J Basic Clin Physiol Pharmacol 2018; aop
Andriy L. Zagayko, Tetiana Ye. Kolisnyk*, Olena I. Chumak, Olena A. Ruban
and Oleh M. Koshovyi
Evaluation of anti-obesity and lipid-lowering
properties of Vaccinium myrtillus leaves powder
extract in a hamster model
https://doi.org/10.1515/jbcpp-2017-0161
Received September 19, 2017; accepted May 23, 2018
Abstract
Background: Vaccinium myrtillus leaves are known to be
rich in phenols and have been used in traditional medi-
cine as an antidiabetic remedy. This study evaluated the
powder extract of V. myrtillus leaves obtained with the use
of L-arginine and myo-inositol for anti-obesity and lipid-
lowering potential in hamsters.
Methods: Standard phytochemical methods were used
to determine the total phenolic and total flavonoid con-
tents of the extract. The obesity condition was induced in
Syrian hamsters by feeding them with highly palatable fat-
and sugar-rich diet (40.3 kcal% fat) for 12 weeks. From the
10th week of diet feeding, the obese hamsters were treated
with the powder extract of V. myrtillus leaves (15, 25 and
35 mg/kg/day, respectively) and “Styfimol” (6.2 mg/kg/day
of hydroxycitric acid) as a positive control drug. At the end
of the treatment period, the biochemical parameters as
well as visceral fat mass were determined.
Results: Vaccinium myrtillus leaves powder extract at 25
and 35 mg/kg/day caused a significant reduction in body
weight gain and visceral fat mass in obese hamsters. Serum
triacylglycerols, free fatty acids, total cholesterol and low-
density lipoprotein cholesterol (LDL-C) levels were also
significantly lower. Besides, the hamsters treated with
powder extract at 25 and 35  mg/kg/day had the closest
intact value ratio of high-density lipoprotein cholesterol
and LDL-C compared with positive control animals.
*Corresponding author: Tetiana Ye. Kolisnyk, Department of
Industrial Technology of Drugs, National University of Pharmacy,
Kharkiv, Ukraine, Phone: +380682474033, +380572678852,
E-mail: kolisnyktatyana@gmail.com. http://orcid.org/0000-0002-
2682-0360
Andriy L. Zagayko and Olena I. Chumak: Department of Biological
Chemistry, National University of Pharmacy, Kharkiv, Ukraine
Olena A. Ruban: Department of Industrial Technology of Drugs,
National University of Pharmacy, Kharkiv, Ukraine
Oleh M. Koshovyi: Department of Pharmacognosy, National
University of Pharmacy, Kharkiv, Ukraine
Conclusions: The results showed that V. myrtillus leaves
powder extract is a promising therapeutic agent for the
treatment of obesity and obesity-induced diseases.
Keywords: dyslipidemia; hamsters; herbal medicine;
obesity; Vaccinium myrtillus leaves.
Introduction
Obesity is a major component of metabolic syndrome
(MS) and an important risk factor for type 2 diabetes
mellitus (T2DM) and atherosclerosis. These pathologies
are strongly interrelated. Excess body weight impairs
insulin sensitivity of tissue cells, which in time can result
in hyperglycemia and diabetes. Clearly, most individu-
als with insulin resistance have truncal obesity [1, 2].
Another feature of obesity is dyslipidemia characterized
by increased triacylglycerols (TAGs), decreased high-den-
sity lipoprotein cholesterol (HDL-C) levels and abnormal
low-density lipoprotein cholesterol (LDL-C) composition.
In turn, obesity-induced dyslipidemia greatly contributes
to the development of cardiovascular pathologies [3, 4].
Currently, prevalence of obesity as well as diabetes
mellitus is dramatically increasing worldwide and for now
has reached epidemic proportions. That is why there is an
urgent need for new strategies not only to combat obesity
but also to prevent related disorders and complications.
Along with diet and physical exercises, dietary and herbal
plants are the most advantageous components of such
strategies. Plant-derived supplements are usually well tol-
erated even for long-term therapy and much less likely to
cause serious side effects compared with synthetic drugs;
nevertheless, they are effective due to multiple mecha-
nisms of action [5, 6]. Besides, in medicinal preparations,
constituents of plant origin may be successfully combined
with other substances of health benefits, such as vita-
mins, amino acids, etc.
The effectiveness of natural products of plant origin in
metabolic disorders is considered to be related to phenolic
compounds such as hydroxybenzoic and hydroxycinnamic
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2      Zagayko et al.: Anti-obesity and lipid-lowering properties of Vaccinium myrtillus leaves powder extract
acids, flavonoids, stilbenes and others [7, 8]. One of the
most known examples of plant source for weight loss
nutraceuticals and preparations is Garcinia cambo-
gia. The major biologically active component present in
G. cambogia fruits – hydroxycitric acid (HCA) – was found
to be effective in decreasing appetite, inhibiting fat syn-
thesis, lowering serum TAG level and reducing body
weight [9, 10]. Another promising source of plant phenols
is the bilberry (Vaccinium myrtillus) leaf, which has a long
history of use in folk medicine as an anti­diabetic remedy.
For now, hypoglycemic and lipid-lowering properties of
extracts of V. myrtillus leaves are confirmed in different
experimental models [11–13].
As it is known, herbal preparations such as dry extracts
are highly hygroscopic and prone to caking. Thereby,
appropriate solid carrier substrates (e.g. maltodextrin, cel-
lulose, lactose) and/or anticaking agents may be added to
native extracts to obtain powder extracts with improved
flowability [14]. Recently, such preparation method for
V. myrtillus leaves powder extract (VMLPE) has been devel-
oped under the supervision of Dr. O. Koshovyi (Depart-
ment of Pharmacognosy, National University of Pharmacy,
Kharkiv, Ukraine) [15]. This method proposes the use of
L-arginine and myo-inositol as carriers of health benefits
in MS and T2DM treatments. L-arginine was chosen as a
donor of nitric oxide – an endogenous vasodilator that
plays an important role in the prevention of endothelial
dysfunction [16]. Besides, L-arginine stimulates lipolysis
in adipocytes and promotes oxidation of long-chain fatty
acids in insulin-sensitive tissues [17]. On the other hand,
L-arginine is also described to increase solubility of poorly
soluble aromatic compounds [18]. Myo-inositol was chosen
because of its insulin mimetic properties [19, 20]. As a
result, in animal testing, VMLPE obtained by this method
had shown better hypoglycemic and insulin-sensitizing
activities compared to those of native extract [15, 21]. Thus,
as obesity and dyslipidemia are very common conditions
in MS and T2DM, the aim of the present work was to evalu-
ate in vivo the anti-obesity and lipid-lowering potential of
VMLPE containing L-arginine and myo-inositol.
Materials and methods
Chemicals and drugs
L-arginine was purchased from Kyowa Hakko Co. Ltd. (Tokyo, Japan).
Myo-inositol, Folin-Ciocalteu reagent, sodium carbonate, aluminum
chloride hexahydrate and analytical standard pyrogallol were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA). USP reference stand-
ard rutin trihydrate (Sigma-Aldrich, Steinheim, Germany) was used.
Hard gelatin capsules of “Styfimol” (Kyiv Vitamin Factory,
Ukraine) intended for excess weight treatment were used as a posi-
tive control drug in animal testing. The composition of active ingre-
dients per capsule was as follows: 100  mg of G. cambogia extract
(equivalent to 50 mg of HCA), 0.1 mg of chrome picolinate, 50 mg of
L-tyrosin, 15 mg of L-carnitine and 0.037 mg of iodine (in the form of
brown seaweed dry extract) [22].
Plant material and extract preparation
Herbal material consisting of dry V. myrtillus leaves with stem parts
was purchased from an herbalist at a local market of Kharkiv, Ukraine,
in September 2016 and identified by a qualified botanist of the Depart-
ment of Pharmacognosy, National University of Pharmacy, Kharkiv,
Ukraine. Dry leaves of V. myrtillus were separated from stem parts and
powdered to particle size of 1–2 mm. Powdered leaves were extracted
at room temperature by triple maceration with 50% (v/v) ethanol
[plant material to total solvent ratio was 1:10 (w/v)]. The resulting
extracts were combined, allowed to settle for 24  h and filtered. The
filtrate was evaporated to dryness using a rotary vacuum evaporator
by adding the mixture of L-arginine and myo-inositol in the amount
equal to the dry residue of the crude liquid extract [European Phar-
macopeia (Ph.Eur.) 2.8.16 [23]], wherein L-arginine was taken in three
equimolar amount to the total phenolic content (TPC) expressed as
pyrogallol. The obtained powder extract was stored at 4 °C until use.
Total phenolic content
The TPC in the crude liquid extract and powder extract was deter-
mined with Folin-Ciocalteu reagent according to Ph.Eur. 2.8.14 [23].
Briefly, 2  mL of sample (250-fold diluted solution of the crude liq-
uid extract or 0.25  mg/mL solution of powder extract) or standard
(0.025 mg/mL solution of pyrogallol) was mixed with 1 mL of Folin-
Ciocalteu reagent and 10 mL of water and then adjusted to 25.0 mL
with a 290  mg/mL solution of sodium carbonate. The mixture was
incubated for 30 min, and absorbance was measured at 760 nm by
a UV-Vis spectrophotometer Carry 100 (Agilent Technologies Inc.,
Santa-Clara, CA, USA). All determinations were performed in trip-
licate, and TPC was expressed as milligrams of pyrogallol equiva-
lent (PE) per milliliter of crude liquid extract or per gram of powder
extract dry weight.
Total flavonoid content
Total flavonoid content (TFC) in the powder extract was determined
by using a colorimetric aluminum chloride method as follows: 5 mL
of sample [5  mg/mL powder extract solution in 50% (v/v) ethanol]
or 1  mL of standard [5  mg/mL rutin solution in 50% (v/v) ethanol]
was mixed with 5 mL of 50% (v/v) ethanol and 5 mL of 2% aluminum
chloride solution in 50% (v/v) ethanol. After 10 min, 1 mL of 3% ace-
tic acid was added, and the mixture was adjusted to 25 mL with 50%
(v/v) ethanol. The mixture was maintained at room temperature for
30 min, and then absorbance was measured at 410 nm by a UV-Vis
spectrophotometer Carry 100 (Agilent Technologies Inc., Santa-
Clara, CA, USA). All determinations were performed in triplicate, and
TFC was expressed as milligrams of rutin equivalent (RE) per gram of
powder extract dry weight.
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 Zagayko et al.: Anti-obesity and lipid-lowering properties of Vaccinium myrtillus leaves powder extract      3

Experimental animals LDL-C levels were determined using HDL-C and LDL-C kits (Leadman,
China).

Sixty male 6-week-old Syrian hamsters (Mesocricetus auratus)

weighing 70–90 g were used for this study. Animals were kept in the Statistical analysis
vivarium of the Central Research Laboratory, National University of
Pharmacy (Kharkiv, Ukraine), according to the Guide for the Care and
Statistical analysis was carried out by the variation-statistical
Use of Laboratory Animals of the National Research Council (National
method using non-parametric analysis (Mann-Whitney U-test) with
Academies, USA, 8th edition, 2011). All experiments were conducted
Statisticа 7.0 (StatSoft Inc., Tulsa, OK, USA) and Microsoft Excel 2007
in accordance with the European Community Council Directive of
software (Microsoft Corporation, Seattle, WA, USA). Results are pre-
24 November 1986 (86/609/EEC).
sented as mean value ± SD. Significant levels were defined at p < 0.05.

Induction of obesity and experimental design

Results
Experimental obesity in hamsters was induced by highly palatable
fat- and sugar-rich (HPFS) diet for 12  weeks [24]. Animals received
about 29 g of fat and 33 g of carbohydrates per 100 g of diet, while the Phenolic constituents and carrier
energy value of fat was 40.3%. In addition, each animal consumed in ­composition of VMLPE
the diet at least 1 g of free fructose per 100 g of body weight (b.w.) per
day, which not only contributed to the disruption of glucose utiliza-
The TPC of the crude liquid extract was 5.44 ± 0.07  mg
tion but also caused voluntary hyperphagia [25, 26]. Animals of the
intact control (IC) group were kept for 12  weeks on a standard bal-
PE/mL and the dry residue was 27.88 ± 0.10 mg/mL. Based
anced diet for hamsters. on that, calculations of the quantity of L-arginine and
From the 10th week of the diet, the animals were randomly myo-inositol were done as described above. Thus, the con-
divided into six groups (n = 10). Group I included IC hamsters who tents of L-arginine and myo-inositol were 40.41 and 9.59%,
were fed with standard balanced diet; group II included obese con- respectively, of the dry weight of the powder extract
trol (OC) hamsters who were fed with HPFS diet; groups III, IV and
obtained. The TPC and TFC for the powder extract were
V included obese hamsters treated with VMLPE at different doses
(15, 25 and 35 mg/kg b.w., respectively) and group VI included obese 99.29 ± 1.21 mg PE/g and 19.28 ± 0.15 mg RE/g, respectively.
hamsters treated with “Styfimol” at a dose of 25 mg/kg b.w., which
corresponds to 6.2  mg/kg b.w. of HCA (animal equivalent doses
were calculated based on the average daily therapeutic doses for Effect of VMLPE on body weight
humans and interspecies difference in mass and body surface area
[27]). Treating agents were dispersed in 1 mL of distilled water and
administered intragastrically using a special probe once a day during
No significant differences were observed in the initial
3 weeks. Animals of the IC and OC groups received 1 mL of distilled body weight of the experimental animals (data not
water in order to reproduce equal experimental conditions instead of shown). After 12 weeks of diet feeding, HPFS diet hamsters
treating agents. After the start of treatment, the diet of all experimen- presented a significant increase in body weight compared
tal groups remained unchanged. The body weight of each hamster with the IC group. However, the administration of VMLPE
was measured once a week on an empty stomach.
at doses of 25 and 35 mg/kg b.w. resulted in a body weight
After the 12th week, the animals were sacrificed by decapitation
under chloralose-urethane anesthesia, and blood samples were col- reduction of 4% for both groups, while the treatment with
lected to determine the serum lipid profile. Visceral fat was dissected “Styfimol” was the most effective and reduced the body
and weighed directly. weight by 6% compared with the OC group (p < 0.05). The
treatment with VMLPE at a dose of 15 mg/kg b.w. did not
show any effect on body weight gain (Table 1).
Biochemical assays

The level of important diagnostic and pathogenic markers for obe-

sity-induced lipid metabolism disorders, including free fatty acids
(FFAs), TAGs, HDL-C (α-cholesterol), LDL-C (β-cholesterol) and total
cholesterol (TC), was determined in the animal serum. Determina-
tions were carried out using the commercially available kits. FFA
level was measured by the reaction with diethyl dithiocarbamate
using an assay kit (Felicit diagnostic, Ukraine). TAG level was deter-
mined using a glycerol oxidase assay kit (Kone, Finland). HDL-C and
LDL-C were separated by turbidimetric method, and the levels of cho-
lesterol fractions were determined. TC level was measured using a
cholesterol oxidase assay kit (Lachema, Czech Republic). HDL-C and
Effect of VMLPE on visceral fat mass
A twofold increase (p < 0.05) in the visceral fat mass was
observed for the OC group as compared with the IC group
(Table 1). The obese hamsters treated with 15 mg/kg b.w.
of VMLPE did not show any significant visceral fat mass
change (slightly decreased) as compared with the OC
group. However, the administration of VMLPE at doses
of 25 and 35  mg/kg b.w. decreased visceral fat mass by

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4      Zagayko et al.: Anti-obesity and lipid-lowering properties of Vaccinium myrtillus leaves powder extract

Table 1: Effect of VMLPE on body weight and visceral fat mass.

Treatment groupsa Body weight, g Visceral fat mass (at the end of
12th week)
9th week (before the treatment) 12th week (at the end of the treatment)

Intact control 109.20 ± 2.49 114.90 ± 2.13 6.5 ± 0.4

Obese control 133.80 ± 3.80b 152.10 ± 3.33b 13.5 ± 0.9b
VMLPE, 15 mg/kg b.w. 135.20 ± 4.00b 153.10 ± 3.62c 12.8 ± 0.6c
VMLPE, 25 mg/kg b.w. 134.10 ± 4.38b 145.90 ± 2.64b,c 10.5 ± 0.6b,c
VMLPE, 35 mg/kg b.w. 133.50 ± 3.40b 146.20 ± 2.89b,c 10.4 ± 0.4b,c
Positive control with “Styfimol” 132.90 ± 3.43b 143.10 ± 2.64b 9.0 ± 0.3b

VMLPE, Vaccinium myrtillus leaves powder extract. an = 10 per group; values are expressed as mean ± SD. bp < 0.05 compared with intact
control animals. cp < 0.05 compared with positive control animals.

approximately 3 g per 100 g of body weight. The best result
in visceral fat mass reduction was demonstrated by the
positive control drug “Styfimol”: the visceral fat mass was
reduced by 4.5 g per 100 g of animal body weight.
Effect of VMLPE on serum lipid profile
HPFS diet during the 12  weeks resulted in a persistent
hyperlipidemia in obese hamsters. Specifically, 1.57-fold,
2.36-fold, 1.55-fold and 2.41-fold (p < 0.05) increases were
observed for TAGs, FFAs, TC and LDL-C, respectively,
in untreated obese hamsters (Figure 1). Treatment with
15  mg/kg b.w. of VMLPE did not lead to a significant
decrease of serum TAGs and FFAs levels compared to
the OC group, but resulted in a slight decrease in TC con-
centration (by 9.3%), both due to HDL-C and LDL-C. On
the other hand, treatment with 25  mg/kg b.w. of VMLPE
reduced TAGs and FFAs by 21.3 and 19.6%, respectively,
and TC level was observed to decrease by 18.1%, primar-
ily due to a nearly 1.5-fold (p < 0.05) decrease in LDL-C.
Administration of 35 mg/kg b.w. of VMLPE to obese ham-
sters tended to be the most noticeably normalizing serum
lipid profile among all treated groups, and the percent-
age of HDL-C and LDL-C was the closest to intact values
(Figure 2). Treatment with “Styfimol” normalized serum
TAGs to the intact levels (p < 0.05) and reduced serum FFA
level by 43.6%. However, the effect of “Styfimol” on the
reduction of TC and LDL-C, as well as on the normaliza-
tion of the HDL-C and LDL-C ratio, was slight (but statisti-
cally significant, p < 0.05) and similar to VMLPE at a dose
of 15 mg/kg b.w.
Discussion
In the present study, the anti-obesity and lipid-lower-
ing properties of VMLPE obtained with the addition of

Figure 1: Effect of VMLPE on serum TAGs, FFAs, TC, HDL-C and LDL-C levels in intact and obese hamsters.
Values are mean ± SD with n = 10. p < 0.05 compared with OC animals.

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 Zagayko et al.: Anti-obesity and lipid-lowering properties of Vaccinium myrtillus leaves powder extract      5
Figure 2: The percentage of HDL-C and LDL-C in the serum of intact and obese hamsters.
L-arginine and myo-inositol were evaluated on a hamster
model of HPFS diet-induced obesity. As it was demon-
strated by other researches, experimental models of
obesity induced by high-calorie diets rich in fat and car-
bohydrates result in a significant body weight gain in
rodents [28], and the addition of palatable components
(in this case, fructose) to animal diet can cause volun-
tary hyperphagia, which leads to a more relevant obesity
model than standard purified diets with high fat content
[26]. Such diets have shown relevance as realistic models
of human obesity [24, 28]. Besides, in hamsters, unlike
mice and rats, high-fat diets generally do not cause dia-
betes mellitus on the background of obesity (although
impaired glucose tolerance and hyperinsulinemia occur).
Therefore, such a choice of experimental animals allows
assessing the effect of test samples directly on obesity,
uncomplicated by other pathologies caused by similar ali-
mentary models.
In the current experiment, the development of obesity
in male hamsters was not affected by pharmacological
agents during the 9 weeks. At this time, the body weight
of animals with induced pathology significantly differed
from that of IC animals, while the mean body weight of all
HPFS diet animals was similar and almost did not differ
between the obese groups before treatment.
We first examined the anti-obesity properties of
VMLPE. It was found to be ineffective at a dose of 15 mg/kg
b.w., while doses of 25 and 35 mg/kg b.w. possess a mild
anti-obesity effect, which was practically at the same
level for both of them. However, it should be empha-
sized that none of the treated groups, including the posi-
tive control, restored the body weight to IC levels, but
showed only the slowdown of weight gain. First of all,
this is because during the treatment animals continued to
receive a constant hypercaloric HPFS diet. This approach
allows screening test samples for pharmacological activ-
ity. However, in clinical practice, non-pharmacological
treatment for obesity, such as diet and physical exercises,
along with these therapeutic agents can greatly potentiate
the effectiveness of the latter.
One of the key factors for the development of obesity-
related diseases, such as diabetes mellitus, cardiovascular
diseases, non-alcoholic fatty liver disease and generalized
vascular arteriosclerosis, is visceral fat accumulation.
The amount of visceral adipose tissue in obese individu-
als directly linked to dyslipidemic disorders and insulin
resistance [29, 30]. In the present experiment, HPFS diet
feeding caused a significant increase in visceral adipose
tissue mass, which did not reduce to normal levels under
the treatment. However, the results obtained in this test
correlated with the effect of treating agents against weight
gain. That is, the lowest effect was observed in animals
treated with 15  mg/kg b.w. The groups received 25 and
35  mg/kg b.w. VMLPE showed a mild but marked reduc-
tion of visceral fat mass.
We further investigated the serum lipid profiles in
obese hamsters. Alterations in lipid fraction levels are
one of the major biochemical markers for diseases whose
pathogenesis is associated with lipid metabolism. In the
initial stages of isolated visceral obesity, dyslipidemia
manifests itself much earlier than the forced fat accu-
mulation in the subcutaneous tissue, and improving the
serum lipid profile in patients with obesity may indicate
regression of the disease even before body weight loss
occurs. Among all lab rodent models, Syrian hamster
lipid metabolism is the closest to human; in addition, the
lipid metabolism of this species is susceptible to exces-
sive intake of edible fats, which is the basis of the chosen
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6      Zagayko et al.: Anti-obesity and lipid-lowering properties of Vaccinium myrtillus leaves powder extract
alimentary model of obesity. It should be noted that in
contrast to humans, hamster serum levels of HDL-C are
greater than LDL-C; however, despite this, the hamster
lipoprotein system is more similar to that of humans than
to rats, due to the high proportion of circulating lipopro-
teins and similar mechanisms of its regulation. Dyslipi-
demia in hamsters leads to a marked increase in serum
LDL-C level, and treatment with hypolipidemic drugs pri-
marily results in a decrease of β-cholesterol, which allows
assessing the effectiveness of the test drug [31].
During the experiment, none of the treating agents
reduced all serum lipid fractions to IC levels. However, in
this test, VMLPE possessed a dose-dependent hypocho-
lesterolemic effect and markedly reduced serum levels of
TAGs, FFAs and TC (primarily by lowering LDL-C) at doses
of 25 and 35 mg/kg b.w. Administration of these doses of
VMLPE was found to be more effective against obesity-
induced dyslipidemia than positive control treatment.
The findings of this study generally correspond to the
earlier ones on the lipid-lowering activity of V. myrtillus
leaves hydroalcoholic extracts [11, 12]; however, this is the
first report on the anti-obesity effect of V. myrtillus leaves.
Obviously, the anti-obesity properties of VMLPE may be
attributed to chlorogenic acid and rutin, which were found
to be its dominant phytochemical constituents [21]. Previ-
ously, it was demonstrated that pure chlorogenic acid, as
well as pure rutin, significantly lowered body weight gain
and adipose tissue mass in high-fat diet-induced obese
animals [32, 33]. The addition of L-arginine and myo-inosi-
tol appeared to potentiate the therapeutic action of VMLPE
phytoconstituents, though carrier doses were unlikely
enough to cause the observed effects by themselves.
In conclusion, the administration of VMLPE mark-
edly inhibited weight gain and visceral fat accumulation
and also improved the abnormalities of lipid metabolism
in obese hamsters. Thus, taking into account previously
established hypoglycemic activity of VMLPE obtained
with the use of L-arginine and myo-inositol [15, 21], it
may be considered as a promising therapeutic agent for
obesity, MS and T2DM treatment.
Author contributions: All the authors have accepted
responsibility for the entire content of this submitted
manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played
no role in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; or in the
decision to submit the report for publication.
References
1. Codario RA. Obesity and the metabolic syndrome. In: Codario
RA, editor. Type 2 diabetes, pre-diabetes, and the metabolic
syndrome. Clifton: Humana Press, 2011:67–92.
2. Jung UJ, Choi MS. Obesity and its metabolic complications: the
role of adipokines and the relationship between obesity, inflam-
mation, insulin resistance, dyslipidemia and nonalcoholic fatty
liver disease. Int J Mol Sci 2014;15:6184–223.
3. Zlobine I, Gopal K, Ussher JR. Lipotoxicity in obesity and
diabetes-related cardiac dysfunction. Biochim Biophys Acta
2016;1861:1555–68.
4. Arca M. Dyslipidemia and cardiovascular risk in obesity. In:
Lenzi A, Migliaccio S, Donini L, editors. Multidisciplinary
approach to obesity. Cham: Springer, 2015:121–30.
5. Hanhineva K, Törrönen R, Bondia-Pons I, Pekkinen J, Kole-
hmainen M, Mykkänen H, et al. Impact of dietary polyphenols on
carbohydrate metabolism. Int J Mol Sci 2010;11:1365–402.
6. Munir KM, Chandrasekaran S, Gao F, Quon MJ. Mechanisms for
food polyphenols to ameliorate insulin resistance and endothe-
lial dysfunction: therapeutic implications for diabetes and its
cardiovascular complications. Am J Physiol Endocrinol Metab
2013;305:E679–86.
7. Quideau S, Deffieux D, Douat-Casassus C, Pouységu L. Plant
polyphenols: chemical properties, biological activities, and
synthesis. Angew Chem Int Ed Engl 2011;50:586–621.
8. Carpene C, Gomez-Zorita S, Deleruyelle S, Carpene MA.
Novel strategies for preventing diabetes and obesity
complications with natural polyphenols. Curr Med Chem
2015;22:150–64.
9. Rao RN, Sakariah KK. Lipid-lowering and antiobesity effect of
(–)-hydroxycitric acid. Nutr Res 1988;8:209–12.
10. Semwal RB, Semwal DK, Vermaak I, Viljoen A. A comprehen-
sive scientific overview of Garcinia cambogia. Fitoterapia
2015;102:134–48.
11. Cignarella A, Nastasi M, Cavalli E, Puglisi L. Novel lipid-
lowering properties of Vaccinium myrtillus L. leaves, a
traditional antidiabetic treatment, in several models of rat
dyslipidaemia: a comparison with ciprofibrate. Thromb Res
1996;84:311–22.
12. Sidorova Y, Shipelin V, Mazo V, Zorin S, Petrov N, Kochetkova A.
Hypoglycemic and hypolipidemic effect of Vaccinium myrtillus
L. leaf and Phaseolus vulgaris L. seed coat extracts in diabetic
rats. Nutrition 2017;41:107–12.
13. Bljajić K, Petlevski R, Vujić L, Čačić A, Šoštarić N, Jablan J, et al.
Chemical composition, antioxidant and α-glucosidase-inhibiting
activities of the aqueous and hydroethanolic extracts of Vac-
cinium myrtillus leaves. Molecules 2017;22:E703.
14. McKenna DJ, Jones K, Hughes K. Botanical medicines: the desk
reference for major herbal supplements. New York: Haworth
Herbal Press, 2002.
15. Koshovyi OM, Zagayko AL, Kolychev IO, Fylymonenko VP,
Komisarenko AM, inventors; National University of Pharmacy,
assignee. Method for producing hypoglycemic composition from
leaves of blueberry (Vaccinium myrtillus L.). UA patent 111134.
March 25, 2016. Ukrainian.
16. Kohli R, Meininger CJ, Haynes TE, Yan W, Self JT, Wu G. Dietary
L-arginine supplementation enhances endothelial nitric oxide
synthesis in streptozotocin-induced diabetic rats. J Nutr
2004;134:600–8.
Authenticated | kolisnyktatyana@gmail.com author's copy
Download Date | 7/28/18 10:43 AM
 Zagayko et al.: Anti-obesity and lipid-lowering properties of Vaccinium myrtillus leaves powder extract      7
17. Tan B, Li X, Yin Y, Wu Z, Liu C, Tekwe CD, et al. Regulatory roles
for L-arginine in reducing white adipose tissue. Front Biosci
2012;17:2237–46.
18. Ariki R, Hirano A, Arakawa T, Shiraki K. Arginine increases the
solubility of alkyl gallates through interaction with the aromatic
ring. J Biochem 2011;149:389–94.
19. Muscogiuri G, Palomba S, Laganà AS, Orio F. Inositols in
the treatment of insulin-mediated diseases. Int J Endocrinol
2016;2016:3058393.
20. Mancini M, Andreassi A, Salvioni M, Pelliccione F, Mantellassi
G, Banderali G. D-chiro inositol in improving insulin resist-
ance in obese male children: preliminary data. Int J Endocrinol
2016;2016:8720342.
21. Koshovyi OM, Zagayko AL, Kolychev IO, Akhmedov EYu, Komis-
sarenko AN. [Phytochemical study of the dry extract from
­bilberry leaves]. Azerbaijan Pharm Pharmacother J 2016;1:
18–23. Russian.
22. Ministry of Health of Ukraine. State register of drugs of Ukraine.
Informational fund. Available at: http://www.drlz.kiev.ua.
Accessed: 4 Sep 2017. Ukrainian.
23. European Pharmacopoeia 9th ed. Strasbourg: Council of Europe,
2016.
24. Dalbøge LS, Pedersen PJ, Hansen G, Fabricius K, Hansen HB,
Jelsing J, et al. A hamster model of diet-induced obesity for
preclinical evaluation of anti-obesity, anti-diabetic and lipid
modulating agents. PLoS One 2015;10:e0135634.
25. Briand F, Thiéblemont Q, Muzotte E, Sulpice T. High-fat and
fructose intake induces insulin resistance, dyslipidemia, and
liver steatosis and alters in vivo macrophage-to-feces reverse
cholesterol transport in hamsters. J Nutr 2012;142:704–9.
26. Higa TS, Spinola AV, Fonseca-Alaniz MH, Evangelista FS.
­Comparison between cafeteria and high-fat diets in the induc-
tion of metabolic dysfunction in mice. Int J Physiol Pathophysiol
Pharmacol 2014;6:47–54.
27. Nair AB, Jacob S. A simple practice guide for dose conversion
between animals and human. J Basic Clin Pharm 2016;7:27–31.
28. Sampey BP, Vanhoose AM, Winfield HM, Freemerman AJ,
­Muehlbauer MJ, Fueger PT, et al. Cafeteria diet is a robust model
of human metabolic syndrome with liver and adipose inflam-
mation: comparison to high-fat diet. Obesity (Silver Spring)
2011;19:1109–17.
29. Wajchenberg BL. Subcutaneous and visceral adipose tis-
sue: their relation to the metabolic syndrome. Endocr Rev
2000;21:697–738.
30. Berings M, Wehlou C, Verrijken A, Deschepper E, Mertens I,
Kaufman JM, et al. Glucose intolerance and the amount of
visceral adipose tissue contribute to an increase in circulating
triglyceride concentrations in Caucasian obese females. PLoS
One 2012;7:e45145.
31. Dillard A, Matthan NR, Lichtenstein AH. Use of hamster as a
model to study diet-induced atherosclerosis. Nutr Metab (Lond)
2010;7:89.
32. Cho AS, Jeon SM, Kim MJ, Yeo J, Seo KI, Choi MS, et al. Chlo-
rogenic acid exhibits anti-obesity property and improves lipid
metabolism in high-fat diet-induced-obese mice. Food Chem
Toxicol 2010;48:937–43.
33. Seo S, Lee MS, Chang E, Shin Y, Oh S, Kim IH, et al. Rutin
increases muscle mitochondrial biogenesis with AMPK
activation in high-fat diet-induced obese rats. Nutrients
2015;7:8152–69.
Authenticated | kolisnyktatyana@gmail.com author's copy
Download Date | 7/28/18 10:43 AM