Biomedical effects of Laurus nobilis L. leaf extract on vital organs in streptozotocin-induced diabetic rats: Experimental research

Abstract

Diabetes mellitus (DM) has been treated with herbs for centuries and many herbs reported to exert antidiabetic activity. Laurus nobilis is an aromatic herb belonging to the Lauraceae family, commonly known as bay. This study aimed to investigate the activity of Laurus nobilis leave extracts on histopathological and biochemical changes in β-cells of streptozotocin (STZ)-induced diabetic rats. Thirty healthy adult male albino rats were included in the study and divided equally into 5 groups for 4 weeks, control group (C), diabetic group (D), diabetic Laurus nobilis extract group (DLN), Laurus nobilis extract group (LN) and diabetic acarbose (DA) group. The results revealed that the administration of LN to diabetic rats significantly decreased (p < 0.05) blood glucose within 4 weeks of treatment. Additionally, LN also showed protection of liver and kidney functions. The glucose concentration decreased significantly in both diabetic rats treated with L. nobilis and acarbose (p < 0.05), the levels of aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT) and alanine aminotransferase (ALT) enzyme were insignificantly decreased in both diabetic rats treated with L. nobilis and acarbose (p ˃ 0.05). Outcomes of this study said that leave extracts of L. nobilis has valuable effect on blood glucose level and ameliorative effect on regeneration of pancreatic islets, it also restored the altered liver enzymes, urea, creatine kinase, total protein levels, calcium and ferritin to near normal.

Full text

Annals of Medicine and Surgery 61 (2021) 188–197
Available online 21 November 2020
2049-0801/© 2020 The Authors. Published by Elsevier Ltd on behalf of IJS Publishing Group Ltd. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Biomedical effects of Laurus nobilis L. leaf extract on vital organs in
streptozotocin-induced diabetic rats: Experimental research
Rebin Rafaat Mohammed
a
,
*
, Abdullah Khalid Omer
b
, Zabit Yener
c
, Ahmet Uyar
d
,
Avin Kawa Ahmed
a
a
Sulaimani Veterinary Directorate, Chamchamal Veterinary Hospital, Sulaimani, Iraq
b
Department of Food Hygiene and Quality Control, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
c
Department of Pathology, Faculty of Veterinary Medicine, Van Yuzuncu Yil University, Van, Turkey
d
Department of Pathology, Faculty of Veterinary Medicine, Hatay Mustafa Kemal University, Hatay, Turkey
ARTICLE INFO
Keywords:
Diabetes mellitus
Laurus nobilis
Streptozotocin
Histopathology
Blood glucose
ABSTRACT
Diabetes mellitus (DM) has been treated with herbs for centuries and many herbs reported to exert antidiabetic
activity. Laurus nobilis is an aromatic herb belonging to the Lauraceae family, commonly known as bay. This study
aimed to investigate the activity of Laurus nobilis leave extracts on histopathological and biochemical changes in
β-cells of streptozotocin (STZ)-induced diabetic rats. Thirty healthy adult male albino rats were included in the
study and divided equally into 5 groups for 4 weeks as follow; control group (C), diabetic group (D), diabetic
Laurus nobilis extract group (DLN), Laurus nobilis extract group (LN) and diabetic acarbose (DA) group. Histo-
pathologically, D group rats exhibited various degenerative and necrotic changes in their liver, pancreas and
kidney, whereas the DLN rats had nearly normal histology. Insulin immunostaining in the pancreatic beta cells
was decreased in the D group compared to the C group, whereas the DLN group was similar to the C group. The
glucose concentration decreased signicantly in both diabetic rats treated with L. nobilis and acarbose (p <0.05).
Additionally, the levels of aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT) and alanine
aminotransferase (ALT) enzyme were signicantly decreased in both diabetic rats treated with L. nobilis and
acarbose, compared to the D group (p ˃ 0.05). Outcomes of this study said that leave extracts of L. nobilis has
valuable effect on blood glucose level and ameliorative effect on regeneration of pancreatic islets, it also restored
the altered liver enzymes, urea, creatine kinase, total protein levels, calcium and ferritin to near normal.
1. Introduction
Diabetes mellitus (DM) is a chronic endocrine disorder of multiple
etiologies distinguished by hyperglycemia resulting from defects in in-
sulin secretion, insulin action, or both [1]. The clinical diagnosis of
diabetes is often indicated by the presence of symptoms such as polyuria,
polydipsia, unexplained weight loss, and is conrmed by documented
hyperglycemia [2,3]. Diabetes complications can be classied as
microvascular complications such as nervous system damage (neurop-
athy), renal system damage (nephropathy) and eye damage (retinop-
athy), and macrovascular complications for example cardiovascular
disease, stroke, and peripheral vascular disease [4–6].
Natural supplements are widely used around the world to treat
diabetes, but medical research does not support their effectiveness. DM
has been treated with herbs for centuries and there are many herbs that
have been reported to exert antidiabetic activity [7–9]. Traditional
plants have been used as a cure for diabetes for a long time before the
introduction of modern medicine. Historically, traditional herbal treat-
ments have been shown to possess successful pharmacological activity,
such as in the case with metformin, isolated from Galega ofcinalis
[10–12].
The dried leaves of Laurus nobilis are used as a spice or avoring
agent in the culinary and food industries [13]. The essential oil (EO)
prepared from the leaves has been reported to have antibacterial, anti-
oxidant and anti-inammatory activities [14,15]. Further leaves of
Laurus nobilis have been also used to treat rheumatism, neuralgia, and
scabies [16,17].
The main volatile compounds in laurel herb extract are usually 1,8-
cineole, methyl eugenol,
α
-terpinyl acetate.
α
-pinene, β-pinene, sabi-
nene, and linalool. Generally, leaves and berries are widely utilized,
* Corresponding author.
E-mail address: rebinpatho@gmail.com (R.R. Mohammed).
Contents lists available at ScienceDirect
Annals of Medicine and Surgery
journal homepage: www.elsevier.com/locate/amsu
https://doi.org/10.1016/j.amsu.2020.11.051
Received 6 October 2020; Received in revised form 18 November 2020; Accepted 18 November 2020

Annals of Medicine and Surgery 61 (2021) 188–197
189
oxygenated monoterpene 1,8-cineole is one of the major constituents of
leaves and berry fruits essential oil obtained from Laurus nobilis [18].
The leaves contains about 1.3% essential oils. The EOs obtained of berry
fruit depending on provenance and storage conditions. The oil extracted
from berries contain fatty acids, which include lauric (54%) linoleic
(17%) oleic (15%) and palmitic (5%) and volatile compounds such as
β-ocimene (22%), 1,8-cineole (9.5%), bicyclogermacrene (4.5%) and
β-elemene (2%) [19,20]. The bioactive components in bay leaves have
been shown to have effects on insulin sensitivity, glucose uptake, anti-
oxidant status, inammatory response, and glucose emptying.
Laurus nobilis is an aromatic herb belonging to the Lauraceae family
known as bay, daphne, bay laurel, true bay, or sweet bay. It is an
evergreen tree or shrub, is a native plant from the Southern Mediterra-
nean region, found in warm climate regions with high rainfall, especially
distribution in Turkey, Greece, Spain, Portugal, Italy, France, Morocco
and Mexico [21,22]. In Turkey, Laurus nobilis grows in the Marmara,
Aegean and Mediterranean regions [23,24]. It’s one of the oldest known
spices, widely used as a condiment and spice. With relevant medicinal
properties due to its important chemical composition and its potential
therapeutic effects [14]. A et al. [25] reported that aqueous extracts
of Laurus nobilis seeds were effective in reducing ethanol-induced gastric
ulcer in rats.
Traditionally bay leave extract have been used orally to treat the
symptoms of gastrointestinal problems, such as epigastric bloating,
impaired digestion, eructation, and atulence [25]. As a traditional
medicine, the decoction or tea of bay leaves is often used as therapy,
intestinal and gastric antispasmodic, against diarrhea, for rheumatic
pains, also diseases of the respiratory tract, as a cough, asthma, and
cardiac diseases [26].
Bay leaves have also shown that display insulin-enhancing activity in
vitro [27], however these enhance glucose metabolism and the overall
condition of individuals with diabetes not only by hypoglycemic effects
but also by improving lipid metabolism, antioxidant status, and capillary
function [28]. Bay leaves reduced serum glucose, total cholesterol, LDL
cholesterol and triglycerides, and increased HDL-cholesterol levels in
people with type 2 diabetes [27]. This study aimed to evaluate the ef-
fects of Laurus nobilis leave extracts on biochemical and histopatholog-
ical changes of pancreas, liver and kidney on diabetic rats.
2. Materials and methods
2.1. Materials
2.1.1. Animals
All experimental protocols were approved by the Experimental An-
imal Center of Yuzuncu Yil University, Van, Turkey. In the present study,
thirty male Wistar rats, weighting about 160–200 g with averagely 7
weeks old were randomly divided into ve groups based on treatment
each group containing 6 animals. All animals were housed under safe
laboratory conditions in a temperature-controlled room (22–24
̊
C) and
kept on a 12 h light/dark cycle. Blood glucose and body weight were
monitored before treatment once a week throughout 4 weeks of exper-
imental period.
2.1.2. Equipments
All measurements were performed by using Automatic tissue pro-
cessor (LEICA TP 1020 Semi-enclosed Benchtop), Centrifuge (Hettich
TD4, Shanghai, China), Nikon digital camera (DXM-1200F), Glucometer
(Accu-Chek, Taiwan), Tissue imbedded parafn (LEICA Eg115◦H
Shanghai, China).
2.2. Methods
2.2.1. Preparation of plant material and diet
The bay leave purchased from traditional herbal markets, a specimen
was deposited at the herbarium of the Hatay. The laurus nobilis leaves
were ground to powder by electric grinder and extracted with ethanol,
and the extraction oils were stored at room temperature in dark place.
200 mg kg
−1
of bay oils was administered daily orally using intragastric
tube at the time of work [29].
2.2.2. Diabetes model with streptozotocin (STZ)
Diabetes mellitus was induced by single intraperitoneal (IP) injection
of freshly prepared STZ (Sigma-aldrich, Saint Louis, MO) at dose of 70
mg kg
−1
b.w. dissolved in 0.01 M citrate buffer, pH 4.5 [30]. After 72 h
of STZ injection, and overnight fast, blood was taken from tail artery of
the rats. Accu-Chek monitoring used to rapidly changing blood glucose
level, when rats with blood glucose higher than 250 mg dl
−1
were
selected for the diabetic groups and involved to the examination. Strict
monitoring of all diabetic group rats was done for blood glucose after
24–48 h of STZ administration.
Injection of STZ and attack on pancreas cause hypersecretion of in-
sulin and this lead to intensive hypoglycemia and this may cause death
to many animals, to avoid this, drinking water containing 10% dextrose
were given to rats directly after I.P of STZ. In addition for taken care
about rats, blood glucose was measured at 3rd, 15th and 28th days of
throughout experimental model in blood taken from tail artery.
2.2.3. Experimental protocols
Experimental animals were randomly divided into 5 groups; each
group was included 6 animals. The examination period was continuous
for four weeks as below:
1. Control group (C): did not receive any other kind of co-
supplementation. Rats were given a standard diet.
2. Diabetes group (D): in this group diabetes was induced by adminis-
tered 70 ml/kg single dose of STZ IP injection [30], and given
standard diet.
3. Diabetes treated with Laurus nobilis leave extract group (DLN) given
200 mg kg
−1
of bay extract that administered every day orally using
intragastric tube for 28 days during the examination [29].
4. Laurus nobilis extract group (LN): 200 mg kg
−1
of bay leave was
administered every day orally using intragastric tube [29].
5. Diabetes with drug (Acarbose) group (DA): The rats of this group
were treated with 150 mg kg
−1
dose of Acarbose tablet (Glucobay),
(Bayer Türk Kimya San) each day orally using intragastric tube [31].
2.2.4. Blood sample collection and biochemical analysis
At the end of the treatment period, all rats were fasted for 18 h,
weighed and then anaesthetized via IP injection of ketamine hydro-
chloride (50 mg kg
−1
b.w.) and xylazine (8 mg kg
−1
b.w.). Blood
samples were collected from the heart puncture of rats and transferred to
suitable tubes for biochemical analysis using Merck commercial diag-
nostic kits (Darmstadt, Germany) on a blood chemistry analyzer (BTS-
350, BioSystems S.A. Barcelona, Spain).
2.2.5. Histopathological studies
Histopathological investigation were performed at last day of the
experiment. Pancreas, liver and kidneys were removed and kept in 10%
formaldehyde and embedded in parafn. Blocks of the preserved tissues
were sectioned (3–5
μ
m) on a microtome (Leica RM 2135: Leica Bio-
systems Nussloch GmbH, Nussloch, Germany) and mounted on glass
slides. Hematoxylin and eosin (H&E) staining was done and histopath-
ological examination was done in accordance with the method adopted
by Nagy and Ewais [32].
2.2.6. Immunohistochemical investigation
Insulin expressions were stained using the streptavidin-peroxidase
method (ABC), with the streptavidin / biotin immunoperoxidase kit
(Histostain-Plus Bulk Kit; Zymed, South San Francisco, CA, USA) in
accordance with the staining procedures of the manufacturer com-
panies. After the sections taken in 4–5 micron thick by microtome had
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Annals of Medicine and Surgery 61 (2021) 188–197
190
been placed on adhesive slides, they were passed through xylene and
alcohol series. In order to remove the endogenous peroxidase activity,
the sections were kept in 3% Hydrogen peroxide (H
2
O
2
) for 20 minutes
after being washed with PBS (phosphate buffer solution). After placing
the antigen in the retrieval solution (citrate buffer), it was heated twice
in the microwave oven for 20 minutes. Then it was taken out of the oven
and it was left to cool until it reached room temperature. After being
washed again with PBS, the sections were blocked by protein blocking
(non-immune serum) for 20 minutes. Insulin antibody (Catalog no: ab-
181547; Abcam, Toronto, Canada, diluted 1:1.000) were dropped into
each tissue and left overnight at +4◦C. Sections were washed again with
PBS and incubated for 20 minutes at room temperature with bio-
tinylated secondary antibody. The sections washed again with PBS were
left in streptavidin-peroxidase for 20 minutes and then washed in the
same way as PBS. After washing, 3,3’-Diaminobenzidine (DAB) was
dropped and left for 1–2 minutes. Then all sections were kept in Mayer’s
hematoxylin (Bio-Optika, 05-06002E) for 1–2 minutes and washed in
tap water. Sections were passed through of 70%, 80%, 90%, 96%
alcohol for 3 minutes, respectively, and 100% alcohol for 10 minutes
and of the xylol series for 5 minutes were closed using entellan. Negative
controls reacted with PBS were used instead of primary antibodies to
conrm staining. Sections were examined and photographed under a
light microscope. The immunoreactivity staining intensities of the
testicular samples obtained from the groups with primary antibodies
were scored as mild (+), moderate (++) and strong (+++).
2.2.7. Statistical analysis
Statistical analyses were performed by using ‘IBM SPSS Alan C.
Elliott software. ANOVA test was applied to analysis the signicant
differences in all groups. (Kruskal-Wallis) huc Dunn’s test was done to
compare among groups, differences were considered signicant when
the p <0.05.
3. Results
3.1. Effect on body weight
The diabetic rats exhibited profound body weight loss as compared
to normal rats. The initial, half-way and nal body weights of the rats
are shown in (Table 1).
The nal body weights in diabetic group (D), diabetes Laurus nobilis
group (DLN), and diabetic Acarbose group (DA) were signicantly
decreased when they compared with measuring at 14 days and also
higher decreased compared with baseline weight at the beginning of the
study. In contrast, body weights in control group (C) were signicantly
higher in comparison to other groups during experimental period while
laurus nobilis group (LN) was not signicantly changed in body weight.
Diabetes seemed to be more effective in decreasing body weight
(Table 1).
3.2. Effect on serum glucose
The serum glucose concentration (mg dl
−1
) in the Group C was
signicantly (p <0.05) lower, whereas Group D showed signicantly (p
<0.05) higher concentration as compared to other groups throughout
the experiment (Table 2). On the other hand, L. nobilis-fed group rats
showed relative to control (C) rats during the rst, second and fourth
week, respectively.
The two diabetic treated Groups (DLN and DA) showed signicant (p
<0.05) decrease in glucose concentration on last week and as compared
to Group C; the decrease was the highest in Groups C and LN. All Higher
blood glucose concentrations were observed in group D, DLN and DA
group after 72 h of STZ injection, and gradually decreased on day 14th
and 28th also signicant difference between group D and treated dia-
betes groups (DLN, DA). Laurus nobilis inhibited the development of
diabetes induced by STZ treatment (Table 2).
3.3. Histopathological ndings
3.3.1. Liver
The normal histological view of the liver was observed in the Control
and LN groups (Fig. 1-A and D). Degeneration and necrosis in the he-
patocytes were detected in Diabetes group. Additionally, disrupted he-
patic cords and sinusoidal architecture were detected. Varying in size
vacuoles were determined in the cytoplasm of degenerated hepatocytes
(Fig. 1-B). These ndings were found to be signicantly reduced in the
liver of rats in DLN group such as degeneration and necrosis (Fig. 1-C).
Similar histological appearance to the Control group were found in DA
group except for slight hydropic degeneration and dilation of sinusoids
(Fig. 1-E).
3.3.2. Pancreas
Histological views in cells of Langerhans islet in a Control and LN
groups were normal (Fig. 2-A and D). The diabetic rats had degenerative
and necrotic changes in the cells of Langerhans islet. As a result, atro-
phied islets which is cells with degenerative and picnotic nucleus had
deteriorated and shrunken architecture (Fig. 2-B). Partially, Langerhans
islet were preserved in rats in DLN group (Fig. 2-C). A signicant re-
covery in DA group was observed in the islets (Fig. 2-E).
3.3.3. Kidney
Kidney had normal histological view in both Control and LN groups
(Fig. 3-A and D). Severe balloon degeneration and necrosis of tubular
epithelium, vascular congestion, degeneration and necrosis of podocytes
of glomeruli of kidney of diabetic rats. Inammatory cells were focally
detected in the periglomerular areas. It were adhesion Bowman capsule
in some Glomeruli (Fig. 3-B). Slight degeneration and necrosis were
determined in some parts of the tubular epithelial cells and dilation of
some lymphatic in the kidney of rats in DLN group. Additionally, seldom
adhesions in bowman capsule of some glomeruli were observed (Fig. 3-
C). DA group were similar to DLN group (Fig. 3-E).
3.4. Immunohistochemical evaluation
The insulin secreting β-cells represented the major cell population of
Table 1
Results of body weight (g) in different groups (mean ±Standard deviation).
G 1st day 15th day 28th day
C 195.33 ±15.31
a
233.66 ±17.90
b
268.00 ±13.91
b
D 204.33 ±19.28
b
195.83 ±33.18
a,b
189.66 ±24.76
a
DLN 217.33 ±19.00
a
211.66 ±10.38
a
214.33 ±10.46
a
LN 212.33 ±13.10
a
229.83 ±17.26
b
262.16 ±21.16
b
DA 215.66 ±18.85
a
213.83 ±24.11
a,b
211.16 ±34.56
a
*G; groups were C (control group), D (diabetic group), DLN (diabetic with Laurus
nobilis trated group), DA (diabetic with drug treated group), LN (Laurus nobilis
fed group).
Data are means ±S.D.
b,c,d
Signicantly different from Control group in initial.
Table 2
Results of blood glucose (mg dl
−1
) in different groups (mean ±Standard
deviation).
G 1st day 15th day 28th day
C 128.16 ±10.87
a
157.50 ±30.73
a
138.33 ±24.23
a
D 600.00 ±0.00
d
592.50 ±18.37
c
457.66 ±170.57
c
DLN 563.16 ±53.21
c
479.16 ±164.36
b
287.33 ±109.83
b
LN 120.00 ±4.97
a
127.33 ±20.61
a
140.00 ±7.29
a
DA 521.66 ±14.26
b
587.33 ±18.61
c
316.16 ±70.00
b
*G; groups were C (control group), D (diabetic group), DLN (diabetic with Laurus
nobilis trated group), DA (diabetic with drug treated group), LN (Laurus nobilis
fed group).
Data are means ±S.D.
b,c,d
Signicantly different from Control group.
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Annals of Medicine and Surgery 61 (2021) 188–197
191
the islets, occupying mainly the central zone in the Langerhans islets.
Positive insulin expression was seen in the form of dark brown granules
present in the cytoplasm of β-cells. Strong insulin immunoreaction were
found in the Langerhans islets of Control and LN groups (Fig. 4-A and D).
Immunoreactivity in the Diabetes group had dramatically reduced ac-
cording to the control group and only a few β-cells displayed minimal
insulin immunoreaction (Fig. 4-B). In diabetic rats treated L. nobilis
extracts was increased in the number and percentage area of reactive
β-cells an apparent observed, as compared with the diabetic group.
Moderate insulin immunoreaction in the DLN group were detected
compared to diabetic rats. (Fig. 4-C). DA group showed signicantly
recovery compared to Diabetes group (Fig. 4-E).
Fig. 1. Hematoxylin and eosin-stained sections of liver. A) Control group: Normal histological appearance. B) Diabetic group: Disseminated vacuolization (arrows) in
the hepatocytes and dilation of sinusoids (stars). C) Diabetic +L. nobilis treated group: Slight hydropic degeneration in the some hepatocytes (arrows) and dilation of
sinusoids (star). D) L. nobilis-fed group: Almost normal histological appearance of the liver. E) Diabetic +drug- treated group: rat administered with acarbose
showing slight hydropic degeneration (arrows) and dilation of sinusoids. Bar =100
μ
m.
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Annals of Medicine and Surgery 61 (2021) 188–197
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Fig. 2. Hematoxylin and eosin-stained sections of pancreas. A) Control group: Normal histological appearance of islets of Langerhans. B) Diabetic group: Note that
islets of Langerhans is atrophic, and there are hydropic degeneration and necrosis (arrows) of some cells of islets of Langerhans. C) Diabetic +L. nobilis treated group:
Almost normal histological appearance of islets of Langerhans. D) L. nobilis group: Normal histological appearance of pancreas. E) Diabetic +Acarbose treated group:
Showing slight hydropic degeneration and a few necrotic cells of islets of Langerhans. Bar =20
μ
m.
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Annals of Medicine and Surgery 61 (2021) 188–197
193
Fig. 3. Hematoxylin and eosin-stained sections of kidney. A) Control group: Normal histological appearance. B) Diabetic group: Showing adhesions (arrows) be-
tween the glomerulus and Bowman capsule, and severe degeneration (stars) of tubular epithelium. C) Diabetic +L. nobilis treated group: Almost normal histological
appearance of tubular epithelium and glomerulus. D) L. nobilis group: Normal histological appearance of tubular epithelium and glomerulus. E) Diabetic +Acarbose
treated group: showing degenerative and necrotic changes (arrows) in some tubular epithelium. Bar =20
μ
m.
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Annals of Medicine and Surgery 61 (2021) 188–197
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3.5. Biochemical results
The glucose concentration decreased signicantly in both diabetic
rats treated with L. nobilis and acarbose (p <0.05) (Table 3). The levels
of alanine aminotransferase (ALT) enzyme were signicantly (p <0.05)
decreased in L. nobilis diabetic treated rats compared to diabetic group
rats. Also ALT level was signicantly (p <0.05) lower in acarbose dia-
betic treated group as compared to that of diabetes group (Table 3).
Fig. 4. Immunohistochemical staining of islets of Langerhans of pancreas by streptavidin-peroxidase (ABC) method. A) Control group: Strong insulin immunore-
activity in β cells, which occupy most of the islet. B) Diabetic group: Weak insulin immunoreactivity in a few β cells (arrows). C) Diabetic +L. nobilis treated group:
Moderate insulin immunoreactivity in β cells. D) L. nobilis group: Strong insülin immunoreactivity in β cells. E) Diabetic +Acarbose treated group: Moderate insulin
immunoreactivity in β cells. Bar =20
μ
m.
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Annals of Medicine and Surgery 61 (2021) 188–197
195
The mean values of aspartate aminotransferase (AST) activities were
signicantly decreased in both diabetic rats treated with L. nobilis and
acarbose (p <0.05), the level of AST was lower in L. nobilis treated rats
as comparison other rat groups (Table 3). As shown in (Table 3), the
mean values of Gamma-glutamyltransferase (GGT) activities were
signicantly decreased in all groups when compared with diabetes
group (p <0.05). However, the differences within other groups were not
signicant (p ˃ 0.05).
A statistically non-signicant decrease (p >0.05) found in alkaline
phosphatase (ALP) level L. nobilis diabetes treatment group in compar-
ison with diabetes groups. In addition, statistically signicant decrease
(p <0.05) can be seen in both diabetes drug treatment acarbose and
undiabetes treatment of L. nobilis extracts as compared with diabetes
group (Table 3).
The mean values of Urea level were signicantly decreased in DLN
extracts group as compared with diabetic groups (p <0.05). Diabetic
treated group of rats showed a signicant high level of calcium as
compared to other group of rats (p <0.05). The L. nobilis-fed group was
no signicant when compared with control group (Table 3). No signif-
icant difference in magnesium level was observed in treated rats group
with the diabetic group (p >0.05), (Table 3). In the phosphor (p) level
did not show any signicant difference in L. nobilis extract compared to
both diabetic and control group of rats (p >0.05) (Table 3).
Statistically non-signicant and slight reduction found in the total
protein (TP) levels as compared with diabetic group. L. nobilis leave
extract and control group are relative and signicant when compared
with diabetic group (p >0.05), (Table 3).
A insignicantly (p >0.05) increased can be seen in the Albumin
(ALB) level in the L. nobilis leave extract group in comparison with
diabetic group (Table 3). The level of creatinine kinase (CK) was
decreased signicantly (p <0.05) in the diabetic L. nobilis group in
comparison with the diabetic group (Table 3). Iron (Fe) levels in the
diabetic rats were treated with L. nobilis signicantly increased when
compared to diabetic group rats, there was no signicant difference in
the treated diabetes groups with control group in the levels of Fe (p >
0.05), (Table 3).
4. Discussion and conclusion
Conventional therapies for DM have many side effects and high rate
of secondary failure. On the other hand herbal extracts are expected to
have similar efcacy with fewer side effects than conventional drugs
[33]. Nowadays, more than 1200 plant species are used to treat symp-
toms of DM, the hypoglycemic property of almost 50% of these tradi-
tionally consumed medicines has been experimentally tested [34].
In our study detected weight loss in all STZ-induced rats group when
comparisons with control group, and exhibited hyperglycaemia in STZ-
induced rats with decrease in serum glucose levels in the treated diabetic
groups. L. nobilis leaves extracts (LNLE) inhibit the development of
diabetes induced by STZ and decrease serum glucose levels. LNLE
treatment did not induce a signicant change in the body weight of the
diabetic rats, however, LNLE had a signicant decrease in the blood
glucose levels for 28 days of the diabetic rats’ treatment group.
In this study, we observed histopathological and biochemical
changes in STZ-induced rats and these changes were reduced with LNLE
treatment. The promising mechanism by which LNLE mediated its
antidiabetic effect could be by potentiation of pancreatic secretion of
insulin from existing β-cells of islets, as was evident by the signicant
decrease in the level of glucose in the extract treated animals.
The hypoglycemic activity of LNLE was compared with acarbose, a
standard hypoglycemic drug, since the results of the present study, it
may be suggested that the mechanism of action of L. nobilis may be
similar to acarbose action. In this essay indicated that the pathological
effects in rats liver and pancreas tissues induced by STZ were reversible
and normalized by received extracts bay leaves for 4 weeks of experi-
ment, this could be attributed to its antidiabetic effects. Several studies
have reported that the hepatocytes of STZ-induced diabetic rats showed
cytoplasmic alterations, sinusoidal dilation and congestion, periportal
inammation, showed kupffer cells activation, cytoplasmic vacuoliza-
tion of hepatocytes and necrosis [35].
The hepatocyte nuclei were generally enlarged and sometimes
showed irregular contours and intranuclear inclusions [36]. In pancre-
atic sections of untreated diabetic rats disclosed that the islets were
comparatively small and shrunken and severe degenerative changes in
the pancreatic islets, mainly at the center of the islets and karyolysis of
the nuclei was visible [37]. The L. nobilis treated diabetic rats were
reversed and the normalization of pancreatic architecture revealed
vacuolations of β-cells was observed.
In addition regarding the immunohistochemistry, different sizes of
islets of pancreas were observed with increasing immunoreaction to
insulin antibody in β-cells treated with L. nobilis leaves extracts com-
parison to diabetic group, langerhans with increasing insulin immuno-
reactivity in cytoplasm of its β-cells, in the number and percentage area
of reactive β-cells, areas of dark brown staining (strong positive) for
insulin antibody were seen in islet β-cells cytoplasm from diabetic
treated by L. nobilis.
In STZ-induced diabetic rats the liver was necrotized. An increase in
the activities of ALT, AST and GGT in plasma might be mainly an indi-
cation of the hepatotoxic effect of STZ [38]. ALT, AST and GGT were
signicantly decreased in diabetic bay leaves treatment, and related to
normal control group. In contrast diabetic group signicantly higher
level of enzymatic liver function test observed [39]. Al Chalabi et al.
Table 3
Changes in the serum of Glucose(mg dL
−1
), ALT(U l
−1
), AST(U l
−1
), GGT(U l
−1
), ALP(U l
−1
), Urea(mg dL
−1
), Calcium(mg dL
−1
), Magnesium(mg dL
−1
),
Phosphor(mg dL
−1
), Total protein(g dL
−1
), Albumin(mg dL
−1
), Creatinine(mg
dL
−1
), and Iron(
μ
mol L
−1
) levels in different groups under study (mean ±
standard error).
BC G
C D DLN DA LN
ALT 49.16 ±
1.07
a
251.83 ±
46.91
b
51.83 ±
10.99
a
34.16 ±
1.53
a
77.66 ±
23.41
a
AST 136.50 ±
7.53
a
547.33 ±
139.46
b
98.50 ±
6.92
a
110.33 ±
1.28
a
216.33 ±
41.20
a
GGT 0.51 ±
.0.14
a
9.16 ±2.08
b
2.18 ±1.16
a
0.16 ±
0.16
a
1.08 ±
.0.40
a
ALP 203.16 ±
9.14
a
678.50 ±
120.73
b
654.33 ±
139.94
b
281.16 ±
25.29
a
366.50 ±
79.07
a,b
Urea 31.61 ±
0.91
c
53.76 ±
1.22
d
19.90 ±
0.90
b
15.05 ±
0.27
a
30.56 ±
2.69
c
Ca 11.10 ±
0.12
a
10.21 ±
0.22
a
12.58 ±
0.93
b
11.35 ±
0.25
a,b
10.98 ±
0.30
a
Mg 3.52 ±
0.08
b
3.50 ±0.18
b
3.13 ±
0.39
a,b
2.64 ±
0.04
a
3.51 ±0.22
b
P 9.03 ±
0.11
b
9.10 ±0.45
b
7.39 ±1.59
b
4.49 ±
0.13
a
9.75 ±1.02
b
TP 6.64 ±
0.07
c
6.23 ±
0.10
a,b
5.90 ±0.13
a
5.87 ±
0.05
a
6.57 ±0.21
b,c
ALB 3.65 ±
0.02
b
3.17 ±
0.07
b
3.56 ±0.10
b
3.35 ±
0.01
a
3.58 ±0.09
b
CK 725.6 ±
85.10
b
664.1 ±
60.77
b
282.1 ±
51.01
a
331.6 ±
16.01
a
809.6 ±
52.38
b
Fe 32.08 ±
1.57
b
17.20 ±
3.55
a
32.20 ±
6.95
b
35.15 ±
3.60
b
40.30 ±
5.60
b
*BC; biochemical tests were ALT (alanine aminotransferase), AST (aspartate
aminotransferase), GGT (gamma-glutamyltransferase), ALP (alkaline phospha-
tase), Ca (calcium), Mg (magnesium), P (phosphor), TP (total protein), ALB
(albumin), CK (creatinine kinase), Fe (iron).
*G; groups were C (control group), D (diabetic group), DLN (diabetic with Laurus
nobilis trated group), DA (diabetic with drug treated group), LN (Laurus nobilis
fed group).
*a - c Means with the same column are not signicantly different P ˃ 0.05. *
a-c
Means with different column are statistically signicant P ˂ 0.05.
R.R. Mohammed et al.

Annals of Medicine and Surgery 61 (2021) 188–197
196
[40] reported that the bay leave extract resulted in a decrease in fasting
blood glucose and a higher level of fasting insulin, also LDL, ALT, and
AST were also decreased, whereas HDL and body weight increased in
groups of diabetic rats relative to control not treated groups. In another
study recommended that the L. nobilis tea consumption in healthy vol-
unteers can improve blood lipid prole (HDL level increased and a small
decrease in levels of LDL and triglycerides) and this implies a possible
positive impact on the risk reduction of coronary heart disease [41].
Similar results were reported by Casamassima et al. [42] and investi-
gated a substantial reduction in blood lipid prole, glycemic prole and
liver enzymes, with decreased levels of LDL, ALT and AST, and increased
HDL, has resulted from dietary incorporation of dried bay leaves meal.
Treatment of the diabetic rats with LNLE reduced the activity of
these enzymes in plasma compared to the diabetic untreated group and
consequently alleviated liver damage caused by STZ-induced diabetes
and indicated the hepato protective role in preventing diabetic com-
plications. The diabetic rats treated with bay leave extract displayed a
statistically signicant reduction in the urea and creatinine levels with
respect to diabetic rats. In this sense, kidney failure is manifested by
increasing urea and creatinine but a decrease indicates clinical
improvement [43].
There’s still clearly a signicant dispute about the use of natural or
cultivated plants, which has both positive and negative aspects in bio-
physical terms, as well as in terms of economics. L. nobilis is a signicant
socioeconomic evergreen tree belonging to the Lauraceae family.
L. nobilis are used as antihyperglycaemic herbs, used to treat bacterial
and fungal contaminations, to treat eructation, atulence and gastro-
intestinal problems. It also exhibits anti-inammatory, anticonvulsive,
antiepileptic and antioxidant properties [44,45].
In conclusion, from this study, based on the experimental ndings, it
was suggested that administration of L. nobilis leave extracts, at a safe
dose level, signicantly suppressed STZ-induced diabetic rats. We
believe that further preclinical research into the utility of L. nobilis
treatment may indicate its suitability as a potential treatment in diabetic
patients, our results expressed that leave extracts of L. nobilis has valu-
able effect on blood glucose level and ameliorative effect on regenera-
tion of pancreatic islets. It also restored the altered liver enzymes (ALT,
AST, and GGT), urea, creatine kinase, total protein levels calcium and Fe
to near normal. It may be used as a therapeutic agent in the management
of diabetes mellitus.
Author contributions
All authors contribute to this research study.
Rebin R. Mohammed, Abdullah K. Omer, Avin K, Ahmed: Data
collection and analysis, original draft preparation, Zabit Yener, Ahmet
Uyar: Study design, conceptualization and writing, review and editing.
5. Provenance and peer review
Not commissioned, externally peer reviewed.
Funding
The authors wish to acknowledge Van Yuzȕncu Yıl University for the
support (Grant number 2015-SBE-YL309).
Ethical approval
All experimental protocols were approved by the Experimental An-
imal Center of Van Yȕzȕncȕ Yıl University, Turkey.
Consent
All experimental protocols were approved by the Experimental An-
imal Center of Van Yȕzȕncȕ Yıl University, Turkey.
Guarantor
Rebin R.Mohammed (on behalf of all authors).
Declaration of competing interest
There is no conict of interest.
Acknowledgements
The authors wish to acknowledge Van Yȕzȕncȕ Yıl University for the
support (Grant number 2015-SBE-YL309).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.amsu.2020.11.051.
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