Phytomedicine Plus 1 (2021) 100109

Contents lists available at ScienceDirect

Phytomedicine Plus
journal homepage: www.sciencedirect.com/journal/phytomedicine-plus

Fruits of Rosa laevigata and its bio-active principal sitostenone facilitate

glucose uptake and insulin sensitivity in hepatic cells via AMPK/
PPAR-γ activation
K.J. Senthil Kumar a, b, Chun Lin a, Yen-Hsueh Tseng a, c, Sheng-Yang Wang a, d, *
a
Department of Forestry, National Chung Hsing University, Taichung 402, Taiwan
b
Bachelor Program of Biotechnology, National Chung Hsing University, Taichung 402, Taiwan
c
Taiwan Forestry Research Institute, Taipei 100, Taiwan
d
Agricultural Biotechnology Research Institute, Academia Sinica, Taipei 128, Taiwan

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Fruits of Rosa laevigata Michx is commonly used in traditional Chinese medicine for treating of
Rosa laevigata spleen deficiency, chronic diarrhea, and chronic urinary tract infection. Recently, fruits of R. laevigate have been
Rosaceae shown to have renal protective effects in diabetic rats. However, up to now, no studies have been reported on the
Sitostenone
anti-hyperglycemic or anti-diabetic effect of R. laevigata or its derived compounds.
Insulin resistance
Insulin sensitivity
Purpose: Therefore, this study investigated the anti-diabetic effect of ethanol extract of R. laevigata (EtRL) fruits,
Gluconeogenesis its derivate sub-fractions, and pure compounds in terms of glucose-lowering effect on hyperglycemia-induced
hepatic cells in vitro.
Methods: We investigated the anti-hyperglycemic effect of EtRL and its derivate sub-fractions (water, n-butanol,
ethyl acetate, and n-hexane), and its major bioactive compound, sitostenone, in normal and high glucose-induced
insulin-resistant hepatic HepG2 cells using in vitro DNS glucose uptake and Western blotting assays.
Results: Treatment with EtRL and its derivate sub-fractions significantly increased glucose uptake by hepatic cells.
Besides, co-treatment with insulin further improved glucose uptake in insulin-resistant cells. Notably, compared
with all soluble fractions, the n-hexane sub-fraction displayed the strongest activity in the context of glucose
consumption. Furthermore, sitostenone, a primary bioactive compound was isolated from n-hexane sub-fraction
by bioactivity-guided fractionation procedure. Sitostenone treatment significantly increased glucose uptake,
whereas there was no further increase when co-treatment with insulin. Indeed, sitostenone significantly
increased glucose uptake and promote insulin sensitivity in insulin-resistant cells. Further analysis revealed that
sitostenone activates proteins involved in the insulin signal transduction pathway, including insulin receptor
substrate-1 (IRS-1), AKT, and glycogen synthase kinase 3 β (GSK3β). It was also found that sitostenone provoked
glucose uptake in insulin-resistant cells via peroxisome proliferator-activated receptor-γ (PPAR-γ) and AMP-
activated protein kinase (AMPK) activation, which facilitates up-regulation of glucose transporters, GLUT2
and GLUT4 in the cell membrane and down-regulation of Forkhead box protein O1 (FOXO1) in the nucleus.
Conclusion: This study demonstrating that sitostenone, a steroid-like compound from fruits of R. laevigata has a
promising ability to promote glucose uptake and repairs insulin resistance in hepatic cells.

Introduction diabetes, about 90% of cases belong to type-2 diabetes (WHO, 2020).
According to World Health Organization report, the number of patients
Diabetes mellitus is a chronic disease caused by insufficient insulin or with diabetes increased from 108 million to 422 million between 1980
insulin resistance in the human body. Diabetes patients often cause and 2014 (WHO, 2020). Among them, the prevalence of diabetes among
blindness, kidney failure, heart disease, stroke, and other diseases and people over 18 increased from 4.7% to 8.5%. In 2016, diabetes caused
lead to lower limb amputation (Tabish, 2007). In all patients with 1.6 million deaths directly, and it has been listed as one of the four major

* Corresponding author at: Department of Forestry, National Chung Hsing University, No. 145, Xinda Rd, Taichung 402, Taiwan.
E-mail address: [email protected] (S.-Y. Wang).

https://doi.org/10.1016/j.phyplu.2021.100109
Received 8 April 2021; Received in revised form 15 May 2021; Accepted 9 August 2021
Available online 11 August 2021
2667-0313/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

non-communicable diseases (WHO, 2020). fruits of R. laevigata, among them 15 compounds exhibited strong
At present, the drugs’ mechanism for treating diabetes is reducing anti-oxidant activity in DPPH and FRAP assays, and β-carotene linoleate
the body’s absorption of glucose, promoting insulin secretion, using model system (Li et al., 2012). Another study has found that fruits of
insulin sensitizers, and direct injection of insulin. However, these drugs R. laevigata possessed strong anti-inflammatory activity as evidenced by a
also bring many side effects while improving diabetes, such as significant reduction in particulate matter-10 (PM-10)-induced expres­
α-glucosidase inhibitors that inhibit polysaccharides decomposition in sion of pro-inflammatory genes including TNF-α, IL-1β, IL-6, IL-13, and
the small intestine and block sugars absorption which can cause IL-17 in human lung epithelial cells (Ko et al., 2020). Since, the fruits of
gastrointestinal discomfort under long-term use (Derosa and Maffioli, R. laevigata possessed strong anti-oxidant and anti-inflammatory proper­
2012). α-Glucosidase inhibitors-associated side effects include diarrhea, ties, we hypothesize that fruits of R. laevigata and its derived pure com­
sulfonylureas, glibenclamide, which stimulates pancreatic β-cells to pounds may have anti-diabetic effect. Also, so far, there is no study has
secrete insulin, which increase mortality risk from cardiovascular dis­ been conducted on the anti-diabetic activity of R. laevigata or its derived
eases (Derosa and Maffioli, 2012). Thiazolidinedione (TZDs) that in­ pure compounds. Therefore, this study explored the anti-diabetic prop­
creases insulin sensitivity can cause weight gain and edema (Ko et al., erties of ethanol extract of R. laevigata fruits and its bioactivity
2017). Besides, high levels of insulin in circulation increase various compounds.
forms of cancer incidence (Dankner et al., 2012; Orgel and Mittelman,
2013). Therefore, it is an important research topic to find compounds Materials and methods
that improve diabetic conditions or treat diabetes with minimal side
effects. Plant materials
Oxidative stress plays a pivotal role in the pathogenesis of type-2
diabetes and its complications (Giacco and Brownlee, 2010). When the Fresh fruits of R. laevigata were collected from the Hui-Sun Experi­
imbalance between excessive production of reactive oxygen species and mental Forest Station of National Chung Hsing University (Nantou
a limited number of endogenous anti-oxidants that this damage can County, Taiwan) in July 2015. The species was identified and confirmed
become debilitatinged and cumulative. Various factors involved in by Prof Yen-Hsueh Tseng (Department of Forestry, National Chung
oxidative stress control, among them the detoxifying enzymes including Hsing University). The voucher specimen (YHT0021 [TCF]) was
catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase deposited in the herbarium of the same university.
(GSH-Px), hemeoxygenase-1 (HO-1), and NAD(P)H quinone
dehydrogenase-1 (NQO-1) are the first line of defense. The variation in Extraction and purification
the levels of these enzymes makes the tissue susceptible to oxidative
stress and leading to the development of diabetic complications. In such Air-dried R. laevigata fruits (3.87 kg) were extracted with 70% EtOH
conditions, exogenous anti-oxidants has a remarkable role (Birben et al., (30 L) at ambient temperature and concentrated under vacuum to yield
2012). Indeed, herbal extracts and natural products are potent the EtOH extract (EtRL; 412.3 g). Based on a previous work, the EtRL
anti-oxidants, which counterpart the activity of the endogenous anti­ extract was partitioned between n-hexane, ethyl acetate, and n-butanol
oxidant defense. Accumulating evidence suggests that plant polyphenols with water to give n-hexane-soluble (19.5 g), ethyl acetate-soluble (20.5
possess high anti-oxidant activity through direct free radical scavenging g), n-butanol-soluble (150.3 g), and water-soluble (53.8 g) fractions (Li
or activating the endogenous anti-oxidant defense mechanism (Pandey et al., 2008). HPLC further separated the n-hexane-soluble fraction with
and Rizvi, 2009). Additionally, researchers have identified that a mixture of ethyl acetate and n-hexane solution. 0 to 5 min, ethyl ac­
inflammation is strongly associated with diabetes. The levels of etate:n-hexane = 5: 95; 10 to 15 min, ethyl acetate: n-hexane = 10 : 90;
pro-inflammatory molecules, including nitric oxide (NO), prostaglandin 20 to 30 min ethyl acetate : n-hexane = 15 : 85 at a flow rate of 3
E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and mL/min. The wavelength of the UV detector set at 254 nm, and the
IL-6 were often found higher in people with type-2 diabetes compared to obtained compounds were analyzed by LC-MS and 400 MHz NMR. A
people without diabetes. Indeed, abnormal inflammation alters insulin’s bioactive compound, sitostenone, was isolated at the retention of 23 to
activity and contributes to disease progression. Alternatively, when 25 min following bioactivity-guided isolation protocol. The structure of
body becomes less sensitive to insulin, in such condition called insulin the compound was analyzed by electrospray ionization (ESI) and LC-MS
resistance leads to inflammation (Xie and Du, 2011). A vast array of was used to detect the molecular weight (m/z) of the compound and its
phytochemicals derived from medicinal plants or herbs exhibited fragments. For structural identification, the compound was dissolved in
notable anti-inflammatory properties in vitro and in vivo and these deuterated chloroform (d-chloroform; CDCl3) for detection, and the
phytoconstituents play a functional role in lowering blood glucose and hydrogen nucleus (1H NMR) or carbon nucleus (13C NMR) in the com­
controlling inflammation or limiting pro-inflammatory molecules pro­ pound was detected under a magnetic field. The difference in resonance
duction and breaking insulin resistance (Kong et al., 2021). frequency between tetramethylsilane and tetramethylsilane (called
Rosa laevigata (Rosaceae), also known as Cherokee rose, is a fragrant chemical shift, and expressed in δ value (ppm)) was analyzed. Alterna­
rose native to southern China and Taiwan. The fruits of R. laevigata are tively, 2D-NMR analysis, such as distortionless enhancement by polari­
widely used as a functional food in Asian countries to prepare marmalade, zation transfer (DEPT) , heteronuclear single quantum coherence
garnishes, fruit pies, and wines (Zhan et al., 2020). In traditional Chinese (HSQC) and heteronuclear multiple bond correlation (HMBC) was per­
medicine, used the fruits to treat arterial sclerosis, urinary incontinence, formed to determine the chemical structure of the sample. The spectral
chronic cough, and menstrual irregularities (Tao et al., 2016). Pharma­ data of sitostenone was correspondent to a previous report (Li et al.,
cological studies have revealed that R. laevigata possessed various 2008).
bio-pharmacological activities, including hypolipidemic (Zhang et al.,
2020); immunomodulatory (Zhan et al., 2020); hepato-protection (Dong Chemicals and reagents
et al., 2018); neuroprotection (Liu et al., 2018) and cardio-protection
(Luo et al., 2016). These biological activities are attributed to the pres­ Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum
ence of various bioactive compounds, including rutin, quercetin, (FBS), penicillin, and streptomycin were purchased from Gibco/Invi­
kaempferol, luteolin, apigenin, liquiritigenin, isorhamnetin, chlorogenic trogen (Carlsbad, CA). 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl
acid, 4-hydroxy-3-methoxybenzoic acid, kaempferide-3-O-glucoside, tetrazolium bromide (MTT) and metformin were purchased from Sig­
quercetin-3-rhamnoside, isorhamnetin-3-O-β-rutinoside, and so on (Li ma–Aldrich (St. Louis, CA). Antibodies against AKT, IRS-1, PPARγ,
et al., 2012; Yan et al., 2013; Zeng et al., 2011). A previous study has GSK3β, FOXO1, GLUT4, phospho-IRS-1Tyr895, phospho-AKTSer473,
demonstrated that 18 polyphenol compounds were isolated from the phospho-GSK3βSer9, phospho-AMPKαT172, and HRP-conjugated goat

2
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

anti-mouse IgG were obtained from Cell Signaling Technology (Denvers, insulin resistance. After insulin resistance induction, the cells were
MA). Antibodies against AMPKα and HRP-conjugated goat anti-rabbit washed with PBS to remove excess glucose, and then the subsequent
IgG were purchased from Millipore (Temecula, CA). The antibody experiments were performed.
against β-actin was purchased from Santa Cruz Biotechnology (Dallas,
TX). The antibody against GLUT2 was bought from Proteintech (Rose­ Determination of reducing sugar content
mont, IL). All other chemicals were of the highest grade commercially
available and supplied either by Merck (Darmstadt, Germany) or Sigma. 3,5-Dinitrosalicylic acid (DNS) can react with reducing sugars or
other compounds with reducing ability to form 3-amino-5-nitrosalicylic
Cell culture and cell viability assay acid, which can be absorbed in a maximum absorption of a wavelength
of 540 nm as described previously (Garriga et al., 2017). After cell
Human hepatocellular carcinoma (HepG2) cell line was purchased treatment, the culture supernatant was deprived. To measure the
from the American Type Culture Collection (ATCC, Manassas, VA) and glucose concentration in the culture medium, 200 µL of the culture
cultured in low-glucose Dulbecco’s modified Eagle’s medium (DMEM), medium and 200 µL of DNS reagent was added to the 1.5 mL eppendorf
supplemented with 10% feral bovine serum (FBS), 1 mM sodium pyru­ and the mixture was heated at 100◦ C for 5 min. After cooling to room
vate, glucose (5.5 mM), penicillin (100 U/mL) and streptomycin (100 temperature, the absorbance of the supernatant was measured at 540
µg/mL). Cells were grown in 10-cm culture dishes and incubated in a nm (OD540) using ELISA micro-plate reader. The relative glucose con­
humidified atmosphere containing 5% CO2 at 37◦ C. For cell viability sumption in cells is calculated according to the following formula.
assay, HepG2 cells were treated with increasing concentrations of EtRL Glucose consumption (%) = [1-(A – B)/B] × 100; were as A is the OD540
(20, 40, and 80 μg/mL), soluble fractions (10, 20 and 40 μg/mL) or of medium of cell culture, B is the OD540 of medium without cell.
sitostenone (10, 20, 25, 30, 50, 100, 250, 500, 750, 1000 μM). All the
test samples were dissolved in dimethyl sulfoxide and an equal volume Statistical analysis
of DMSO (0.1%) was used as vehicle control. Cell viability was assessed
by MTT colorimetric assay as described previously (Senthil Kumar et al., Data are expressed as mean ± SD. All data were analyzed using the
2012). statistical software Graphpad Prism version 6.0 for Windows (GraphPad
Software, La Jolla, CA). Statistical analysis was performed using one-
Protein extraction and Western blotting way ANOVA followed by Dunnett’s test for multiple comparison. P
values of less than 0.05*, 0.01**, and 0.001*** were considered statis­
HepG2 cells (1 × 106 cells/dish) were seeded in 6-cm dish and tically significant for the control vs samples treatment groups. P value of
incubated overnight, after culture media were replaced with serum-free less than 0.001Δ was considered statistically significant for the control vs
medium for 24 h. Then the cells were treated with sitostenone or met­ high glucose (insulin-resistant) group. P value of less than 0.001Φ was
formin (2 mM) in the presence or absence of insulin (100 nM) for 6-24 h. considered statistically significant for the control vs insulin alone
Cells were lysed with either mammalian protein extraction reagent (M- treatment group.
PER) or radio-immuno precipitation assay (RIPA) buffer (Pierce
Biotechnology, Rockford, IL). Protein concentrations were determined Results and discussion
using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules,
CA). Equal amounts of protein samples (60-100 μg) were separated by 8- Many studies are confirming that Rosaceae plants can improve the
12% SDS-PAGE and the separated proteins were transferred onto poly­ symptoms of diabetes. For example, Malus toringoides can improve the
vinylidene fluoride (PVDF) membrane overnight. The transferred pro­ symptoms of diabetes in rodents by reducing blood glucose and
tein membranes were blocked with 5% nonfat skim milk for 30 min, increasing insulin (Li et al., 2014); Agrimonia pilosa reduces high-fat
followed by incubation with specific primary antibodies overnight, and diet-induced inflammation in rats through regulating blood sugar bal­
then incubated with either horseradish peroxidase-conjugated anti- ance (Jang et al., 2020); the pentacyclic triterpenoids from Eriobotrya
rabbit or anti-mouse or anti-goat antibodies for 2 h. Immunoblots were japonica break the insulin resistance and reduce fat accumulation in
developed with the enhanced chemiluminescence reagents (Advansta high-fat diet fed mice (Shih et al., 2010); phenolic compounds from
Inc., San Jose, CA), images were captured by ChemiDoc XRS+ docking Prunus avium exhibited blood sugar lowering, anti-oxidation, α-amylase,
system, and the protein bands were quantified using Imagelab software and α-glucosidase inhibitory activity, and increase in glucose uptake and
(Bio-Rad). insulin secretion (Noratto et al., 2018); also, phenolic compounds of
strawberries and cranberries can increase insulin sensitivity (Paquette
Glucose uptake assay et al., 2017). These studies provided a strong base that the Rosaceae
plants have great potential for the develpment of anti-diabetic agents .
HepG2 cells were seeded into 48-well cell culture plate with a cell Additionally, the role of oxidative stress and inflammation in the path­
density of 1.5 × 105 cells/well in low glucose (5.5 mM) DMEM con­ ogenesis of type-2 diabetes and its complications were confirmed by
taining 10% FBS. The culture plate was incubated for overnight to interventional studies. It has been shown that Chinese medicinal herbs
ensure cell attachment. Then the cells were replaced with serum-free reduce the incidence of type-2 diabetes and its vascular complications
low glucose DMEM. After cells were starved for 24 h, cells were partially via anti-inflammatory mechanisms. Xie and Du (2011) sys­
treated with samples for 6 h. Insulin or metformin was served as positive tematically listed most frequently used Chinese herbs including, Radix
drug control. Glucose levels in the culture supernatant were measured astragali, Radix rehmanniae, Radix trichosanthis, Panax ginseng, Fructus
using commercially available glucose assay kit (Abcam, Cambridge, schisandrae, Radix ophiopogonis, Rhizoma anemarrhenae, Radix puerariae,
UK). Fructus lycii, Poria sp, Rhizoma coptidis, Rhizoma dioscoreae, Rhizoma
polygonati, Radix salviae miltiorrhizae, Radix glycyrrhizae, Semen trig­
Insulin resistance model onellae, Momordica charantia, Allium sativum, Opuntia stricta, Aloe vera,
Cortex Cinnamomi, Rhizoma Curcumae longae for the treatment of dia­
HepG2 cells were seeded into 48-well cell culture plate with a cell betes through its anti-inflammatory action. These herbs exhibit
density of 1.5 × 105 cells/well in low glucose (5.5 mM) DMEM con­ anti-inflammatory effects in various in vitro and in vivo models by
taining 10% FBS. The culture plate was incubated for overnight to inhibiting the expression of inducible nitric oxide synthase (iNOS),
ensure cell attachment. Then the cells were replaced with serum-free cyclooxygenase-2 (COX-2), IL-1β, IL-6, and TNF-α, while also reducing
high glucose (30 mM) DMEM for 24 h to induce the cells to produce production of NO and PGE2. Their anti-inflammatory effects is

3
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

attributed to the inactivation of their corresponding transcriptional plumbagin, a known cytotoxic drug, significantly induced cell death in
factors including, nuclear factor -κB (NF-κB) and/or mitogen-activated HepG2 cells (Fig. 1F). These data are consistent with other observations
protein kinases (MAPKs). Morvaridzadeh et al (2020) systematically that aqueous extract of R. laevigata does not show cytotoxicity to HepG2
reviewed and reported that supplementation of ginger, one of the cells (Ko et al., 2015), human lung epithelial cells (Lee et al., 2020), and
common ingredients in Chinese herbal medicine markedly reduced human lens epithelial cells (Liu et al., 2015) up to a maximum con­
C-reactive protein (CRP) and TNF-α levels, but not IL-6 in diabetic pa­ centration of 300 μg/mL, 1000 mg/mL and 10 mg/mL, respectively.
tients compared to non-diabetic patients. Additionally, ginger supple­
mentation was found to be reduce blood sugar and ameliorate blood
lipids through its anti-oxidant property. Another detailed analysis EtRL and its soluble fractions promote glucose uptake in hepatic cells
indicating that lemon balm intake significantly reduced blood pressure
and inflammatory markers, including CRP, whereas serum lipid profile Type-2 diabetes is characterized by insufficient secretion of insulin
and glycemic parameters were not significantly altered by lemon balm from β-cells of the pancreatic islets resulted in hyperglycemia (glucose
(Heshmati et al., 2020). A recent study has shown that R. laevigata in­ accumulation in the circulation). The most direct way to improve the
hibits expression levels of inflammatory genes, including IL-1β, IL-6, symptoms of hyperglycemia is to inject insulin to encourage the target
IL-13, IL-17, and TNF-α in PM-10-challanged human lung epithelial cells, such as muscle, fat and liver cells to uptake glucose to achieve the
cells (Ko et al., 2020). In this study, we demonstrated that ethanol purpose of lowering blood sugar (Cantley and Ashcroft, 2015). How­
extract of R. laevigata (EtRL) fruits, its derivate sub-fractions, and its ever, several in vitro and in vivo experiments revealed that high insulin
major bioactive compound sitostenone promote glucose uptake and concentrations can also result in impaired insulin action in targeted
repairs insulin resistance in hyperglycemia-induced hepatic cells in vitro. tissues, a condition called insulin resistance (Wilcox, 2005). Therefore,
any chemical agent with similar insulin function can be used to replace
insulin and also reduce insulin-associated complications. Many plant
Cytotoxicity of EtRL on hepatic cells extracts, and phytochemicals were reported to mimic insulin (Broad­
hurst et al., 2000; Patel et al., 2012; Teoh and Das, 2018). A previous
Before the experiment, the EtRL’s cytotoxicity against human hep­ study by Zhou et al. (2012) reported that the aqueous extract of
atoma HepG2 cells was determined by MTT colorimetric assay. As R. laevigata protects rat’s kidneys from streptozotocin-induced oxidative
shown in Figure 1, treatment with either EtRL or its derivative soluble stress and diabetic nephropathy through the inhibition of the NF-κB
fractions, including water, n-butanol, ethyl acetate, and n-hexane does signaling pathway. Another study by Liu et al. (2015) revealed that
not show cytotoxicity against HepG2 cells up to a treatment concen­ aqueous extract of R. laevigata protects human lens epithelial cells from
tration of 80 μg/mL. The observed cell survival rates were 101.5%, hyperglycemia-induced oxidative stress via up-regulation of
106.8%, 100.8%, 107.8%, and 106%, by EtRL (Fig. 1A), water (Fig. 1B), Nrf-2-mediated anti-oxidant genes in vitro. However, there is no report
n-butanol (Fig. 1C), ethyl acetate (Fig. 1D), and n-hexane (Fig. 1E) indicating the insulin-mimetic effect of R. laevigata or its derived com­
fractions, respectively, indicating that the ethanol extract and the sol­ pounds. Thus, this study investigated whether the ethanol extract of
uble fractions are not cytotoxic to HepG2 cells. Cells exposed to R. laevigata has an insulin-like ability to promote glucose uptake.

Fig. 1. Cytotoxic effects of ethanol extract of R. laevigata fruits (EtRL) and its derivative sub-fractions on human hepatic cells. HepG2 cells were incubated with
increasing concentrations of (10, 20, 40 and 80 μg/mL) EtRL (A), and its souluble fractions, including water (B), n-butanol (C), ethyl acetate (D), n-nexane (E) and a
positive drug control plumbagin (25, 50 and 100 μM) for 24 h. The cell survival was analyzed by MTT colorimetric assay. Values represent the mean ± SD of three
independent experiments. Statistical significance ***P < 0.001 was compared to control vs. sample treatment groups.

4
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

Fig. 2. The ethanol extract of R. laevigata fruits (EtRL) and its derivative souluble fractions promote glucose uptake in hepatic cells. (A) HepG2 cells were treated with
different concentrations of EtRL (20, 40 and 80 μg/mL) for 6 h or various concentrations of (10, 20, 40 μg/mL) EtRL soluble fractions, such as water (B), n-butanol
(C), ethyl acetate (D), and n-hexane for 6 h. 100 nM of insulin was subjected as positive control. After treatment, culture supernatant was collected and glucose
concentration was tested by DNS method. Values represent the mean ± SD of three independent experiments. Statistical significance **p < 0.01, ***p < 0.001 were
compared to control vs. sample treatment groups.

Glucose concentration in the culture media was determined by DNS insulin resistance in hepatic cells (Leclercq et al., 2007). It was well
assay. As shown in Figure 2A, a significant increase in glucose uptake demonstrated that hepatic cells were exposed to high glucose provoking
was observed after treatment with EtRL for 6 h. Compared to that of insulin resistance in vitro (Boonloh et al., 2015). Therefore, next, we
basal uptake (30.5%), cells incubated with 20, 40, and 80 μg/mL of EtRL sought to examine whether EtRL and its derived soluble fractions could
increased glucose uptake to 31.2%, 34.4% and 36.2%, respectively. improve glucose uptake in insulin-resistant hepatic cells. Insulin resis­
Alternatively, cells exposed to 100 nM insulin significantly increased tance in HepG2 cells was induced by high concentration of glucose (30
glucose uptake to 41.1%. These data strongly suggest that EtRL can mM) for 24 h; then the cells were washed with PBS and incubated with
promote glucose uptake as well as a mimic insulin. increasing concentrations of EtRL or its derived soluble fractions for 6 h.
Next, to further evaluate the glucose uptake activity of EtRL-derived Glucose concentration in the culture media was determined by DNS
soluble fractions, cells were incubated with increasing concentrations of assay. The result showed that a significant decrease in glucose con­
water, n-butanol, ethyl acetate, and n-hexane fractions for 6 h, and the sumption was noted in insulin-resistant HepG2 cells compared with
glucose concentration in the culture media was determined by DNS control cells (Fig. 3A). This result indicates that high glucose concen­
assay. Results showed that the basal glucose consumption by the control tration can produce insulin resistance in HepG2 cells, and this assay can
was noted as 29.4%, while treatment with water (Fig. 2B), n-butanol be used to determine insulin impedance in vitro. This result is also well
(Fig. 2C), ethyl acetate (Fig. 2D), and n-hexane (Fig. 2E) fractions correlated with other observations that 30 mM glucose induces insulin
significantly increased glucose uptake in HepG2 cells. Particularly, cells resistance in HepG2 cells (Xuguang et al., 2019).
exposed to high dosages of ethyl acetate and n-hexane fractions (40 μg/ Next, we found that insulin treatment significantly increased glucose
mL) increased glucose uptake to 35.5% and 39.2%, respectively. uptake in insulin-resistant cells. In contrast, a profound increase in
Notably, the effect of glucose uptake by n-hexane fraction was compa­ glucose uptake was noted when cells were dose-dependently co-incu­
rable to that of the insulin group, which showed an increase of 39.3%. bated with EtRL (Fig. 3A). Compared with the insulin resistance group
(25.5%), glucose consumption was significantly provoked in high doses
EtRL and its soluble fractions promote glucose uptake insulin-resistant of EtRL (40 and 80 μg/mL) treated cells. The effect of EtRL on glucose
hepatic cells uptake in insulin-resistant cells was highly comparable to that of met­
formin, a biguanide agent that reduces hyperinsulinaemia and improved
Insulin-resistant glucose uptake is a prominent hallmark of type-2 hepatic insulin resistance (Li et al., 2019). This result strongly suggests
diabetes, characterized by reducing sensitivity or responsiveness of that it can improve insulin resistance in human hepatic cells.
muscle, liver, and adipose tissue to the metabolic actions of insulin in Next, we examined the effect of EtRL derived soluble fractions on
human and experimental models of diabetes in rodents (Czech, 2017). glucose uptake in insulin-resistant cells. The result showed that
This defect is evident in vitro as reduced glucose uptake responds to compared with insulin-resistant cells, co-treatment of insulin and water

5
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

Fig. 3. The ethanol extract of R. laevigata fruits (EtRL) and its derivative soluble fractions promote glucose uptake in insulin-resistant hepatic cells. To induce insulin-
resistant, HepG2 cells were pre-incubated with 30 mM of D-glucose for 24 h, then the cells were treated with different concentrations of (20, 40, and 80 μg/mL) EtRL
(A) for 6 h or various concentrations (10, 20, and 40 μg/mL) of EtRL soluble fractions, such as water (B), n-butanol (C), ethyl acetate (D), and n-hexane for 6 h with or
without 100 nM of insulin. 2 mM of metformin was utilized as positive drug control. After treatment, culture supernatant was collected and glucose concentration
was tested by DNS method. Values represent the mean ± SD of three independent experiments. Statistical significance Δp < 0.001 compared to control vs. high
glucose treatment (insulin-resistant) group; *p < 0.05, **p < 0.01, ***p < 0.001 were compared to insulin-resistant vs. sample treatment groups.

(Fig. 3B) or n-butanol (Fig. 3C) or ethyl acetate (Fig. 3D) or n-hexane fraction, HPLC was performed to separate the compounds in the soluble
(Figure 3E) soluble fractions significantly improved glucose uptake in n-hexane fraction. The isolated compounds were tested their biological
insulin-resistant HepG2 cells. These fractions increased glucose con­ activity based on DNS glucose uptake assay. Among them, a compound
sumption from 24.5% to 26.9%, 30.2%, 29.9%, and 31.6%, respectively. appearing in the retention time between 23 and 25 min was found to
Indeed, all the fractions significantly improved insulin resistant. The n- have potent glucose uptake activity. We further purified and obtained a
hexane soluble fraction produced high effect, which is close to the colorless solid component. The molecular ion peak measured by ESI-MS
metformin, a positive drug control, improved insulin resistance by is [M+1]+ m/z 413, and the molecular weight of the compound is 412
provoking increased glucose uptake (31.1%). along with NMR analysis, the compound was identified as sitotenone (Li
et al., 2008) (Fig. 4A). To quantify the amount of sitostenone in
R. laevigata, further quantitative analysis of sitostenone was conducted.
Separation, identification and quantification of sitostenone
According to the quantitative results, it was found that 1 g of the EtRL
containing 4.9 mg of sitostenone (0.49%; w/w), i.e. 1 kg of R. laevigata’
To obtain a major active compounds from the n-hexane-soluble

Fig. 4. Chemical structure and cytotoxic effect of sitostenone on human hepatic cells. (A) Chemical structure of sitostenone. (B) HepG2 cells were incubated with
increasing doses of sitostenone (25, 50, 100, 250, 500, 750, and 1000 μM) for 24 h. The cell survival was analyzed by MTT assay. Values represent the mean ± SD of
three independent experiments. Statistical significance **p < 0.01, ***p < 0.001 were compared to control vs. sample treatment groups.

6
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

fruits contain 518 mg sitostenone. side-chain, attached at C–24. For example, 4–methyl and 24–ethyl
conjugate at C-24 of cholesterol and produce campesterol and sitosterol,
The cytotoxic effect of sitostenone on hepatic cells respectively (Tapiero et al., 2003). Phytosterols are highly regarded for
their cholesterol-lowering effect by competitively blocking cholesterol
To determine the cytotoxicity of sitostenone, HepG2 cells were absorption from the intestinal lumen (Tapiero et al., 2003). Tanaka
incubated with increasing concentrations of sitostenone for 24 h, the cell et al. (2006) reported five sterols (lophenol, 24-methyl-lophenol,
viability was determined by MTT colorimetric assay. As shown in 24-ethyl-lophenol, cycloartanol, and 24-methylene-cycloartanol) from
Figure 4B, treatment with sitostenone (25, 50, 100, 250, 500, 750, and Aloe vera gel reduced blood glucose in the diabetic mice. A recent study
1000 μM) for 24 h has no significant cytotoxicity in HepG2 cells, the cell by Ramalingam et al. (2020) found that treatment with β-sitosterol
survival rates were noted as 104.38%, 96.7%, 96.3%, 92.8%, 89.0%, significantly reduced the plasma glucose level in high-fat diet and
84.6%, and 80.4%, respectively. The cell survival rate was significantly streptozotocin-induced diabetic mice. Many studies have reported
reduced over a concentration of 500 μM, however the cell survival rate is bio-pharmaceutical activities, including the anti-diabetic activity of
still greater than 80% at a concentration of 1000 μM. It was previously phytosterols (Miras-Moreno et al., 2016). However, no study indicates
demonstrated that when the cell viability is above 80%, the drug can be the anti-diabetic properties of phyto-stenones, which are the byproducts
thought to have no significant cytotoxicity on cell growth (López-García of sterol degradation (Dai et al., 2005). The result from
et al., 2014). Therefore, we concluded that sitostenone has no significant bioactivity-guided fractionation analysis revealed that sitostenone
cytotoxicity toward HepG2 cells. possessed significant glucose-lowering effect in vitro. Briefly, HepG2
cells were incubated with increasing concentrations of sitostenone (10,
20, and 30 μM) for 6 h, then the glucose level in the culture media was
Sitostenone promotes glucose uptake in hepatic cells analyzed by the DNS method. As shown in Figure 5A, treatment with
sitostenone significantly promoted glucose uptake from 30.5% to
Phytosterols are bioactive natural products, e.q. β-sitosterol, cam­ 33.7%, 35.8%, and 37.3% by 10, 20, and 30 μM, respectively. The de­
pesterol, and stigmasterols are abundant plant sterols with a structure gree of glucose consumption by 30 μM sitostenone was highly compa­
similar to cholesterol (Trautwein and Demonty, 2007). Phytosterols are rable with the insulin treatment group (40.0%). This data provides
characterized by an extra one or two carbon substituents on the

Fig. 5. Sitostenone promotes the glucose consumption in normal and insulin-resitant hepatic cells. (A) HepG2 cells were treated with sitostenone (10, 20, and 30 μM)
or insulin (100 nM) for 6 h. (B) Cells were incubated with a combination of sitostenone and 100 nM insulin for 6 h. (C) To induce insulin-resistant, cells were pre-
incubated with 30 mM D-glucose for 24 h, then the cells were treated with different concentrations of sitostnone or metformin in the presence of insulin for 6 h. (D)
Insulin-resistant cells were incubated with various concentrations of sitostenone or 2 mM of metformin for 6 h. After treatment, culture supernatant was collected and
glucose concentration was tested by DNS method. Values represent the mean ± SD of three independent experiments. Statistical significance Δp < 0.001 compared to
control vs. high glucose treatment (insulin-resistant) group; *p < 0.05, **p < 0.01, ***p < 0.001 were compared to insulin-resistant vs. sample treatment groups.

7
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

positive feedback that under normal physiological conditions, sitoste­ Sitostenone up-regulates IRS-1 and AKT activation in hepatic cells
none mimics insulin and provokes glucose consumption in hepatic cells.
Next, we compared the glucose uptake ability of sitostenone along with It has been well demonstrated that activation of insulin signaling
insulin. Interestingly, compared with control groups, sitostenone pathway, which includes phosphorylation of insulin receptor substrate-1
co-treatment with insulin remarkably increased glucose uptake from (IRS-1) and PI3K/AKT are the critical regulators of glucose uptake,
30.1% to 41.5%, 42.3%, and 44.7% by 10, 20, and 30 μM sitostenone protein synthesis, and gene expression in many cell types, including
with insulin, respectively (Fig. 5B). However, the glucose uptake was liver, muscle, and adipocytes (Boucher et al., 2014). Therefore, to
not statistically significant from sitostenone alone versus co-treatment further elucidate the molecular mechanism of the effect of sitostenone in
with insulin. It is speculated that under normal conditions, sitostenone hepatic cells, we determined the IRS-1 and AKT phosphorylation levels
slightly inhibits insulin’s ability to promote glucose consumption failed using Western blotting. As shown in Figure 6A, the control cells had a
to increase insulin sensitivity in hepatic cells. lower expression of phosphorylated IRS-1Y895, while treatment with
sitostenone significantly increased IRS-phosphorylation. Indeed, treat­
ment with 20 μM sitostenone exhibited the most vital IRS-1 activation
Sitostenone promotes glucose uptake in insulin-resistant hepatic cells then that of 30 μM sitostenone or 100 nM of insulin-treated cells.
Additionally, sitostenone significantly and dose-dependently activate
The above result indicates that sitostenone failed to increase insulin the AKTSer473, a down-stream regulator IRS-1 in hepatic cells (Fig. 6B).
sensitivity under normal condition, whereas it significantly promotes These data are consistent with other observations that β-sitosterol, a
glucose uptake. To further clarify this mechanism, the glucose uptake relative compound to sitostenone activates IRS-1 and AKT in
ability of sitostenone was determined under insulin-resistant condition. sucrose-induced diabetic mice (Babu et al., 2020).
As shown in Figure 5C, glucose consumption in control cells was
observed as 30.1%, which was markedly increased to 41.6% after the
addition of 100 nM insulin. Alternatively, glucose consumption by the Sitostenone up-regulates IRS-1, AKT, and GSK3β activation in insulin-
control group was noted as 21.1% under insulin-resistant conditions. resistant hepatic cells
Although insulin treatment failed to provoke glucose uptake in insulin-
resistant cells, glucose consumption was noted as 23.2%, which is It has become increasingly apparent that chronic accumulation of
significantly lower than that of un-induced and insulin-treated groups. glucose in the bloodstream (hyperglycemia) played a crucial role in the
Interestingly, when the insulin-resistant cells were co-incubated with impairment of insulin-stimulated glucose uptake in obesity and type-2
10, 20, and 30 μM sitosterone and 100 nM insulin significantly increased diabetic patients (Czech, 2017). Hepatic cells exposed to high
glucose uptake from 21.1% to 27.4%, 31.1%, and 35.5%, respectively. glucose-impaired glycogen biosynthesis and glucose usage (Huang et al.,
The 30 μM sitostenone-mediated glucose uptake in insulin-resistant cells 2015). Furthermore, prolonged exposure of D-glucose wound the insulin
was highly comparable with the positive drug control metformin signaling molecules by decreasing the phosphorylation of IRS-1 and AKT
(35.7%). These data strongly suggest that sitostenone can promote and down-regulate the expression and membrane export of GLUT-4,
glucose uptake in insulin-resistant cells. which eventually resulted in the defect of glucose uptake, in such a
To further explore the role of sitostenone on glucose uptake in condition called insulin-resistance (Boucher et al., 2014; Huang et al.,
insulin-resistant cells without the addition of insulin, the insulin- 2015). Consistent with the above studies, our results also demonstrated
resistant hepatic cells were incubated with increasing concentrations that 30 mM D-glucose significantly reduced insulin-induced phosphor­
of sitostenone for 6 h, and then the contents of glucose in the culture ylation of IRS-1 and AKT levels in hepatic cells. To further observe the
medium were determined. As shown in Figure 5D, compared with effect of sittostenone on insulin signaling molecules under insulin
insulin-resistant control cells, treatment with sitostenone markedly resistance conditions, we examined the phosphorylation of IRS-1 and its
improved glucose uptake from 21.4% to 23.3%, 26.6%, and 28.8% by down-stream molecule AKT in high glucose-induced insulin-resistant
10, 20, and 30 μM, respectively. Indeed, 20 and 30 μM sitostenone hepatic cells. As shown in Figure 7A, treatment with sitostenone
significantly provoked glucose in insulin-resistant cells, which is similar significantly recovered the phosphorylation of IRS-1under stimulation
to the positive drug control metformin (32.2%). These data strongly with insulin. The total level of IRS-1 was also increased dramatically by
suggest that sitostenone can simultaneously promote glucose con­ sitotenone in a dose-dependent manner. Besides, treatment with sitos­
sumption and increase insulin sensitivity in hepatic cells. tenone significantly prevented the diminution in the phosphorylation of

Fig. 6. Sitostenone promotes phosphorylation

of IRS-1 and AKT in hepatic cells. Cells were
treated with various doses of sitostenone or 100
nM of insulin for 10 min. Western blot analysis
was performed to determine total and phos­
phorylated protein levels of IRS-1 (A) and AKT
(B). β-actin was served as an internal control.
Histogram indicate relative protein expression
of the corresponding immunoblot. The phos­
phorylated levels of IRS-1 and AKT were
normalized with their corresponding total
levels. Values represent the mean ± SD of three
independent experiments. Statistical signifi­
cance **p < 0.01, ***p < 0.001 were compared
to control vs. sample treatment groups.

8
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

Fig. 7. Sitostenone promotes phosphorylation of IRS-1 and AKT in insulin-resistant hepatic cells. (A-E) HepG2 cells were exposed to high glucose (30 mM) for 24 h to
produce insulin resistance, then the cells were treated with various doses of sitostenone along with or without100 nM of insulin for 10 min. Western blot analysis was
performed to determine total and phosphorylated protein levels of IRS-1, AKT and GSK3β. β-actin was served as an internal control. Histogram indicate relative
protein expression of the corresponding immunoblot. The phosphorylated levels of IRS-1, AKT and GSK3β were normalized with their corresponding total levels.
Values represent the mean ± SD of three independent experiments. Statistical significance Φp < 0.001 compared to control vs. insulin alone treatment group; Δp <
0.001 compared to control vs. high glucose treatment (insulin-resistant) group; *p < 0.05, **p < 0.01, ***p < 0.001 were compared to insulin-resistant vs. sample
treatment groups.

AKT caused by high glucose. In contrast, there was no significant impact hepatic cells has been well characterized, with specific PPAR-γ activa­
on the total forms of IRS-1 and AKT levels (Fig. 7B). Furthermore, we tion to recover high glucose-induced insulin resistance and promotes
found that treatment with sitostenone alone (without insulin) does not glucose uptake (Kim and Ahn, 2004). Therefore, we examined whether
protect the high glucose-induced reduction of IRS-1 and AKT phos­ the sitostenone-mediated increase in insulin sensitivity in hepatic cells
phorylation (Fig. 7C, D). Next, a down-stream target of AKT, the phos­ was caused by PPAR-γ activation, the protein levels of PPAR-γ was
phorylation level of GSK3β, was determined. Compared with control, determined by western blotting. As shown in Figure 8A, treatment with
high glucose concentration significantly diminished GSK3β phosphory­ sitostenone significantly and dose-dependently increased PPAR-γ pro­
lation, whereas sitostenone treatment significantly provoked GSK3β tein level in insulin-resistant cells. Compared with unstimulated control
phosphorylation in insulin-resistant cells. The restoration ability of cells, a significant increase of PPAR-γ was found in high glucose-induced
sitostenone was identical to the effect of metformin (Fig. 7E). All these insulin-resistant cells. This result is consistent with other’s observation
results indicate that sitostenone restrained the IRS-1 and AKT pathway that high glucose treatment resulted with PPAR-γ up-regulation in
inhibition, which constitutes a route in the insulin signaling pathway. human kidney cells (Panchapakesan et al., 2004).
The forkhead box O 1 (FOXO1), a nuclear transcription factor, which
Sitostenone up-regulates PPAR-γ in insulin-resistant hepatic cells mediates the inhibitory action of insulin or insulin-like growth factor on
key functions in diverse pathways, including cell metabolism, prolifer­
Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a member ation, differentiation, oxidative stress, cell survival as well as senes­
of the nuclear hormone receptor superfamily ligand-activated tran­ cence, autophagy, and aging in mammals (Lee and Dong, 2017). The
scription factor that regulates cell growth, inflammation, lipid meta­ expression of FOXO1 in the nucleus was significantly increased after
bolism, and insulin sensitivity (Panchapakesan et al., 2004). The cells exposed to a high concentration of glucose, which is consistent with
thiazolidinedione family of compounds that are now commonly used as a previous study that described high glucose concentration forces
insulin-sensitizing agents to improve glucose tolerance, enhancing in­ FOXO1 in mesangial cells (Das et al., 2014). Interestingly, treatment
sulin sensitivity, and restoring the function of β-cells in patients with with sitostenone significantly decreased FOXO1 expression in the nu­
type-2 diabetes (Soccio et al., 2014). The functional role of PPAR-γ in clear fraction, and the FOXO1 inhibitory effect of 30 μM sitostenone was

9
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

Fig. 8. Sitostenone up-regulates PPAR-γ and down-regulates FOXO1 expression in insulin-resistant hepatic cells. HepG2 cells were exposed to high glucose (30 mM)
for 24 h to produce insulin resistance, then the cells were treated with various doses of sitostenone or 2 mM metformin for 6 h. Western blot analysis was performed
to determine protein levels of PPAR-γ (A) and FOXO-1 (B). β-actin was served as an internal control. Histogram indicate relative protein expression of the corre­
sponding immunoblot. Values represent the mean ± SD of three independent experiments. Statistical significance Δp < 0.001 compared to control vs. high glucose
treatment (insulin-resistant) group; *p < 0.05, **p < 0.01, ***p < 0.001 were compared to insulin-resistant vs. sample treatment groups.

highly comparable with metformin (Figure 8B). These data suggest that Substantial evidence suggesting that numerous natural products activate
sitostenone-mediated improvement of glucose uptake and insulin AMPK, either directly or indirectly (Coughlan et al., 2014). Thus, in this
resistance was associated with up-regulation of PPAR-γ and study, the effect of sitostenone on the phosphorylation of AMPK was
down-regulation of FOXO-1 in hepatic cells. evaluated by Western blotting. In accordance with other’s observation
(Zang et al., 2004), hepatic cells exposed to high glucose significantly
reduced AMPK phosphorylation compared with control cells, whereas
Sitostenone prevents high glucose-induced down-regulation AMPK sitostenone treatment significantly and dose-dependently restored the
activation in hepatic cells phosphorylation of AMPK in insulin-resistant hepatic cells (Fig. 9A).
Notably, a sitostenone-mediated increase in AMPK phosphorylation was
5′ AMP-activated protein kinase (AMPK) is an energy-sensing highly comparable with known anti-diabetic drug metformin.
enzyme activated when cellular energy levels are low, and it signals to
stimulate glucose uptake, insulin sensitivity, fatty acid oxidation, and
mitochondrial biogenesis in skeletal muscles, liver, and adipocytes. Sitostenone restores high glucose-induced down-regulation GLUT2 and
AMPK is down-regulated in humans and animals with diabetes, and the GLUT4 in hepatic cells
AMPK activation can improve insulin sensitivity and glucose uptake via
glucose transporters such as GLUT2 and GLUT4 (Coughlan et al., 2014). To further determine the effect of sitostenone on AMPK’s down-

Fig. 9. Sitostenone activates AMPK and up-regulates GLUT2, GLUT4 levels in insulin-resistant hepatic cells. HepG2 cells were exposed to high glucose (30 mM) for
24 h to produce insulin-resistance, then the cells were treated with various doses of sitostenone or 2 mM metformin for 6 h. The membrane fraction was prepared and
western blot was performed to determine total and phosphorylated level of AMPK (A) and the protein levels of GLUT2 (B) and GLUT4 (C). β-actin was served as an
internal control. Histogram indicate relative protein expression of the corresponding immunoblot. The phosphorylated level of AMPK was normalized with its
corresponding total levels. Values represent the mean ± SD of three independent experiments. Statistical significance Δp < 0.001 compared to control vs. high glucose
treatment (insulin-resistant) group; *p < 0.05, **p < 0.01, ***p < 0.001 were compared to insulin-resistant vs. sample treatment groups.

10
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

stream targets, such as GLUT2 and GLUT4, in insulin-resistant hepatic Das, F., Ghosh-Choudhury, N., Dey, N., Bera, A., Mariappan, M.M., Kasinath, B.S., Ghosh
Choudhury, G., 2014. High glucose forces a positive feedback loop connecting Akt
cells, cells were incubated with increasing concentrations of sitostenone
kinase and FoxO1 transcription factor to activate mTORC1 kinase for mesangial cell
and 2 mM metformin for 6 h. GLUT2 and GLUT4 expression levels in the hypertrophy and matrix protein expression. J. Biol. Chem. 289, 32703–32716.
membrane fraction was determined by Western blotting. Data demon­ Derosa, G., Maffioli, P., 2012. α-Glucosidase inhibitors and their use in clinical practice.
strated that compared with control cells, cells exposed to high glucose Arch. Med. Sci. 8, 899–906.
Dong, L., Han, X., Tao, X., Xu, L., Xu, Y., Fang, L., Yin, L., Qi, Y., Li, H., Peng, J., 2018.
significantly reduced the protein expression levels of GLUT2 and GLUT4 Protection by the total flavonoids from Rosa laevigata Michx fruit against
in hepatic cells, whereas treatment with sitostenone significantly and lipopolysaccharide-induced liver injury in mice via modulation of FXR signaling.
dose-dependently restored GLUT2 and GLUT4 protein levels in the Foods. 7, 88.
Garriga, M., Almaraz, M., Marchiaro, A., 2017. Determination of reducing sugars in
membrane (Fig. 9B). These results indicate that sitostenone promotes extracts of Undaria pinnatifida (harvey) algae by UV-visible spectrophotometry (DNS
glucose uptake via up-regulation of GLUT2 and GLUT4 in the membrane method). Acta Ingen. 3, 173–179.
through the AMPK pathway. Giacco, F., Brownlee, M., 2010. Oxidative stress and diabetic complications. Circ. Res.
107, 1058–1070.
Heshmati, J., Morvaridzadeh, M., Sepidarkish, M., Fazelian, S., Rahimlou, M., Omidi, A.,
Conclusion Palmowski, A., Asadi, A., Shidfar, F., 2020. Effects of Melissa officinalis (Lemon Balm)
on cardio-metabolic outcomes: A systematic review and meta-analysis. Phyto Ther.
Res. 24, 3113–3123.
In this study, we first demonstrated that ethanol extract of Huang, Q., Chen, L., Teng, H., Song, H., Wu, X., Xu, M., 2015. Phenolic compounds
R. laevigata fruits and its derivative sub-fractions promotes glucose up­ ameliorate the glucose uptake in HepG2 cells’ insulin resistance via activating
take and alleviates hepatic insulin resistance, as they had low cytotox­ AMPK: Anti-diabetic effect of phenolic compounds in HepG2 cells. J. Funct. Food 19,
487–494.
icity in hepatic cells. Bioactivity-guided fractionation analysis revealed Jang, H.H., Bae, J.H., Kim, M.J., Park, M.Y., Kim, H.R., Lee, Y.M., 2020. Agrimonia pilosa
that sitostenone, a steroid-like compound exhibited a pronounceable Ledeb. Ameliorates Hyperglycemia and Hepatic Steatosis in Ovariectomized Rats
effect on hepatic cells. As sitostenone treatment remarkably increased Fed a High-Fat Diet 12, 1631.
Kim, H.I., Ahn, Y.H., 2004. Role of peroxisome proliferator-activated receptor-γ in the
glucose uptake and insulin sensitivity in hepatic cells, which was
glucose-sensing apparatus of liver and β-cells. Diabetes 53, S60–S65.
seemingly mediated via IRS-1 and AKT cascade activation in normal Ko, H., Jegal, K., Song, S., Kim, N., Kang, J., Byun, S., Kim, Y., Cho, I., Kim, S., 2015.
hepatic cells. Also, sitostenone treatment restores insulin-induced Water Extract of Rosa laevigata Michx. Protects hepatocytes from arachidonic acid
and iron-mediated oxidative stress. Korea J. Herbol. 30, 7–15.
phosphorylation of IRS-1 and AKT in high glucose-induced insulin-
Ko, H.M., Choi, S.-H., Kim, Y., An, E.J., Lee, S.H., Kim, K., Jung, H.J., Jang, H.J., 2020.
resistant cells. Furthermore, high glucose-induced dephosphorylation of Effect of Rosa laevigata on PM10-induced inflammatory response of human lung
IRS-1, AKT, GSK3β, and AMPK, and down-regulation of GLUT2 and epithelial cells. Evid. Based Compl. Alternat. Med. 2020, 2893609.
GLUT4 were significantly restored by sitostenone. To conclude, this Ko, K.D., Kim, K.K., Lee, K.R., 2017. Does weight gain associated with thiazolidinedione
use negatively affect cardiometabolic health? J. Obes. Metab. Syndr. 26, 102–106.
study strongly suggests the concept that activation GLUT2/4 by IRS-1- Kong, M., Xie, K., Lv, M., Li, J., Yao, J., Yan, K., Wu, X., Xu, Y., Ye, D., 2021. Anti-
AKT via PPAR-γ/AMPK signaling pathways is involved in sitostenone- inflammatory phytochemicals for the treatment of diabetes and its complications:
mediated improvement in insulin-resistant cells. However, further in Lessons learned and future promise. Biomed. Pharmacother. 133, 110975.
López-García, J., Lehocký, M., Humpolíček, P., Sáha, P., 2014. HaCaT keratinocytes
vivo investigation of this activity is necessary to elaborate the mecha­ response on antimicrobial atelocollagen substrates: extent of cytotoxicity, cell
nisms and permit full exploitation of its promise, specifically the role of viability and proliferation. J. Funct. Biomater. 5, 43–57.
altered signal transduction. Leclercq, I.A., Da Silva Morais, A., Schroyen, B., Van Hul, N., Geerts, A., 2007. Insulin
resistance in hepatocytes and sinusoidal liver cells: mechanisms and consequences.
J. Herpatol. 47, 142–156.
Declaration of Competing Interest Lee, S.H., Choi, S.H., Lee, I.S., Kim, Y., An, E.J., Jang, H.J., 2020. Anti-inflammatory
effect of Rosa laevigata extract on in vitro and in vivo model of allergic asthma via the
suppression of IgE and related cytokines. Mol. Cell Toxicol. 16, 119–127.
The authors declare that there is no conflict of interest. Lee, S., Dong, H.H., 2017. FoxO integration of insulin signaling with glucose and lipid
metabolism. J. Endocrinol. 233, R67–R79.
Li, D., Peng, C., Xie, X., Mao, Y., Li, M., Cao, Z., Fan, D., 2014. Antidiabetic effect of
Acknowledgment flavonoids from Malus toringoides (Rehd.) Hughes leaves in diabetic mice and rats.
J. Ethnopharmacol. 153, 561–567.
This study was supported by the Ministry of Science and Technology, Li, M., Hu, X., Xu, Y., Hu, X., Zhang, C., Pang, S., 2019. A possible mechanism of
metformin in improving insulin resistance in diabetic rat models. Int. J. Endocrinol.
Taiwan, (106-2313-B-005 -012 -MY3). 2019, 3248527-3248527.
Li, W.H., Chang, S.T., Chang, S.C., Chang, H.T., 2008. Isolation of antibacterial
References diterpenoids from Cryptomeria japonica bark. Nat. Prod. Res. 22, 1085–1093.
Li, X., Cao, W., Shen, Y., Li, N., Dong, X.P., Wang, K.J., Cheng, Y.X., 2012. Antioxidant
compounds from Rosa laevigata fruits. Food Chem. 130, 575–580.
Babu, S., Krishnan, M., Rajagopal, P., Periyasamy, V., Veeraraghavan, V., Govindan, R.,
Liu, X., Gao, Y., Li, D., Liu, C., Jin, M., Bian, J., Lv, M., Sun, Y., Zhang, L., Gao, P., 2018.
Jayaraman, S., 2020. Beta-sitosterol attenuates insulin resistance in adipose tissue
The neuroprotective and antioxidant profiles of selenium-containing polysaccharides
via IRS-1/Akt mediated insulin signaling in high fat diet and sucrose induced type-2
from the fruit of Rosa laevigata. Food Funct. 9, 1800–1808.
diabetic rats. Eur. J. Pharmacol. 873, 173004.
Liu, Y., Luo, W., Luo, X., Yong, Z., Zhong, X., 2015. Effects of Rosa laevigata Michx.
Birben, E., Sahiner, U.M., Sackesen, C., Erzurum, S., Kalayci, O., 2012. Oxidative stress
extract on reactive oxygen species production and mitochondrial membrane
and antioxidant defense. World Allergy Organ J. 5, 9–19.
potential in lens epithelial cells cultured under high glucose. Int. J. Clin. Exp. Med. 8,
Boonloh, K., Kukongviriyapan, U., Pannangpetch, P., Kongyingyoes, B., Senggunprai, L.,
15759–15765.
Prawan, A., Thawornchinsombut, S., Kukongviriyapan, V., 2015. Rice bran protein
Luo, W., Liu, Y., Luo, X., Zhang, J., 2016. Heme oxygenase-1 mediates the protective
hydrolysates prevented interleukin-6- and high glucose-induced insulin resistance in
effect of Rosa Laevigata Michx on doxorubicin induced myocardial cell injury. Int. J.
HepG2 cells. Food Funct. 6, 566–573.
Clin. Exp. Med. 9, 18012–18018.
Boucher, J., Kleinridders, A., Kahn, C.R., 2014. Insulin receptor signaling in normal and
Miras-Moreno, B., Sabater-Jara, A.B., Pedreño, M.A., Almagro, L., 2016. Bioactivity of
insulin-resistant states. Cold Spring Harb. Perspect. Biol. 6, a009191.
phytosterols and their production in plant in vitro cultures. J. Agric. Food Chem. 64,
Broadhurst, C.L., Polansky, M.M., Anderson, R.A., 2000. Insulin-like biological activity of
7049–7058.
culinary and medicinal plant aqueous extracts in vitro. J. Agric. Food Chem. 48,
Morvaridzadeh, M., Fazelian, S., Agah, S., Khazdou, M., Rahimlou, M., Agh, F., Potter, E.,
849–852.
Heshmati, S., Heshmati, J., 2020. Effect of ginger (Zingiber officinale) on
Cantley, J., Ashcroft, F.M., 2015. Q&A: insulin secretion and type 2 diabetes: why do
inflammatory markers: A systematic review and meta-analysis of randomized
β-cells fail? BMC Biol. 13, 33-33.
controlled trials. Cytokine 135, 155224.
Coughlan, K.A., Valentine, R.J., Ruderman, N.B., Saha, A.K., 2014. AMPK activation: a
Noratto, G.D., Lage, N.N., Chew, B.P., Mertens-Talcott, S.U., Talcott, S.T., Pedrosa, M.L.,
therapeutic target for type 2 diabetes? Diabetes Metab. Syndr. Obes. 7, 241–253.
2018. Non-anthocyanin phenolics in cherry (Prunus avium L.) modulate IL-6, liver
Czech, M.P., 2017. Insulin action and resistance in obesity and type 2 diabetes. Nat. Med.
lipids and expression of PPARδ and LXRs in obese diabetic (db/db) mice. Food
23, 804–814.
Chem. 266, 405–414.
Dai, J., Sun, M.Y., Culp, R.A., Noakes, J.E., 2005. Changes in chemical and isotopic
Orgel, E., Mittelman, S.D., 2013. The links between insulin resistance, diabetes, and
signatures of plant materials during degradation: Implication for assessing various
cancer. Curr. Diab. Rep. 13, 213–222.
organic inputs in estuarine systems. Geophys. Res. Lett. 32, L13608.
Panchapakesan, U., Pollock, C.A., Chen, X.M., 2004. The effect of high glucose and
Dankner, R., Shanik, M.H., Keinan-Boker, L., Cohen, C., Chetrit, A., 2012. Effect of
PPAR-γ agonists on PPAR-γ expression and function in HK-2 cells. Am. J. Physiol.
elevated basal insulin on cancer incidence and mortality in cancer incident patients:
Renal. Physiol. 287, F528–F534.
the Israel GOH 29-year follow-up study. Diabet Care 35, 1538–1543.

11
K.J.S. Kumar et al. Phytomedicine Plus 1 (2021) 100109

Pandey, K.B., Rizvi, S.I., 2009. Plant polyphenols as dietary antioxidants in human health Tapiero, H., Townsend, D.M., Tew, K.D., 2003. Phytosterols in the prevention of human
and disease. Oxid. Med. Cell Longev. 2, 270–278. pathologies. Biomed. Pharmacother. 57, 321–325.
Paquette, M., Medina Larqué, A.S., Weisnagel, S.J., Desjardins, Y., Marois, J., Pilon, G., Teoh, S.L., Das, S., 2018. Phytochemicals and their effective role in the treatment of
Dudonné, S., Marette, A., Jacques, H., 2017. Strawberry and cranberry polyphenols diabetes mellitus: a short review. Phytochem. Rev. 17, 1111–1128.
improve insulin sensitivity in insulin-resistant, non-diabetic adults: a parallel, Trautwein, E.A., Demonty, I.J.O., 2007. Phytosterols: natural compounds with
double-blind, controlled and randomised clinical trial. Br. J. Nutr. 117, 519–531. established and emerging health benefits. Oilseed Fat Crop. Lipid. 14, 259–266.
Patel, D.K., Prasad, S.K., Kumar, R., Hemalatha, S., 2012. An overview on antidiabetic WHO, 2020. Diabetes. https://www.who.int/news-room/fact-sheets/detail/diabetes.
medicinal plants having insulin mimetic property. Asian Pac. J. Trop. Biomed. 2, Wilcox, G., 2005. Insulin and insulin resistance. Clin. Biochem. Rev. 26, 19–39.
320–330. Xie, W., Du, L., 2011. Diabetes is an inflammatory disease: evidence from traditional
Ramalingam, S., Packirisamy, M., Karuppiah, M., Vasu, G., Gopalakrishnan, R., Chinese medicines. Diabet. Obesity Metabol. 13, 289–301.
Gothandam, K., Thiruppathi, M., 2020. Effect of β-sitosterol on glucose homeostasis Xuguang, H., Aofei, T., Tao, L., Longyan, Z., Weijian, B., Jiao, G., 2019. Hesperidin
by sensitization of insulin resistance via enhanced protein expression of PPRγ and ameliorates insulin resistance by regulating the IRS1-GLUT2 pathway via TLR4 in
glucose transporter 4 in high fat diet and streptozotocin-induced diabetic rats. HepG2 cells. Phytother. Res. 33, 1697–1705.
Cytotechnology 72, 357–366. Yan, M., Zhu, Y., Zhang, H.J., Jiao, W.H., Han, B.N., Liu, Z.X., Qiu, F., Chen, W.S., Lin, H.
Senthil Kumar, K.J., Liao, J.W., Xiao, J.H., Gokila Vani, M., Wang, S.Y., 2012. W., 2013. Anti-inflammatory secondary metabolites from the leaves of Rosa
Hepatoprotective effect of lucidone against alcohol-induced oxidative stress in laevigata. Bioorg. Med. Chem. 21, 3290–3297.
human hepatic HepG2 cells through the up-regulation of HO-1/Nrf-2 antioxidant Zang, M., Zuccollo, A., Hou, X., Nagata, D., Walsh, K., Herscovitz, H., Brecher, P.,
genes. Toxicol. in Vitro 26, 700–708. Ruderman, N.B., Cohen, R.A., 2004. AMP-activated protein kinase is required for the
Shih, C.-C., Lin, C.-H., Wu, J.-B., 2010. Eriobotrya japonica improves hyperlipidemia and lipid-lowering effect of metformin in insulin-resistant human HepG2 cells. J. Biol.
reverses insulin resistance in high-fat-fed mice. Phytother. Res. 24, 1769–1780. Chem. 279, 47898–47905.
Soccio, R.E., Chen, E.R., Lazar, M.A., 2014. Thiazolidinediones and the promise of insulin Zeng, N., Shen, Y., Li, L.Z., Jiao, W.H., Gao, P.Y., Song, S.J., Chen, W.S., Lin, H.W., 2011.
sensitization in type 2 diabetes. Cell Metab. 20, 573–591. Anti-inflammatory triterpenes from the leaves of Rosa laevigata. J. Nat. Prod. 74,
Tabish, S.A., 2007. Is diabetes becoming the biggest epidemic of the twenty-first 732–738.
century? Int. J. Health Sci. 1, V–VIII. Zhan, Q., Wang, Q., Lin, R., He, P., Lai, F., Zhang, M., Wu, H., 2020. Structural
Tanaka, M., Misawa, E., Ito, Y., Habara, N., Nomaguchi, K., Yamada, M., Toida, T., characterization and immunomodulatory activity of a novel acid polysaccharide
Hayasawa, H., Takase, M., Inagaki, M., Higuchi, R., 2006. Identification of five isolated from the pulp of Rosa laevigata Michx fruit. Int. J. Biol. Macromol. 145,
phytosterols from aloe vera gel as anti-diabetic compounds. Biol. Pharmaceutical. 1080–1090.
Bull. 29, 1418–1422. Zhang, X., Hu, Y., Jin, C., Wu, W., 2020. Extraction and hypolipidemic activity of low
Tao, X., Sun, X., Xu, L., Yin, L., Han, X., Qi, Y., Xu, Y., Zhao, Y., Wang, C., Peng, J., 2016. molecular weight polysaccharides isolated from Rosa laevigata fruits. Biomed. Res.
Total flavonoids from rosa laevigata michx fruit ameliorates hepatic ischemia/ Int. 2020, 2043785.
reperfusion injury through inhibition of oxidative stress and inflammation in rats. Zhou, Y., Liao, Q., Luo, Y., Qing, Z., Zhang, Q., He, G., 2012. Renal protective effect of
Nutrients 8, 418. Rosa laevigata Michx. by the inhibition of oxidative stress in streptozotocin-induced
diabetic rats. Mol. Med. Rep. 5, 1548–1554.

12