Biomedicine & Pharmacotherapy 108 (2018) 1015–1021

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Biomedicine & Pharmacotherapy

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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.

Diabetes mellitus is a group of metabolic syndrome characterized by
fasting hyperglycemia, postprandial hyperglycemia and hyperlipi-
demia, resulting from defects in carbohydrate, fat and protein meta-
bolism [1–3]. It is recognized as the wide-reaching chronic disorder
affecting almost people of all age groups. According to a report of IDF
2017, there are 422 million people experiencing diabetes in the world
and this figure is expected to rise 629 million in 2045 [4]. Therefore,
the management of diabetes mellitus is considered as a global problem
Nowadays, several synthetic drugs with anti-diabetic effects in-
cluding oral synthetic hypoglycemic agents like sulfonylureas group,
insulin treatment given parenterally and specific enzyme inhibitors
such as acarbose, miglitol are used for patients. However, these drugs
are expensive and commonly associated with side-effects and draw-
backs like insulin resistance, anorexia nervosa, brain atrophy, hepato-
toxicity, abdominal pain, and flatulence, which limit their applications
[5–7]. Besides these treatments, a couple of scientific data have in-
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.
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
century is switching towards naturopathy especially in developing
countries where resources are in scanty and the cost of conventional
medicines is a burden to the population [9,10].
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
like analgesic, antioxidant, antimicrobial, antidiarrhoeal and hepato-
protective have been reported from different parts of P. cineraria
[11–15].
In our previous investigations, we 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. Their chemical
structures, including absolute configurations were established on the
basis of detailed physical data analysis methods viz. IR, 1H NMR, 13C
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
but still the mode of action by which it mediates antidiabetic effects is
not clearly defined [17]. Therefore, the present study was designed to
assess the in vitro and in vivo antidiabetic activity of chloroform fraction
of P. cineraria (stem bark) in streptozotocin (STZ) induced experimental
diabetes and their possible mode of action.
2. Materials and methods
2.1. Chemicals
Porcine pancreatic alpha-amylase (EC 3.2.1.1) was purchased from
Sigma-Aldrich, USA. Streptozotocin and glibenclamide were procured
from Himedia Laboratories, Mumbai, India while acarbose tablet was
1016
Biomedicine & Pharmacotherapy 108 (2018) 1015–1021
purchased from local market, manufactured by BAL Pharm Ltd, India.
The estimation of biochemical parameters was carried out using kits
(Accurex Biomedical Pvt. Ltd. Mumbai, India). Other chemicals and
reagents used in the study were analytical grade.
2.2. Plant material
The stem barks of P. cineraria (L.) were collected from the locality of
Bagru, Jaipur, Rajasthan, India. The material was confirmed tax-
onomically by Mr. Vinod Kumar Sharma and a voucher specimen
(RUBL 211353) has been deposited at the Department of Botany,
University of Rajasthan, Jaipur, India for future reference.
2.3. Extraction and fractionation
The stem barks of P. cineraria were shade dried and powdered in a
mechanical grinder. The powdered material (3 kg) was extracted in 10 L
methanol (MeOH) using a Soxhlet apparatus at an ambient temperature
for 72 h. Later, the extract was filtrated under vacuum, concentrated in
a rotary evaporator and then lyophilized to yield 300 g solid brown
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
fractionation by chromatographed over silica gel (60–120 mesh,
6.5 cm × 120 cm),) with chloroform as a mobile phase solvent. The
chloroform fraction was filtered through Whatman No. 1 filter paper.
The fractions were evaporated in rotavapor stored at −20 °C for further
use.
L.K. Soni et al.
2.4. In vivo antidiabetic activity
2.4.1. Animals
Thirty, colony-bred, adult, male albino rats of Wistar strain
(190 ± 10 g) were used for the antidiabetic study. They were main-
tained under standard conditions of humidity (55 ± 5%), temperature
(25 ± 3 °C), and light (12-h light: 12-h dark cycle). The animals were
fed with conventional diets (commercial pelleted diet procured from
Aashirwad Industries, Chandigarh, India) and water ad libitum.
2.4.2. Dose preparation
The chloroform fraction of P. cineraria (CfPc) was concentrated in
vacuo using a rotary flash evaporator and dried in a desiccator. The
material that remained after successive solvent extraction was con-
sidered as a residual fraction. CfPc was suspended in 0.5% DMSO in
distilled water for the assessment of Antidiabetic activity.
2.4.3. Acute toxicity studies
The acute toxicity study of CfPc was performed as per guidelines of
the OECD-423 received from the Committee for the Purpose of Control
and Supervision of Experiments on Animals (CPCSEA). The CfPc was
administrated to rats by gavage using a stomach tube in increasing dose
levels of 500, 1000 and 2000 mg/kg b.wt, respectively [18]. All animals
were observed for gross behavioral, neurological, autonomic and toxic
effects at short intervals of time for 5 h after administration and then for
next 24 h. Food consumption and body weights were recorded daily for
7 days. On the 7th day, the animals were sacrificed and all the organs
were removed for gross pathological examination [19].
2.4.4. Induction of diabetes
Diabetes was induced by a single intraperitoneal injection of STZ
(55 mg/kg b.wt), freshly prepared in 0.1 M sodium citrate buffer (pH
4.5) after overnight fasting [20]. Animals were fed with 5% glucose
solution for 12 h to avoid hypoglycaemia. On the 4th day of STZ ad-
ministration, blood glucose level was measured through Glucometer
(one touch, Johnson & Johnson) and the rats with moderate diabetes,
having hyperglycemia (blood glucose range of above 250 mg/dl) were
considered as diabetic and were employed in the study.
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
(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
b.wt, respectively, Group 5: Positive control received a reference
standard drug Glibenclamide (5 mg/kg b.wt). All treatments were given
orally after the 4th day of STZ administration (except normal control)
for 21 days. The body weight was recorded initially and after the end of
treatment whereas blood glucose level was measured by Glucometer
(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-
ture and allowed to clot at room temperature, further centrifuged at
4 °C at 3000 rpm for separation of serum. The animals were sacrificed
using mild anesthesia and liver was dissected, rinsed with ice cold
saline and stored at −20 °C for further studies.
2.4.6. Estimation of blood glucose and glycosylated hemoglobin (HbA1c)
After the completion of treatment, the rats were fasted overnight
and blood samples for fasting blood glucose and glycosylated he-
moglobin (HbA1c) were obtained from the tail vein under mild ether
anesthesia. Fasting blood glucose was measured by Glucometer (one
touch, Johnson & Johnson) and glycosylated hemoglobin (HbA1c) was
estimated using the method of Nayak and Pattabiraman [21].
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Biomedicine & Pharmacotherapy 108 (2018) 1015–1021
2.4.7. Estimation of Serum lipid profile and insulin
Serum lipid profile markers like total cholesterol (TC) triglycerides
(TG), LDL-C and HDL-C were determined spectrophotometrically using
commercially available kits (Accurex Biomedical Pvt. Ltd. Mumbai,
India). The concentrations of serum insulin were assessed according to
the standard protocol using radioimmunoassay (RIA).
2.4.8. Estimation of glycogen content in liver
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
tissue was homogenized in 1.5 mL of 5% potassium hydroxide and
boiled for 30 min. Later, 5 mL of 95% ethanol was added to precipitate
glycogen. After precipitation, test tubes were centrifuged and the su-
pernatant decanted. Finally, the pellet was dissolved in anthrone re-
agent and change in green color was measured at 620 nm.
2.5. In vitro α-amylase inhibition assay
The α-amylase inhibitory activity was determined by using soluble
starch as a substrate in a colorimetric reaction by the method of
Bernfield [23]. The assay mixture containing 500 μl of 0.02 M sodium
phosphate buffer (pH 6.9 with 0.006 M sodium chloride), containing
25 μl alpha-amylase solution and CfPc in various concentrations (20,
40, 60 and 80 μg/ml in DMSO) were incubated at 37 °C for 10 min.,
After pre-incubation, 500 μl of a (1% w/v) starch solution in 0.02 M
phosphate buffer (pH 6.9 with 0.006 M sodium chloride) was added to
each tube and further incubated at 37 °C for 10 min at timed intervals.
The reaction was stopped by adding 1 mL of DNS color reagent (12.0 g
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
heated in a boiling water bath for 5 min. After 15 min, the reaction
mixture was removed from the water bath and cooled thereafter, di-
luted with 9 mL distilled water and the absorbance value determined at
540 nm using spectrometer (Systronics digital type 105). Control in-
cubations, representing 100% enzyme activity were conducted in an
identical fashion replacing CfPc with DMSO. For blank incubations (to
allow for absorbance produced by the CfPc), the enzyme solution was
replaced with distilled water and the same procedure was carried out as
above. The % inhibition was calculated according to the formula
Inhibition (%) = [(A540Control − A540-Sample) / A540Control] × 100%.
2.6. Ethical aspects
The ethical committee, Center for Advanced Studies, Department of
Zoology, University of Rajasthan, Jaipur (India), approved the study.
The Indian National Sciences Academy, New Delhi, guidelines were
followed for the maintenance of animals during the experiment.
2.7. Statistical analysis
Statistical analysis was performed using SPSS, version 21.0 (SPSS,
Chicago, IL). All the results were expressed as mean ± SEM for six rats
in each group, and statistical analysis was performed by one-way ana-
lysis of variance (ANOVA) followed by the Duncan’s multiple compar-
ison test. The values of p < 0.05 were considered as statistically sig-
nificant.
3. Results
3.1. Acute toxicity
In acute toxicity study, oral administration of CfPc at the dose of
500, 1000 and 2000 mg/kg b.wt did not induce any changes in
L.K. Soni et al. Biomedicine & Pharmacotherapy 108 (2018) 1015–1021

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).

1018
L.K. Soni et al.
Fig. 4. Effect of CfPc and glibenclamide on Serum insulin in STZ-induced dia-
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.
3.6. Effect of CfPc on serum lipid profile
The Antidiabetic effect of CfPc was observed through determination
of lipid profile in serum. We observed that STZ-induced diabetic rats
displayed a significant increase in the levels of TC, TG and LDL-C and a
significant decrease in the level HDL-C when compared to normal
control rats. Administration of CfPc brought back the levels of serum
lipids to near normal values, signified the hypolipidemic activity of CfPc
(Fig. 5).
3.7. Effect of CfPc on liver glycogen content
Table 1 presents the effect of CfPc on liver glycogen content in
normal and treated rats. Observation showed that there was a massively
decreased (65.31%) in the liver glycogen content of diabetic rats when
compared to normal control rats. After administration of CfPc to dia-
betic rats for 21 days, liver glycogen content increased significantly
(P < 0.05).
3.8. In vitro α-amylase inhibition assay
In the present study, CfPc showed a significant inhibition of α-
amylase enzyme activity in a concentration dependent manner. CfPc at
the concentrations 20, 40, 60, 80 and 100 μg/ml showed 33.14, 56.17,
61.94, 73.28 and 84.09% inhibition of α-amylase enzyme activity, re-
spectively with an IC50 value 40.29 μg/ml. The acarbose used as a re-
ference standard at the same concentrations showed 39.46, 61.22,
Fig. 5. Effect of CfPc and glibenclamide on Serum lipid profile 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
groups: bp < 0.05.
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Biomedicine & Pharmacotherapy 108 (2018) 1015–1021
Fig. 6. Effect of CfPc and acarbose in the in vitro α-amylase inhibition model.
69.63, 81.91 and 88.46% inhibition of α-amylase activity with an IC50
value 29.43 μg/ml (Fig. 6)
4. Discussion
The associated drawbacks with insulin and oral hypoglycemic
agents have led to stimulation in the research for locating natural re-
sources showing antidiabetic activity. Therefore there is a need to find
safer and more effective antidiabetic drugs in the form of the herbal
based regimen. A survey of the literature reveals that not much scien-
tific evaluation has been conducted to check the antidiabetic potential
of P. cineraria. In our previous studies, we had isolated two new com-
pounds (Methyl 5-tridecyloctadec-4-enoate and Nonacosan-8-one), to-
gether with three known compounds (Lupeol, β-sitosterol and
Stigmasterol.) from CfPc. Their chemical structures, including absolute
configurations were established on the basis of detailed physical data
analysis methods viz. IR, 1H NMR, 13C NMR and Mass spectra [16].
Therefore, CfPc was used in this study to evaluate the in vitro and in vivo
antidiabetic activity in STZ induced diabetic rats.
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-
perlipidemic conditions [24]. The mechanism by which streptozotocin
brings about its diabetic state includes generation of nitric oxide and
free radicals which impair the mitochondrial function of beta cells [25].
In our study, oral administration of CfPc lowered the increased blood
glucose level in STZ-induced diabetic rats. The possible mechanism
behind hypoglycemic activity of CfPc might be due to promoting glu-
cose uptake metabolism by inhibiting hepatic gluconeogenesis in adi-
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].
The reduced serum insulin level in the STZ diabetic rats was due to
destruction of the pancreatic beta cells as observed in earlier studies
[31,32]. CfPc treatment caused a marked increase of serum insulin in
STZ diabetic rats and it might have either by stimulating the remaining
L.K. Soni et al.
beta cells to secrete insulin or by regeneration of pancreatic beta cells.
Our results are similar to those reported by previous researcher who
reported the insulin releasing activity of plant extracts by above pos-
sible mechanisms [33,34].
The synthesis of hepatic glycogen content is reduced during diabetes
[35], and it might be due to lack of insulin in the diabetic state which
results in the inactivation of glycogen synthase systems. The reduction
in glycogen synthesis in diabetes has been also observed in earlier
studies [36]. The significant increase in the glycogen content of CfPc
treated rats indicating that the drug stimulates the activity of glycogen
synthase enzyme by enhancing insulin production from beta cells.
Dyslipidemia is a secondary complication accompanied by long
term effect of diabetes and had been discussed many times during the
past decades [37,38]. During diabetes, the levels of TC, TG and LDL-C
increase and the HDL-C levels decline significantly [39]. In the diabetic
model, abnormal concentrations of serum lipids might be due to a
disturbance in hormone sensitive enzyme, lipase. Since insulin defi-
ciency causes its increase concentration that allows the mobilization of
free fatty acid from peripheral fat repository [40]. The administration
of CfPc significantly restored TC, TG, LDL-C and HDL-C levels to their
normal values in diabetic rats and it might be due to its lipid lowering
activity [41]. Therefore, CfPc also plays a significant role against dia-
betes associated complications.
The treatment goal of the diabetic subject is to maintain near
normal levels of glycemic control in post-prandial conditions. Many
natural sources have been investigated with respect to suppression of
glucose production from the carbohydrates in the gut [42]. Alpha-
amylase catalyses the hydrolysis of alpha-1,4-glycosidic linkages of
starch, glycogen and various oligosaccharides. The inhibition of their
activity in the digestive tract of humans is considered to be an effective
tool to control diabetes. In addition, these effects may lead to dimin-
ished absorption of monosaccharides [43]. Therefore, effective and
non-toxic inhibitors of alpha-amylase have long been sought. Our in
vitro study showed an appreciable inhibition of alpha-amylase enzyme
catalytic activity against starch as a substrate.
Literature review revealed that Lupeol and β-sitosterol dramatic
lowered blood glucose level in STZ-induce diabetic model via stimula-
tion of pancreatic regeneration [44,45]. On the other hand, Stigmas-
terol also improves antioxidant status and insulin level, reported by
Panda et al. [46]. Therefore, the presence of these compounds in our
drug may be attributed for its antidiabetic property.
5. Conclusion
In conclusion, the present study provides an evidence of the anti-
diabetic activity of CfPc in STZ-induced diabetic rats. The possible
mechanisms of its antidiabetic action include inhibition of carbohy-
drate hydrolytic enzymes, enhancement of glycogen regulatory en-
zymes expression in the liver and glucose uptake by tissues and adi-
pocytes as well as stimulation of pancreatic insulin release. In addition,
CfPc treatment also showed an improvement in lipid profile of diabetic
rats which signifies its protective action against dyslipidemic compli-
cation occurs in diabetes. It is also inferred from our previous studies
that CfPc possesses various phytocompounds that may be responsible
for its antidiabetic property. Therefore, it can be concluded that the
CfPc might be used safely as an adjunct therapy in the management of
diabetes and its associated complications. Furthermore, comprehensive
pharmacological investigations are needed to elucidate the exact me-
chanism of action of the antidiabetic effect.
Conflict of interest
The authors declare that there are no conflicts of interest.
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Biomedicine & Pharmacotherapy 108 (2018) 1015–1021
Acknowledgments
Mr. Lokesh Kumar Soni, Mr. Dharmendra Arya and Miss. Kiran
Bhagour are thankful to Council of Scientific and Industrial Research
(CSIR), New Delhi, India and University Grants Commission, New
Delhi, India, for providing senior research fellowship. Authors are also
grateful to the Head and Coordinator (CAS), Department of Zoology,
University of Rajasthan, Jaipur (India), for providing the necessary
facilities.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.biopha.2018.09.099.
References
[1] American Diabetes Association (ADA), Diagnosis and classification of diabetes
mellitus, Diabetes Care 37 (2014) S81–S90.
[2] M.R. Taskinen, Diabetic dyslipidemia, Atheroscler. Suppl. 3 (2002) 47–51.
[3] A. Ceriello, Postprandial hyperglycemia and diabetes complications, Diabetes 54
(2005) 1–7.
[4] N.H. Cho, J.E. Shaw, S. Karuranga, Y. Huang, J.D. da Rocha Fernandes,
A.W. Ohlrogge, B. Malanda. IDF Diabetes Atlas: global estimates of diabetes pre-
valence for 2017 and projections for 2045, Diabetes Res. Clin. Pract. 1 (2018)
271–281.
[5] G. Piedrola, E. Novo, F. Escobar, R. Garcia-Robles, White blood cell count and in-
sulin resistance in patients with coronary artery disease, Ann. Endocrinol. 62 (2001)
7–10.
[6] J.A. Yaryura‐Tobias, A. Pinto, F. Neziroglu, Anorexia nervosa, diabetes mellitus,
brain atrophy, and fatty liver, Int. J. Eat. Disord. 30 (2001) 350–353.
[7] R.F. Coniff, J.A. Shapiro, T.B. Seaton, G.A. Bray, Multicenter, placebo-controlled
trial comparing acarbose (BAY g 5421) with placebo, tolbutamide, and tolbuta-
mide-plus-acarbose in non-insulin-dependent diabetes mellitus, Am. J. Med. 98
(1995) 443–451.
[8] G. Philomena, Concerns regarding the safety and toxicity of medicinal plants - an
overview, J. Appl. Pharm. Sci. 1 (2011) 40–44.
[9] C. Bodeker, G.Bodeker C.K. Ong, C.K. Grundy, G. Burford, K. Shein, WHO Global
Atlas of Traditional, Complementary and Alternative Medicine, World Health
Organization, Geneva, Switzerland, 2005.
[10] M. Ekor, The growing use of herbal medicines: issues relating to adverse reactions
and challenges in monitoring safety, Front. Pharmacol. 4 (2014) 177.
[11] S.T. Khan, N. Riaz, N. Afza, A. Nelofar, A. Malik, E. Ahmed, S. Hussain, Studies on
the chemical constituents of Prosopis cineraria, J. Chem. Soc. Pakistan 28 (2006)
619–622.
[12] V. Velmurugan, G. Arunachalam, V. Ravichandran, Anthelmintic potential of
Prosopis cineraria (Linn.) druce stem barks, Asian J. Plant Sci. Res. 1 (2011) 88–91.
[13] S. Sumathi, B. Dharani, J. Sivaprabha, K.S. Raj, P.R. Padma, Cell death induced by
methanolic extract of Prosopsis cineraria leaves in MCF-7 breast cancer cell line, Int.
J. Pharm. Sci. Invent. 2 (2013) 21–26.
[14] N.D. Naik, R. Malothu, R.G. Reddy, B.C. Naadella, P. Jayasri, A. Elumalai,
Evaluation of in-vivo anti-diarrhoeal activity of Prosopis Cineraria linn stem bark,
Int. J. Biol. Pharm. Res. 3 (2012) 317–319.
[15] V. Velmurugan, G. Arunachalam, Hepatoprotective activity of methanol extract of
stem bark of Prosopis cineraria Linn against carbon tetrachloride induced hepato-
toxicity, Int. J. Pharm. Pharm. Sci. 6 (2014) 491–503.
[16] L.K. Soni, A. Basak, P. Parasher, M.P. Dobhal, Isolation and structure elucidation of
two new compounds from stem bark of Prosopis cineraria, Chem. Sci. Rev. Lett. 4
(2015) 777–782.
[17] D. Sharma, Y.P. Singla, Evaluation of antihyperglycemic and antihyperlipidemic
activity of Prosopis cineraria (Linn.) in wistar rats, J. Sci. Innov. Res. 2 (2013)
751–758.
[18] M.N. Gosh, Toxicity studies, Fundamentals of Experimental Pharmacology,
Scientific Book Agency, Calcutta, 1984, pp. 153–158.
[19] R.A. Turner, Screening Method in Pharmacology, Academic Press, New York, 1965,
pp. 22–41.
[20] A. Gajdosik, A. Gajdosikova, M. Stefek, J. Navarova, R. Hozova, Streptozotocin-
induced experimental diabetes in male Wistar rats, Gen. Physiol. Biophys. 18
(1999) 54–62.
[21] S.S. Nayak, T.N. Pattabiraman, A new colorimetric method for the estimation of
glycosylated hemoglobin, Clin. Chim. Acta 109 (1981) 267–274.
[22] N.V. Carroll, R.W. Longley, J.H. Roe, The determination of glycogen in liver and
muscle by use of anthrone reagent, J. Biol. Chem. 220 (1956) 583–593.
[23] P. Bernfeld, Amylases, alpha and beta, in: S.P. Colowick, N.O. Kaplan (Eds.),
Methods in Enzymology, 1 Academic Press, New York, 1955, pp. 149–158.
[24] A. Akbarzadeh, D. Norouzian, M.R. Mehrabi, S.H. Jamshidi, A. Farhangi,
A.A. Verdi, B.L. Rad, Induction of diabetes by streptozotocin in rats, Indian J. Clin.
Biochem. 22 (2007) 60–64.
[25] N. Rakieten, M.N. Rakieten, M.V. Nadkarni, Studies on the diabetogenic action of
streptozotocin (NSC-37917), Cancer Chemother. Rep. 29 (1963) 91–98.
L.K. Soni et al.
[26] L. Ali, A.K. Azad Khan, M.I.R. Mamun, M. Mosihuzzaman, N. Nahar, M.N.E. Alan,
B. Rokeya, Studies on the hypoglycemic effects of fruits pulp, seed and whole plant
of Momordica charantia on normal and diabetic model rats, Planta Med. 59 (1993)
408–412.
[27] A.M. Gray, Y.H.A. Abdel-Wahab, P.R. Flatt, The traditional plant treatment, Sabucus
nigra (Elder), exhibits insulin-like and insulin releasing actions in vitro, J. Nutr. 130
(2000) 15–20.
[28] M.T. Pepato, R.H. Migliorini, A.L. Goldberg, I.C. Kettelhut, Role of different pro-
teolytic pathways in degradation of muscle protein from streptozotocin-diabetic
rats, Am. J. Physiol. Endocrinol. Metab. 271 (1996) E340–347.
[29] M.J. Sampson, D.A. Hughes, M.J. Carrier, I.R. Davies, Status of HbA1c during acute
hyperglycemia in type 2 diabetes, Diabetes Care 25 (2002) 537–541.
[30] K. Baskaran, B.K. Ahamath, K.R. Shanmugasundaram, E.R.B. Shanmugasundaram,
Antidiabetic effect of a leaf extract from Gymnema sylvestre in non-insulin-depen-
dent diabetes mellitus patients, J. Ethnopharmacol. 30 (1990) 295–305.
[31] S. Kurup, R.R. Bhonde, Combined effect of nicotinamide and streptozotocin on
diabetic status in partially pancreatectomized adult BALB/c mice, Horm. Metab.
Res. 32 (2000) 330–334.
[32] R. Chandran, T. Parimelazhagan, S. Shanmugam, S. Thankarajan, Antidiabetic ac-
tivity of Syzygium calophyllifolium in Streptozotocin-Nicotinamide induced Type-2
diabetic rats, Biomed. Pharmacother. 82 (2016) 547–554.
[33] J. Eliza, P. Daisy, S. Ignachimuthu, V. Duraipandiyan, Antidiabetic and antilipi-
demic effect of eremanthin from Costus speciosus (Koen.)Sm., in STZ-induced dia-
betic rats, Chem. Biol. Interact. 1 (2009) 67–72.
[34] D.B. Asante, E. Effah-Yeboah, P. Barnes, H.A. Abban, E.O. Ameyaw, J.N. Boampong,
E.G. Ofori, J.B. Dadzie, Antidiabetic effect of young and old ethanolic leaf extracts
of i: a comparative study, J. Diabetes Res. 2016 (2016) 1–13.
[35] L. Rossetti, A. Giaccari, Relative contribution of glycogen synthesis and glycolysis to
insulin-mediated glucose uptake. A dose-response euglycemic clamp study in
1021
Biomedicine & Pharmacotherapy 108 (2018) 1015–1021
normal and diabetic rats, J. Clin. Invest. 85 (1990) 1785–1792.
[36] P.D. Whitton, D.A. Hems, Glycogen synthesis in the perfused liver of streptozotocin
diabetic rats, Biochem. J. 150 (1975) 153–165.
[37] Y. Zhang, X. Li, D. Zou, W. Liu, J. Yang, N. Zhu, L. Huo, M. Wang, J. Hong, P. Wu,
G. Ren, Treatment of type 2 diabetes and dyslipidemia with the natural plant al-
kaloid berberine, J. Clin. Endocrinol. Metab. 93 (2008) 2559–2565.
[38] A.D. Mooradian, Dyslipidemia in type 2 diabetes mellitus, Nat. Clin. Pract.
Endocrinol. Metab. 5 (2009) 150–159.
[39] K. Arvind, R. Pradeepa, R. Deepa, V. Mohan, Diabetes and coronary artery disease,
Indian J. Med. Res. 116 (2002) 163–176.
[40] J.D. Schofield, Y. Liu, P. Rao-Balakrishna, R.A. Malik, H. Soran, Diabetes dyslipi-
demia, Diabetes Ther. 7 (2016) 203–219.
[41] P.G. Jain, S.J. Surana, Hypolipidemic activity of Prosopis cineraria L (Druce) fruit
extract and molecular modeling study with farnesoid X receptor (FXR), Trop. J.
Pharm. Res. 14 (2015) 1621–1628.
[42] T. Matsui, T. Tanaka, S. Tamura, A. Toshima, Tamaya K, Y. Miyata, K. Matsumoto,
α-Glucosidase inhibitory profile of catechins and theaflavins, J. Agric. Food Chem.
55 (2007) 99–105.
[43] Y. Hara, M. Honda, The inhibition of α-amylase by tea polyphenols, Agric. Biol.
Chem. 54 (1990) 1939–1945.
[44] R. Gupta, A.K. Sharma, M.P. Dobhal, M.C. Sharma, R.S. Gupta, Antidiabetic and
antioxidant potential of β‐sitosterol in streptozotocin‐induced experimental hy-
perglycemia, J. Diabet. 3 (2011) 29–37.
[45] R. Gupta, A.K. Sharma, M.C. Sharma, M.P. Dobhal, R.S. Gupta, Evaluation of an-
tidiabetic and antioxidant potential of lupeol in experimental hyperglycaemia, Nat.
Prod. Res. 26 (2012) 1125–1129.
[46] S. Panda, M. Jafri, A. Kar, B.K. Meheta, Thyroid inhibitory, antiperoxidative and
hypoglycemic effects of stigmasterol isolated from Butea monosperma, Fitoterapia
80 (2009) 123–126.