Journal of Ethnopharmacology 175 (2015) 229–240

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Journal of Ethnopharmacology
journal homepage: www.elsevier.com/locate/jep

Extract of Woodfordia fruticosa flowers ameliorates hyperglycemia,

oxidative stress and improves β-cell function in
streptozotocin–nicotinamide induced diabetic rats
Aditya Arya a,n, Mazen M. Jamil Al-Obaidi a, Rustini Binti Karim a, Hairin Taha c,
Ataul Karim Khan a, Nayiar Shahid a, Abu Sadat Sayem a, Chung Yeng Looi b,
Mohd Rais Mustafa b, Mustafa Ali Mohd b, Hapipah Mohd Ali c
a
Department of Pharmacy, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
b
Department of Pharmacology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia
c
Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia

art ic l e i nf o a b s t r a c t

Article history: Ethnopharmacological relevance: The art of Ayurveda and the traditional healing system in India have
Received 3 December 2014 reflected the ethnomedicinal importance of the plant Woodfordia fruticosa Kurtz, which demonstrates its
Received in revised form vast usage in the Ayurvedic preparations as well as in the management of diabetes by the traditional
7 August 2015
healers.
Accepted 31 August 2015
Aims of study: The study aimed to ascertain the antidiabetic potential of W. fruticosa flower methanolic
Available online 2 September 2015
extract (WF) on Streptozotocin (STZ)–nicotinamide-induced diabetic rat model.
Keywords: Materials and methods: Diabetes was induced in Sprague Dawley (SD) rats by STZ–nicotinamide and
Woodfordia fruticosa thereafter diabetic rats were treated with three different doses of WF (100, 200 and 400 mg/kg body
Antidiabetic
weight) respectively and glibenclamide as a positive control. Biochemical parameters such as blood
Antioxidant
glucose, serum insulin and C-peptide levels were measured with oxidative stress markers. Furthermore,
Insulin
Glycogen histology of liver and pancreas was carried out to evaluate glycogen content and β-cell structures.
Bcl-2 Moreover, immunohistochemistry and western blot analysis were performed on kidney and pancreas
tissues to determine renal Bcl-2, pancreatic insulin and glucose transporter (GLUT-2, 4) protein ex-
pression in all the experimental groups.
Results: The acute toxicity study showed non-toxic nature of all the three doses of WF. Further, studies
on diabetic rats exhibited anti-hyperglycemic effects by upregulating serum insulin and C-peptide levels.
Similarly, WF shown to ameliorate oxidative stress by downregulating LPO levels and augmenting the
antioxidant enzyme (ABTS). Furthermore, histopathological analysis demonstrate recovery in the
structural degeneration of β-cells mass of pancreas tissue with increase in the liver glycogen content of
the diabetic rats. Interestingly, protective nature of the extract was further revealed by the im-
munohistochemical study result which displayed upregulation in the insulin and renal Bcl-2 expression,
the anti apoptosis protein. Moreover, western blot result have shown slight alteration in the GLUT-2 and
GLUT-4 protein expression with the highest dose of WFc treatment, that might have stimulated glucose
uptake in the pancreas and played an important role in attenuating the blood glucose levels.
Conclusion: The overall study result have demonstrated the potential of WF in the management of
diabetes and its related complications, thus warrants further investigation on its major compounds with
in depth mechanistic studies at molecular level.
& 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction diabetes mellitus (DM) and its associated complications due to the
unabated increase in the incidence of this metabolic syndrome. At
There is a growing research interest in the management of present DM is one of the alarming global threat giving rise to
complications such as retinopathy, neuropathy, heart attack and
n
Corresponding author.
atherosclerotic vascular disease (Gandhi et al., 2014; Irudayaraj
E-mail addresses: aditya@um.edu.my, adityaarya18@gmail.com (A. Arya). et al., 2012; Laakso, 1999). The impairment in carbohydrate, lipid

http://dx.doi.org/10.1016/j.jep.2015.08.057
0378-8741/& 2015 Elsevier Ireland Ltd. All rights reserved.
230 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240
and protein metabolism and defects in insulin signaling leads to
hyperglycemia, a known characteristic of DM, which is associated
with oxidative stress by increasing the formation of reactive oxy-
gen species (ROS) causing reduction in the antioxidant levels.
Under the oxidative stress condition, an increased level of mal-
ondialdehyde (MDA), a highly toxic by-product is released by lipid
peroxidation and ROS and consequently causing oxidative damage
in the pancreas, liver and kidney (Evans, 2007).
According to WHO estimation, herbal medicinal plants have
started to gain more attention in recent years for the management
of diabetes owing to their less side effects compared to the syn-
thetic pharmaceutical drugs which are more costly (Chavre et al.,
2010). These ethnomedicinal plant species are known to exert
anti-diabetic effect by ameliorating blood glucose levels and oxi-
dative stress and improving the pancreatic expressions of insulin
and glucose transporter proteins (Taha et al., 2014). The presence
of glycosides, alkaloids, terpenoids, flavonoids, carotenoids, etc
account for the antidiabetic properties of the plants. As the search
for more effective and less expensive anti-diabetic plant extract is
still ongoing, there is a renewed research interest on the tradi-
tionally used antidiabetic plants. One such plant is Woodfordia
fruticosa, the flower of which have been traditionally used in the
Beed district of Maharashtra as anti-diabetic agent (Patil et al.,
2011; Chavre et al., 2010; Das et al., 2007).
W. fruticosa Kurtz, the well-known plant belong to the family
Lythraceae and located abundantly in the tropical and subtropical
region of India growing upto an altitude of 1500 m and have been
in use for a long time by the traditional practitioners of South East
Asian countries. The flower fulfills huge demand of both domestic
and international markets even though other parts of the plant
also behold some medicinal values. The flowers which is specia-
lized in the preparation of herbal medicines are brilliant red in
color, pungent, acrid and it exerts uterine sedative and anthel-
mintic effect. The other therapeutic values of the dried flowers of
W. fruticosa act against dysentery, sprue, bowel complaint, rheu-
matism, dysuria, hematuria, wounds, bleeding injuries, otorrhoea,
leucorrhea and dysmenorrhea. The flowers also contribute in the
preparation of Ayurvedic fermented drugs named as ‘Aristhas’ and
‘Asavas’ and was made patented against diabetics and calorie
conscious non-diabetics by Brindavanam et al. (2003). The anti-
diabetic property of the plant was due to the phytochemicals such
as tannins, flavonoids, anthraquinone glycosides and polyphenols
present in flowers and leaves (Chavre et al., 2010; Das et al., 2007).
The immunomodulatory activity of the Ayurvedic drug ‘Nimba
Aristha’, possessing W. fruticosa flowers was demonstrated by
Kroes et al. (1993). The presence of glycosides, quercetin and
myricetin in WF attribute to the anti-inflammatory effect of the
plant and it is validated by its ability to inhibit the two pivotal
enzymes, lipoxygenase and cycloxygenase of pelargonidin, which
play dominant role in regulating the pathophysiology of
inflammation.
Although various interesting pharmacological aspects have
been identified in previous studies to reflect the antioxidant, anti-
inflammatory, anti-microbial and anticancer properties of differ-
ent parts of this plant, two recent studies demonstrated the anti-
hyperglycemic potential of the methanolic and ethanolic extract of
the flower on alloxan induced diabetic mice and STZ-induced
diabetic rats (Bhatia and Khera, 2013; Verma et al., 2012). How-
ever, lack of scientific report on the anti-diabetic mechanism in-
cluding the pancreatic expression of insulin and glucose trans-
porters by the methanolic extract of W. fruticosa flower (WF)
warrants further investigation. Keeping this in view, the present
study attempts to understand the mechanism involved in anti-
hyperglycemic activity of the flower methanolic extract, WF, as-
sociated with important biochemical parameters, oxidative stress
markers, hepatic glycogen content, pancreatic β-cell mass, insulin
and targeted GLUT-2 and 4 protein expressions and the renal Bcl2
protein analysis in STZ–nicotinamide induced diabetic rat model.
2. Materials and methods
2.1. Chemicals
Streptozotocin (STZ), Nicotinamide and Glibenclamide were
purchased from Sigma-Aldrich (USA).
2.2. Collection of plant materials
Dried W. fruticosa (1 kg) flowers were procured from Amritum
Bio-Botanica Herbs Research Laboratory Pvt. Ltd (Madhya Pradesh,
India). The identification and authentication of W. fruticosa flower
was carried out by the company's Quality Control Department.
Voucher specimen (PH-12W7) had been deposited with Depart-
ment of Pharmacy, Faculty of Medicine, University of Malaya, Kuala
Lumpur, Malaysia, 50603.
2.3. Extraction of W. fruticosa flower
The dried flowers of W. fruticosa (500 g) were coarsely pow-
dered using grinder for the purpose of extraction with Soxhlet
extractor. The powder was first extracted with 100% n-hexane
using hot extraction at 43 °C with a Soxhlet extractor for 48 h.
After completing the extraction with n-hexane, the obtained de-
fatted residue was further extracted using 100% chloroform and
lastly with 100% methanol. The solvents from each crude fraction
were dried by rotary evaporator (BUCHI, R-215) under reduced
pressure at a maximum temperature of 40 °C to get the extract of
W. fruticosa flower. The final fraction was then stored at
20 °C
until further use.
2.4. Phytochemical analysis of WF by LCMS/MS-QTOF
The chemical components in WF were analyzed through de-
replication method based on MS published data (Alali and Tawaha,
2009). This strategy proved to be effective and useful in analyzing
crude extracts. The analytical HPLC (Spark Holland Symbiosis
PICO) system was coupled with a hybrid quadrupole-time of
flightmass spectrometry (Applied Biosystems, Triple TOF 4600, CA)
with an electrospray ionization (ESI) source. A hypersil ODS C18
column (4.6 mm i.d.  100 mm, 3 mm particle diameter) was used
for this analysis. Solid Phase Extraction (SPE) CEC18 cartridge (UCT,
Bristol, PA) was used for sample clean-up. The mobile phase
consisted of solvent A (water with 0.1% formic acid) and solvent B
(acetonitrile with 0.1% formic acid). The HPLC method gradient
and mass spectrometry parameters were followed by previous
method for the determination of phenolic compounds with some
modifications. Analyst software was used for data processing and
acquisition.
2.5. Experimental animals
Healthy adult Sprague Dawley (SD) male rats were procured
from the Experimental Animal House, Faculty of Medicine, Uni-
versity of Malaya. The rats weighing between 250 and 300 g were
caged in polypropylene crates and preserved at normal pathogen
free light controlled conditions, consisting of 12 h light/dark cycle
at room temperature (25 72 °C) with humidity level of 35–60%
and provided with normal rat pellet diet with distilled water. The
animal experiment was performed in accordance with the animal
experimentation guidelines issued by the Experimental Animal
House committee, Faculty of Medicine, University of Malaya
 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240
(Ethics number: 2014-07-01/PHARM/R/NS).
2.6. Experimental design
The experimental rats were randomly assigned into six groups
(n ¼6), containing of six rats in each group as follows:
Group 1, Normal Control (NC): Rats fed with distilled water.
Group 2, Diabetic Control (DC): STZ–nicotinamide induced
diabetic rats.
Group 3, WFa: STZ–nicotinamide induced diabetic rats, treated
with 100 mg/kg WF
Group 4, WFb: STZ–nicotinamide induced diabetic rats, treated
with 200 mg/kg WF
Group 5, WFc: STZ–nicotinamide induced diabetic rats, treated
with 400 mg/kg WF
Group 6, Positive Control (PC): STZ–nicotinamide induced
diabetic rats, treated with Glibenclamide (2.5 mg/kg).
The rats were orally administered once a day with WF (100,
200 and 400 mg/kg body weight) and Glibenclamide (2.5 mg/kg
body weight) by dissolving in distilled water by intra-gastric tube.
The animals were sacrificed, 24 h after 45 days of last treatment
dose.
2.7. Acute toxicity study
Acute toxicity test was carried out according to the guidelines
of the Organisation for Economic Co-Operation and Development
(OECD) on normal rats with three different doses of WF. The rats
were fasted overnight and the next morning fed with single dose
of 500, 1000 and 3000 mg/kg body weight to three different
groups respectively (n ¼6). All the rats were continually examined
for 2 h to check any abnormalities in behavior of the animals and
further continued to monitor and examine the rats for 24 and 72 h.
2.8. Diabetes induction by streptozotocin–nicotinamide
Type 2 diabetes mellitus was induced in overnight fasted rats
by a single intraperitoneal injection of Streptozotocin at a dose of
45 mg/kg body weight. After 15 min, 120 mg/kg Nicotinamide was
injected intraperitoneally. The glucose level of blood was mea-
sured from the tail vein after 72 h of STZ–nicotinamide injection
and hyperglycaemic rats (blood glucose level 416.7 mmol/L) were
considered to be diabetic rats.
2.9. Collection of blood sample
The rats were moderately anesthetised by combination of Ke-
tamine 50 mg/kg and Xylazine 5 mg/kg at the end of 45 day of
treatment and 3 ml of blood samples were collected from the in-
ner canthus of the eye by using capillary tubes to collect the ser-
um. For the preparation of serum, blood samples were collected in
vacutainer tube and allowed to clot at ambient temperature for
15–30 min. After that, the clot was removed by centrifuging at
4000 rpm for 10 min in a refrigerated centrifuge and serum was
collected and stored in
80 °C until further use.
2.10. Assessment of biochemical parameters
The fasting blood glucose levels and body weights of all the
groups were measured on days 7, 21 and 45. Blood glucose level
was determined by the portable glucometer (Accu-Check Nano
Performa). Serum Insulin and C-peptide levels in all the treated,
diabetic and normal groups were measured by Enzyme Linked
Immunosorbent Assay (ELISA Kit, Item number 589501, Cayman,
USA) and (Rat ELISA Kit K4757, Bio Vision Corporation, USA) fol-
lowing manufacturer's protocol.
231
2.11. Antioxidant status
The Cayman Chemical Antioxidant Assay Kit determines the
total antioxidant capacity (SOD, Catalase, GPx) by measuring the
level of ABTS in the serum of diabetic, normal and treatment
groups. However, the cumulative effect of all the antioxidant en-
zymes can be measured using ABTS. The ability of the extract in
preventing the oxidation of ABTS (2, 2′-azino-di-[3-ethyl-
benzthiazoline sulfonate]) to ABTS by metmyoglobin was mea-
sured using the kit (Item number 709001) and was compared to
that of Trolox, a water-soluble tocopherol analogue and was then
quantified as molar Trolox equivalents. To determine the lipid
peroxidation (LPO) levels, the hydroperoxides level in the serum of
diabetic, normal and treatment groups were measured directly in
the serum by utilizing the redox reactions with ferrous ions using
LPO assay kits (Item number 705003). Both the assays were per-
formed based on Cayman manufacturer instructions (Cayman
Chemical, Ann Arbar, USA).
2.12. Histological study
At the end of experiment the liver, kidney and pancreas were
dissected and washed with normal saline and preserved in 10%
formalin for 7 days at room temperature. After that, the samples
were washed and dehydrated with 70–100% of ethanol and
cleansed with xylene, and inserted in paraffin blocks. The samples
were then cut as thin as 5 mm pieces, with a revolving microtome.
Pancreas sections were stained with hematoxylin and eosin dye;
and the liver tissue sections were stained with Periodic acid Schiff
(PAS) staining for 10 min (for demonstration of glycogen content),
and then photomicrographs were obtained under light
microscope.
2.13. Immunohistochemistry analysis
For the purpose of immunohistochemistry analysis, 5 μm sec-
tions of pancreas and kidney tissue blocks were placed on poly-L-
lysine coated and the slide sections were immersed in target re-
trieval solution (DAKO, Lot 10069393) and heated in microwave
oven at 98 °C for 20 min, and then cooled at room temperature.
The sections of pancreas were incubated for 1 h in Insulin (Item
no: ab46716, 1/50 dilution) primary antibody and anti-BcL-2 (Item
no: ab7973, 1/100) antibody was used for kidney tissue incubation
at room temperature. After that, three times rinsing was done with
Dako Tris-buffered saline (TBS), the sections were incubated with
biotinylated secondary antibody (LSAB systems2-HRP, Item no:
10069908) for 1 h at room temperature. Following TBS rinses, the
sections were incubated for 30 min at room temperature with
streptavidin–horseradish peroxidase conjugate, followed by a
course of incubation in Diaminobenzidine (DAB) (DAKO, Item no:
10067468). Control immunohistochemistry reactions were per-
formed to evaluate the specificity of the labels by omitting the
primary antibody. Staining with hematoxylin was performed and
used as a reference of the cytoarchitecture of the tissue.
2.14. Histomorphometric analysis
Positive signals of the target protein were determined by using
a high resolution inverted microscope (Nikon Ti series). Further-
more, Image J software was used to evaluate the integral optical
density (IOD) of immunohistochemistry stain. All the single pixels
in the digital image signified the IOD of the target protein, which
was more precise, according to the strength of stain and the la-
beled surface areas.
232 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240
400
Week 1
B o dy We i g ht/g
300
$ *
200
100
0
Fa
Fc
C
Fb
N
W
W
W
Fig. 1. Effect of WF on the body weight of normal and diabetic rats during 45 days treatment. All the values are expressed as the mean 7 SD. * Indicates the significant
difference when compared to the NC group (P o0.05). $ Indicates the significant difference when compared to the DC group (P o0.05).
2.15. Western blot
The samples of rats' pancreas were segregated with 4–20%
sodium dodecyl sulfate (SDS-PAGE) gels to fill the polyvinylidene
fluoride (PVDF) membranes with proteins. The membranes were
blocked with 5% non-fat milk and as well as primary antibodies
recognized as anti-glucose transporter GLUT-2 antibody (Item no:
ab54460, 1/200) and anti-glucose transporter GLUT-4 antibody
(Item no: ab33780, 1/1000). After that, the membranes were in-
cubated at 4 °C overnight then cleansed and again incubated for
1 h with Horseradish peroxidase (HRP)-conjugated goat anti rabbit
IgG. The quantities were normalized with the overall volume of
protein filled in each well, which was identified by densitometric
analysis of PVDF membranes, with the staining of Amersham ECL
Prime Western Blotting Detection Reagent (GE Healthcare). The
pictures of proteins were taken by chemiluminescence (UVP, Bio
Spectrum, USA) and the quantities of particular bands were
measured by densitometry by ImageJ 1.37 software (NIH, USA).
2.16. Statistical analysis
One way analysis of variance (ANOVA) was used followed by
Tukey's Multiple Comparison Test (Graph Pad version 5.0; Graph
Pad Software Inc., San Diego, CA, USA) to analyze the effect of WF
on all experimental rat groups. Experimental differences were
considered statistically significant if P o 0.05.
3. Results
3.1. Extract yield
The extract yield of W. fruticosa flower was 2.92 g for hexane
(WH), 3.44 g for chloroform (WC) and 151.65 g for methanol (WF).
On the basis of preliminary screening of all the extracts, WF with
the highest percentage yield possesses hypoglycemic effects on
different biochemical assays (data not shown). Thus, the extract
was chosen for further studies.
3.2. Acute toxicity study
The acute toxicity study represents the non-toxic nature of WF
at all the three doses tested. The animals did not show any leth-
ality and behavioral changes after 24 and 72 h of the treatment.
3.3. LCMS-QTOF analysis
The qualitative analysis of chemical compounds in WF were
Week 3 Week 6
* *
* * * * * * *
* *
$ $
Fa
Fc
Fa
Fc
PC
PC
PC
C
Fb
Fb
D
W
W
W
assesed by LCMS-QTOF method in which 4 flavonoid glycosides
(Quercetin glucoside, Quercetin 3-O-(6″galloyl)-β-D-galactopyr-
anoside, Naringenin 7-glucoside, Kaempferol 3-O-glucoside),
3 flavonoid aglycones (Queretin, Naringenin, Kaempferol) and
2 phenolic acids (gallic acid, ellagic acid) were identified from WF
based on mass fragmentation patterns and compared to the pre-
vious literature. In this analysis, identification of phenolic com-
pounds and characterization of the compounds were achieved by
the negative ion-mode ionization. All the compounds were iden-
tified and structurally elucidated in previous studies on WF. Fig. S1
shows the total ion chromatogram (TIC) of WF, which demon-
strated the presence of polar compounds in the extract.
Quercetin 3-O-(6″- galloyl)-β-D-galactopyranoside was identi-
fied based on the fragment ions at m/z 615 [M–H]
similarly
identified Quercetin at m/z 300 [M–H]
and Quercetin glucoside
at m/z 463 [M–H]
. The presence of a galloyl unit was indicated by
the loss of 152 Da from the base peak at m/z 615 in Fig. S2. Mass
spectra data of the fragment ions was compatible with previous
literature. Likewise, the mass spectra of Naringenin and Nar-
ingenin 7-glucoside (Fig. S3), Kaempferol and Kaempferol 3-O-
glucoside (Fig. S4), gallic acid (Fig. S5) and ellagic acid (Fig. S6) was
shown.
3.4. Effect of WF on body weight
The effect of WF on body weight in normal and diabetic rats at
week 1, 3 and 6 are shown in Fig. 1. The untreated type 2 diabetic
rats exhibited signficant reduction of body weight compared to
that of the NC group (P o0.05). Nevertheless, upon treatment with
three different doses of WF, the diabetic rats demonstrated sig-
nificant improvement in their body weight as compared to DC
group (P o0.05).
3.5. Effect of WF on blood glucose level
Fig. 2 illustrates the effect of PC and WF on blood glucose level
of normal, diabetic and diabetic treated rats at week 1, 3 and 6.
Monitoring of the rats' fasting blood glucose levels for 45 days
treatment period revealed significant reduction in the elevated
blood glucose levels of diabetic rats treated with PC and WF
compared to that of the DC group (P o0.05). However, there was a
higher reduction in the blood glucose level with WFc compared to
the other two doses of WFa and WFb in diabetic rats.
3.6. Effect of WF on insulin and C-peptide level
Fig. 3 demonstrates that the serum insulin and C-peptide levels
in the untreated diabetic rats were markedly declined compared to
 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240 233

Week 1 Week 3 Week 6

20 $

G l uc o s e Le ve l mmo l /L
$ $
15 * *
*
* * *
* *
10 * * *
*

Fa

Fc

Fa

Fc

Fa

Fc
C

PC

PC

PC
Fb

Fb

Fb
N

D
W

W
W

W
W

W
Fig. 2. Effect of WF extract on the blood glucose level of normal and diabetic rats during 45 days treatment. All the values are expressed as the mean7 SD. * Indicates the
significant difference when compared to the NC group (Po 0.05). $ Indicates the significant difference when compared to the DC group (Po 0.05).

Fig. 3. Effect of WF extract on the serum Insulin (A) and C-peptide (B) levels of
normal and diabetic rats after 45 days treatment. All the values are expressed as the
mean 7 SD. * Indicates the significant difference when compared to the NC group
(Po 0.05). $ Indicates the significant difference when compared to the DC group
(Po 0.05).
that of the NC group (P o0.05). The WFa, WFb and WFc groups
displayed significant increase in the serum insulin and C-peptide
levels after the six-week treatment period as compared to DC
group (Po0.05). However, the diabetic rats treated with WFc
group exhibited the highest increase in these parameters com-
pared with those observed in the DC group and was quite similar
to that in the PC group, suggesting a dose-dependent improve-
ment in the glycemic control.
Fig. 4. Effect of WF extract on the antioxidant ABTS (A) and LPO (B) levels in
normal diabetic rats after 45 days treatment. All the values are expressed as the
mean7 SD. * Indicates the significant difference when compared to the NC group
(P o0.05). $ Indicates the significant difference when compared to the DC group
(P o0.05).
3.7. Effect of WF on antioxidant (ABTS) and LPO levels
Fig. 4 represents the effect of WF on oxidative stress in normal
and diabetic rats. As demonstrated in Fig. 4A, the antioxidant
(ABTS) level was significantly reduced in DC group against NC
group (P o0.05) and this effect was recovered by WF in a dose-
dependent manner. PC and WFc showed higher antioxidant level
when compared to the other lower doses of WF. On the other
hand, DC group displayed a significant rise in the LPO level when
234 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240
Fig. 5. Effect of three different doses of WF extract on the pancreas of STZ–nicotinamide induced diabetic and normal rats after 45 days treatment. H & E staining of pancreas
(400  ). A: NC; B: DC: C: WFa; D: WFb, E: WFc and F: PC.
compared to that of NC group (Fig. 4B). In contrast, diabetic rats
treated with PC and WFc group attained highest reduction in the
LPO level compared to that of WFa and WFb groups, indicating the
dose-dependent effect of WF.
3.8. Effect of WF on pancreas tissue
Fig. 5 demonstrates the impact of WF on pancreas structure in
normal and diabetic rats. After 45 days of treatment, the pancreas
section of rats were stained with hematoxylin and eosin as de-
scribed in method section. Panel (A) showed a normal and round
rat islet of Langerhans with surrounding of normal structure and
normal exocrine acini tissues. Panel (B) which corresponds to the
STZ–nicotinamide induced diabetic rats, showed severe β-cell
damage and deformation with atrophic and vacuolated pancreatic
islet. In addition, focal necrosis, congestion in central vein and
infiltration of lymphocytes were examined. In Panel (C), the WFa
treatment exerted minimal restoration of pancreatic endocrine
cells and expansion of pancreatic islets, which showed a promi-
nent hyperplastic islet. Panel (D) showed that WFb exhibited
protective effects over the pancreatic islet, as evidenced by the
absence of vacuolization. Although less degranulation was ob-
served in the β-cells, the cell structure was disorganized with a
reduced-sized islet and also, degeneration of some of the pan-
creatic acini was still observed. In Panel (E), the effect of WFc on
diabetic rats has shown considerable reduction of lesion and
normal β-cell structure. Panel (F) displays the protective role of
Glibenclamide on the pancreas of diabetic rats against the de-
structive effect of STZ–nicotinamide.
3.9. Effect of WF on liver glycogen content
Fig. 6 illustrates the effect of three doses of WF on Glycogen
expression in STZ–nicotinamide induced diabetic rats after 45 days
of treatment. Rats' liver sections were stained with PAS staining as
mentioned in the method section. Stored glycogen was visualized
as purple particles. In NC group, the increased level of purple
particles represented abundant glycogen and normal cellular
metabolism when compared to that of DC group which was lightly
stained due to the less number of stored glycogen. It can be seen
that the hepatic glycogen content has elevated with PC and in-
creasing dose of WF, whereby the diabetic rats treated with WFc
 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240 235

Fig. 6. Effect of three different doses of WF extract on the liver glycogen content in STZ–nicotinamide induced diabetic and normal rats after 45 days treatment. PAS staining
of liver where glycogen is indicated by arrows (400  . A: NC; B: DC; C: WFa; D: WFb, E: WFc and F: PC.

has retained higher glycogen content reflecting its positive mod-
ulation on hepatic carbohydrate metabolism compared to that of
DC group.
3.10. Effect of WF on Bcl-2 expression in kidney tissue
Fig. 7 reveals the effect of WF on Bcl-2 expression in normal
and diabetic rats. According to Fig. 7(I), the expression level of Bcl-
2 was found to be decreased in DC group against NC group
(P o0.05) and this effect was reversed with three doses of WF after
45 days of treatment. This immunohistochemical observation was
confirmed by histomorphometric study as shown in Fig. 7(II)
which demonstrates that the recovery of Bcl-2 expression was
dose dependent, as the highest Bcl-2 expression was achieved
with the highest dose, which was WFc.
3.11. Effect of WF on insulin expression in pancreas tissue
showed higher level of insulin expression compared to that of WFa
and WFb. These results were confirmed by histomorphometric
study as displayed in Fig. 8(II). However, both the figures de-
monstrate significant enhancement of insulin expression level in
diabetic rats treated with PC and three different doses of WF
against DC group (Po 0.05).
3.12. Effect of WF on GLUT-2 and GLUT-4 expression levels
The determination of GLUT-2 and GLUT-4 expression level in
rat's pancreas following WF treatment was presented in the
western blot analysis in Fig. 9. The untreated diabetic rats ex-
hibited significant reduction in the total amount of these two
proteins as compared to that of the NC group (P o0.05). Interest-
ingly, WF treatment led to slight alteration in the GLUT-2 and
GLUT-4 proteins expression, reflecting the protective nature of
WFc, among the tested doses (Fig. 9(II) a and b).

Fig. 8 shows the changes in insulin expression level after

treatment with WF and PC in diabetic rats. The reduced expression 4. Discussion
of insulin level in DC group was restored gradually with increasing
dose of WF as illustrated in Fig. 8(I). The diabetic rats in WFc group In the present study, we have scientifically evaluated the
236 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240

A B

C D

E F

II 250
*
B c l 2 Ki dne y (DOI)

200 *
*
*
150

100 $

50

0
Fa

Fb

Fc

PC
C

C
N

W
W

Fig. 7. (I) Effect of WF on the kidney Bcl-2 expression level in STZ-nicotinamide induced diabetic and normal rats after 45 days treatment (400  ). A: NC; B: DC; C: WFa; D:
WFb, E: WFc and F: PC. (II) Bcl-2 expression level were quantified by Image J analysis software as mean7 SD and analyzed by one-way ANOVA (Tucky). * Indicates the
significant difference when compared to the NC group (Po 0.05). $ Indicates the significant difference when compared to the DC group (Po 0.05).

traditional claim for the use of W. fruticosa flower in the man- The acute toxicity study results demonstrated non-toxic nature
agement of diabetes mellitus and its associated complications by of WF with no lethality at different dosages. Similarly, previous
its antioxidant and protective nature against the liver, pancreas study have also shown that W. fruticosa flower extract was not
and kidney of STZ–nicotinamide induced diabetic rats. toxic to mice and rats (Verma et al., 2012). Oral administration of
 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240 237

II
400
Insl ul i n Expr e s si o n (IOD)

300 *
*

200 * *
$

100

0
Fa

Fb

Fc

PC
C

C
N

W
W

Fig. 8. (I) Effect of WF on the pancreas insulin expression level in STZ–nicotinamide induced diabetic and normal rats after 45 days treatment (400  ). A: NC; B: DC; C: WFa;
D: WFb; E: WFc and F: PC. (II) Insulin expression level were quantified by Image J analysis software as mean7 SD and analyzed by one-way ANOVA (Tucky). * Indicates the
significant difference when compared to the NC group (Po 0.05). $ Indicates the significant difference when compared to the DC group (Po 0.05).

three doses of WF (WFa, WFb and WFc) to diabetic rats for 45
consecutive days exhibited attenuation in the elevated blood
glucose level and thus augmented serum insulin levels accom-
panied by rise in serum pro-insulin, C-peptide which serves as a
linker between A and B chains of insulin (Al-Numair et al., 2009).
The outcomes of our result is in agreement with the previous
study that reported ethanolic extract of WF leaves was found to be
effective in increasing plasma insulin in dexamethasone induced
insulin resistance in mice (Bhujbal et al., 2012). The up-regulation
in serum insulin and C-peptide levels could be due to the
improvement in β-cell function which is indirectly/directly asso-
ciated with the antioxidative and free radical scavenging proper-
ties of the extract as evidenced by a rise in antioxidant defense
system, ABTS and reduction in LPO levels. The hyperglycemia
mediated oxidative stress contributes to the accelerated formation
of ROS by glycating and inactivating the antioxidant defense en-
zymes, SOD, catalase and GPx (Arunachalam and Parimelazhagan,
2013). In consistence to our results, (Verma et al., 2012; Singh and
Kakkar, 2013) showed that catalase, superoxide dismutase, glu-
tathione peroxidase and glutathione reductase activities were
238 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240

Fig. 9. (I) Effect of WF on pancreas tissue of STZ–nicotinamide induced diabetic and normal rats after 45 days treatment. A: NC; B: DC; C: WFa; D: WFb; E: WFc and F: PC.
Tissue was prepared and subjected to western blotting using an antibody specific for GLUT-2 and GLUT-4, where β-actin was used as an internal control. The data were
analyzed by density color and presented as mean 7 S.D (II). Histogram (a and b) denotes band intensity percentage of GLUT-2 and GLUT-4.

improved and lipid peroxidation was declined in diabetic rats
treated with WF.
Moreover, the degeneration of pancreatic β-cell caused by STZ-
induced oxidative stress as shown in the histological analysis is
associated with reduced level of insulin in the serum of diabetic
rats. Diabetic rats treated with WF have shown regeneration of
insulin-producing islet cells and upregulation of insulin expression
in the pancreas. This restoration of β-cell mass and its structure by
reducing the lesion and acini developed in the diabetic state could
be due to the antioxidant nature of WF by scavenging the free
radicals as determined in the immuno-histochemical study. Thus,
the present investigation extends our postulation on the existing
polyphenolic compounds in WF as identified by LCMS-QTOF that
might have shown antidiabetic effects by improving the de-
generated β-cell mass in the diabetic rats which is in agreement
with the study results of Taha et al. (2014). The better regulation of
hyperglycemic state may also support the hypothesis that WF di-
rectly modulates insulin release from remaining β-cells which
eventually restores hepatic glycogen storage by potentiating glu-
cose metabolism and/or peripheral glucose uptake.
Based on these findings, we determined the hepatic glycogen
content in diabetic rats by PAS staining and observed that it was
significantly restored in case of WF treated animals. The impaired
capacity of liver to store glycogen is associated with reduced ac-
tivity of glycogen synthase and enhanced glycogen phosphoryla-
tion due to lack of insulin in the diabetic state (Shirwaikar et al.,
2004). The mechanism resulting hyperglycemia in diabetes
mellitus was due to the excessive hepatic glycogenolysis and
gluconeogenesis associated with decreased glucose uptake by the
tissues (Qamar and Qureshi, 2013; Wang et al., 2010) reflecting the
pivotal role of liver in maintaining the normal concentrations of
blood glucose (Onkaramurthy et al., 2013). The hepatic damage as
observed in the present histopathology analysis (Fig. 6) displays
the deleterious effects of hyperglycemia-induced oxidative stress.
The existing flavonoids in WF, such as quercetin and quinic acid
would have played important role in preventing hepatic damage in
diabetic rats which is in line with the histological studies reported
in our previous study and by other researchers where quercetin
reduced the severity of hepatic injuries by increasing the anti-
oxidant enzymes activity and by scavenging the free radicals (Arya
et al., 2014; Bashir et al., 2014; Florence et al., 2014). The anti-
oxidative protection rendered by WF may likewise functioned in
safeguarding the hepatic system in the presence of elevated serum
insulin level, a regulator of glycogen metabolism in mammals
(Gandhi et al., 2014). Thus, diabetes reduces liver glycogen stores
and insulin reverses this effect of hepatic damage independently
and WF beside ameliorating hepatic glycogen content also plays a
protective role against the hepatic complications in diabetes.
The corelation between insulin secretion and glucose uptake
was further investigated by pancreatic protein determination as
evidenced by upregulation in GLUT-2 and GLUT-4 expression in
the WF treated diabetic rats. The improved glucose uptake in the
insulin-signaling cascade is managed by glucose transporter
(GLUT) proteins, GLUT-2 and GLUT-4 which translocates the
 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240
insulin-regulated glucose into the cells (Hajiaghaalipur et al., 2015)
and has impact on whole-body glucose homeostasis (Zhao et al.,
2014). The availability of polyphenols in WF might have triggered
glucose ingestion by modulating the glucose transporter proteins
(Li et al., 2015). Interestingly, many researchers have identified the
potential role of polyphenolic enriched herbal extracts in attenu-
ating hyperglycemia mediated insulin resistance and thus modu-
lated uptake of glucose by GLUT-2 and GLUT-4 expression in
pancreas of the diabetic rats (Hajiaghaalipur et al., 2015). The
present result together with those of the previous study suggest
that the ability of WF to improve cellular glucose uptake was as-
sociated with the upregulation of GLUT-2 and GLUT-4 expressions
in the pancreas which was due to the restoration of β-cell mass
(Bähr et al., 2012; Ong et al., 2011; Olson, 2012).
The impairment in renal function is one of the major compli-
cations associated with diabetes as caused by the inability of the
kidney to clean the blood vessels due to the increased glucose and
fatty acids accumulation in the blood (Zhang et al., 2014; Bernard
et al., 1999). The diabetic kidney cells undergo apoptosis and
widening of Bowman's space as observed in previous studies and
this severe pathological condition can be regulated by the anti-
apoptotic nature of Bcl-2 protein which support the treatment of
diabetes via the prevention of apoptosis (Junhua et al., 2015; Arya
et al., 2014). According to our renal immunohistochemical analysis,
the Bcl-2 protein expression was found to be downregulated in the
diabetic rats which was then restored with WF treatment. This
effect of WF on the kidney proteins was probably due to the im-
proved blood glucose caused by restoration of β-cell mass. It is the
anti-apoptotic nature of Bcl-2 proteins which play vital role in β-
cell physiology and apoptosis and hence cause increased survival
of the cell (Gurzov and Eizirik, 2011). Interestingly according to
(Kaeidi et al., 2011) the olive leaf extract which contains high level
of phenolic compounds have enhanced the expression level of Bcl-
2 in diabetic rats. Research with polyphenolic compounds atte-
nuated the diabetes-induced kidney damage by improving Bcl-2
expression. Hence, the presence of this apoptosis regulator within
a cell is thought to be critical in the way the cell responds to
apoptotic stimuli and determines cell death or survival (Gurzov
and Eizirik, 2011; Zhang et al., 2004).
Our findings revealed the pivotal role of WF in protecting the
pancreas, liver and kidney by restoring the antioxidant enzymes,
pancreatic insulin, hepatic glycogen content and renal BCl-2 with
slight alteration in GLUT-2 and GLUT-4 protein expressions. Thus,
we may suggest that the exhibiting antioxidant and anti-
hyperglycemic properties of WF could be due to the combinatorial
effect of the existing polyphenolic compounds.
5. Conclusion
The data from this study supports the traditional use of W.
fruticosa flower in the management of diabetes. The outcomes of
this study on STZ–nicotinamide induced diabetic rats indicates the
protective nature of WF on the pancreas by increasing β-cell mass
and the liver hepatic glycogen content. Altogether, the study result
suggests the possible usage of WF in diabetes and its related
complications, thus, requires further validation through in-depth
study models at molecular level.
Acknowledgments
The accomplishment of this research was made possible by the
support of High Impact Research Grant (UM-MOHE UM.C/625/1/
HIR/MOHE/09) and the Fundamental Research Grant (FRGS):
239
FP021-2014A from the Ministry of Higher Education, Malaysia and
the University of Malaya Research Grant (UMRG Grant no:
RP001D-13BIO).
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.jep.2015.08.057.
References
Alali, F.Q., Tawaha, K., 2009. Dereplication of bioactive constituents of the genus
hypericum using LC-( þ,
)-ESI-MS and LC-PDA techniques: Hypericum triqu-
terifolium as a case study. Saudi Pharm. J. 17 (4), 269–274.
Al-Numair, K.S., Pugalendi, K.V., Chandramohan, G., 2009. Effect of 3-hydro-
xymethyl xylitol on hepatic and renal functional markers and protein levels in
streptozotocin-diabetic rats. Afr. J. Biochem. Res. 3 (5), 198–204.
Arunachalam, K., Parimelazhagan, T., 2013. Antidiabetic activity of Ficus amplissima
Smith. bark extract in streptozotocin induced diabetic rats. J. Ethnopharmacol.
147 (2), 302–310.
Arya, A., Jamil, M.M., Shahid, N., Noordin, M.I., Looi, C.Y., Wong, W.F., Rais Mustafa,
M., 2014. Synergistic effect of quercetin and quinic acid by alleviating structural
degeneration in the liver, kidney and pancreas tissues of STZ-induced diabetic
rats: a mechanistic study. Food Chem. Toxicol. 71, 183–196.
Bähr, I., Wutschke, I.B., Wolgast, S., Hofmann, K., Streck, S., Mühlbauer, E., Peschke,
E., 2012. GLUT4 in the endocrine pancreas – indicating an impact in pancreatic
islet cell physiology? Horm. Metab. Res. 44 (06), 442–450.
Bashir, S.O., Morsy, M.D., Sakr, H.F., Refaey, H.M., Eid, R.A., Alkhateeb, M.A., Defallah,
M.A., 2014. Quercetin ameliorates diabetic nephropathy in rats via modulation
of renal Na þ , K þ -ATPase expression and oxidative stress. Am. J. Pharmacol.
Toxicol. 9 (1), 84–95.
Bernard, C., Berthault, M.F., Saulnier, C., Ktorza, A., 1999. Neogenesis vs. apoptosis
asmain components of pancreatic beta cell ass changes in glucose-infused
normal and mildly diabetic adult rats. J. Fed. Am. Soc. Exp. Biol. 13, 1195–1205.
Bhatia, A., Khera, N., 2013. Hypoglycemic activity of orally administered Woodfordia
fruticosa flower extract in alloxan-induced diabetic mice. Int. J. Life Sci. Bio-
technol. Pharm. Res. 2, 72–84.
Bhujbal, S.S., Providencia, C.A., Nanda, R.K., Hadawale, S.S., Yeola, R.R., 2012. Effect of
Woodfordia fruticosa on dexamethasone induced insulin resistance in mice.
Braz. J. Pharmacogn. 22 (3), 611–616.
Brindavanam, N., Katiyar, C., Rao, Y., 2003. A process for preparing asava or arista
composition. Ind. Patent, 191246.
Chavre, B.W., Jadhav, D.S., Markandeya, S.K., 2010. Traditionally used forest plants
for the treatment of diabetes in beed district of Mahrashtra. Int. Q. J. Ethno Soc.
Sci. 2 (3–4), 15–17.
Das, P.K., Goswami, S., Chinniah, A., Panda, N., Banerjee, S., Sahu, N.P., Achari, B.,
2007. Woodfordia fruticosa: traditional uses and recent findings. J. Ethno-
pharmacol. 110 (2), 189–199.
Evans, J.L., 2007. Antioxidants: do they have a role in the treatment of insulin re-
sistance? Indian J. Med. Res. 125 (3), 355–372.
Florence, N.T., Benoit, M.Z., Jonas, K., Alexandra, T., Désiré, D.D., Pierre, K., Théophile,
D., 2014. Antidiabetic and antioxidant effects of Annona muricata (Annonaceae),
aqueous extract on streptozotocin-induced diabetic rats. J. Ethnopharmacol.
151 (2), 784–790.
Gandhi, G.R., Vanlalhruaia, P., Stalin, A., Irudayaraj, S.S., Ignacimuthu, S., Paulraj, M.
G., 2014. Polyphenols-rich Cyamopsis tetragonoloba (L.) Taub. beans show hy-
poglycemic and β-cells protective effects in type 2 diabetic rats. Food Chem.
Toxicol. 66 (0), 358–365.
Gurzov, E.N., Eizirik, D.L., 2011. Bcl-2 proteins in diabetes: mitochondrial pathways
of β-cell death and dysfunction. Trends Cell Biol. 21 (7), 424–430.
Hajiaghaalipur, F., Khalilpourfarshbafi, M., Arya, A., 2015. Modulation of glucose
transporter protein by dietary flavonoids in Type 2 diabetes mellitus. Int. J. Biol.
Sci. 11 (5), 508–524.
Irudayaraj, S.S., Sunil, C., Duraipandiyan, V., Ignacimuthu, S., 2012. Antidiabetic and
antioxidant activities of Toddalia asiatica (L.) Lam. Leaves in Streptozotocin in-
duced diabetic rats. J. Ethnopharmacol. 143, 515–523.
Junhua, H., Yang, Z., Yang, H., Wang, L., Wu, H., Fan, Y., Wang, W., Fan, X., Li, X., 2015.
Regulation of insulin sensitivity, insulin production, and pancreatic β cell sur-
vival by angiotensin-(1–7) in a rat model of streptozotocin-induced diabetes
mellitus. Peptides 64 (0), 49–54.
Kaeidi, A., Esmaeili-Mahani, S., Sheibani, V., Abbasnejad, M., Rasoulian, B., Hajiali-
zadeh, Z., Afrazi, S., 2011. Olive (Olea europaea L.) leaf extract attenuates early
diabetic neuropathic pain through prevention of high glucose-induced apop-
tosis: in vitro and in vivo studies. J. Ethnopharmacol. 136 (1), 188–196.
Kroes, B., Van den Berg, A., Abeysekera, A., De Silva, K., Labadie, R., 1993. Fermen-
tation in traditional medicine: the impact of Woodfordia fruticosa flowers on the
immunomodulatory activity, and the alcohol and sugar contents of Nimba
arishta. J. Ethnopharmacol. 40 (2), 117–125.
Laakso, M., 1999. Hyperglycemia and cardiovascular disease in type 2 diabetes.
Diabetes 48 (5), 937–942.
240 A. Arya et al. / Journal of Ethnopharmacology 175 (2015) 229–240
Li, L., Xu, J., Mu, Y., Han, L., Liu, R., Cai, Y., Huang, X., 2015. Chemical characterization
and anti-hyperglycaemic effects of polyphenol enriched longan (Dimocarpus
longan Lour.) pericarp extracts. J. Funct. Foods 13 (0), 314–322.
Olson, A.L., 2012. Regulation of GLUT4 and insulin-dependent glucose flux. ISRN
Mol. Biol. 2012, 12 856987.
Ong, K.W., Hsu, A., Song, L., Huang, D., Tan, B.K.H., 2011. Polyphenols-rich Vernonia
amygdalina shows anti-diabetic effects in streptozotocin-induced diabetic rats.
J. Ethnopharmacol. 133, 598–607.
Onkaramurthy, M., Veerapur, V.P., Thippeswamy, B.S., Madhusudana Reddy, T.N.,
Rayappa, H., Badami., S., 2013. Anti-diabetic and anti-cataract effects of Chro-
molaena odorata Linn., in streptozotocin-induced diabetic rats. J. Ethno-
pharmacol. 145, 363–372.
Patil, S.B., Naikwade, N.S., Magdum, C.S., Awale, V.B., 2011. Some medicinal plants
used by people of Sangli district, Maharashtra. Asian J. Pharm. Res. 1 (2), 42–43.
Qamar, A., Qureshi, I.Z., 2013. Anti-hyperglycemic and anti-nephropathic effects of
Woodfordia fruticosa Linn. in Alloxan-induced iabetic ats. ISESCO J. Sci. Technol.
9 (15), 33–38.
Shirwaikar, A., Rajendran, K., Kumar, C.D., Bodla, R., 2004. Antidiabetic activity of
aqueous leaf extract of Annona squamosa in streptozotocin-nicotinamide type
2 diabetic rats. J. Ethnopharmacol. 91, 171–175.
Singh, J., Kakkar, P., 2013. Modulation of liver function, antioxidant responses, in-
sulin resistance and glucose transport by Oroxylum indicum stem bark in STZ
induced diabetic rats. Food Chem. Toxicol. 62, 722–731.
Taha, H., Arya, A., Paydar, M., Looi, C.Y., Wong, W.F., Vasudeva Murthy, C., Hadi, A.H.
A., 2014. Upregulation of insulin secretion and downregulation of pro-in-
flammatory cytokines, oxidative stress and hyperglycemia in STZ–nicotina-
mide-induced type 2 diabetic rats by Pseuduvaria monticola bark extract. Food
Chem. Toxicol. 2, 7–9.
Verma, N., Amresh, G., Sahu, P.K., Rao, C., Singh, A.P., 2012. Antihyperglycemic ac-
tivity of Woodfordia fruticosa (Kurz) flowers extracts in glucose metabolism and
lipid peroxidation in streptozotocin-induced diabetic rats. Indian J. Exp. Biol.
50, 351–358.
Wang, L., Zhang, X.T., Zhang, H.Y., Yao, H.Y., Zhang, H., 2010. Effect of Vaccinium
bracteatum Thunb. leaves extract on blood glucose and plasma lipid levels in
streptozotocin-induced diabetic mice. J. Ethnopharmacol. 130, 465–469.
Zhang, Z., Lapolla, S.M., Annis, M.G., Truscott, M., Roberts, G.J., Miao, Y., 2004. Bcl-2
homodimerization involves two distinct binding surfaces, a topographic ar-
rangement that provides an effective mechanism for Bcl-2 to capture activated
Bax. J. Biol. Chem. 279 (42), 43920–43928.
Zhang, Y., Chunjiu, R., Guobing, L., Zhimei, M., Weizheng, C., Huiju, G., Yanwen, W.,
2014. Anti-diabetic effect of mulberry leaf polysaccharide by inhibiting pan-
creatic islet cell apoptosis and ameliorating insulin secretory capacity in dia-
betic rats. Int. Immunopharmacol. 22 (1), 248–257.
Zhao, K., Liu, H.Y., Zhou, M.M., Zhao, F.Q., Liu, J.X., 2014. Insulin stimulates glucose
uptake via a phosphatidylinositide 3-kinase-linked signaling pathway in bovine
mammary epithelial cells. J. Dairy Sci. 97 (6), 3660–3665.