histopathological changes of pancreatic tissues in
hyperglycemic male rats treated with mixture of plants
extracts
maysaa ? adil hadi, haider kamil zaidan, ali hmood al-saadi
department of biology, college of science, university of babylon/ hilla-iraq
abstract : the present study aims to evaluate the hypoglycemic, and protective effects of
mixture of methanol-watery extracts of five selective medicinal plants: trigonella faenumgraecum
seeds, nigella sativa seeds, zingiber officinale rhizomes, olea europeae leaves and
fraxinus ssp. seeds, and determine the efficacy of this mixture in the treatment of diabetes
mellitus type 2.
animals were randomly divided into six groups: group i: normal negative control, group ii:
diabetic control, group iii: normal rats treated with mixture of plants extracts for 60 days,
group iv, v, vi: diabetic rats treated with mixture of plants extracts for 45, 60, 75 days
respectively.
the results revealed significant decrease (p<0.05) in body weight of the diabetic rats, diabetic
rats treated with mixture of plants extracts for 45, 60 days as compared with normal control,
and significant increase (p<0.05) in the diabetic rats treated for 75 days as compared with the
diabetic control, while normal rats treated with mixture of plants extracts for 60 days which
showed significant decrease (p<0.05) as compared with normal control, but it was considered
significant increase as compared with diabetic rats. the result of fasting blood glucose levels
showed significant decrease (p<0.05) in all treated groups as compared with diabetic control.
on the other hand, significant decrease (p<0.05) was shown in serum insulin levels and
pancreas/body weight ratios in diabetic group as compare with negative control while treatment
with mixture of plants extracts for different periods caused non significant differences in all
treatment periods as compared with negative control and returned pancreas/body weight ratio
near the normal value. treatment of normal rats with mixture of plants extracts for 60 days
caused non significant differences in fasting blood glucose, insulin and pancreas / body weight
ratio.
histological sections of diabetic pancreas revealed degeneration, vacuolization of the islets of
langerhans and the exocrine pancreas manifested inflammatory cells infiltration, and vascular
congestion, while treated groups exhibited normal appearance of islets of langerhans especially
?-cells and pancreatic acini and some pancreatic sections showed with inflammatory cells
infiltration.
in conclusion, type ?? dm caused histopathological changes in pancreas, many of these changes
could be prevented or reduced by using mixture of plants extracts used in this study. the effect
of this mixture had more positive effects when given orally for longer period (75 days).
keywords : histopathology, pancreas, hyperglycemic rats, plants extracts.
international journal of chemtech research
coden (usa): ijcrgg, issn: 0974-4290, issn(online):2455-9555
vol.9, no.06 pp 501-513, 2016
maysaa ? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 502
introduction
diabetes mellitus (dm) is a common heterogeneous metabolic disorder of multiple causes characterized
by chronic hyperglycemia and disorders of carbohydrate, fat and protein metabolism associated with defect in
insulin secretion (type 1) or resistance to insulin action (type 2)(1) with micro and macro vascular complications
that results in significant morbidity and mortality in the world(2). dm is a multifactorial disease involving
interaction of genetic and environmental factors(3-6). it is caused by inherited and/or acquired deficiency in the
production of insulin by the ?-cells of pancreas, or by ineffectiveness of insulin produced (an absolute or
relative insulin deficiency)(7). hyperglycemia and hyperlipidemia thereafter exert additional damaging or toxic
effect on the ?-cell(3).
s everal pathogenic processes are involved in the development of dm. these range from autoimmune
destruction of the ?-cells of the pancreas with consequent insulin deficiency to abnormalities that result in
resistance to insulin action and becomes insufficient to control systemic glucose levels(8).
diabetic patients are at risk of the micro and microvascular complications through the body(9,10). the
microvascular complications, include nephropathy, retinopathy, neuropathy, and macrovascular complications
including heart disease and stroke(9). diabetes has been associated with reproductive impairment in both men
and women(10). the mild thyroid dysfunction in dmt2 was linked to significantly changes in body weight ,lipid
profile and likely represents risk factor for healthy and obesity11.
although insulin treatment and other chemical therapies can control many aspects of diabetes,
numerous complications are common in the disease(12,13). as continuous oral administration of insulin can
culminate in many side effects and toxicity(14). also, the use of biguanides, sulphonylurea and other drugs are
valuable in the treatment of diabetes mellitus their use, however, is restricted by their limited action,
pharmacokinetic properties, secondary failure rates and side effects(15).
recently, attention has been paid to the search of effective drugs in the field of traditional medicine(10).
medicinal plants are frequently considered to be less toxic and free from side effects than the synthetic ones(16).
this study was designed to use the mixture of plant extracts of five medicinal plants to treatment of dm and
aimed to evaluate the possible hypoglycemic, protective effect and determine the efficiency of this mixture in
the treatment of type 2 dm which may occur against harmful effect and damage in pancreatic tissues of
alloxan-induced diabetes in rats.
2. materials and methods
2.1: preparation of solutions(17).
2.1.1: formalin fixative(10%): each 1ml of formalin 40% added to 9 ml of tap water.
2.1.2: haematoxylin-harris stain: the solution was prepared by dissolving 1g of haematoxylin in 10 ml ethyl
alcohol. 20g of potassium alum dissolved in 200 ml of dw and then boiled. haematoxylin then added and the
solution boiled for ½ minutes. 0.5g of mercuric oxide added. the solution cooled rapidly and a few dropings of
acetic acid were added it is optional but its inclusion gives more precise and selective staining of nuclei.
2.1.3: eosin stain: prepared by mixing 1 gm of eosin y in 99 ml ethanol.
2.1.4: acid alcohol 1%: prepared by mixing 1ml of hcl in 100 ml of 70% ethanol.
2.2: plants collection and identification
the plants used in this study were purchased from a local herbal markets except olive leaves were
collected from gardens of babylon university. the plants were identified by plant harbarium /college of
science/ university of babylon. the plants parts used in this study were trigonella faenum-graecum seeds,
nigella sativa seeds, zingiber officinale rhizomes, olea europeae leaves, and fraxinus ssp.seeds. oilve leaves
were rinsed with water to remove dust, insecticides, and contaminated materials then dried in dark. all plants
materials were grained into fine powder.
maysaa ? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 503
2.3: preparation of plant extracts: the plants extracts were prepared according to sato et al. (1990). the plant
powder was extracted with mixture of methanol and distilled water in a ratio of 20 % methanol: 80% distilled
water (v/v) in average of 1 gm of plant powder :3 gm of mixture using blender for 30 min at room temperature.
the suspension were filtered by guase and the filtrate concentrated in oven at 45 ?c. the crude extracts were
stored at 4 ?c until use.
2.4: animals of experiments: adult albino male rats aged 2-3 months were used in this study. the overall
number of animals used was 36. the animals were provided with food and water ad libitum. after adaptation
animals were used for experimental studies. after the induction of t2 dm, the animals were divided into
different groups that included 6 animals in each group. some of diabetic rats died without treatment.
2.5: detection of plants extracts doses:
treatment by mixture started by giving different oral doses of plants extracts (100-1000 mg/kg body
weight) to normal and diabetic rats. the treatment performed by mixing 0.5 ml of each plant extract
immediately and administrated orally by orogastric tube.
2.6: induction of type 2 dm:
diabetes was induced by injecting the animals with 3 doses of alloxan 120 mg/kg dissolved in 0.5 ml
normal saline immediately for the induction of t2dm. fastig blood glucose level (fbg) of fasting rats were
measured weekly by using the glucometer and rats with fbg > 200 mg /dl were considered diabetic and used in
this study(14).
2.7: experimental design of the study: rats were randomly divided into six groups:
1- normal control group( negative control): included healthy intact animals which were given normal saline
intraperitonialy ( i.p. ) and distilled water orally by orogastric tube.
2- diabetic control group: included animals which had been given alloxan i.p. and distilled water orally but
did not treated with mixture of plants extracts (35 days).
3- normal group treated with mixture of plants extracts: included healthy intact animals treated orally with
mixture of plant extracts only for 60 days.
4- treatment groups: included 18 rats. they were treated with alloxan i.p. and then, after the induction of
dm, they treated orally with mixture of plants extracts. these diabetic treated animals were subdivided into
three subgroups (n=6) treated for 45, 60 and 75 days.
through the study period glucose and body weight of animals were measured. the percentage of
weight changes was calculated according to the formula:
x 100
2.8: blood sampling:
through the course of the study, blood was obtained by puncturing the caudal vein by a sterile needle,
then a blood droping was put in contact with the strip of glucometer to measure the blood glucose level. after one
week from the end of experiment, the animals were sacrificed and blood was collected directly by heart
puncture. also, pancreas were removed and weighted to calculate pancreas/ body weight ratio by using the
formula:
organ /body weight ratio (%) =
body weight after experiment (gm)
final body weight – initial body weight
initial body weight
organ weight (gm) x 100
maysaa
? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 504
2.9: serum insulin measurement:
insulin level (ng/dl) was measured according to rat insulin elisa kit ( cosabio biotech co.)
2.10: histological processing and staining:
ordinary histological processing are prepared for pancreas in order to study the histopathological
changes. the pancreas was harvested and trimmed of fat. its macroscopic appearance was recorded and the
gland was fixed in 10% formaldehyde. dehydration, clearing, embedding in paraffin, sectioning at 5 ? and
stained with hematoxylin and eosin according to bancroft and steven (1982) was carried out.
2.11: statistical analysis: analysis of data was performed by using statistical package for social science
(spss) system/ version 17. results expressed as mean ± s.e .the analysis of variance (anova) and the paired
sample t- test were used for this purpose.
3. results
3.1: body weight: results of this study showed significant decrease (p<0.05) in the body weight of diabetic
group from (212.67 ± 16.65) gm to (187.67± 14.03) gm compared to negative control which increased from
(216 ± 8.103) gm to (255 ± 7.302) gm. in normal group administrated with mixture of plants extracts for 60
days, there was a little increase in body weight which statistically considered as significant decrease (p<0.05) in
the body weight from (305 ± 5.477) gm to(312.33 ± 7.60) gm compared to negative control. diabetic groups
treated with mixture of plants extracts registered a body weight gain with increasing period of treatment.
diabetic group treated with mixture of plants extracts for 45 days still had significant decrease in their body
weight from (299 ± 35.704) gm to (280 ±29.828) gm when compared to negative control and non significant
difference as compared to diabetic control, but there was significant increase (p<0.05) in the weight gain from
(329.33 ± 4.77) gm to (333. 66 ± 6.296) gm and from (323 ± 2.19) gm to (354 ± 7.669) gm in diabetic groups
treated with mixture of plants extracts for 60 and 75 days respectively when compared to diabetic control.
diabetic group treated for 75 days could revert to normal body weight gain as in negative control as shown in
table 1.
table (1): changes of body weight in experimental rats
different letters refer to significant difference between groups
similar letters refer to non significant difference.
n= 6 for each group s.e :standard error
3.2: fasting blood glucose (fbg) & fasting serum insulin
the fbg levels in negative control and all experimental groups were analyzed (table 2) and variation
of fbg was observed (figure 2). alloxan injection caused significant increase (p<0.05) in mean of fbg levels
(471 ± 37.98) mg/dl as compared to negative control (124 ± 4.219) mg/dl. furthermore, there was decrease in
weight change body weight (mean ± s.e) group
final body weight
( gm)
initial body weight (gm)
39 a 255 ± 7.302 216 ± 8.103 negative control
-25 b 187.67± 14.030 212.67 ± 16.650 diabetic control
7 c 312.33±7.600 305 ± 5.477 plant extracts mixture
(60 days)
-19 bcd 280 ± 29.828 299 ± 35.700 dm + plants extracts
mixture (45 days)
4 ce 333. 66 ± 6.296 329.33 ± 4.770 dm + plants extracts
mixture(60 days)
31 a 354 ± 7.669 323 ± 2.191 dm + plants extracts
mixture (75 days)
maysaa ? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 505
mean of fbg levels but was non significant in normal rats treated with mixture of plants extracts for 60 days as
compared to negative control while this group had significant reduction in mean of fbg level (79.66± 4.168)
mg/dl as compared to diabetic control. diabetic groups which were treated with mixture of plants extracts for
45, 60, 75 days showed significant reduction (p<0.05) in mean of fbg levels as compared to diabetic control
and could revert fbg to normal value in this groups compared to negative control.
also, there was no significant differences in means of insulin level (0.193 ± 0.012) ng/dl in normal rats
treated with mixture of plants extracts for 60 days as compared to negative control while the same group caused
non significant increase in insulin level as compared to diabetic control (table 2).
serum level of insulin was significantly reduced (0.162 ± 0.003) ng/dl in alloxan –induced diabetic rats
compared to negative control (0.303 ± 0.072) ng/dl. this alteration was ameliorated by administration of
mixture of plants extracts which caused significant increase (p<0.05) in insulin level (0.4± 0.073 ) ng/ dl and
(0.43 ± 0.11) ng/ dl in diabetic rats treated with mixture of plants extracts for 45 and 60 days, respectively. the
75 days treatment caused preservation of insulin level near normal value as compared to negative control (table
2).
figure (2): variation in fasting blood glucose (mg/dl) of experimental rats.
table (2): changes of fbg (mg/dl)& fasting serum insulin (mg/dl) in experimental rats (mean ±s.e).
different letters refer to significant difference between groups
similar letters refer to non significant difference
n=6 for each group s.e: standard error
3.3: pancreas / body weight ratio
as shown in table 3, injection of alloxan markedly decreased tissue weight of pancreas and caused
significant decrease (p<0.05) in pancreas / body weight ratio (0.330 ± 0.01) % as compared to negative control
(0.905 ± 0.158) %. although mixture of plants extracts caused non- significant difference in pancreas / body
fasting serum insulin (ng/dl) fbg (mg/dl) group
0.303 ± 0.07 a 124 ± 4.22 a negative control
0.162 ±0 .003 b 471 ± 37.9 b diabetic control
0.193 ± 0.012 abc 79.66 ± 4.17 ac mixture of plants extracts (60 days)
0.4 ± 0.07 a 107 ± 2.56 acd dm + mixture of plants extracts
(45 days)
0.43 ± 0.11 a 137.66 ± 12.08 ad dm + mixture of plants extracts
(60 days)
0.31 ± 0.01 abc 140 ± 5.84 ad dm + mixture of plants extracts
(75 days)
time of treatment (weeks)
maysaa ? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 506
weight ratio in normal rats as compared to normal control, yet it could return pancreas / body weight ratio near
normal value in treated groups and caused significant increasing (p<0.05) in pancreas / body weight ratio (0.778
± 0.034) % and (0.856 ± 0.097) % in diabetic rats treated with mixture of plants extracts for 45 and 60 days,
respectively and non- significant increasing in diabetic rats treated for 75 day as compared to diabetic control.
table (3): changes of pancreas / body weight ratios in experimental rats (mean ± s.e).
pancreas / body weight ratio group
0.905 ± 0.16 a negative control
0.330 ± 0.01 b diabetic control
0.775 ± 0.07 ac mixture of plants extracts
(60 days)
0.778 ± 0.03 ac dm + mixture of plants extracts
(45 days)
0.856 ± 0.09 ac dm + mixture of plants extracts
(60 days)
0.728 ± 0.07 ac dm + mixture of plants extracts
(75 days)
different letters refer to significant difference between groups
similar letters refer to non significant difference
n=6 each group , s.e: standard error
3.4:histopathological study of pancreas
histological section of the negative control group showed that the pancreas composed of the hormone
–producing cells of the pancreas which is islet of langerhans containing several secretory cell types and
exocrine part composed of acini as seen in figure (3 a). the pancreata of the normal group treated with mixture
of plants extracts for 60 days had normal islets and normal acini (figure 3 b).
histologically, some changes of the pancreata of experimental rats have been observed in sections
stained hematoxylin /eosin stain. in diabetic control, there was degenerative changes in both exocrine and
endocrine pancreas. the most characteristic changes of the endocrine pancreas was a decrease in the size and
number of pancreatic islets, abnormal appearance of many islets which had less number of cells compared to
islets of negative control group. islets consisted of many islet cells with vacuolated or shrunken cytoplasm
indicating the degranulation of the islet cells (figures 4 a&b). also, another islets of langerhans had severe
damage with presence of inflammatory cells and fibroblast in islets (figure 4c). exocrine pancreas had
moderate vascular congestion and degenerative acini. the presence of a few lymphocyte between acini and
connective tissue in the pancreata of the diabetic rats was observed (figures 4 d&e). diabetic groups which
were treated with mixture of plants extracts for different periods (45, 60, 75 days respectively ) showed
regeneration of both islets and acini and had less number of inflammatory cells. these groups histologically
had normal architecture of many islets and acini (figures 5 a). few islet still had few vascular congestion
(figures 5 b) and degeneration (figure 5 c&d) while other islets showed a bigining of regeneration (figures 5
e). also, some islets showed presence of a few inflammatory cells at boundaries of islets (figure 5f).
maysaa ? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 507
figure (3): sections of pancreatic tissues show: a- negative control group shows normal islets of
langerhans (il) and normal acini (a) h&e (100x). b- normal group treated with plants extracts
mixture for 60 days shows normal islets of langerhans (il) and acini (a) h&e(40x).
figure 4: sections in pancreatic tissues of diabetic group show :a & b: degenerative changes of islets of
langerhans (il) and vacuolation of cytoplasm (v). h&e (400x&1000x). c- islet of langerhans (il)
with sever damage and presence of inflammatory cells (?) and fibroblast (f) in islets. h&e (400x). ddegenerated
islet of langerhans (il) with presence of vascular congestion (vc). h & e (400x). edegenerated
acini with presence of inflammatory cells between acini and connective tissue. h&e
(100x).
il
maysaa ? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 508
figure 5: sections in pancreatic tissues of diabetic group treated with mixture of plants extracts: a:
treatment for 45 days showed normal islets of langerhans (il) and acini (a), duct (d). b: few vascular
congestion (vc). c&d: some degenerated islets of langerhans (il) and normal acini (a). e- treatment
for 60 days shows bigining of regeneration of islets of langerhans (il) and normal acini (a). ftreatment
for 45 days shows inflammatory cell at the boundaries of normal islets (il) and normal acini
(a),vessels(v), duct(d). h&e(400x).
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4. discussion
results of the present study showed that the alloxan-induced group exhibited a significant decrease in
the whole body weight compare with negative control group. this result was expected because it is well known
that diabetes causes decrease in the whole body weight which is considered one of the most important
diagnostic symptoms of diabetes(8) (ada, 2014). in addition, in diabetes there is inability to store fat and
protein along with breakdown of existing fat and protein stores (ravi et al., 2005) reflected in decreasing body
weight. the decreased mean body weight in diabetic rats is due to excessive break down of tissue protein
(ganesh et al., 2010). this result is consistent with other studies that showed a decrease in body weight in
diabetic rats(12)
the data showed in table (2) and table (3) demonstrated significant increase (p<0.05) of fbg and
significant decrease (p<0.05) of fasting serum insulin levels and pancreas/body weight ratio in diabetic group
and persistent hyperglycemia in diabetic animals may be due to the partial destruction of pancreatic ?-cells by
the direct effect of alloxan, because alloxan is well known to cause selective hydropingic degeneration,
degranulation, necrosis of the pancreatic ?-cells, and fibrosis of the islets in a dose depending pattern. weight
reduction may be due to the effects of alloxan which causes degeneration and necrotic effects (singh and gupta,
2007).
decreasing both ?-cell function and number can contribute to insulin deficiency in type 2 diabetes ,the
average of ?-cell mass is about 39% lower in type 2 diabetes subjects compared to matched control (rahier et
al.,2008). electron microscopic observation demonstrated the presence of more dead ? -cells in islets of
diabetics than from non-diabetic controls. the type of cell death observed seems to be apoptosis or autophagyassociated
cell death, altered levels or impaired function of autophagy, possibly defects in the process of
lysosome fusion and/or proteolytic enzyme activation, may contribute to the reduced ?-cell mass by
accelerating ? -cell death (masini et al. 2009). also, alloxan-induced diabetes cause degenerative changes in
endocrine and exocrine pancreas which may reflected in its weight. various tissues in the diabetic state are
more prone to oxidative damage resulting in various complications of dm(15).the reduction of pancreas area
and size may assist finding of this study about pancreas reduced weight.
histopathological observation in diabetic control showed degenerative changes in both endocrine and
exocrine pancreas such as atrophy of islets, vacuolization of islets cells, vascular congestion, and presence of
inflammatory cells. a probable explanation may be related to oxidative stress resulting from hyperglycemia
decreases the antioxidants levels and increases ros (al-kufaishi, 2012 shen and pierce, 2015) and the
activities of antioxidant enzyme (three primary scavenger enzymes: super oxide dismutase, catalase, and
glutathione peroxidase) were altered in diabetic rats (arulselvan and subramanian, 2006) and these effects
further exacerbate the development and progression of diabetes complications through the damage of protein,
lipids, and dna, and then the cells (johansen et al., 2005), and these may represent the causes of degeneration
revealed in histological sections.
the presence of chronic inflammatory cells may refer to the presence of inflammation because
hyperglycemia, which may have increased inflammation by increasing inflammatory markers such as migration
inhibitory factor (mif) and c-reactive protein (crp), and decreases the antioxidant endothelia nitric oxide
(sojoholm and nystrom, 2006). the vascular congestion may be due to specific defect in the blood capillaries
that nourish the pancreas, or it may be a result from increased capillary blood pressure which may be due to
increased systemic blood pressure because diabetes are usually associated with hypertension as one of diabetic
complications, as it was referred previously that people with type 2 diabetes also have high rates of high blood
pressure(8) (ada, 2014). this results is in agreement with shaffie et al (2010) who noticed that the islets of
langerhans showed severe necrotic changes, congestion and increase in connective tissue leading to relative
reduction in size of islets.
the results of this study found out that the group of normal animals treated with mixture for 60 days
showed a little increase in body weight that is statistically considered as significant decrease in the body weight,
and this mixture of plants extracts may affect on metabolism when given to normal cell in normal group
primarily. this action is not correlated to the toxicity of the mixture of plants extracts, since no sign of toxicity
detected in this study.
maysaa ? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 510
the treatment for 60 and 75 days caused significant increase in the body weight which may be due to
the enhancement of the blood glucose reflecting on enhancement of carbohydrates, protein, and fat metabolism
due to the longer period of treatment. many mechanisms explain enhancement of body weight as the mixture
increase appetite. besides, this may be because it contains high protein content. the increasing body weight
may be due to increase in pancreas weight as we have seen in this study and increasing of other organs such as
spleen (bajallan, 2006), testis and epididymus (al-salammi,2004) after fenugreek extracts administration and
increasing testis and epididymus weight when z. officinale extract used (bahar, 2011).or this may be because
olea leaves and fenugreek seeds increased glycogen content in liver and liver /body weight ratio which due to
induction significant insulin release (al-hamadani, 2002). also, petit et al (1995) reported that saponin isolated
from fenugreek seeds increased food intake and body weight of rats and suggested that fenugreek caused
hypoglycemic effect and activation of glycogenesis in the liver which may be reflected in increasing body
weight. this steroid saponin converted in the body to corticosteroids which caused hypertrophy of pancreas and
stimulate insulin secretion. in addition in vivo administration of dry olive leaf extract significantly reduced
clinical signs of t1dm (hyperglycemia and body weight loss) and led to complete suppression of histological
changes in pancreatic islets (cvjeticanin et al.,2009).this property of antidiabetic attributed to oleorupein and
oleonolic acid (sato et al., 2007).
there was non significant and significant decrease in fbg level in the normal and three diabetic groups
treated for different periods respectively. this reflects the efficiency of this mixture in the treatment of
hyperglycemia and it can revert fbg to normal value in diabetic group treated for 75 days. the significant
effect of this mixture on hyperglycemia may be due to its effect in the enhancement of insulin sensitivity and
increase cellular uptake then decreasing it´s level in plasma or decreasing hepatic glucose protection. this
hypoglycemic and normoinsulinimic activity of mixture may be due to the ability of hypoglycemic compound
in the mixture to act synergistically to improve diabetic status. the mixture may enhance and increase the
release of endogenous insulin from pancreatic ?-cells and induce regeneration of pancreas. they also promote
and facilitate peripheral tissue uptake and utilization of glucose. alternatively, its action can be related to
possessing insulin-like action or glucagon inhibition, which increases in dm and stimulates gluconeogenesis in
liver, leading to enhancement of glucose metabolism which is impaired in dm.
also, the mixture contain compound that have antidiabetic/insulin mimetic effects as fenugreek seeds.
also, broca et al. (2000) reported that 4-hydroxyisoleucine (4-oh-ile), an amino acid extracted and purified
from fenugreek seeds, displays an in vitro insulinotropic activity and cause improvement of the diabetic state.
for instance, flavonoids are reported to regenerate the damaged pancreatic ?-cells in diabetic animals and have
been reported to activate ppars (saravanan and leelavinothan, 2006).
these results are consistent with previous studies who reported that the administration of trigonella
foenum-graecum seeds powder to diabetic animals has been shown to lower blood glucose levels and partially
restore the activities of key enzymes of carbohydrates and lipid metabolism to near normal levels in various
animal models (vats et al. 2003).
however, earlier studies have shown the presence of saponin compounds diasgenin, alkaloids and
trigonelline – inhibit intestinal glucose uptake in vitro (al-habori et al. 2001). the treatment with trigonella
seeds powder (tsp) and vanadate could increase glucose utilization and reduce glycosylation of proteins, ros
formation and lipid peroxidation by controlling hyperglycaemia (baqure et al., 2011). in addition, inhibition in
renal glucose reabsorption by fraxinus excelsior was reported (donga et al., 2011).
different mixture phytoconstituents had proven to reduce fbg as oleuropein extracted from olive
leaves (omar, 2010) and [6]-gingerol of the ginger rhizome (ali et al.,2008), flavanoids, , saponins,
polyphenolic compounds, tannins, glycosides, alkaloids and terpenoids (tiwari and rao, 2002 baeshen et al.,
2010) had proven effective against diabetes. preliminary study showed the presence of different
phytoconstituents which may be beneficial for the anti-hyperglycemic activity of this mixture (al-joubori et al.,
2013).
in treatment groups, insulin levels returned near normal value, this result reflects the hypoglycemic
effect of the mixture suggesting that the hypoglycaemic effect may be mediated through potentiation of
pancreatic secretion of insulin from ?-cell of islets. another possible hypoglycaemic mechanism of the mixture
of plants extracts may increase the sensitivity of tissue to available insulin. this effect of mixture may be
maysaa ? adil hadi et al /international journal of chemtech research, 2016,9(6),pp 501-513. 511
because containing natural antioxidant compound which reduce ros formation in ?-cell induced by alloxan
and enhance the defense antioxidant mechanism against ros production in diabetes type 2.
in all treatment study groups, pancreas / body weight ratio significantly increased and the mixture can
reverted the pancreas / body weight ratio near normal value .this may be due to ability of the mixture to
enhancement the pancreas function as induction of b-cells repair or decreasing apoptosis. also, it acted to
reduce degenerative changes in histological structure of pancreas and induce regeneration of islets of
langerhans as shown in histopathological study. thus, in addition to fbg lowering effect, histopathological
observation also supports the idea that this mixture of plants extracts produces significant antihyperglycenic
activity by protecting pancreas and b-cell against alloxan action and enhance defence mechanisms. hence the
possible mechanism by which mixture of plants extracts brings about its antihyperglycemic action may be by
the stimulation of surviving ?-cells to release more insulin. this was clearly evidenced by the increased level of
insulin in diabetic rats treated with the mixture.
this ability of this mixture in regeneration pancreatic tissue may be due to the precense of n.sativa
which had a positive effect on the regenerative of langerhans islets and attenuates the damage to ?-cell of the
pancreas initially distorted by stz, was observed at the end of the experiment period (17 days) of treatment
with thymoquinone (25mg/kg bw/day) or neutral lipid fraction (100mg/kg bw/day) (widad et al., 2011). also,
there was increased number of ? cells in the islets of langerhans when treatment with crude nigella for 21 days
in adult male rats (khanam and dewan, 2008). in addition, fenugreek oil significantly improved blood glucose
levels and insulin, less pancreatic islet and ?-cells damage were observed after the administration of fenugreek
oil to diabetic rats and reveals the efficacy of fenugreek oil in the amelioration of diabetes, hematological status,
and renal toxicity which may be attributed to its immunomodulatory activity and insulin stimulation action
along with its antioxidant potential (hamden et al.,2010).
reducing inflammatory cells may be because the mixture contains many plants acts as antiinflammatory
as z. officinale (raji et al.,2002). zingiber officinale dried rhizomes ethanol extract and gingerols,
the most biologically active components of ginger, produces its antiinflammatory effect by inhibiting the
release, synthesis and/or production of inflammatory mediators, including polypeptide kinins, prostaglandins
and so forth, like diclofenac (jiang et al., 2006).
these enhancement in both glucose and insulin levels which reverted near normal levels are also seen
in histopathological study of pancreas which indicates protective effect of the mixture of plants extracts used in
this study on pancreatic tissues. this results in consistent with previous studies which reported that dm has
negative effect in male testis and epidydimis of alloxan-induced rats and may contribute in reduction of fertility
while the same mixture of plants extracts used in this study can reduce most degerenative changes occurred in
testes and epidydimis in diabetic groups(10). also, cytogenetic study showed significant increasing (p<0.05) in
both mitotic index and chromosomes aberrations in diabetic group while treatment with the same mixture of
plants extracts significantly reduced mitotic index and chromosomes aberrations in all treatment groups and for
different period of study (45, 60 and 75 days)( al-joubori et al.,2014)..
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