 1.7% in concentrations of 250, 500, and 1000 µg/mL,
respectively. In the case of the UA extract, each treatment concentration showed 79.8 ± 2.2,
86.9 ± 1.9, and 96.3 ± 1.6% of α-glucosidase inhibitory activity. Acarbose, a positive control,
showed 80.0 ± 1.8, 88.9 ± 1.0, and 98.2 ± 1.2% of inhibitory activity at each treatment
concentration, respectively. Both extracts showed α-glucosidase inhibitory activity in a
dose-dependent manner, and the α-glucosidase inhibitory activity of UA extract was similar
to that of acarbose (Figure 1).

3.2. Cell Viability of HepG2 and IR-HepG2

To confirm the cytotoxic effects of the SH and UA extracts, HepG2 cells were treated
with different concentrations of the extract (125, 250, 500, and 1000 µg/mL). The results of
the cell viability assay after 24 h of treatment showed 100% viability of cells treated with
the extract at concentrations of 250 µg/mL or lower (Figure 2A,B). Thus, the concentrations
used in the present study did not cause cytotoxicity.
Curr. Issues Mol. Biol. 2023, 45 7498

Figure 1. α-glucosidase inhibitory activity of Sargassum horneri and Ulva australis extracts. Acarbose
was used as a positive control in the glucosidase inhibition assay. S. horneri is denoted as SH, and U.
australis is denoted as UA. U. australis showed higher glucosidase inhibitory activity than S. horneri.
Data are expressed as mean ± standard deviation (n = 3).

Figure 2. Cell viability of HepG2 and IR-HepG2 treated with Sargassum horneri and Ulva australis
extracts. Evaluation of HepG2 cell viability after treatment with (A) S. horneri and (B) U. australis
ethanol extracts. Significant differences: ** p < 0.01 vs. the control group. Evaluation of IR-HepG2 cell
viability after treatment with (C) S. horneri and (D) U. australis ethanol extracts. Cell viability was
measured using a WST-1 assay. Data are expressed as mean ± standard deviation (n = 3). Significant
differences: ** p < 0.01 vs. IR group; ## p < 0.01 vs. NC group.

After induction of insulin-resistant HepG2 (IR-HepG2) cells, cell viability was con-
firmed by treatment with the extract and metformin. The results showed that cells treated
with the extract at a concentration of 250 µg/mL or lower and the positive control group
treated with 1 mM metformin demonstrated a viability of approximately 90%, which was
higher than that of IR-HepG2 cells (Figure 2C,D).

3.3. Effect of Extract Treatment on ROS Content

Because IR and hyperglycemia are known to increase cellular ROS levels due to
oxidative stress, we analyzed the intracellular ROS content. After preparing IR-HepG2
cells, SH and UA extracts were administered for 24 h at concentrations that did not exhibit
Curr. Issues Mol. Biol. 2023, 45 7499

cytotoxicity, and the ROS content was analyzed. The results revealed that the ROS content
of IR-HepG2 cells increased by approximately 22% compared to that of normal cells, and the
ROS levels of the experimental group treated with the extract decreased in a concentration-
dependent manner. The experimental group treated with 250 µg/mL extract had ROS
content similar to that of the positive control group treated with 1 mM metformin (Figure 3).

Figure 3. Effects of Sargassum horneri and Ulva australis treatment on the intracellular ROS content
in IR-HepG2 cells. ROS content in IR-HepG2 cells was detected using a fluorescence microplate.
ROS content after treatment with (A) S. horneri and (B) U. australis extracts. The ROS content in the
IR-HepG2 group was significantly higher than that in the control group. Treatment with the extract
and metformin significantly lowered ROS levels. Data are expressed as mean ± standard deviation
(n = 3). Significant differences: * p < 0.05, ** p < 0.01 vs. IR group; # p < 0.05 vs. NC group.

ROS fluorescence was confirmed using a fluorescence microscope, and the fluorescence
level of IR-HepG2 cells was stronger than that of the extract- and metformin-treated groups
(Figure 4). These results indicated that the SH and UA extracts reduced oxidative stress in
IR-HepG2 cells.

Figure 4. Analysis of ROS level of IR-HepG2 cells according to Sargassum horneri and Ulva australis
extracts treatment using fluorescence microscopy. After the induction of IR-HepG2, the extract
(250 µg/mL) and metformin (1 mM) were added and cultured for 24 h, and then the cells were
stained using 10 µM DCFH-DA. When the fluorescence level of the cells was confirmed using a
fluorescence microscope, IR-HepG2 cells showed overall fluorescence, whereas the fluorescence
expression of the extract and metformin decreased (magnification ×400).

3.4. Effect of Seaweed Extract on Glucose Uptake in IR-HepG2 Cells

After inducing insulin resistance in HepG2 cells, the extract and metformin were
administered for 24 h and glucose uptake was analyzed using 2-NBDG. The glucose uptake
in IR-HepG2 cells was reduced by approximately 50% compared with that in normal cells.
Treatment with SH extract at 62.5, 125, and 250 µg/mL led to glucose uptake rates of
69.7%, 87.4%, and 90.2%, respectively. The UA extract concentrations were 70.7, 86.9, and
93.2%, respectively. The positive control group treated with 1 mM metformin showed 94.9%
glucose uptake. Both the extracts increased the glucose uptake rate in a concentration-
dependent manner, and UA extract treatment led to a glucose uptake rate similar to that of
metformin (Figure 5).
Curr. Issues Mol. Biol. 2023, 45 7500

Figure 5. Effects of Sargassum horneri and Ulva australis treatment on glucose uptake in IR-HepG2
cells. Percentage of cellular glucose uptake (%) after (A) S. horneri extract and (B) U. australis extract
treatment. The glucose uptake rate of the IR-HepG2 group was significantly lower than that of the
NC group, and the glucose uptake rate significantly increased with extract and metformin treatment.
Data are expressed as mean ± standard deviation (n = 3). Significant differences: ** p < 0.01 vs. IR
group; ## p < 0.01 vs. NC group.

After 2-NBDG staining to confirm the glucose uptake rate, and Hoechst 33342 staining
to confirm the number of cells, the degree of fluorescence was evaluated using a fluorescence
microscope. The results confirmed that the fluorescence intensity of the IR-HepG2 group
was lower than that of the extract- and metformin-treated groups (Figure 6). These results
indicated that SH and UA help mitigate the glucose uptake ability of the cells.

Figure 6. Cellular glucose uptake rate in IR-HepG2 cells with Sargassum horneri and Ulva australis
extracts treatment. After induction of IR-HepG2, the extract (250 µg/mL) and metformin (1 mM)
were added and cultured for 24 h. The cells were then stained with 40 µM 2-NBDG, and the
fluorescence intensity of 2-NBDG was used to determine the extent of glucose uptake in control and
insulin-resistant HepG2 cells. The number of cells was evaluated using Hoechst 33342 staining and
confirmed using a fluorescence microscope (magnification ×400). Fluorescence intensity images were
merged using ImageJ software V1.52.
Curr. Issues Mol. Biol. 2023, 45 7501

3.5. Glycogen Content of IR-HepG2 Cells According to Extract Treatment

HepG2 cells were treated with the extract and metformin for 24 h after inducing IR,
and the glycogen content was analyzed. The glycogen content of IR-HepG2 cells was
reduced by approximately 40% compared to that of normal cells. Positive controls treated
with 1 mM metformin showed glycogen levels similar to normal cells. At the same time,
treatment with SH and UA extracts at concentrations of 62.5, 125, and 250 µg/mL increased
the glycogen content in a concentration-dependent manner (Figure 7).

Figure 7. Effect of Sargassum horneri and Ulva australis on glycogen contents in IR-HepG2 cells.
Intracellular glycogen content of IR-HepG2 cells following treatment with (A) S. horneri extract and
(B) U. australis extract. The IR-HepG2 group exhibited significantly lower levels than the control
group. Compared to the IR-HepG2 group, metformin and the extract at 1 mM were significantly
higher at a concentration of 250 µg/mL. Data are expressed as mean ± standard deviation (n = 3).
Significant differences: * p < 0.05, ** p < 0.01 vs. IR group; ## p < 0.01 vs. NC group.

3.6. Effect of Extract Treatment on IRS-1/AKT/GLUT4 Gene Expression

To investigate whether insulin resistance could be alleviated by SH and UA extracts,
the expression levels of IRS-1/AKT/GLUT4 transcription factors were measured. IR-
HepG2 cells were treated with SH and UA extracts (125 and 250 µg/mL, respectively) and
metformin (1 mM), which served as a positive control, and cultured for 24 h for subsequent
analysis. The mRNA expression levels of IRS-1 and AKT were downregulated in IR-HepG2
cells compared with those in normal cells. In the extract and metformin treatment groups,
the mRNA levels were upregulated, and the results confirmed that the mRNA level in
the UA extract treatment group was higher than that in the SH extract treatment group
(Figure 8).
In addition, we analyzed the mRNA expression levels of the GLUT4 gene found in
skeletal muscle, hepatocytes, and adipocytes. The expression level of the GLUT4 gene
decreased in IR-HepG2 cells, and the mRNA level significantly increased in the extract-
and metformin-treated groups (Figure 8C,F).

3.7. Effect of Glycogen-Metabolism-Related Gene Expression by Extract Treatment

To confirm the regulation of glycogen metabolism in IR-HepG2 cells following treat-
ment with SH and UA extracts, the expression of FoxO1, PEPCK, and GSK-3β genes was
investigated. The mRNA levels of FoxO1 and PEPCK, which are gluconeogenesis genes
regulated by AKT, were examined. The expression levels of FoxO1 and PEPCK genes in IR-
HepG2 cells were higher than those in normal cells, and, after treatment with the extract and
metformin, mRNA levels significantly decreased compared to IR-HepG2 cells (Figure 9).
In addition, the mRNA expression of GSK-3β, which regulates glycogen synthesis in
response to insulin, was analyzed. IR-HepG2 cells showed upregulated mRNA expression
compared to normal cells, whereas these changes were suppressed in the extract- and
metformin-treated groups (Figure 9C,F). These results confirmed that the mRNA levels of
genes related to glucose metabolism in the groups treated with the SH and UA extracts
were suppressed to a level similar to that in the group treated with metformin.
Curr. Issues Mol. Biol. 2023, 45 7502

Figure 8. Effects of Sargassum horneri and Ulva australis on the expression of IRS-1/AKT and GLUT4 in
IR-HepG2 cells. (A–C) IRS-1, AKT, and GLUT4 gene expression analysis according to S. horneri extract
treatment; (D–F) IRS-1, AKT, and GLUT4 gene expression analysis by U. australis extract treatment.
The mRNA expression level was measured by qRT-PCR and normalized using the housekeeping gene
β-actin. Data are expressed as mean ± standard deviation (n = 3). Significant differences: * p < 0.05,
** p < 0.01 vs. IR group; # p < 0.05 vs. NC group.

Figure 9. Effects of Sargassum horneri and Ulva australis on the expression of FoxO1, PEPCK, and
GSK-3β in IR-HepG2 cells. (A–C) FoxO1, PEPCK, and GSK-3β gene expression analysis according to
S. horneri extract treatment; (D–F) FoxO1, PEPCK, and GSK-3β gene expression analysis by U. australis
extract treatment. The mRNA expression level was measured by qRT-PCR and normalized using
the housekeeping gene β-actin. Data are expressed as mean ± standard deviation (n = 3). Significant
differences: * p < 0.05, ** p < 0.01 vs. IR group; ## p < 0.01 vs. NC group.

3.8. Changes in Body Weights

The effects of the SH and UA extracts on body weight changes in animals with diabetes
induced by the administration of streptozotocin (40 mg/kg) for 5 days were analyzed.
Before the start of the experiment, the body weight was 31.0 to 32.5 g, and there was no
difference in body weights between the experimental groups. Body weight was analyzed
for six weeks after the substance was administered, and the weight gain rate of the diabetic
Curr. Issues Mol. Biol. 2023, 45 7503

animals was found to be lower than that of the normal group. In the group administered
metformin and the extract, weight increased from the third week, but, in the diabetes group,
weight gradually decreased (Figure 10).

Figure 10. Changes in body weight for 6 weeks in diabetic mice induced by streptozotocin following
oral administration of the extracts. Weekly changes in body weight. NC, normal control; DC, diabetic
control; Met, metformin 100 mg/kg; SH, Sargassum horneri extract 500 mg/kg; UA, Ulva australis
extract 500 mg/kg. Each value represents the mean ± standard deviation (n = 5).

The body weight gain was 6.93 ± 1.22 g, −0.86 ± 0.57 g, 1.28 ± 0.45 g, 1.12 ± 0.36 g,
and 2.54 ± 0.43 g in the normal, diabetic, metformin-, SH-, and UA-treated groups, respec-
tively. The group administered metformin and the extract showed significantly higher
weight gain than the diabetic group, with the highest weight gain observed in the UA-
treated group (Table 2). Food intake was approximately 4 g per animal in all groups tested,
and no differences were found between groups. Therefore, the weight regain in animals
treated orally with metformin, SH, and UA is likely due to substance administration.

Table 2. Changes in body weight gain of streptozotocin-induced diabetic mice following oral admin-
istration of the seaweed extract.

Initial Weight Final Weight Body Weight Gain

Groups
(g) (g) (g)
NC 31.03 ± 0.61 38.44 ± 1.22 6.93 ± 1.22
DC 31.96 ± 0.78 30.24 ± 1.06 −0.86 ± 0.57 ##
Met 31.88 ± 0.79 33.07 ± 1.08 1.28 ± 0.45 *
SH 32.54 ± 0.47 33.33 ± 0.88 1.12 ± 0.36 *
UA 32.49 ± 0.95 35.65 ± 0.93 2.54 ± 0.43 **
Data are expressed as mean ± standard deviation (n = 5). Significant differences: * p < 0.05, ** p < 0.01 vs. DC
group; ## p < 0.01 vs. NC group.

3.9. Changes in Blood Glucose at 10-Day Intervals According to the Administration of the Extract
Blood was collected from the tail and a blood glucose analyzer was used to analyze
the changes in blood glucose levels during the oral administration of SH and UA extracts
for 6 weeks. The blood glucose level in the control group was 160 mg/dL. In contrast, in
animals with STZ-induced diabetes, the blood sugar level was 430–440 mg/dL, and there
was no difference between the experimental groups. The results of blood glucose levels
10 days after administration of the extract showed that the blood glucose level increased
to 516.8 ± 43.3 mg/dL in the diabetic group. In the metformin-, SH-, and UA-treated
groups, blood glucose levels decreased by 422.4 ± 43.6 mg/dL, 424.0 ± 37.6 mg/dL, and
423.0 ± 41.3 mg/dL, respectively. Subsequently, it was confirmed that blood glucose was
Curr. Issues Mol. Biol. 2023, 45 7504

significantly reduced in the metformin-, SH-, and UA-administered groups compared to

the diabetic group (Table 3).

Table 3. Changes in blood glucose at 10-day intervals in diabetic mice induced by streptozotocin
following oral administration of the extracts.

Date of the Blood Glucose Measurement

Groups
0 10 20 30 40
NC 160.00 ± 12.01 157.50 ± 13.83 146.25 ± 9.86 128.50 ± 11.61 124.00 ± 12.71
DC 440.50 ± 48.74 ## 516.75 ± 43.27 ## 521.75 ± 33.93 ## 531.25 ± 55.43 ## 547.75 ± 36.62 ##
Met 437.00 ± 36.40 422.40 ± 43.63 * 414.25 ± 23.16 ** 418.00 ± 43.47 * 452.25 ± 47.27 *
SH 435.50 ± 51.50 424.00 ± 37.30 * 391.50 ± 42.80 ** 392.30 ± 46.80 ** 446.30 ± 33.00 *
UA 434.17 ± 57.22 423.00 ± 41.32 * 359.75 ± 32.96 ** 336.25 ± 29.79 ** 387.00 ± 42.07 **
Data are expressed as mean ± standard deviation (n = 5). Significant differences: * p < 0.05, ** p < 0.01 vs. DC
group; ## p < 0.01 vs. NC group.

3.10. Analysis of Insulin Tolerance Test and Oral Glucose Tolerance Test
Blood glucose levels measured 30 min after insulin injection were higher in the diabetic
and drug-administered groups than in the normal group; however, insulin resistance in the
drug-administered groups tended to improve compared to that in the diabetic group. The
area under the curve (AUC) was determined using an insulin tolerance test. The diabetic
group showed a significantly higher AUC than the normal group, whereas the metformin-,
SH-, and UA-treated groups showed significantly lower AUC values than the diabetic
group (Figure 11A,B).

Figure 11. Analysis of insulin and oral glucose tolerance test in STZ-diabetic mice following the
administration of the extracts. (A) insulin tolerance test (ITT), (B) area under the curve (AUC) of ITT,
(C) oral glucose tolerance test (OGTT), and (D) area under the curve (AUC) of OGTT. NC, normal
control; DC, diabetic control; Met, metformin 100mg/kg treatment; SH, Sargassum horneri extract
500mg/kg treatment; UA, Ulva australis extract 500 mg/kg treatment. Each value represents the
mean ± standard deviation (n = 5). Significant differences: ** p < 0.01 vs. DC group; ## p < 0.01 vs.
NC group.

The blood glucose levels 30 min after glucose administration were 900 mg/dL in
the diabetic group and 800 mg/dL in the groups administered metformin, SH, and UA
extracts. After 60 min, the blood glucose levels were the lowest in the UA-administered
group. Analysis of the AUC using the oral glucose tolerance graph confirmed that the
group treated with metformin, SH, and UA extracts showed significantly decreased glucose
tolerance compared with the diabetic group (Figure 11C,D).
Curr. Issues Mol. Biol. 2023, 45 7505

3.11. Serum Biochemical Analysis

The AST and ALT levels were significantly higher in all groups than in the normal
group. However, the ALT levels in the UA-administered group were significantly lower
than those in the diabetic group. In the case of ALP and TP levels, there were no significant
differences in the values of the normal group among the experimental groups with diabetes.
The TG levels were significantly higher in all groups than in the normal group. The
corresponding values amounted to 258.4 ± 26.0 mg/dL, 78.4 ± 17.3 mg/dL, 78.9 ± 16.5
mg/dL, and 79.1 ± 12.3 mg/dL in the diabetes group, metformin-, SH-, and UA-treated
groups, respectively. Compared with the diabetic group, the levels were significantly
lower in the groups administered metformin or the extract. Total cholesterol significantly
decreased in all substance-administered groups compared with that in the diabetic group.
HDL cholesterol levels were significantly higher in the metformin- and UA-treated groups
than in the diabetic group.
Blood glucose levels were significantly higher in animals inoculated with streptozo-
tocin compared to those in the normal group (98.0 ± 11.2 mg/dL). The diabetic group
demonstrated blood glucose levels of 748.0 ± 38.8 mg/dL, which were significantly higher
than those in the groups administered metformin or seaweed extracts. In addition, we
found no significant differences in the BUN and creatinine levels between the experimental
groups (Table 4).

Table 4. Changes in serum biochemical parameters in mice with streptozotocin-induced diabetes.

NC DC Met SH UA
AST
131.3 ± 21.6 249.6 ± 27.0 ## 247.8 ± 19.8 236.5 ± 25.5 238.7 ± 22.1
(U/L)
ALT
23.5 ± 3.3 64.5 ± 8.7 ## 62.5 ± 9.4 54.5 ± 7.8 36.8 ± 7.5 **
(U/L)
ALP
93.1 ± 9.9 130.5 ± 28.0 128.3 ± 15.0 110.2 ± 16.3 111.5 ± 16.0
(U/L)
T-P
5.0 ± 0.3 4.9 ± 0.5 4.6 ± 0.5 5.0 ± 0.6 5.0 ± 0.4
(g/dL)
TG
16.1 ± 3.0 258.4 ± 26.0 ## 78.4 ± 17.3 ** 78.9 ± 16.5 ** 79.1 ± 12.3 **
(mg/dL)
CHO
90.0 ± 8.6 125.5 ± 12.7 ## 93.5 ± 9.3 * 96.3 ± 8.7 * 97.0 ± 8.4 *
(mg/dL)
HDL-C
112.5 ± 6.9 83.7 ± 7.0 # 117.4 ± 14.5 * 92.6 ± 7.3 116.0 ± 12.9 *
(mg/dL)
GLU
98.0 ± 11.2 748.0 ± 38.8 ## 624.0 ± 38.5 ** 658.0 ± 38.1 ** 602.0 ± 34.8 **
(mg/dL)
BUN
30.5 ± 2.6 39.4 ± 8.5 28.7 ± 3.0 28.4 ± 3.4 28.2 ± 3.7
(mg/dL)
CRE
0.4 ± 0.1 0.7 ± 0.2 0.5 ± 0.1 0.5 ± 0.1 0.5 ± 0.1
(mg/dL)
Data are expressed as mean ± standard deviation (n = 5). Significant differences: * p < 0.05, ** p < 0.01 vs. DC
group; # p < 0.05, ## p < 0.01 vs. NC group.

3.12. Tissue Weight and Liver Index Analysis

At the end of the experiment, weights of the liver, kidneys, pancreas, spleen, and
heart were measured and compared. The weight of the liver significantly increased by
2.04 ± 0.24 g in the diabetic group compared to 1.34 ± 0.08 g in the normal group. The
weights of the liver in the metformin-, SH-, and UA-treated groups were 1.69 ± 0.09 g,
1.72 ± 0.08 g, and 1.68 ± 0.09 g, respectively. Liver weights in the metformin and UA
groups were significantly lower than those in the diabetic group. The kidney, pancreas,
spleen, and heart weights were similar to those of animals in the normal group (Table 5).
The liver index, calculated using the body and liver weights at the end of the animal
experiment, was significantly higher in the diabetic group than in the normal group. The
metformin-, SH-, and UA-treated groups demonstrated significantly lower values than the
diabetic group, with the UA group showing the highest significance level (Figure 12).
Curr. Issues Mol. Biol. 2023, 45 7506

Table 5. Tissue weight changes in diabetic animals by treatment with seaweed extract.

NC DC Met SH UA
Liver 1.34 ± 0.08 2.04 ± 0.24## 1.69 ± 0.09 * 1.72 ± 0.08 1.68 ± 0.09 *
Kidney 0.59 ± 0.05 0.58 ± 0.09 0.58 ± 0.06 0.55 ± 0.03 0.56 ± 0.03
Pancreas 0.18 ± 0.02 0.21 ± 0.05 0.20 ± 0.02 0.18 ± 0.01 0.17 ± 0.02
Spleen 0.09 ± 0.01 0.11 ± 0.02 0.08 ± 0.02 0.11 ± 0.04 0.09 ± 0.02
Heart 0.18 ± 0.02 0.16 ± 0.01 0.15 ± 0.01 0.16 ± 0.01 0.17 ± 0.01
Each value represents the mean ± standard deviation (n = 5). Significant differences: * p < 0.05 vs. DC group;
## p < 0.01 vs. NC group.

Figure 12. Changes in liver index in STZ-diabetic mice. Liver index after administration of Sargas-
sum horneri and Ulva australis extracts. NC, normal control; DC, diabetic control; Met, metformin
100 mg/kg treatment; SH, S. horneri extract 500 mg/kg treatment; UA, U. australis extract 500 mg/kg
treatment. Each value represents the mean ± standard deviation (n = 5). Significant differences:
* p < 0.05, ** p < 0.01 vs. DC group; ## p < 0.01 vs. NC group.

3.13. Histopathological Analysis

Histological analyses of liver and pancreatic tissues were performed using hematoxylin
and eosin staining. In the case of the liver tissue, the lobular structure was well maintained
in the normal group, and hepatocytes were formed in a single layer centered on the central
vein. However, the diabetic group exhibited severe hepatocellular degeneration, including
fatty acid changes and swelling. In contrast, in the group administered metformin, SH, and
UA, the hepatic lobules were well maintained, and the degree of fatty degeneration was
significantly reduced compared to that in the diabetic group (Figure 13A–E).

Figure 13. Effect of Sargassum horneri and Ulva australis on microphotographs of the liver and pancreas
in STZ-diabetic mice. (A–E) Hematoxylin and eosin (H,E) stain of liver tissue; (F–J) hematoxylin and
eosin (H,E) stain of pancreas tissue (magnification ×200). NC, normal control; DC, diabetic control;
Met, metformin 100 mg/kg treatment; SH, S. horneri extract 500 mg/kg treatment; UA, U. australis
extract 500 mg/kg treatment.
Curr. Issues Mol. Biol. 2023, 45 7507

In the case of the pancreas, it was confirmed that, in the normal group, the shape of
the islets of Langerhans was conspicuously well preserved, the endocrine and exocrine
structures were well maintained, and acini cells were abundant. However, in the diabetic
group, atrophied cells were observed, the structure of the islets of Langerhans was abnor-
mal, and severe inflammatory cells were observed around the blood vessels and islets. In
the group administered metformin, SH, and UA, the size of the islets of Langerhans tended
to shrink compared to that in the normal group, but the number of inflammatory cells was
lower than that in the diabetic group (Figure 13F–J).

4. Discussion
Diabetes is a group of chronic diseases associated with hyperglycemia and character-
ized by excessive concentrations of circulating glucose in the blood. Patients with diabetes
are over four times more likely than healthy controls to suffer from diseases such as hyper-
tension, vascular disease complications, blindness, and renal failure [36]. Drugs used for the
treatment of diabetes include oral hypoglycemic agents and insulin; however, these drugs
have been reported to cause many side effects related to the gastrointestinal system [37,38].
Accordingly, diet and medication have been reported to be the most commonly used meth-
ods to treat diabetes. For example, consuming fruits, whole grains, and vegetables can
prevent or reduce type 2 diabetes [39]. In addition, research is being conducted to develop
new drugs using natural products with fewer side effects to prevent and treat diabetes and
to use them as dietary supplements [40].
Seaweed, one of the foods used to prevent diabetes, contains polyphenols, carotenoids,
vitamins, phycobilins, and physiologically active compounds in the form of polysaccha-
rides [41,42]. Seaweeds used as food are suitable for diabetes management because of their
low-calorie content and richness in dietary fiber, unsaturated fatty acids, and vitamins [43].
However, SH and UA, which are abundant in East Asia, are not consumed domestically,
and have been identified as the cause of coastal pollution on Jeju Island. In the present
study, extracts were prepared from these two types of marine waste, which cause envi-
ronmental problems and are generated in volumes of approximately 10,000 tons per year.
Subsequently, their anti-diabetic effects were analyzed in insulin-resistant HepG2 cells and
in animals with STZ-induced diabetes.
Alpha-glucosidase is an important enzyme involved in carbohydrate digestion that
catalyzes the breakdown of ingested polysaccharides into monosaccharides, which are then
absorbed. Inhibitors of this enzyme are important targets for the treatment of diabetes
because they inhibit carbohydrate digestion and glucose absorption [44]. The results of our
analysis of α-glucosidase inhibitory ability confirmed that SH and UA extract increased
α-glucosidase inhibitory ability in a concentration-dependent manner. Furthermore, the
UA extract exhibited an effect similar to that of acarbose, the positive control. This suggests
a higher efficacy of SH compared to the 93.2% inhibitory effect of the Sargassum fulvellum
hexane fraction at a concentration of 2 mg/mL [45]. Notably, UA showed a higher efficacy
than the 88.0% inhibitory ability reported for a concentration of 4 mg/mL Ulva lactuca [46].
Thus, SH and UA increased α-glucosidase inhibition in vitro to levels similar to the positive
control drug acarbose, but further analysis is needed to replace acarbose, which is known
to cause abdominal pain in patients [47].
The liver plays a significant role in regulating blood sugar levels via glycogen synthesis
and glucose metabolism [48]. HepG2 cells derived from human liver cancer tissues exhibit
biological activities similar to those of normal hepatocytes and are extensively used in
insulin resistance studies because exposure to insulin causes cellular damage [49]. The
efficacy of SH and UA extracts was analyzed after inducing insulin resistance in HepG2
cells. In cells with induced insulin resistance, intracellular ROS levels increase significantly,
thereby suppressing tissue antioxidant defenses and causing oxidative stress [50]. Therefore,
ROS levels were analyzed after treatment with the two extracts. Following treatment with
SH and UA extracts, the ROS levels tended to decrease in a concentration-dependent
manner and were similar to the corresponding trends observed in the metformin-treated
Curr. Issues Mol. Biol. 2023, 45 7508

group, indicating that the extracts can help relieve oxidative stress. As insulin resistance is
associated with difficulties in regulating glucose production owing to impaired glucose
absorption and glycogen synthesis, the therapeutic effects of SH and UA extracts have been
confirmed [51]. Glucose uptake and glycogen content decreased in insulin-resistant HepG2
cells but were alleviated in a concentration-dependent manner in the experimental group
treated with the extract. This was similar to the metformin-treated positive control group,
suggesting that SH and UA can help alleviate insulin resistance.
In general, the IRS-1/PI3K/AKT signaling pathway plays an essential role in regu-
lating glucose metabolism, including glycogen synthesis, promotion of glucose synthesis,
and inhibition of angiogenesis in the liver [52]. The activation IRS-1 further activates other
signaling pathways, such as the PI3K/AKT pathway, followed by the activation of targets
such as GLUT4, GSK-3β, and PEPCK [53]. However, when insulin resistance is induced,
the signaling of IRS-1, an insulin receptor substrate, and AKT, which regulates glucose
and lipid metabolism, is downregulated, thereby affecting the expression of downstream
factors. Treatment of insulin-resistant HepG2 cells with SH and UA extracts upregulated
IRS-1/AKT and GLUT4 expression. These findings are consistent with those of a previ-
ous report showing that monounsaturated fatty acids in seaweeds can help preserve the
IRS/PI3K insulin pathway and increase GLUT4 translocation [54]. In addition, FoxO1, a
significant target of AKT, lowers the glucose level and induces the expression of PEPCK
to enhance gluconeogenesis [55,56], and GSK-3β is known to regulate glycogen synthase,
an enzyme that stores glycogen [57]. In the present study, as the expression of AKT was
upregulated by treatment with SH and UA extracts, the gene expression of FoxO1, a major
target, was downregulated, similar to that of PEPCK and GSK-3β. These results suggest
that the two seaweed extracts inhibit or improve the expression of key transcription factors
involved in insulin resistance.
Diabetic mice with STZ-induced diabetes are a model similar to human diabetes; there-
fore, they have been widely used to investigate antidiabetic activity [58]. Streptozotocin
has been previously reported to induce diabetes by causing the death of pancreatic β cells
by alkylation of DNA, reducing insulin synthesis and release [59]. In the present study, the
positive control substances, metformin, SH, and UA, were orally administered for 6 weeks
to confirm their antidiabetic effects on streptozotocin-induced ICR mice. As weight loss is
a common symptom of diabetes, no significant weight change was observed in the experi-
mental group with induced diabetes until the second week. Weight gain in the diabetic
group decreased by 0.86 g; the metformin- and SH-treated groups showed an increase of
1.25 g and 1.12 g, respectively, while the UA group showed the highest increase rate of 2.54
g. These findings are consistent with previous findings that Sargassum wightii methanol
extract and Caulerpa racemose extract prevent weight loss in diabetic animals [60,61].
Insulin resistance and oral glucose tolerance tests are routinely used to assess glycemic
control in diabetic animals [62]. Insulin and oral glucose tolerance according to the admin-
istration of the extract were compared using the AUC of the blood glucose graph; the AUC
of the substance-administered group was significantly lower than that of the diabetic group,
indicating an improvement in glucose tolerance. Seaweeds of the genus Sargassum contain
phlorotannins and phloroglucinol, which regulate blood glucose levels by delaying carbo-
hydrate absorption in streptozotocin-induced diabetic mice [63]. Polysaccharides extracted
from Ulva lactuca inhibit important enzymes involved in carbohydrate and fat metabolism
in blood and the small intestine [64]. The decrease in blood glucose levels following SH
and UA administration was thought to be caused by components of the seaweed.
In patients with diabetes, the accumulation of cholesterol and triglycerides is accom-
panied by a reduction in high-density lipoprotein cholesterol levels; these changes are
major risk factors for coronary artery disease [65]. In addition, persistent hyperglycemia
affects the permeability of the hepatocyte membrane, causing hepatotoxicity or glomerular
filtration dysfunction; therefore, serological analysis is an important indicator [66,67]. AST
and ALT levels, which are indicators of liver function, were significantly higher in all
groups than in the normal group. At the same time, the ALT levels were significantly lower
Curr. Issues Mol. Biol. 2023, 45 7509

in the UA group compared to those in the diabetic group. These results suggest that UA
alleviates chronic hyperglycemia-induced liver damage more effectively than SH. Among
the blood lipid test parameters, triglyceride and cholesterol levels were significantly lower
in the metformin, SH, and UA groups than in the diabetic group. HDL cholesterol levels
significantly increased only in the metformin and UA groups. These results are believed to
be attributable to the finding that high omega-6 fatty acids in Ulva can reduce insulin resis-
tance through other mechanisms, including anti-inflammatory action, and triglyceride and
low-density lipoprotein reduction [68]. Several studies have reported hepatomegaly due to
hepatic steatosis and glycogen accumulation in patients with diabetes [69]. Comparing the
changes in liver tissue weight after treatment with the two seaweed extracts, we found that
hepatomegaly was significantly reduced in the UA and metformin groups compared to
that in the diabetic group.
Therefore, the results of the present study confirmed that SH and UA possess alpha-
glucosidase inhibitory activity in vitro, suggesting that the two seaweed extracts may
be candidates to improve intracellular insulin resistance. In addition, compared with
metformin, a positive control substance in diabetic animal models, both seaweeds could
control blood sugar, but the effect of UA was found to be more profound. These results
suggest that SH and UA, recognized as environmental pollutants in Jeju, can be used as
functional additives for the treatment of diabetes and as healthy food candidates. However,
in future studies, it will be necessary to investigate the active ingredients in SH and UA by
analyzing fractions prepared using various organic solvents.

Author Contributions: Conceptualization, K.-S.C., H.-S.K. and H.-R.K.; methodology, Y.-H.L. and
M.-H.Y.; software, S.-C.K., H.-B.H. and Y.-M.H.; validation, Y.-H.J. and M.-H.Y.; formal analysis,
Y.-H.L. and H.-S.K.; investigation, H.-R.K.; resources, Y.-H.J.; data curation, Y.-H.L. and K.-S.C.;
writing original draft preparation, Y.-H.L., H.-R.K. and K.-S.C.; writing—review and editing, S.-C.K.,
H.-B.H. and Y.-M.H.; funding acquisition, Y.-H.J. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was supported by the Jeju Special Self-Governing Province’s Organic Waste
Industrialization Project and the National Research Foundation of Korea (NRF) project funded by the
Korean government (MSIT) (RS-2022-00165637 to H.-R.K.).
Institutional Review Board Statement: The animal study protocol was approved by the Animal
Experimentation Ethics Committee of the Catholic University of Pusan (CUP AEC 2022-003).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
DM: Diabetes mellitus
IR: insulin resistance
SH: Sargassum horneri
UA: Ulva australis
ROS: Reactive oxygen species
STZ: streptozotocin
NC: Normal control
DC: Diabetic control
Met: Metformin
AUC: area under the curve
AST: aspartate aminotransferase
ALT: alanine aminotransferase
ALP: alkaline phosphatase
T-P: total protein
TG: triglycerides
Curr. Issues Mol. Biol. 2023, 45 7510

CHO: total cholesterol

HDL-C: high-density lipoprotein cholesterol
GLU: glucose
BUN: blood urea nitrogen
CRE: creatinine

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