Biomedicine & Pharmacotherapy 108 (2018) 1015–1021

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Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

In vitro and in vivo antidiabetic activity of isolated fraction of Prosopis T

cineraria against streptozotocin-induced experimental diabetes: A
mechanistic study
Lokesh Kumar Sonia, Mahabeer Prasad Dobhala, Dharmendra Aryab, Kiran Bhagourb,
⁎
Pradeep Parasherc, R.S. Guptab,
a
Natural Products Laboratory, Department of Chemistry, University of Rajasthan, Jaipur, Rajasthan, 302004, India
b
Reproductive Physiology and Endocrinology Section, Department of Zoology, University of Rajasthan, Jaipur, Rajasthan, 302004, India
c
Department of Chemistry, Govt. P.G. College, Jhalawar, Rajasthan, 326001, India

A R T I C LE I N FO A B S T R A C T

Keywords: A rapidly increasing incidence of Diabetes mellitus throughout the world is a major concern in both developed
Prosopis cineraria and developing countries and the drawbacks associated with currently available treatments led to switching
Streptozotocin researcher’s attention towards naturopathy. Since ancient time, herbal plants have been traditionally used for
Antidiabetic the treatment of diabetes as they consider to be less toxic and free from side effects than synthetic ones. In our
Alpha-amylase
previous studies, we had isolated two new compounds (Methyl 5-tridecyloctadec-4-enoate and Nonacosan-8-
one), together with three known compounds (Lupeol, β-sitosterol and Stigmasterol) from chloroform fraction of
stem bark of P. cineraria (CfPc). The present study aimed to determine the in vivo and in vivo antidiabetic activity
of CfPc in streptozotocin induced experimental diabetes and also evaluated their possible mode of action. CfPc
was orally administrated to STZ (55 mg/kg b.wt) induced diabetic rats at the doses of 50 and 100 mg/kg b.wt for
21 days. Treatment of CfPc significantly (p < 0.05) lowered the level of blood glucose, glycosylated hemoglobin
and also restored body weight, liver glycogen content and serum insulin level in diabetic rats in a dose-de-
pendent manner. A significant (p < 0.05) reduction in serum lipid profile markers and elevation in HDL-C after
treatment with CfPc, also signifying the protective effects of CfPc in diabetes-associated complications. In ad-
dition, CfPc also promoted a significant inhibition of α-amylase enzyme activity with an IC50 value of 40.29 μg/
ml. Results indicate that CfPc possess a potential in vitro and in vivo antidiabetic activity and this effect could be
due to multitarget mode of action that includes antihyperglycemic, postprandial hypoglycemic, hypolipidemic
and insulin secretory actions. Therefore, it could be used as a safer complementary drug in the management of
diabetes and associated complications.

1. Introduction and successful treatment is yet to be discovered.

Nowadays, several synthetic drugs with anti-diabetic effects in-
Diabetes mellitus is a group of metabolic syndrome characterized by cluding oral synthetic hypoglycemic agents like sulfonylureas group,
fasting hyperglycemia, postprandial hyperglycemia and hyperlipi- insulin treatment given parenterally and specific enzyme inhibitors
demia, resulting from defects in carbohydrate, fat and protein meta- such as acarbose, miglitol are used for patients. However, these drugs
bolism [1–3]. It is recognized as the wide-reaching chronic disorder are expensive and commonly associated with side-effects and draw-
affecting almost people of all age groups. According to a report of IDF backs like insulin resistance, anorexia nervosa, brain atrophy, hepato-
2017, there are 422 million people experiencing diabetes in the world toxicity, abdominal pain, and flatulence, which limit their applications
and this figure is expected to rise 629 million in 2045 [4]. Therefore, [5–7]. Besides these treatments, a couple of scientific data have in-
the management of diabetes mellitus is considered as a global problem dicated that medicinal plant and their products possess antidiabetic

Abbreviations: CfPc, chloroform fraction of stem bark of P. cineraria; STZ, streptozotocin; b.wt, body weight; DMSO, dimethyl sulfoxide; HbA1C, glycosylated
hemoglobin; TC, total cholesterol; TG, triglyceride; LDL-C, low density lipoprotein cholesterol; HDL-C, high density lipoprotein cholesterol; DNS, 3,5-dinitrosalicylic
acid
⁎
Corresponding author.
E-mail address: gupta_rs@hotmail.com (R.S. Gupta).

https://doi.org/10.1016/j.biopha.2018.09.099
Received 21 May 2018; Received in revised form 15 September 2018; Accepted 18 September 2018
0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
L.K. Soni et al. Biomedicine & Pharmacotherapy 108 (2018) 1015–1021

Fig. 1. Structure of Compound A (Methyl 5-tridecyloctadec-4-enoate), Compound B (Nonacosan-8-one) and Compound C (Lupeol) isolated from chloroform fraction
of Prosopis cineraria.

properties with less toxicity and side-effects [8]. Therefore, the present purchased from local market, manufactured by BAL Pharm Ltd, India.
century is switching towards naturopathy especially in developing The estimation of biochemical parameters was carried out using kits
countries where resources are in scanty and the cost of conventional (Accurex Biomedical Pvt. Ltd. Mumbai, India). Other chemicals and
medicines is a burden to the population [9,10]. reagents used in the study were analytical grade.
Prosopis cinerarium (L.), commonly known as ‘Khejri’, is a flowering
plant species belonging to Fabaceae family, generally found in Western
Asia and the Indian subcontinent. Several pharmacological activities 2.2. Plant material
like analgesic, antioxidant, antimicrobial, antidiarrhoeal and hepato-
protective have been reported from different parts of P. cineraria The stem barks of P. cineraria (L.) were collected from the locality of
[11–15]. Bagru, Jaipur, Rajasthan, India. The material was confirmed tax-
In our previous investigations, we isolated two new compounds onomically by Mr. Vinod Kumar Sharma and a voucher specimen
(Methyl 5-tridecyloctadec-4-enoate and Nonacosan-8-one), together (RUBL 211353) has been deposited at the Department of Botany,
with three known compounds (Lupeol, β-sitosterol and Stigmasterol) University of Rajasthan, Jaipur, India for future reference.
from chloroform fraction of stem bark of P. cineraria. Their chemical
structures, including absolute configurations were established on the
basis of detailed physical data analysis methods viz. IR, 1H NMR, 13C 2.3. Extraction and fractionation
NMR and Mass spectra (Fig. 1.) [16]. To our best knowledge, only a few
studies have been reported on the antidiabetic activity of P. cineraria The stem barks of P. cineraria were shade dried and powdered in a
but still the mode of action by which it mediates antidiabetic effects is mechanical grinder. The powdered material (3 kg) was extracted in 10 L
not clearly defined [17]. Therefore, the present study was designed to methanol (MeOH) using a Soxhlet apparatus at an ambient temperature
assess the in vitro and in vivo antidiabetic activity of chloroform fraction for 72 h. Later, the extract was filtrated under vacuum, concentrated in
of P. cineraria (stem bark) in streptozotocin (STZ) induced experimental a rotary evaporator and then lyophilized to yield 300 g solid brown
diabetes and their possible mode of action. crude extract. Then, the obtained residue was fractionated by liquid-
liquid partition successively using petroleum ether (7 g), chloroform
(20 g) and ethyl acetate (32 g). Finally, the defatted residue was again
2. Materials and methods
fractionation by chromatographed over silica gel (60–120 mesh,
6.5 cm × 120 cm),) with chloroform as a mobile phase solvent. The
2.1. Chemicals
chloroform fraction was filtered through Whatman No. 1 filter paper.
The fractions were evaporated in rotavapor stored at −20 °C for further
Porcine pancreatic alpha-amylase (EC 3.2.1.1) was purchased from
use.
Sigma-Aldrich, USA. Streptozotocin and glibenclamide were procured
from Himedia Laboratories, Mumbai, India while acarbose tablet was

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L.K. Soni et al. Biomedicine & Pharmacotherapy 108 (2018) 1015–1021

2.4. In vivo antidiabetic activity 2.4.7. Estimation of Serum lipid profile and insulin
Serum lipid profile markers like total cholesterol (TC) triglycerides
2.4.1. Animals (TG), LDL-C and HDL-C were determined spectrophotometrically using
Thirty, colony-bred, adult, male albino rats of Wistar strain commercially available kits (Accurex Biomedical Pvt. Ltd. Mumbai,
(190 ± 10 g) were used for the antidiabetic study. They were main- India). The concentrations of serum insulin were assessed according to
tained under standard conditions of humidity (55 ± 5%), temperature the standard protocol using radioimmunoassay (RIA).
(25 ± 3 °C), and light (12-h light: 12-h dark cycle). The animals were
fed with conventional diets (commercial pelleted diet procured from 2.4.8. Estimation of glycogen content in liver
Aashirwad Industries, Chandigarh, India) and water ad libitum. Glycogen content was determined in the liver by anthrone reagent
method as adapted by Carrol et al. [22]. In brief, 500 mg of fresh liver
2.4.2. Dose preparation tissue was homogenized in 1.5 mL of 5% potassium hydroxide and
The chloroform fraction of P. cineraria (CfPc) was concentrated in boiled for 30 min. Later, 5 mL of 95% ethanol was added to precipitate
vacuo using a rotary flash evaporator and dried in a desiccator. The glycogen. After precipitation, test tubes were centrifuged and the su-
material that remained after successive solvent extraction was con- pernatant decanted. Finally, the pellet was dissolved in anthrone re-
sidered as a residual fraction. CfPc was suspended in 0.5% DMSO in agent and change in green color was measured at 620 nm.
distilled water for the assessment of Antidiabetic activity.
2.5. In vitro α-amylase inhibition assay
2.4.3. Acute toxicity studies
The α-amylase inhibitory activity was determined by using soluble
The acute toxicity study of CfPc was performed as per guidelines of
starch as a substrate in a colorimetric reaction by the method of
the OECD-423 received from the Committee for the Purpose of Control
Bernfield [23]. The assay mixture containing 500 μl of 0.02 M sodium
and Supervision of Experiments on Animals (CPCSEA). The CfPc was
phosphate buffer (pH 6.9 with 0.006 M sodium chloride), containing
administrated to rats by gavage using a stomach tube in increasing dose
25 μl alpha-amylase solution and CfPc in various concentrations (20,
levels of 500, 1000 and 2000 mg/kg b.wt, respectively [18]. All animals
40, 60 and 80 μg/ml in DMSO) were incubated at 37 °C for 10 min.,
were observed for gross behavioral, neurological, autonomic and toxic
After pre-incubation, 500 μl of a (1% w/v) starch solution in 0.02 M
effects at short intervals of time for 5 h after administration and then for
phosphate buffer (pH 6.9 with 0.006 M sodium chloride) was added to
next 24 h. Food consumption and body weights were recorded daily for
each tube and further incubated at 37 °C for 10 min at timed intervals.
7 days. On the 7th day, the animals were sacrificed and all the organs
The reaction was stopped by adding 1 mL of DNS color reagent (12.0 g
were removed for gross pathological examination [19].
of sodium potassium tartrate tetrahydrate in 8 mL of 2 M NaOH and
96 mM 3, 5- dinitrosalicylic acid solution) and the test tubes were then
2.4.4. Induction of diabetes heated in a boiling water bath for 5 min. After 15 min, the reaction
Diabetes was induced by a single intraperitoneal injection of STZ mixture was removed from the water bath and cooled thereafter, di-
(55 mg/kg b.wt), freshly prepared in 0.1 M sodium citrate buffer (pH luted with 9 mL distilled water and the absorbance value determined at
4.5) after overnight fasting [20]. Animals were fed with 5% glucose 540 nm using spectrometer (Systronics digital type 105). Control in-
solution for 12 h to avoid hypoglycaemia. On the 4th day of STZ ad- cubations, representing 100% enzyme activity were conducted in an
ministration, blood glucose level was measured through Glucometer identical fashion replacing CfPc with DMSO. For blank incubations (to
(one touch, Johnson & Johnson) and the rats with moderate diabetes, allow for absorbance produced by the CfPc), the enzyme solution was
having hyperglycemia (blood glucose range of above 250 mg/dl) were replaced with distilled water and the same procedure was carried out as
considered as diabetic and were employed in the study. above. The % inhibition was calculated according to the formula

Inhibition (%) = [(A540Control − A540-Sample) / A540Control] × 100%.

2.4.5. Experimental design
A total of 30 rats (6 normal; 24 STZ-diabetic rats) were assigned to
the study. The rats were randomly divided into five groups of six ani-
mals each. Group 1: Normal control rats (NC) received vehicle only 2.6. Ethical aspects
(0.5% DMSO; 2 ml/kg b.wt), Group 2: Diabetic control rats (DC), Group
3 and 4: Diabetic rats received CfPc at the dose of 50 and 100 mg/kg The ethical committee, Center for Advanced Studies, Department of
b.wt, respectively, Group 5: Positive control received a reference Zoology, University of Rajasthan, Jaipur (India), approved the study.
standard drug Glibenclamide (5 mg/kg b.wt). All treatments were given The Indian National Sciences Academy, New Delhi, guidelines were
orally after the 4th day of STZ administration (except normal control) followed for the maintenance of animals during the experiment.
for 21 days. The body weight was recorded initially and after the end of
treatment whereas blood glucose level was measured by Glucometer 2.7. Statistical analysis
(one touch, Johnson & Johnson) on 0, 7, 14 and 21 day of the study. On
the 21st day, the blood was collected from the heart via cardiac punc- Statistical analysis was performed using SPSS, version 21.0 (SPSS,
ture and allowed to clot at room temperature, further centrifuged at Chicago, IL). All the results were expressed as mean ± SEM for six rats
4 °C at 3000 rpm for separation of serum. The animals were sacrificed in each group, and statistical analysis was performed by one-way ana-
using mild anesthesia and liver was dissected, rinsed with ice cold lysis of variance (ANOVA) followed by the Duncan’s multiple compar-
saline and stored at −20 °C for further studies. ison test. The values of p < 0.05 were considered as statistically sig-
nificant.
2.4.6. Estimation of blood glucose and glycosylated hemoglobin (HbA1c)
After the completion of treatment, the rats were fasted overnight 3. Results
and blood samples for fasting blood glucose and glycosylated he-
moglobin (HbA1c) were obtained from the tail vein under mild ether 3.1. Acute toxicity
anesthesia. Fasting blood glucose was measured by Glucometer (one
touch, Johnson & Johnson) and glycosylated hemoglobin (HbA1c) was In acute toxicity study, oral administration of CfPc at the dose of
estimated using the method of Nayak and Pattabiraman [21]. 500, 1000 and 2000 mg/kg b.wt did not induce any changes in

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Table 1
Effect of CfPc and glibenclamide on body weight and liver glycogen content in STZ-induced diabetic rats.
Groups Body Weight (g) Liver glycogen (mg/g tissue)

Initial Final % Change in body weight

Group-I Normal Control 197 ± 5.30 201 ± 5.62 2.03 51.22 ± 6.77
Group-II 195 ± 4.56 158 ± 5.09a −18.97 17.61 ± 5.91a
Diabetic control
Group-III 198 ± 5.80 184 ± 4.77b 7.07 43.46 ± 5.68b
Diabetic + CfPc
(50 mg/kg b.wt)
Group-IV 193 ± 4.40 198 ± 5.10b 2.59 48.97 ± 6.01b
Diabetic + CfPc
(100 mg/kg b.wt)
Group-V 195 ± 4.90 197 ± 4.35b 1.025 47.11 ± 4.72b
Diabetic + Glibenclamide
(5 mg/kg b.wt)

Data represented as mean ± SEM (n = 6).

a
= (P < 0.05) statistically significant difference when compared with Normal control.
b
= (P < 0.05) statistically significant difference when compared with Diabetic control.

behavior and no mortality was observed during the study (Table S1).
There was no significant difference in the body weight and food con-
sumption when compared to the vehicle treated group (Fig. S1). On the
7th day, macroscopic pathology observations revealed no visible lesions
in any animals after sacrifice. The observed data suggested that the oral
LD50 value of CfPc is supposed to be greater than 2000 mg/kg b.wt.
Therefore, CfPc can be safely used as a dose up to 2000 mg/kg b.wt for
therapeutic use.

3.2. Effect of CfPc on body weight

Table 1 presents the effect of CfPc and glibenclamide on changes in

body weight. In diabetic control rats, there was a significant decrease
(18.97%) in final body weight when compared to normal control rats.
Treatment with CfPc showed a significant (P < 0.05) increased in body
weight of diabetic rats. Change in body weight was minimal for positive
control (1.02%) whereas CfPc treatments showed moderate improve-
ment in body weight (0.50–2.60%). Fig. 2. Effect of CfPc and glibenclamide on Blood glucose level in STZ-induced
diabetic rats. Data represented as mean ± SEM (n = 6). Statistically significant
differences were observed between NC to DC: ap < 0.05 and DC to drug treated
3.3. Effect of CfPc on blood glucose groups: bp < 0.05.

The blood glucose level was measured in normal and experimental

groups at 0 days, 7th day, 14th and 21st day of treatment. STZ ad-
ministration showed a significant increase (P < 0.05) in the blood
glucose level when compared to normal control group. There was a
dramatical reduction in blood glucose level from 277 mg/dL to 146 mg/
dL after 21 days of treatment with CfPc at the dose of 100 mg/kg b.wt,
signified the hypoglycemic potential of CfPc (Fig. 2).

3.4. Effect of CfPc on glycosylated hemoglobin (HbA1c)

Fig. 3 shows the effect of CfPc treatment and glibenclamide on

HbA1c level in normal and experimental rats. STZ treated rats showed a
significant elevation in HbA1c level (6.55%) as compared with normal
control. Following CfPc and glibenclamide administration to diabetic
rats caused a significant reduction (P < 0.05) in HbA1c level (∼4–6%)
as compared to diabetic control rats.

3.5. Effect of CfPc on insulin level

As depicted in Fig. 4, STZ administration cause a dramatical re- Fig. 3. Effect of CfPc and glibenclamide on Glycosylated hemogloblin (%) in
duction in serum insulin level (57.19%) in rats when compared with STZ-induced diabetic rats. Data represented as mean ± SEM (n = 6).
Statistically significant differences were observed between NC to DC: ap < 0.05
normal control rats. Oral administration of CfPc and glibenclamide
and DC to drug treated groups: bp < 0.05.
brought the serum insulin level back to the normal range (42.14% and
76.85% at the doses of 50 and 100 mg/kg b.wt, respectively).

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Fig. 4. Effect of CfPc and glibenclamide on Serum insulin in STZ-induced dia-

Fig. 6. Effect of CfPc and acarbose in the in vitro α-amylase inhibition model.
betic rats. Data represented as mean ± SEM (n = 6). Statistically significant
differences were observed between NC to DC: ap < 0.05 and DC to drug treated
groups: bp < 0.05. 69.63, 81.91 and 88.46% inhibition of α-amylase activity with an IC50
value 29.43 μg/ml (Fig. 6)
3.6. Effect of CfPc on serum lipid profile
4. Discussion
The Antidiabetic effect of CfPc was observed through determination
of lipid profile in serum. We observed that STZ-induced diabetic rats The associated drawbacks with insulin and oral hypoglycemic
displayed a significant increase in the levels of TC, TG and LDL-C and a agents have led to stimulation in the research for locating natural re-
significant decrease in the level HDL-C when compared to normal sources showing antidiabetic activity. Therefore there is a need to find
control rats. Administration of CfPc brought back the levels of serum safer and more effective antidiabetic drugs in the form of the herbal
lipids to near normal values, signified the hypolipidemic activity of CfPc based regimen. A survey of the literature reveals that not much scien-
(Fig. 5). tific evaluation has been conducted to check the antidiabetic potential
of P. cineraria. In our previous studies, we had isolated two new com-
3.7. Effect of CfPc on liver glycogen content pounds (Methyl 5-tridecyloctadec-4-enoate and Nonacosan-8-one), to-
gether with three known compounds (Lupeol, β-sitosterol and
Table 1 presents the effect of CfPc on liver glycogen content in Stigmasterol.) from CfPc. Their chemical structures, including absolute
normal and treated rats. Observation showed that there was a massively configurations were established on the basis of detailed physical data
decreased (65.31%) in the liver glycogen content of diabetic rats when analysis methods viz. IR, 1H NMR, 13C NMR and Mass spectra [16].
compared to normal control rats. After administration of CfPc to dia- Therefore, CfPc was used in this study to evaluate the in vitro and in vivo
betic rats for 21 days, liver glycogen content increased significantly antidiabetic activity in STZ induced diabetic rats.
(P < 0.05). The STZ induced diabetic model was used in the study since STZ
makes pancreas swell and at last causes degeneration in Langerhans
islet beta cells, leading to hyperglycemia, hypoinsulinemia and hy-
3.8. In vitro α-amylase inhibition assay perlipidemic conditions [24]. The mechanism by which streptozotocin
brings about its diabetic state includes generation of nitric oxide and
In the present study, CfPc showed a significant inhibition of α- free radicals which impair the mitochondrial function of beta cells [25].
amylase enzyme activity in a concentration dependent manner. CfPc at In our study, oral administration of CfPc lowered the increased blood
the concentrations 20, 40, 60, 80 and 100 μg/ml showed 33.14, 56.17, glucose level in STZ-induced diabetic rats. The possible mechanism
61.94, 73.28 and 84.09% inhibition of α-amylase enzyme activity, re- behind hypoglycemic activity of CfPc might be due to promoting glu-
spectively with an IC50 value 40.29 μg/ml. The acarbose used as a re- cose uptake metabolism by inhibiting hepatic gluconeogenesis in adi-
ference standard at the same concentrations showed 39.46, 61.22, pose tissues [26,27], through the stimulation of insulin secretion from
the remaining pancreatic beta cells.
In diabetic rats, reduction in body weight observed might be the
result of degradation of structural proteins and fats due to deficiency of
carbohydrate for the energy metabolism [28]. A significant increase in
body weight of diabetic rats treated with CfPc showed the blood glucose
stabilization effect which in turn prevents the loss of body weight.
STZ causes selectively destruction of pancreatic beta cells that leads
to generation of free radicals by oxidation of excessive glucose, the non-
enzymatic glycation of proteins and subsequent oxidative degradation
of glycation proteins, therefore, increases the HbA1c level [29]. Similar
observations have been also reported by previous researcher in STZ-
induced diabetic rats [30]. In our study, CfPc treatment significantly
reduced the HbA1c level in diabetic rats and it might be due to its
antioxidative activity [17].
Fig. 5. Effect of CfPc and glibenclamide on Serum lipid profile in STZ-induced The reduced serum insulin level in the STZ diabetic rats was due to
diabetic rats. Data represented as mean ± SEM (n = 6). Statistically significant destruction of the pancreatic beta cells as observed in earlier studies
differences were observed between NC to DC: ap < 0.05 and DC to drug treated [31,32]. CfPc treatment caused a marked increase of serum insulin in
groups: bp < 0.05. STZ diabetic rats and it might have either by stimulating the remaining

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beta cells to secrete insulin or by regeneration of pancreatic beta cells. Acknowledgments

Our results are similar to those reported by previous researcher who
reported the insulin releasing activity of plant extracts by above pos- Mr. Lokesh Kumar Soni, Mr. Dharmendra Arya and Miss. Kiran
sible mechanisms [33,34]. Bhagour are thankful to Council of Scientific and Industrial Research
The synthesis of hepatic glycogen content is reduced during diabetes (CSIR), New Delhi, India and University Grants Commission, New
[35], and it might be due to lack of insulin in the diabetic state which Delhi, India, for providing senior research fellowship. Authors are also
results in the inactivation of glycogen synthase systems. The reduction grateful to the Head and Coordinator (CAS), Department of Zoology,
in glycogen synthesis in diabetes has been also observed in earlier University of Rajasthan, Jaipur (India), for providing the necessary
studies [36]. The significant increase in the glycogen content of CfPc facilities.
treated rats indicating that the drug stimulates the activity of glycogen
synthase enzyme by enhancing insulin production from beta cells. Appendix A. Supplementary data
Dyslipidemia is a secondary complication accompanied by long
term effect of diabetes and had been discussed many times during the Supplementary material related to this article can be found, in the
past decades [37,38]. During diabetes, the levels of TC, TG and LDL-C online version, at doi:https://doi.org/10.1016/j.biopha.2018.09.099.
increase and the HDL-C levels decline significantly [39]. In the diabetic
model, abnormal concentrations of serum lipids might be due to a References
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