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Pharmacognostical and pharmacological research on some vegetal species with hypoglycemic virtues1
1. UNIVERSITY OF MEDICINE AND PHARMACY CRAIOVA
Ph.D. THESIS
PHARMACOGNOSTICAL AND PHARMACOLOGICAL
RESEARCHES ON SOME VEGETAL SPECIES WITH
HYPOGLYCEMIC VIRTUES
ABSTRACT
Scientific Supervisor
Professor of Pharmacology Florica Popescu, Ph. D
Ph.D. Candidate
Anca Berbecaru-Iovan
CRAIOVA
2009
2. SUMMARY
1. Introduction
STATE OF KNOWLEDGE
2. Pharmacognostical and pharmacological data on some vegetal
species with hypoglycemic activity
3. General information about diabetes mellitus
PERSONAL CONTRIBUTION
4. Research on obtaining and physical and chemical characterization of
tinctures of hypoglycemic plants
4. 1. Introduction
4. 2. The determination of the identity, purity and quality of plants material
4. 3. Preparation of tinctures by simple percolation method
4. 4. Organoleptic rating
4. 5. Determination of relative density
4. 6. Determination of refractive index
4. 7. Quality conditions
4. 8. Quantitative determination of phenyl-propane compounds
4. 8. 1. Materials and methods
4. 8. 2. Results and discussions
4. 9. Flavonoids and polyphenolic acids analysis by TLC
4. 9. 1. Materials and methods
4. 9. 2. Results and discussions
4. 10. VIZ footprint spectrophotometric
4. 10. 1. Materials and methods
4. 10. 2. Results and discussions
4. 11. IR spectral footprint
4. 11. 1. Materials and methods
4. 11. 2. Results and discussions
4. 12. Conclusions
5. Preliminary studies on hypoglycemic effect of plant species
5. 1. Preliminary studies on hypoglycemic effect of plant species on
experimentally induced hyperglycemia by adrenaline in rats
5. 1. 1. Introduction
5. 1. 2. Materials and methods
5. 1. 3. Results and discussions
5. 1. 4. Conclusions
5. 2. Preliminary studies on the evaluation of Syringae vulgaris flos f.
àlba and Syringae vulgaris flos f. violàcea tinctures on blood glucose
levels when performed oral glucose tolerance test in normal rats
3. 5. 2. 1. Introduction
5. 2. 2. Materials and methods
5. 2. 3. Results and discussions
5. 2. 4. Conclusions
5. 3. Acute and subacute toxicity study of Syringae vulgaris flos f. àlba
and Syringae vulgaris flos f. violàcea tinctures
5. 3. 1. Introduction
5. 3. 2. Materials and methods
5. 3. 3. Results and discussions
5. 3. 4. Conclusions
6. Experimental model to induce diabetes by administration of
streptozotocin in rat
6. 1. Introduction
6. 2. Materials and methods
6. 3. Results and discussions
6. 4. Conclusions
7. Evaluation of hypoglycemic effect of some vegetal species on
streptozotocin-induced diabetes in rats
7. 1. Introduction
7. 2. Materials and methods
7. 3. Results and discussions
7. 4. Conclusions
8. Research on the hypoglycemic effect, cholesterol-lowering and
antioxidant effect of plant species in rats with experimental
streptozotocin-induced diabetes
8. 1. Introduction
8. 2. Materials and methods
8. 3. Results and discussions
8. 4. Conclusions
9. Final conclusions
10. Bibliography
Key words: diabetes mellitus, phytotherapy, vegetal species, hypoglycemic
effect, cholesterol lowering effect, antioxidant effect
4. 1. INTRODUCTION
The worldwide prevalence of diabetes mellitus (DM) has risen
dramatically over the past two decades; based on current trends, more than
360 million individuals will have diabetes by year 2030.
In addition to oral agents and insulin therapy, phytotherapy is an
alternative that offers a wide range of natural resources with hypoglycemic
effects some plants provide materials recommended for people with
diabetes. Herbal remedies are beneficial to patients with type 2 diabetes
and, in addition to the diet prescribed by a doctor, they can help glucose
homeostasis, but they can not replace insulin and oral medication.
In recent years, researchers turned attention specifically to
oxidative stress and the key role it plays, as a common element in the
pathogenesis of diabetes complications. Hyperglycemia generates reactive
oxygen species which, in turn, causes membrane lipid peroxidation and
degradation.
Many of the complications of diabetes, including vascular
atherosclerosis, major cause of mortality in DM, are closely related to
oxidative stress and, thus, antioxidants play an important role in the
treatment of diabetes.
Herbal remedies contain large amounts of antioxidants such as
flavonoids, polyphenolic acids, carotenoids, vitamins C and E;
experimental research has shown that the antioxidant activity may be an
important property of medicinal plants used for their hypoglycemic effect
in the treatment of DM.
STATE OF KNOWLEDGE
2. Pharmacognostical and pharmacological data on some vegetal
species with hypoglycemic activity
Plants with hypoglycemic action from the Romania’s spontaneous
flora are mostly from families: Asteraceae, Cruciferae, Ericaceae,
Fabaceae, Gentianaceae, Lamiaceae, Liliaceae, Moraceae, Polygonaceae,
Rosaceae.
In terms of distribution of active ingredients, most hypoglycemic
medicinal plants include: flavonoids, polyphenolcarboxylic acids, sterols
and triterpenoid, tannins, volatile oils, saponozide, bitter principles,
coumarines, carotenoids, mucilages, micronutrients, alkaloids, aminoacids,
proteins, the therapeutic activity being the result of interaction of several
classes of compounds, respectively of an phytocomplex.
5. Pharmaceutical preparations based on natural products
recommended in DM may contain either a single plant product (powder or
extract) or combination of products with hypoglycemic action or other
properties beneficial to the diabetic patient.
3. General information about diabetes mellitus
Diabetes mellitus refers to a group of common metabolic disorders
that is characterized by hyperglycemia.
Depending on the etiology of diabetes, factors leading to
hyperglycemia include decreased insulin secretion, decreased glucose use
and increased production of glucose. The metabolic dysregulation
associated with DM causes secondary pathophysiologic changes in multiple
organ system that imposes a tremendous burden on the individual with
diabetes and on health system. DM is the leading cause of end-stage renal
disease, nontraumatic lower extremity amputation and adult blind.
Antidiabetic medication seeks to maintain a normal glucose level.
Antidiabetic drug classification by origin, chemical structure and
mechanism of action includes insulin and insulin analogues and oral
antidiabetic agents used in type 2 diabetes. The oral antihyperglycemic
drugs may enhance pancreatic insulin secretion (secretagogues), sensitize
peripheral tissues to insulin (sensitizers) and impair GI absorption of
glucose.
PERSONAL CONTRIBUTION
4. Research on obtaining and physical and chemical characterization of
tinctures of hypoglycemic plants
The main objectives of this chapter were the obtaining and the
physical and chemical characterization of tinctures of hypoglycemic plant
products studied (Cynosbati fructus, Galegae herba, Myrtilli folium,
Myrtilli fructus, Phaseoli sine seminibus fructus (pericarpium), Syringae
vulgaris flos f. álba, Syringae vulgaris flos f. violácea, Trigonellae foenum-
graeci semen, Violae tricoloris herba); qualitative and quantitative analysis
for phenyl-propan compounds from the analised tinctures by thin layer
chromatography (TLC) and spectrophotometric VIZ; flavonoids and
polyphenolic acids analysis to separate and identify them, using appropriate
standards, by TLC.
Analysis of chromatograms obtained confirmed flavonoids and
polyphenolic acids presence in all samples analyzed except the tinctures
6. from Trigonellae foenum-graeci semen and Galegae herba, where was no
evidence of polyphenolic acids.
For the first time were characterized as such, without further
processing, both in terms of absorption peaks and pads in VIZ and specific
vibration in IR the tinctures obtained by simple percolation of the nine
hypoglycemiant plant products, the results contributing to the schemes
which can identify various tinctures and determine their degree of
alteration.
5. Preliminary studies on hypoglycemic effect of plant species
5. 1. Preliminary studies on hypoglycemic effect of plant species on
experimentally induced hyperglycemia by adrenaline in rats
The aim of this study was to investigate the hypoglycemic effect of
the powder of mulberry leaves (Mori folium) after oral administration in
rats with experimentally-induced adrenalin hyperglycemia, compared with
less studied species regarding their hypoglycemic effect, pansy (Violae
tricoloris herba) and acacia leaves (Robiniae pseudoacaciae folium).
The results showed that the Mori folium powder easily decrease the
experimental hyperglycemia induced by adrenalin in rats and promote a
return to baseline of the blood glucose level, proving to be a remedy with
mild hypoglycemic activity. Robiniae pseudoacaciae folium and Violae
tricoloris herba powders marginally reduced hyperglycemia in rats
compared with control, but favoring a return of glucose values close to the
reference.
5. 2. Preliminary studies on the evaluation of Syringae vulgaris flos f.
àlba and Syringae vulgaris flos f. violàcea tinctures on blood glucose
levels when performed oral glucose tolerance test in normal rats
This research aimed to investigate the potential antihyperglycemic
effect on oral glucose tolerance test (OGTT) of tinctures of Syringae
vulgaris flos f. álba and Syringae vulgaris flos f. violácea when given
orally to normal rats one hour prior to the OGTT.
Improving response to exogenous glucose load, performed after the
treatment with tinctures, is demonstrated by a decrease in blood glucose
level at both 30 and 60 minutes, compared with corresponding values from
untreated group (control). This pharmacodynamic effect was not shown so
far from the studied plant products.
7. 5. 3. Acute and subacute toxicity study of Syringae vulgaris flos f. àlba
and Syringae vulgaris flos f. violàcea tinctures
This study aimed to asses the toxic potential after oral
administration of tincture of Syringae vulgaris flos f. alba and Syringae
vulgaris flos f. violácea by conducting in vivo experiments in normal rats.
In determining the toxicity degree of the studied plant products,
were used much higher doses than the therapeutic doses, watching the
emergence of toxic and lethal effects at these doses.
Research conducted on normal rats showed that no animal has died
during the study. Treated animal behaviour was normal and there was no
toxic and lethal effects reported even at doses of 10 or 20 times higher than
the effective dose of tincture of Syringae vulgaris flos f. álba, respectively
Syringae vulgaris flos f. violácea.
6. Experimental model to induce diabetes by administration of
streptozotocin in rat
In addition to presenting the method of induction of experimental
diabetes mellitus by administering streptozotocin (STZ) in normal adult
Wistar rats, this chapter aim is to establish the working protocol and to
present the equipment used to monitor serum glucose level and serum total
cholesterol level.
Intraperitoneal injection of STZ (a synthetic antineoplastic agent)
causes inflammation in the Langerhans islets with pancreatic β-cells
degeneration inducing experimental diabetes mellitus in about 2-4 days.
7. Evaluation of hypoglycemic effect of some vegetal species on
streptozotocin-induced diabetes in rats
The purpose of this study was to investigate the oral hypoglycemic
effect of some vegetal products (Cynosbati fructus, Myrtilli folium, Myrtilli
fructus, Phaseoli sine seminibus fructus, Trigonellae foenum-graeci semen,
Violae tricoloris herba) on streptozotocin-induced diabetes in rats.
The experimental groups were as follows: group 1, non-diabetic
control rats; group 2, diabetic control rats – untreated; group 1 and group 2
were orally administered distilled water daily using an intragastric tube;
groups 3-8, diabetic rats – treated (categorized by alphabetical order of the
vegetal species administered) – fed orally daily by an intragastric tube with
powder (of each vegetal species used, categorized alphabetically)
suspended in distilled water.
8. Statistical analysis: values are presented as means ± SD of 5 rats
in each group. All the data were statistically evaluated by t-value two-sided
test; the limit of statistical significance was set at p-level < 0.05.
The treatment of diabetic animals with the vegetal species powder
produced a significant decrease in blood glucose concentrations as
compared with untreated diabetic control animals; the results indicated the
potent anti-diabetic activity of Myrtilli folium (G4), Myrtilli fructus (G5),
Phaseoli sine seminibus fructus (G6), Trigonellae foenum-graeci semen
(G7) and slightly anti-diabetic activity of Violae tricoloris herba (G8) and
Cynosbati fructus (G3).
A significant decrease in total cholesterol was determined by the
oral administration of the Trigonellae foenum-graeci semen powder (G7),
followed by Cynosbati fructus powder (G3) and a slightly decrease was
observed at animals treated orally with Myrtilli folium powder (G4).
The results indicate the possibility that these vegetal species could
be effective to prevent or retard the development of diabetes complications
due to metabolic disorders.
8. Research on the hypoglycemic effect, cholesterol-lowering and
antioxidant effect of plant species in rats with experimental
streptozotocin-induced diabetes
The aim of this study is the evaluation of hypoglycemic and
cholesterol-lowering effect by determination of serum glucose level and
serum total cholesterol level after oral administration of tinctures derived
from plant products less studied in this respect (Syringae vulgaris flos f.
álba, Syringae vulgaris flos f. violácea) in streptozotocin-induced diabetes
in rats. It was also studied the antioxidant effect of the products listed, as
well as some plant products known for their antidiabetic effect (Myrtilli
folium, Phaseoli sine seminibus fructus) by determining the activity of
antioxidant enzymes (superoxide dismutase – SOD, glutathione peroxidase
– GPx, glutathione reductase - GR) and lipid peroxidation evaluated as
level of reactive substances with tiobarbituric acid. Also, histological
analysis was performed in order to correlate the effect of treatment on
biochemical parameters of interest with changes in the liver and kidney
tissue for the different study groups.
The experimental groups were as follows: group 1, non-diabetic
control rats; group 2, diabetic control rats – untreated; group 1 and group 2
were orally administered distilled water daily using an intragastric tube;
groups 3-6, diabetic rats treated (named according to the initial letters of
the used products): G3 (SV – Syringae vulgaris flos f. violácea), G4 (SA –
9. Syringae vulgaris flos f. álba), G5 (MF – Myrtilli folium) and G6 (PP –
Phaseoli pericarpium) fed orally daily by an intragastric tube with
tinctures 20% suspended in distilled water.
At the end of the six weeks of treatment, after 12 hours of fasting,
animals were sacrificed and the collected blood was used for biochemical
analysis. The liver and kidney tissue were harvested for histological
analysis.
Statistical analysis: values are presented as means ± SD of 5 rats
in each group. All the data were statistically evaluated by t-value two-sided
test; the limit of statistical significance was set at p-level < 0.05.
The treatment of diabetic animals with tinctures from the studied
plants significantly decreased glucose levels compared with untreated diabetic
animals, the results confirming a potent antidiabetic activity for Myrtilli folium
and Phaseoli pericarpium and a moderate antidiabetic activity for Syringae
vulgaris flos f. violácea, respectively Syringae vulgaris flos f. álba.
It was also observed a significant decrease in total cholesterol
levels compared with untreated diabetic animals, the results showing a
significant cholesterol-lowering activity when it was given Myrtilli folium,
respectively Phaseoli pericarpium and a moderate reduction of total
cholesterol in diabetic animals treated with tinctures of Syringae vulgaris
flos f. violácea and Syringae vulgaris flos f. álba.
The research has revealed an important antioxidant effect, with
the increased of SOD, GPx and GR activity and the decreased of lipid
peroxides after the treatment with tinctures of Myrtilli folium and Phaseoli
pericarpium and a capacity of antioxidant enzymes stimulation and
reducing lipid peroxidation for tinctures of Syringae vulgaris flos f.
violácea and Syringae vulgaris flos f. álba.
Regarding the histological analysis of pathological changes
occurring in diabetic animals treated with tinctures from studied plant
products (groups G3-G6), it is noted discrete changes witch confirms
favorable development after six weeks of treatment and beneficial role of
administration of these herbal remedies. We noted a correlation between
changes in biochemical parameters determined and the degree of tissue
deterioration.
10. FINAL CONCLUSIONS
1. Diabetes mellitus has become a major problem for the
individual, medicine and society. The alarming find is that, when diagnosed
type 2 diabetes, a high percentage of people already have chronic
complications and/or morbid associations.
2. Diabetes mellitus is defined as a group of metabolic disorders
characterized mainly by hyperglycemia induced by impaired insulin
secretion, insulin resistance or both entities in varying proportions.
3. Besides oral agents and insulin therapy, phytotherapy is an
alternative source that provides a range of natural resources with
hypoglycemic effects, a range of plants providing raw materials
recommended for people with diabetes.
4. Medicinal plants in different oral formulations were
recommended to the diabetic patient, but the mechanisms for hypoglycemic
activity still remained incompletely understood.
5. Among the most popular medicinal species with hypoglycemic
effect we mentioned: Allium cepa, Allium sativum, Arctium lappa, Brassica
sp., Centaurium umbellatum, Cynara cardunculus subsp. scolymus,
Gentiana sp., Glycyrrhiza glabra, Morus sp., Phaseolus vulgaris, Rubus
sp., Salvia officinalis, Taraxacum officinale, Trigonella foenum-graecum,
Vaccinium myrtillus.
6. Plants with hypoglycemic activity from Romania’s spontaneous
flora, are mostly from families: Asteraceae, Cruciferae, Ericaceae,
Fabaceae, Gentianaceae, Lamiaceae, Liliaceae, Moraceae, Polygonaceae,
Rosaceae.
7. Following pharmacognostical research on obtaining and
chemical and physical characterization of tinctures of some hypoglycemic
plant products (Cynosbati fructus, Galegae herba, Myrtilli folium, Myrtilli
fructus, Phaseoli pericarpium, Syringae vulgaris flos f. álba, Syringae
vulgaris flos f. violácea, Trigonellae foenum-graeci semen, Violae
tricoloris herba), their quality was determined according to Romanian
Pharmacopoeia, 10th Edition and has conducted qualitative and quantitative
analysis for flavonoids and phenyl-propane compounds.
8. Analysis tinctures revealed as active principles flavonoids and
polyphenolic acids as active principles with a remarkable therapeutic
potential and who are giving plant products hypoglycemic and antioxidant
properties.
9. For the first time were characterized as such without further
processing, both in terms of absorption peaks and pads in VIZ and specific
11. vibration in IR, tinctures obtained by simple percolation of the
hypoglycemic plant products taken in study.
10. The preliminary study on hypoglycemic effect on plant species,
experimental hyperglycemia was induced by subcutaneous administration
of adrenaline in normal rat. From research conducted for Mori Folium
powder that turns out to be a remedy with mild hypoglycemic activity,
thereby urging the combination with other hypoglycemic addition of phar-
macodynamic action. Robiniae pseudoacaciae folium and Violae tricoloris
herba powders marginally reduced hyperglycemia in rats compared with
control, but favoring a return of glucose values close to the reference.
11. The evaluation of Syringae vulgaris flos f. àlba and Syringae
vulgaris flos f. violàcea tinctures on blood glucose levels when performed
oral glucose tolerance test in normal rats showed improving response to
exogenous glucose load. This pharmacodynamic effect was not shown so
far from the studied plant products.
12. The study to asses the toxic potential after oral administration
of tincture of Syringae vulgaris flos f. alba and Syringae vulgaris flos f.
violácea by conducting in vivo experiments in normal rats showed that they
are non-toxic products.
13. The research to investigate the oral hypoglycemic effect of
some vegetal products (Cynosbati fructus, Myrtilli folium, Myrtilli fructus,
Phaseoli sine seminibus fructus, Trigonellae foenum-graeci semen, Violae
tricoloris herba) on streptozotocin-induced diabetes in rats proved that the
treatment during four weeks of diabetic animals with the vegetal species
powder produced a significant decrease in blood glucose concentrations as
compared with untreated diabetic control animals. The results indicated the
potent anti-diabetic activity of Myrtilli folium, Myrtilli fructus), Phaseoli
sine seminibus fructus, Trigonellae foenum-graeci semen and slightly anti-
diabetic activity of Violae tricoloris herba and Cynosbati fructus.
14. A significant decrease in total cholesterol was determined by
the oral administration of the Trigonellae foenum-graeci semen powder,
followed by Cynosbati fructus powder and a slightly decrease was observed
at animals treated orally with Myrtilli folium powder.
15. The results indicate the possibility that these vegetal species
could be effective to prevent or retard the development of diabetes
complications due to metabolic disorders.
16. In the research on the hypoglycemic effect, cholesterol-
lowering and antioxidant effect of plant species in rats with experimental
streptozotocin-induced diabetes we studied the effects after oral
administration of tinctures derived from plant products less studied in this
12. respect (Syringae vulgaris flos f. álba, Syringae vulgaris flos f. violácea),
as well as tinctures of some plant products known for their antidiabetic
effect (Myrtilli folium, Phaseoli sine seminibus fructus).
17. The results after six weeks treatment of diabetic animals confirmed a
potent antidiabetic activity for Myrtilli folium and Phaseoli pericarpium and a
moderate antidiabetic activity for Syringae vulgaris flos f. violácea,
respectively Syringae vulgaris flos f. alba and also a significant
cholesterol-lowering activity when it was given Myrtilli folium,
respectively Phaseoli pericarpium and a moderate reduction of total
cholesterol in diabetic animals treated with tinctures of Syringae vulgaris
flos f. violácea and Syringae vulgaris flos f. álba.
18. The research has revealed an important antioxidant effect, with
the increased of SOD, GPx and GR activity and the decreased of lipid
peroxides after the treatment with tinctures of Myrtilli folium and Phaseoli
pericarpium and a capacity of antioxidant enzymes stimulation and
reducing lipid peroxidation for tinctures of Syringae vulgaris flos f.
violácea and Syringae vulgaris flos f. álba.
19. Regarding the histological analysis of pathological changes
occurring in diabetic animals treated with tinctures from studied plant
products, it is noted discrete changes wich confirms favorable development
after six weeks of treatment and beneficial role of administration of these
herbal remedies. We noted a correlation between changes in biochemical
parameters determined and the degree of tissue deterioration.
20. The research on antioxidant status after treatment with powder
and vegetal extracts in rats with experimentally streptozotocin-induced
diabetes by determining the enzymatic activity of SOD, GPx and GR and
the index of lipids peroxidation and also by correlating the changes of
biochemical parameters of interest with changes in tissue liver and kidney,
was not mentioned in the literature of our country, representing the
originality of this thesis.
21. Research carried out shows that diabetic animals are subjected
to oxidative stress and that oral administration of tinctures of plant products
can reduce part imbalance between reactive oxygen species generation and
antioxidant enzymes activity. Therefore, plant remedies are useful both in
prevention and improvement of certain disease like diabetes,
atherosclerosis and the complications caused by them.
22. For the first time was studied the effect of oral administration
of tinctures of Syringae vulgaris flos f. violácea and Syringae vulgaris flos
f. alba in streptozotocin-induced diabetic rats, results showing existing
antidiabetic action and lowering serum total cholesterol, with a ability to
stimulate antioxidant enzymes activity and reduce lipid peroxidation.
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16. CURRICULUM VITAE
Name: BERBECARU-IOVAN
First name: ANCA
Birth date: 15.11.1970
Birth place: Craiova, Dolj
Present: married, one child
Address: Craiova, Ştefan cel Mare street, bloc 13, sc. A, apt. 11
Studies: High School of Mathematics and Physics „Nicolae Bălcescu“,
Craiova, Mathematics and Physics Profile, 1985-1989;
Faculty of Medicine, General Medicine Specialization, University
of Craiova, 1990-1996.
Professional activity:
Resident doctor in Emergency Medicine specialization, 1997-2000;
Specialist doctor in 2000; currently enrolled in the training program in the
second specialization Internal Medicine;
Junior Assistant by competition 2000-2002, University Assistant
by competition 2002-2004, Assistant Professor by competition 2004 -
present at the discipline of Pharmacology, Faculty of Pharmacy, University
of Medicine and Pharmacy from Craiova.
Active member in scientific societies:
Member of Romanian Society of Pharmacology, Therapeutics and
Clinical Toxicology.
Member of the Society of Pharmaceutical Sciences from Romania.
Member of the Society of Pharmacy History.
Postgraduate courses: graduate of Department of Training Teacher
(2006); graduate courses: „Assistant Manager“ of Management Research
improvement programme (2007); „Euroson School, Guidelines in
Gastroenterogical Ultrasound“ (2007); „Theoretical and practical
electrocardiography“ (2007); „Liquid chromatography“ (2008);
„Pharmacotherapeutic education of patient“ (2008); „Pharmacogenetics and
Pharmacogenomics“ (2008); „Insulin therapy in diabetes“ (2008).
Scientific papers published in extenso – 15
Scientific papers at national and international scientific meetings published
in abstracts – 34
Books and educational materials published (co-author) – 3
Attendance at the National and International Congress of Clinical
Pharmacology, Therapeutics and Toxicology – 7
Attendance in other medical and pharmaceutical symposiums - 13
Foreign language: English, French.