Hydroxytyrosol nicotinate, a new multifunctional hypolipidemic and hypoglycemic agent

Article abstract of DOI:10.1016/j.biopha.2018.01.123

Hydroxytyrosol (HT) is a natural polyphenol antioxidant that exists in olive oil. In the study of multifunctional hypolipidemic of nicotinic derivatives, we found that hydroxytyrosol nicotinate (HT-N) incorporation of niacin with HT displayed α-glucosidase inhibitory activities in vitro, such as yeast α-glucosidase (IC₅₀ = 117.72 μM) and rat intestinal α-glucosidases maltase (IC₅₀ = 31.86 μM) and sucrase (IC₅₀ = 22.99 μM), and had a good control of postprandial blood glucose (PBG). HT-N shown significantly hypoglycemic action by 16.9% and protection of pancreatic tissue in type 2 diabetic mellitus (T2DM) mouse model. HT-N also shown a potent antioxidant activity and property of anti-glycation in vitro, which were benefit for ameliorating diabetic complications. Moreover, HT-N exhibited much significant hypolipidemia, lowering plasma triglyceride (TG), total cholesterol (TC), and malonaldehyde (MDA) by 34.6%, 45.8% and 32.1% respectively, in hyperlipidemic mice induced by Triton WR 1339. The results indicated that HT-N has hypolipidemic, hypoglycemic and antioxidant actions. All these properties could be conducive to amelioration of oxidative stress, hyperlipidemia, and diabetes that HT-N may serve as a multifunctional potential therapeutic strategy in diabetic patients with hyperlipidemia.

Full text of DOI:10.1016/j.biopha.2018.01.123

Biomedicine&Pharmacotherapy99(2018)715–724
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Hydroxytyrosol nicotinate, a new multifunctional hypolipidemic and
hypoglycemic agent
Yun-Dong Xiea, Zi-Zhang Chena, Na Lib, Wen-Fang Luc, Yan-Hong Xua, Yuan-Yuan Lina,
Li-Hua Shaoa, Qiu-Tang Wanga, Li-Ying Guoa, Yu-Qiong Gaoa, Guang-De Yanga, Yi-Ping Lia,
Xiao-Li Biana,⁎
a
College of Pharmacy, Xi’an Jiaotong University, No.76 Yanta West Road, Xi’an, Shaanxi Province, 710061, People’s Republic of China
College of Pharmacy, Shaanxi University of Chinese Medicine, Shiji Ave., Xi’an-Xianyang New Ecomic Zone, Shaanxi Province, 712046, People’s Republic of China
Pharmacy Department, Xianyang Central Hospital, No.78 Renmin East Road, Xianyang, Shaanxi Province, 712000, People's Republic of China
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
Hydroxytyrosol (HT) is a natural polyphenol antioxidant that exists in olive oil. In the study of multifunctional
hypolipidemic of nicotinic derivatives, we found that hydroxytyrosol nicotinate (HT-N) incorporation of niacin
with HT displayed α-glucosidase inhibitory activities in vitro, such as yeast α-glucosidase (IC50 = 117.72 μM)
and rat intestinal α-glucosidases maltase (IC50 = 31.86 μM) and sucrase (IC50 = 22.99 μM), and had a good
control of postprandial blood glucose (PBG). HT-N shown significantly hypoglycemic action by 16.9% and
protection of pancreatic tissue in type 2 diabetic mellitus (T2DM) mouse model. HT-N also shown a potent
antioxidant activity and property of anti-glycation in vitro, which were benefit for ameliorating diabetic com-
plications. Moreover, HT-N exhibited much significant hypolipidemia, lowering plasma triglyceride (TG), total
cholesterol (TC), and malonaldehyde (MDA) by 34.6%, 45.8% and 32.1% respectively, in hyperlipidemic mice
induced by Triton WR 1339. The results indicated that HT-N has hypolipidemic, hypoglycemic and antioxidant
actions. All these properties could be conducive to amelioration of oxidative stress, hyperlipidemia, and diabetes
that HT-N may serve as a multifunctional potential therapeutic strategy in diabetic patients with hyperlipidemia.
Hydroxytyrosol nicotinate
α-glucosidase inhibition
Antiglycation
Antioxidant
Hypoglycemia
Hypolipidemia
1. Introduction
(PPAR-α) to modulate lipid metabolism [4]. However, studies to date
have not demonstrated an overall benefit of fibrates for reduction of
The prevalence of diabetes mellitus is increasing at a surprising
rapidity worldwide which is part of the most major public health pro-
blems. It is reported that the type 2 diabetes mellitus (T2DM) patients
are more liable to be dyslipidemia than the ordinary people [1]. The
dyslipidemia of diabetes mellitus plays a main role in the development
of atherosclerosis. It is well known that the elevated triglyceride (TG) is
the most common form of dyslipidemia among patients with T2DM [2].
High TG levels were always regarded as a cardiovascular disease (CVD)
risk factor, including the patients with diabetes mellitus. Although
many recent studies have shown the relationship between high TG le-
vels and the risk factor of CVD in patients with coronary heart disease,
clinical observations to patients with diabetic dyslipidemia have failed
to verify CVD risk decrease with TG lowering drugs in combination with
statins which is the foundation of therapy of dyslipidemia in diabetes
mellitus [3]. For T2DM patients with high TG, the most common setting
is that fibrates should be used as the first-line agent, which appears to
involve the activation of peroxisome proliferator activated receptor-α
cardiovascular or total mortality [5]. The previous literature demon-
strated that the fibrates in patients with T2DM is not tolerated and
failed to meliorate high TG [6].
Niacin, a kind of water-soluble B-complex vitamin, is a potent agent
for lowering plasma TG in pharmacological doses [7]. Niacin has been
previously shown to lessen coronary atherosclerosis and reduce the rate
of coronary mortality [8]. To solve the residual risk in dyslipidemic
diabetic patients, clinical and experimental surveillances have demon-
strated that the combination of niacin with other hypolipidemic agents
may offer potent effects in lowering plasma TG and other lipids [9]. The
recent study revealed that lipid-modifying doses of niacin can be ap-
plied safely to patients with stable and controlled in T2DM. Niacin
treatment may be regarded as an alternative to fibrates and/or statins
agents in patients with dyslipidemic diabetes [6]. However, niacin has
clinically slight negative impact on glycemia that it can lead to an in-
crease in blood glucose [10]. Moreover, niacin has more adverse effects
in clinical application, such as flushing, itchy, gastrointestinal
⁎
Corresponding author.
E-mail address: bianxl@mail.xjtu.edu.cn (X.-L. Bian).
https://doi.org/10.1016/j.biopha.2018.01.123
Received 12 November 2017; Received in revised form 15 January 2018; Accepted 24 January 2018
0753-3322/©2018ElsevierMassonSAS.Allrightsreserved.

Y.-D. Xie et al.
Biomedicine&Pharmacotherapy99(2018)715–724
irritation, stomach ulcers, and so on. Therefore, it is unwilling to apply
to patients with T2DM.
with a Bruker AV400 spectrometer, chemical shifts were recorded in
ppm, tetra-methylsilane was used as the internal standard and coupling
constants (J) were given in Hertz. Mass spectra were obtained on
Shimadzu HPLC-MS-QP2010 instrument. The progress of the reactions
was monitored by TLC using Qingdao Haiyang Chemical Co. Ltd, HG/
T2354-92 Silica Gel GF254. Purifications of compounds were made by
flash column chromatography using Qingdao Haiyang Chemical Co.
Ltd, silica gel (100–200 mesh). Melting points were recorded using
Tech. Instrument Co. Ltd, XT-4 melting point apparatus.
Compound 1a was prepared according to the literature procedure
(Mp: 55–57 °C) [17]. Niacin (1.00 g, 8.12 mmol) was dissolved in di-
chloromethane (CH2Cl2) solution and reacted with oxalyl chloride
(1.05 mL, 10.58 mmol) under ice-bath for 12 h to give 1b. 1a (2.17 g,
6.49 mmol) was dissolved in CH2Cl2, and reacted with 1b by dropwise
adding under ice-bath for 12 h. Then the crude product was in ethanol
solution was mixed with palladium over charcoal (Pd-C) and the mix-
ture was hydrogenated under the atmosphere of hydrogen with mag-
netic stirring. After 24 h at r.t. the catalyst was filtered off and solvent
was evaporated in vacuum, and purified by column chromatography
(chloroform-methanol, 20:1) to yield HT-N as white solid (1.26g, Yield:
74.91%). Mp: 145.1 ∼ 146.2 °C. EI-MS (m/z): 259.1 (M+). 1H NMR
(400 MHz, DMSO-d6) δ 9.06 (d, J = 1.7 Hz, 1H), 8.86 ∼ 8.79 (m, 2H),
8.73 (s, 1H), 8.26 (m, 1H), 7.58 (m, 1H), 6.65 ∼ 6.69 (m,2H), 6.55 (m,
1H), 4.42 (t, J = 6.8 Hz, 2H), 2.86 (t, J = 6.8 Hz, 2H). 13C NMR
Furthermore, as is well known that sustainable hyperglycemia is the
feature of diabetic mellitus, which leads to an activation of oxidative
stress with an overproduction of reactive oxygen species (ROS) such as
superoxide anion, hydroxyl radical, hydrogen peroxide, and peroxyni-
trite [11,12]. ROS has been shown to refer to the formation of advanced
glycation end-products (AGEs). Excessive production of ROS accelerates
non-enzymatic protein glycation reactions. Increased glycation and the
build-up of tissue AGEs have been implicated in diabetes mellitus and
led to diabetic complications. Recently, there were more and more at-
tentions have been paid on the contribution of AGEs to diabetic mel-
litus. In the issue, both high-FBG and AGEs are regarded as the pa-
thological cause of diabetic complications. It has been stated that
antioxidants and reactive free radical scavengers stop these processes
[13]. Additionally, the recent researches have shown that compounds
combination antioxidant and antiglycation activities are more efficient
in administration diabetic mellitus and its complications [14].
HT (3, 4-dihydroxy phenylethyl alcohol) is the major natural phe-
nolic antioxidant present in olives and olive oil. The natural antioxidant
phenolic molecules have antioxidant and α-glucosidase inhibition ac-
tivities [15]. Moreover, HT has shown a diverse biological activities
such as scavenging ROS, inhibition of lipid peroxidation, cardiopre-
ventive property, hypolipidemic and hypoglycemic activities [16]. In
order to address the insufficient effect on increasing FBG of niacin used
in dyslipidemic diabetes, we planned to introduce HT pharmacophore
to niacin and successfully designed and synthesized compound HT-N
(Chart 1). We expected that the new compound HT-N can not only
reduce lipid profiles and FBG, but also decrease the formation of AGEs
and oxidative stress as well as lessen the adverse effects.
(100 MHz, DMSO-d6)
137.24, 129.01, 126.17, 124.40, 120.05, 116.72, 116.01, 66.45, 34.19.
δ 165.10, 154.12, 150.39, 145.61, 144.27,
2.2. Biological evaluation
2.2.1. Antioxidant activities
The hybrid compound HT-N is a case in multi-target agents that
only one chemical entity is found able to adjust more than one target at
the same time. At present, it is meaningful to apply this kind of agents
to administrate the dyslipidemic diabetes. In this paper, we reported the
conclusive results of antioxidant, anti-glycation, and α-glucosidase in-
hibition in vitro as well as hypolipidemic and hypoglycemic activities in
vivo of HT-N.
The antioxidant activities of HT-N were evaluated towards
scavenging free radicals (DPPH% and OH%) and inhibiting lipid perox-
idation. The assay procedures were referred to the previous literatures.
DPPH radical scavenging activity assay [18]: An aliquot (0.5 mL) of
0.08 g/mL DPPH% solution was mixed with 0.5 mL anhydrous alcohol,
0.5 mL sample (anhydrous alcohol reagent or antioxygen) and 0.1 M
acetate sodium acetate buffer solution (pH 5.5). Then the solution
should be mixed well and keep in the dark place at r.t. for 30 min. The
detective wavelength was 517 nm. The DPPH radical scavenging result
was expressed according to the following formula:
2. Experimental protocols
DPPH radical scavenging activity (%) = [(Ao-A1)/Ao)] × 100.
Wherein, Ao was the absorbance at 517 nm of the solvent control
reaction and A1 is the absorbance at 517 nm of varying concentrations
of samples. Each absorbance was rectified by using blank solution
(distilled water instead of the samples). All tests were carried out as
parallel for three times.
2.1. Chemistry
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded
Hydroxyl radical scavenging activity assay: the hydroxyl radical
scavenging activity of target compound HT-N was calculated by the
Fenton reaction [19]. Hydroxyl radicals were produced in the
Fe2+–Phenanthroline–H2O2 system (Fenton reaction). The reaction
solution contained 0.5 mL phenanthroline (2.1 mM), 1.0 mL KH2PO4-
NaOH buffer (50 mM, pH 7.4), 0.5 mL freshly prepared 0.03% H2O2 (v/
v), FeSO4, 0.5 mL ascorbic acid (100 mM), 0.5 mL solvent, and 0.5 mL
varying concentrations of compound HT-N. 10 mL tubes covering re-
action solutions were incubated at 37 °C for 60 min. After that, the
absorbance of the solution was measured and the detective wavelength
was 536 nm with an appropriate blank group (without target compound
and replaced by an equal amount of distilled water). The hydroxyl ra-
dical scavenging activity was calculated as follows:
Hydroxyl radical scavenging activity (%) = [1-(As0 -As1) / (Ab0
-Ab1)] × 100.
Wherein, As0 was the absorbance at 536 nm of the sample reaction
which does not contain the H2O2 solution and As1 was the absorbance at
536 nm of the sample reaction which contains the H2O2 solution and
Ab0 was the absorbance at 536 nm of the control reaction which does
not contain the H2O2 solution and Av1 was the absorbance at 536 nm of
Chart 1. Design of compound HT-N and structures of Niacin and Hydroxytyrosol (HT).
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Y.-D. Xie et al.
Biomedicine&Pharmacotherapy99(2018)715–724
the control reaction which contains the H2O2 solution. All tests were
carried out as parallel for three times.
Sucrase activity inhibition was determined by using sucrose as the
substrate following the same procedure of the test of maltase.
Anti-lipid peroxidation activity [20]: 0.1 mL of compound HT-N,
1.5 mL of 0.2 M sodium phosphate buffer (pH 7.45), 0.2 mL of yolk
suspension, and 0.2 mL of FeSO4 solution (25 mM) were shook well and
incubated in water bath at 37 °C for 30 min. Then 1.0 mL of 20% tri-
chloroacetic acid (TCA) was added. After that, all of the tubes with a
mixture solution were centrifuged at 3500 r/min for 15 min. TBA
(1.0 mL; 0.80%) was added and incubated at 100 °C for 15 min and
cooled at r.t.. The absorbance of the mixture solution was measured at
532 nm. All tests were carried out triply. The effects were calculated as
follows:
2.2.4. Effect of PBG in normal mice
KM male mice (20.0
2.0 g) were adapted to local environmental
conditions for a week and offered normal diet and water ad libitum.
Subsequently all of mice were fasted for 10h (allow water) and then the
PBG were measured by hand-held glucometer [23]. All of the mice were
divided into five groups by PBG: blank group, vehicle; model group,
starch; acarbose group, starch + acarbose; HT-N group, starch + HT-N;
niacin group, starch + niacin. The dosage of starch was 3 g/kg. The
glucose levels of all the groups were respectively measured with a hand-
held glucometer at 0.5 h, 1.0 h, 2.0 h after intragastrical gavage with
corresponding administration. Blood samples were applied directly to
the glucose strip from mice tails to measure the levels of blood glucose.
Anti-lipid peroxidation activity (%) = (A0-AS)/A0 × 100.
Wherein, A0 is the absorbance at 532 nm of the control reaction and
AS is the absorbance at 532 nm of the samples reaction.
2.2.5. Evaluation of HT-N in experimental T2DM mouse model
In the T2DM experimental mouse model, high fat diet (HFD) was
used to induce insulin resistance, besides the destruction of pancreatic β
cells was induced by multiple low doses of STZ and led to a mild im-
pairment of insulin secretion, which was similar to that of the later
stage of T2DM [24,25]. The potent compound HT-N was further eval-
uated for its activities in this T2DM mouse model.
2.2.2. Anti-glycation activity
The glycation reactions of the body were simulated with bovine
serum albumin (BSA)-methylglyoxal (MGO) system and according to
the previous literature with minor modification [20,21]. The AGEs
were determined with the fluorometric spectrophotometry. 2.0 mL BSA
was added to 2.0 mL MGO and shook well, then added the set con-
centrations of samples and 2.0 mL PBS. After that the reaction tubes
were incubated at 55 °C for 40 h. All tests were carried out in thrice and
averaged. The inhibitory rates were calculated as the following for-
mula:
Male KM mice (20.0
2.0 g) were acclimated for a week and allowed
water and normal rodent chow ad libitum. Subsequently they were di-
vided into five groups: blank group, normal diet; model group, HFD + STZ
(high fat diet + streptozotocin); Met-HCl group, HFD + STZ + compound
Inhibition (%) = (F- F)/F × 10 (100)
acarbose; HT-N group, HFD + STZ + compound HT-N; N group,
HFD + STZ + compound niacin. During the test, mice of all groups were
allowed ad libitum the HFD or normal diet. At the end of 6-week, lipid
profiles of HFD diet treated mice were measured and the lipid levels
shown significant difference compared to the blank group (not given
here). At 7-week, STZ was dissolved in 0.1 M citrate buffer (pH 4.5) and
administrated (60 mg/kg) by intraperitoneal injection after 12 h of fasting
to groups HT-N, niacin and acarbose with multiple low doses (60 mg/kg/
day × 7 days). From 8-week to 11-week, compounds were administered
by intragastrical gavage once daily, while similarly, saline was given by
intragastrical gavage to blank and model groups. After the experiment, all
the mice were taken from the eyeball for blood. The serum was prepared
by centrifugation and the pancreas was removed and cleaned; all these
samples were stored at −80 °C. Pieces of pancreas were fixed in 4%
paraformaldehyde solution for histological studies.
Where F0 was the fluorescence intensity of the blank control and F1 was
the fluorescence intensity of the sample.
2.2.3. α-glucosidase inhibitory activityin vitro
The α-glucosidase inhibitory activities of the compound HT-N were
assayed towards yeast α-glucosidase and rat intestinal α-glucosidase
(maltase and sucrase).
Inhibitory activity towards yeast α-glucosidase: The inhibitory ac-
tivity of HT-N towards yeast α-glucosidase was measured as reported in
the literature with a slight modification [22]. In brief, α-glucosidase
was dissolved in phosphate buffer (pH 6.86) at a final concentration of
10 U/mL. About 10 μL enzyme solution was preincubated with 50 μL of
the samples at varying concentrations in phosphate buffer (pH 6.86) at
37 °C for 15 min. The reaction was started by the addition of 20 μL 4-
nitrophenyl-α-D-glucopyranoside (p-NPG, the final concentration of
5.3 mM) and ended with the addition of 50 μL of 0.5 M Na2CO3 after
15 min. The amount of released 4-nitrophenol from p-NPG was de-
termined as the absorbance at 405 nm. In each set of experiments the
assay was conducted in triplicate. The increased absorbance was com-
pared to that of the control containing 50 μL of phosphate buffer in
place of the test solution.
Inhibitory activity towards rat intestinal α-glucosidase of maltase:
The α-glucosidase from rats intestinal brush border membranes were
prepared accordance with literatures method that were used as the
enzyme source of rat intestinal maltase and sucrose [22]. For maltase
activity inhibition, maltose was selected as substrate. The D-glucose
produced from hydrolysis of maltose was measured by the previous
method of Glucose Test Kit (Nanjing Jiancheng). About 10 μL enzyme
solution was preincubated with 50 μL of the samples at varying con-
centrations in phosphate buffer (pH 6.86) at 37 °C for 15 min. Then
20 μL maltose (50 mM) was added, and the mixture was incubated for
another 60 min, and ended by the boiling water bath. Then the above
solution (10 μL) was added with reagent A (500 μL) and reagent B
(500 μL), and then the solution was mixed well and incubated at 37 °C
for 15 min. The absorbance was measured at 505 nm. The test was
conducted in triplicate.
2.2.5.1. Glucose levels. Blood glucose levels were determined by a
hand-held glucometer at the end of the experiment. Blood samples
were applied directly to the glucose strip from mice tails to measure the
levels of blood glucose after 6 h of fasting.
2.2.5.2. Other serum biochemical parameters. At the end of experiment,
lipidemia and oxidative parameters were determined using
commercially available kits (Nanjing Jiancheng). Lipidemic
parameters included TG, TC, HDL-C and LDL-C. Oxidative parameters
included SOD, MDA, GSH-PX, and CAT.
2.2.5.3. Histological study. Pieces of fixed pancreas were embedded in
paraffin and the optimal cutting temperature (OCT). They were cut into
7μm thickness sections and stained with hematoxylin–eosin. All of the
sections were examined by the light microscope (Olympus).
2.2.6. Antihyperlipidemiain vivo
Compound HT-N (0.38 mmol/kg), finely suspended with the help of
CMC-Na (with a final concentration of ∼0.5%) in saline, or saline only,
was administrated by intragastric gavage for one week. Niacin
(0.38 mmol/kg) was selected as a positive agent. At the sixth day, an
aqueous solution of Triton WR 1339 (400 mg/kg) was given to KM male
Inhibitory activity towards rat intestinal α-glucosidase of sucrose:
mice (20.0
2.0 g) by tail vein [26]. After 24 h, blood was taken from
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Biomedicine&Pharmacotherapy99(2018)715–724
Scheme 1. Synthesis route of compound HT-N. Reagents and conditions: (a) BnBr, K2CO3, acetone, reflux; (b) oxalyl chloride, ice-bath, CH2Cl2; (c) ice-bath, CH2Cl2; (d) H2, Pd/C,
ethanol.
the eyeballs and used for the determination of plasma TG, TC, and MDA
levels, using commercially available kits (Nanjing Jiancheng).
2.3. Statistical analysis
Data are expressed as the mean
SEM. The determinations were
performed from 10 animals each group and the differences were ex-
amined by the one-way analysis of variance (ANOVA) followed by the
Dunnett’s post-test, and a statistically significant difference was inferred
if P < .05.
3. Results
3.1. Chemistry
The preparation of compound HT-N was demonstrated in Scheme 1.
HT was transformed into benzyl derivatives 1a by reaction with benzyl
bromide and potassium carbonate in acetone. Niacin was first reacted
with oxalyl chloride under ice-bath to give 1b, then esterified with 1a
to give 2a, which was deprotected to target compound HT-N in hy-
drogen and Pd/C. The product was purified by column chromato-
graphy.
Fig. 1. The antioxidant activities of HT-N were evaluated towards scavenging free radi-
cals (DPPH% and OH%) and inhibiting lipid peroxidation in vitro. Data are shown as the
mean
SEM, n = 3.
3.2. Biological activity
3.2.1. Antioxidant propertiesin vitro
We evaluated antioxidant activities of HT-N in vitro by scavenging
radicals (DPPH• and OH•) and inhibiting lipid peroxidation. The results
are summarized in Fig. 1, both HT-N and HT exhibited strong anti-
oxidant ability, while niacin did not manifest the antioxidant proper-
ties. The results indicated that phenolic hydroxyl is the favorable
structure for scavenging reactive free radicals and anti-lipid peroxida-
tion.
3.2.2. Anti-glycation propertiesin vitro
The effect of HT-N on AGEs formation was showed in Fig. 2. Bovine
serum albumin (BSA)-methylglyoxal (MGO) system was used to simu-
late the glycation reactions of the body [20]. HT-N and HT showed
excellent inhibition of AGEs formation, with IC50 values of 0.27 and
0.09 mM, respectively. While niacin did not show anti-glycation ac-
tivity. It has been reported that free radicals have been shown to par-
ticipate in the formation of AGEs and naturally occurring polyphenols
Fig. 2. Effect of HT-N on anti-glycation activity. Bovine serum albumin (BSA)-methyl-
glyoxal (MGO) system was used to simulate the glycation reactions of the body. The
results are represented as the mean
SEM, n = 3.
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Biomedicine&Pharmacotherapy99(2018)715–724
displayed stronger inhibitory activities than both niacin and HT. From
the structure of HT-N and HT, we can find that both them have pyr-
ocatechol. So they were belong to polyphenols. Previous studies have
reported that polyphenolic compounds possess α-glucosidase inhibitory
activity [30]. Both HT-N and HT have the structure of catechol.
Therefore, the results of this study support the viewpoint reported in
the literature.
3.2.4. Effect of PBG in normal mice
As compound HT-N shown be lower yeast α-glucosidases and
stronger rat intestinal α-glucosidases maltase and sucrose compared to
HT in vitro. In order to further confirm the α-glucosidases inhibitory
activities, we performed the test of α-glucosidases inhibiton by post-
prandial blood glucose. The PBG was measured in normal mice (Fig. 4).
Fig. 4A shown that the glucose levels of all the groups were increased
except blank and acarbose groups between 0 min and 60 min. And then
the glucose levels went down rapidily. While the glucose levels of
acarbose group reached the top at 30 min, then went down and kept
horizontal. During the test, the glucose levels of HT-N and HT groups
still lower than model group. Fig. 4B revealed that the area under the
curve (AUC) of acarbose, HT and HT-N, which shown a significant
difference compared to model group, which indicated that HT-N and
HT have the properties of α-glucosidases inhibition.
Fig. 3. The α-glucosidase inhibitory activities of the compound HT-N were assayed to-
wards yeast α-glucosidase and rat intestinal α-glucosidase (maltase and sucrase). The
results are represented as the mean
SEM, n = 3.
play a valuable role in preventing AGEs formation [27]. The results of
our present study support these viewpoints. Moreover, reports in-
dicated that compounds with combined antiglycation and antioxidant
properties were more effective in protecting against glycation reactions
in the early and late stages of AGEs formation as well as the diabetic
complications [28].
3.2.3. α-glucosidase inhibitory activities
3.2.5. Evaluation of HT-N in experimental T2DM mouse model
3.2.5.1. Hypoglycemic activity. Due to the inhibition of α-glucosidases
in vitro and the significant reduction of PBG in normal mice, we
performed the hypoglycemic action in T2DM mouse model. Fig. 5
shown the FBG. The FBG of model group was significantly increased.
After treatment of HT-N, it was found able to markedly reduce glucose
levels by 16.9% compared with the model group. HT also shown the
potent decreasing blood glucose levels by 9.9%. While niacin did not
display hypoglycemic activity. This findings indicated that the
incorporation of the HT structure into niacin reserves the
hypoglycemic activity of HT.
The activity of compound HT-N was evaluated in vitro against yeast
α-glucosidase from Saccharomyces cerevisiae and rat intestinal α-glu-
cosidase (maltase and sucrase). The α-glucosidase inhibitory activity
was assayed by the method described in the previous literatures
[22,29]. Acarbose was selected as positive control. The results were
summarized in Fig. 3 which shown a comparison of the IC50 values of
acarbose, HT, niacin and HT-N. HT-N revealed an excellent α-gluco-
sidase
(IC50 = 117.72 μM)
inhibitory
activities,
which
including
was stronger
yeast
α-glucosidase
acarbose
than
(IC50 = 230.92 μM) and both rat intestinal α-glucosidases maltase (IC50
=31.86 μM) and sucrase (IC50 = 22.99 μM) which were lower than
acarbose (the IC50 values of maltase and sucrase were 3.06 μM and
1.99 μM, respectively). And HT also shown α-glucosidase inhibitory
activities, and the IC50 values of yeast α-glucosidase, rat intestinal α-
glucosidases maltase and sucrase were 35.71 μM, 42.02 μM, and
72.30 μM, respectively. While niacin did not show the α-glucosidase
inhibitory activities. These findings indicated that the target compound
HT-N exhibits significantly enhanced inhibitory activities on yeast α-
glucosidase compared with niacin and slightly weaken compared with
HT. While on rat intestinal α-glucosidases maltase and sucrase, HT-N
3.2.5.2. Plasma lipid levels. Since there is intense correlation between
dyslipidemia and T2DM, plasma lipids concentrations of TG, TC, HDL-C
and LDL-C were tested at the end of the experiment (Fig. 6). In the
model group, plasma TG, TC, HDL-C, and LDL-C levels apparently
increased. HT-N significantly reduced the TG, TC, and LDL-C levels by
43.8%, 20.6%, and 38.7%, HT lowered these indexes by 18.5%, 12.8%,
and 29.4% and niacin decreased the lipid parameters by 36.9%, 11.0%
and 29.0% compared with the model group. These findings suggested
Fig. 4. The PBG were measured in normal mice. Starch (3 g/kg body weight) was orally administrated to 10 h fasted mice of each group. The glucose levels of all the groups were
respectively measured with a hand-held glucometer at 0.5 h, 1.0 h, 2.0 h after intragastrical gavage with corresponding administration. (A) Glycemic time curve; (B) Area under the curve
###
*
(AUC) of each group. HT-N (0.38 mmol/kg), HT (0.38 mmol/kg), niacin (0.38 mmol/kg) and acarbose (0.039 mmol/kg). Mean
SEM, n = 10,
P < .001 vs model group,
***
P < .05,
P < .001 vs model group.
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activities in vivo, increasing plasma SOD, GSH-PX and CAT levels by
51.1%, 22.4% and 73.0%, respectively, and reducing plasma MDA
levels by 41.3%, compared to the model group. HT exhibited the similar
antioxidant effects as HT-N, elevating plasma SOD, GSH-PX and CAT
levels by 41.9%, 23.8% and 65.2%, respectively, and lowering plasma
MDA levels by 40.4%, While niacin aggrandized plasma SOD levels by
9.9%, decreased plasma MDA, GSH-PX and CAT levels by 3.7%, 13.1
and 11.6%, respectively, which did not show statistical significance
compared to model group. The results indicated that catechol of HT-N
or HT is the favorable structure for ameliorating oxidative stress. Thus,
the potent antioxidant activity of HT-N is confirmed in vivo.
3.2.5.4. Pancreas histology. As shown in Fig. 8, the pancreas of blank
mice shown normal islets (Fig. 8A). In contrast, the β cells destruction
and reduction in the number were found in T2DM mice model (model
group, Fig. 8B). Exhilaratingly, potently protective action of β cells was
recorded in diabetic mice administrated with HT-N (Fig. 8C) and HT
(Fig. 8D). While treatment with niacin and acarbose, β cells did not
show to be improved (Fig. 8E and F, respectively).
Fig. 5. Blood glucose levels in the T2DM mouse model after intragastrical gavage ad-
ministration with HT-N (0.38 mmol/kg), HT (0.38 mmol/kg), niacin (0.38 mmol/kg), and
acarbose (0.039 mmol/kg) as well as saline (blank and model groups). All group mice
were sacrificed after 4 weeks of treatment. The results were expressed as Mean
SEM,
n = 10. Statistically significant difference between groups: ###P ＜ .001 vs blank group;
*P ＜ 0.05, ***P ＜ .001 vs model group.
3.2.6. Antihyperlipidemic activityin vivo
Hypolipidemia property of HT-N was evaluated in hyperlipidemia
mouse model induced by Triton WR-1339 [26]. HT-N was found able to
significantly lower plasma TG and TC levels by 34.6% and 45.8% re-
spectively (Fig. 9). HT-N shown a superior effect on both lipidemia
indices compared to that of HT and niacin, which reduced plasma TG
levels by 17.0% and 28.3%, and TC levels by 30.5% and 7.9% respec-
tively. The results may indicate that HT-N contributes a synergistic
antihyperlipidemic effect through hydrolyzation by esterase to niacin
that HT-N may have both the hypolipidemic effects of HT and niacin.
3.2.5.3. Antioxidant activities. As described in Fig. 7, the activities of
antioxidases such as SOD, GSH-Px, and CAT were observably reduced in
T2DM mice plasma and the production of lipid peroxide byproduct
MDA was memorably increased. HT-N shown intense antioxidant
Fig. 6. Plasma levels of TG, TC, LDL-C, and HDL-C in the T2DM mouse model after intragastrical gavage treatment with 0.38 mmol/kg compounds HT-N, HT and niacin as well as saline
(blank and model groups). All group mice were sacrificed after 4 weeks of treatment. The results were expressed as Mean
groups: ##P ＜ .01, ###P ＜ .001 vs blank group; *P ＜ .05, ***P ＜ .001 vs model group.
SEM, n = 10. Statistically significant difference between
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Biomedicine&Pharmacotherapy99(2018)715–724
Fig. 7. Plasma levels of SOD, MDA, GSH-PX, and CAT in the T2DM mouse model after intragastrical gavage treatment with HT-N (0.38 mmol/kg), HT (0.38 mmol/kg), niacin
(0.38 mmol/kg), and acarbose (0.039 mmol/kg) as well as saline (blank and model groups). All group mice were sacrificed after 4 weeks of treatment. The results were expressed as
Mean
SEM, n = 10. Statistically significant difference between groups: ##P ＜ .01, ###P ＜ .001 vs blank group; **P ＜ .01, ***P ＜ .001 vs model group, △△△P ＜ .001 vs niacin group.
and HT. Furthermore, HT-N reduced MDA level in the plasma of ex-
perimentally induced hyperlipidemia mice by 32.1% (Fig. 10). Thus,
increased potent antioxidant activity in vitro is also confirmed in vivo.
are the most common form of disorder, which brings about coronary
heart disease and strokes attributed to atherosclerosis. Dyslipidemia,
particularly the hypertriglyceride, often involved in the development
and progression of atherosclerosis. It is common knowledge that T2DM
patients are more liable to be dyslipidemic compared to the ordinary
population and dyslipidaemia in patients with T2DM is often char-
acterised by hypertriglyceridaemia and low HDL-C. Dyslipidemia oc-
curs earlier and progresses faster in T2DM patients [31]. Recent studies
4. Discussion
T2DM companied by vascular complications resulted in a con-
siderable morbidity and mortality worldwide. Vascular complications
Fig. 8. Effects of HT-N on pancreas protection in normal (blank) and T2DM mice (H&E 200×). Pancreas were embedded in paraffin and cut into 7μm thickness sections and stained with
hematoxylin–eosin. (A) Blank group; (B) Model group; (C) HT-N group; (D) HT group; (E) Niacin group; (F) Acarbose group; (G) Beta-cell mass. Data indicate the mean of six independent
experiments
SEM. ###P ＜ .001 vs blank group; *P ＜ .05 vs model group; ***P ＜ .001 vs model group.
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Biomedicine&Pharmacotherapy99(2018)715–724
Fig. 9. Percent reduction of TG and TC plasma levels of Triton WR 1339 induced hyperlipidemic mice after intragastrical gavage administration of 0.38 mmol/kg HT-N, HT and niacin as
well as saline (blank and model groups). All group mice were sacrificed after 4 weeks of treatment. The results were expressed as Mean
between groups: ###P ＜ .001 vs blank group; **P ＜ .01, ***P ＜ .001 vs model group.
SEM, n = 10. Statistically significant difference
antioxidant activity and α-glucosidase inhibitory activity. HT-N over-
comes the slight negative impact increased FBG of niacin. HT-N ex-
hibited a strong α-glucosidase inhibitory activity in vitro, inhibiting
yeast α-glucosidase (IC50 = 117.72 μM) and rat intestinal α-glucosi-
dases maltase (IC50 = 31.86 μM) and sucrase (IC50 = 22.99 μM)
(Fig. 3), and had a good control of postprandial blood glucose (PBG) in
normal mice (Fig. 4). Moreover, in T2DM mouse model, HT-N could
significantly reduce the blood glucose levels by 16.9% (Fig. 5).
A series of evidence has been shown that diabetic patients manifest
oxidative stress resulting from hyperglycemia, especially the post-
prandial hyperglycemia, with an overproduction of ROS. The en-
dogenous ROS is generated by activation one of a leading procedure:
increased formation of advanced glycation end-products (AGEs) in
diabetic patients. HT-N shown to be a significant inhibitor of glycation
reaction in vitro at very low concentration (IC50 = 0.27 μM, Fig. 2).
Moreover, ROS are involved in the insulin resistance and the damage of
various cell types, including endothelial (in atherosclerosis) and pan-
creatic β cells (in diabetes), acting by diverse mechanisms, e.g., via lipid
peroxidation [26]. Thus, oxidative stress is seen as responsible for the
progressive degeneration of β cells due to the particularly high sensi-
tivity of these cells to excessive ROS. Compound HT-N was shown to be
potent antioxidant activities both in vitro and in vivo. HT-N displayed a
good scavenging free radicals such as DPPH% (IC50 = 7.58 μM) and OH%
(IC50 = 0.34 mM) as well as an excellent anti-lipid peroxidation
(IC50 = 0.27 μM) in vitro (Fig. 1). The enhanced antioxidant activity of
the target compound HT-N also shown in vivo, as it elevated plasma
SOD, GSH-PX and CAT levels by 51.1%, 22.4% and 73.0%, respectively,
and lowered plasma MDA levels by 41.3% in T2DM mice (Fig. 7).
Moreover, a patent protective action of β cells was recorded in HT-N
group (Fig. 8). Thus, the potential mechanism of activity of HT-N on
hypoglycemia may be possibly explained by the following reasons: (i)
inhibition of glycation; (ii) inhibition of intestinal α-glucosidases mal-
tase and sucrase and decreasing the source of glucose; (iii) protection of
pancreatic β cells from damage and death induced by prolonged oxi-
dative stress. Our results shown that HT-N treatment induced a pro-
tective effect in T2DM experimental mouse. Therefore, our findings
support the recent studies that compounds with combined antioxidant
and antiglycation properties are more effective in treating diabetes
mellitus [36].
Fig. 10. Plasma MDA levels of Triton WR 1339 induced hyperlipidemic mice in the
presence of 0.38 mmol/kg compounds HT-N, HT and niacin as well as saline (blank and
model groups). All group mice were sacrificed after 4 weeks of treatment. The results
were expressed as Mean
SEM, n = 10. Statistically significant difference between
groups: ###P ＜ .001 vs blank group; ***P ＜ .001 vs model group.
outline the role of oxidative stress in combination with hyperlipidemia
in the pathogenesis and development of T2DM. At present, TG has been
a leading target for intervention efforts in the clinical management of
dyslipidemia, which plays a positive part in ameliorating vascular
complications, with fibrates being the predominant choice for ther-
apeutic intervention. However, other than increasing the size and the
number of PPAR-α, fibrates induced the expression of genes encoding
enzymes of the peroxisomal β-oxidation pathway, resulting in a dra-
matic elevation of the activity of peroxisomal acyl-CoA oxidase [32].
The increased production of ROS by this pathway was thought to play a
key role in oxidative stress [33]. The superoxide anion (O2−) and hy-
drogen peroxide (H2O2) were marked increase as well as superoxide
dismutase (SOD) and glutathione peroxidase (GPX) was significantly
decreased in rat liver during treatment with fibrates [34]. Both known
important side effects of fibrates (hepatotoxicity and myotoxicity) have
been associated with oxidative stress induced by elevation of the ac-
tivity of peroxisomal acyl-CoA oxidase. Moreover, studies to date have
not demonstrated an overall benefit of fibrates for reduction of cardi-
ovascular or total mortality [3]. It has been reported that lipid-mod-
ifying doses of niacin have broad lipid-modifying effects and can be
applied safely in T2DM patients with dyslipidemia [1]. Niacin treat-
ment may be regarded as an alternative to fibrates in patients with
dyslipidemic diabetes. However, niacin has clinically slight negative
impact on blood glucose that it could lead to an increase in FBG [35].
The design of target compound HT-N was based on the pathogenesis
and characteristics of type 2 diabetes mellitus (T2DM). Compound HT-
Related literatures have displayed the contribution of oxidative
stress in coupled with dyslipidemia in the development and progression
of both T2DM and atherosclerosis. HT-N exhibited a very strong hy-
polipidemic effect, decreasing plasma concentrations of TG and TC by
34.6% and 45.8% in hyperlipidemic mice (Fig. 9). Moreover, HT-N also
shown a potent antihyperlipidemic activity in T2DM mouse model,
reducing the TG, TC, and LDL-C levels by 43.8%, 20.6%, and 38.7%,
respectively (Fig. 6).
N
(Chart 1) was built on the incorporation of the anti-
hypertriglyceridemia and inhibition of α-glycosidase (polyphenol
compound HT). HT is the major natural antioxidant phenolic and exerts
Niacin has more adverse effects in clinical application, such as
flushing, itchy, gastrointestinal irritation, stomach ulcers, and so on.
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Biomedicine&Pharmacotherapy99(2018)715–724
Research shown that the prodrug of niacin ester derivatives (such as
inositol nicotinate, niceritrol and nicomol) can be converted to niacin to
play the role of lowering blood lipid in vivo and it can reduce the side
effects of niacin. Niacin was in alkyd esterification process to produce
the target compound HT-N. Thus, we infered that HT-N may decrease
the adverse effects in vivo. Moreover, lipo-hydro partition coefficient of
HT-N (LogP = -0.583) was similar to niacin (LogP = −0.797), which
did not describe in the paper. So the absorption capacity of HT-N may
be similar to niacin. Therefore, HT-N will be a potential therapeutic
agent in clinical application.
Niacin can regulate lipids metabolism in vivo. HT shown marked
hypolipidemic and hypoglycemia activities and endowed with sig-
nificantly antioxidant and antiatherogenic activities. The hypolipidemic
mechanism of HT may be due to the inhibition of dietary lipids ab-
sorption in the intestine or its production by liver or stimulation of the
biliary secretion of lipid and lipids excretion in the faeces [37]. The
hypoglycemia mechanism of HT may be due to protection of pancreatic
β cells from oxidative stress and can enhance insulin secretion [38].
These findings agree with previous studies showing that HT decrease
TG and TC in a group of mildly dyslipidemic patients. Therefore, we
hypothesized that niacin and HT hydrolyzed by esterase from HT-N
synergistically suppress the progress of hyperlipidemia with antioxidant
activity and prevent the β pancreatic cells from damage in T2DM mice.
This hypothetic statement also can be supported by the results of re-
lated studies which have reported HT derivatives which were composed
of HT units attached to other compounds via ester were hydrolyzed by
enzymes to release HT and other metabolites [39]. Additionally, the
results of HT-N in vitro displayed significant antioxidant, anti-glycation
and α-glucosidase inhibitory activities. Based on the effects in vivo and
in vitro, the molecular mechanisms of HT-N in experimental mice may
be by the multifunctional effects of HT-N and/or be converted by es-
terases into the bioactive metabolite niacin and HT.
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5. Conclusion
In summary, a multi-target-directed approach in this study led us
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N significantly reduced PBG in normal mice. In T2DM mouse model,
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TG and TC as well as lower MDA in hyperlipidemic mouse model. All of
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Acknowledgement
Financial support from the National Natural Science Foundation of
China (Grant No. 21172177) is gratefully acknowledged.
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