Toxicology Reports 9 (2022) 1213–1221

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Toxicology Reports
journal homepage: www.elsevier.com/locate/toxrep

White saffron (Curcuma mangga Val.) attenuates diabetes and improves

pancreatic β-cell regeneration in streptozotocin-induced diabetic rats
Dwiyati Pujimulyani a, *, Wisnu Adi Yulianto a, Astuti Setyowati a, Prastyo Prastyo a,
Sulkhan Windrayahya b, c, Ali Maruf d, e
a
Faculty of Agroindustry, University of Mercu Buana Yogyakarta, Jl. Wates Km 10, 55753 Yogyakarta, Indonesia
b
Particle and Interfacial Technology Group (PaInT), Department of Green Chemistry and Technology, Ghent University, Coupure Links 653, 9000 Ghent, Belgium
c
Centre for Food and Microbial Technology, KU Leuven, Kasteelpark Arenberg 22, B-3001 Haverlee, Belgium
d
Biotechnology Center, The Silesian University of Technology, Krzywoustego 8, 44-100 Gliwice, Poland
e
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, The Silesian University of Technology, Krzywoustego 4, 44-100 Gliwice, Poland

A R T I C L E I N F O A B S T R A C T

Handling Editor: Lawrence Lash Diabetes is a chronic disease caused by an imbalance of insulin release to the bloodstream in response to
excessive glucose influx, which causes hyperglycemia. White saffron (Curcuma mangga Val.), an Indonesian ar­
Keywords: omatic spice, contains essential phytochemicals and has a number of potential health benefits. Here, we
Diabetes examined the effects of oral administration of white saffron powder (WSP): 1.5 and 4.5 g (WSP 1.5 and WSP 4.5)
Glucose
in streptozotocin (STZ)-induced diabetic rats. WSP was administered orally on a daily basis for one month and its
Insulin
antidiabetic, anti-inflammatory, and antioxidative effects were investigated by measuring the concentrations of
Pancreatic β-cells
White saffron blood glucose, insulin, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin 8 (IL-8), malondialde­
hyde (MDA), and superoxide dismutase (SOD) activity. In response to high WSP intervention (WSP 4.5), treated
rats showed increased insulin level and SOD activity and reduced blood glucose, IL-6, IL-8, TNF-α, and MDA
levels, which were closely related to the positive control (PC) group. In addition, Hematoxylin and Eosin (H&E)
staining of the pancreatic tissues showed that WSP 4.5-treated rats had significant improvement in β-cell
regeneration, which taken together reflected the antidiabetic potential of Curcuma mangga Val.

1. Introduction abnormalities (microangiopathy and macroangiopathy) [3]. The former

Diabetes mellitus, commonly known as diabetes, is a chronic disease
that predominantly changes the metabolism of macronutrients (e.g.,
carbohydrates, proteins, and fats), electrolytes, and water due to
abnormal insulin secretion. The number of population worldwide
suffering from diabetes was estimated around 451 million people in
2017 [1]. Notably, about 80 million of them were in Southeast Asia, and
it was predicted to reach 150 million by 2045 [1]. Data from the World
Health Organization (WHO) in 2016 revealed that the prevalence of
diabetes among Indonesians was around 7.0% [2]. The primary risk
factors, including overweight, obesity, and physical inactivity accounted
for 24.4%, 5.7%, and 22.8%, respectively [2]. Other factors, such as
population growth, increased numbers of elderly people, urbanization,
eating patterns, and unhealthy lifestyles are believed to escalate dia­
betes cases in the future.
Diabetes can cause acute complications, such as vascular
is mainly found in diabetic retinopathy and nephropathy that can cause
blindness and kidney failure [3,4]. Meanwhile, macroangiopathy can be
found in the lower limbs and blood vessels that can cause gangrene and
coronary heart disease [5,6]. Therefore, effective, affordable, and safe
treatments are necessary, especially by using local materials that are
easily obtained.
Recently, various types of medicinal plants have been explored to
cure diabetes, such as Mangifera indica, sambiloto (Andrographis pan­
iculata [Burn.f.] Ness), ngai camphor, Madagascar periwinkle, green tea,
Java plum, Java tea, mahogany (Swietenia mahagoni Jacq), Plectranthus
esculenthus N.E.Br, and Indonesian bay leaf (Syzygium polyanthum
[Wight.] Walp) [7–9]. White saffron (Curcuma mangga Val.), a natural
aromatic spice, is widely grown in Indonesia and commonly used as
traditional medicine as well, which is linked to its rich content in cur­
cuminoid [10].
Due to the increasing popularity of medicinal plants as alternative

* Corresponding author.
E-mail address: dwiyati@mercubuana-yogya.ac.id (D. Pujimulyani).

https://doi.org/10.1016/j.toxrep.2022.05.014
Received 11 April 2022; Received in revised form 13 May 2022; Accepted 16 May 2022
Available online 20 May 2022
2214-7500/© 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
D. Pujimulyani et al. Toxicology Reports 9 (2022) 1213–1221

sources of treatment, many studies have been conducted to explore their

potential for clinical relevance. For instance, the pharmacological
characteristics of phytochemical compounds from different medicinal
plants have been studied to treat different kinds of chronic diseases, such
as diabetes and hypertension [11,12]. The contained bioactive phyto­
chemicals (e.g., phenolic compounds, flavonoids, coumarin, and terpe­
noid) might play a role in glucose-lowering effects [11,12].
In our previous study, we have studied in vitro hypoglycemic activity
of white saffron, which was related to its ability to modulate glucose
transporter type 4 (GLUT4), a transmembrane receptor that is respon­
sible for insulin-dependent glucose uptake [13]. At the same time, white
saffron could reduce peroxisome proliferator activated receptor γ
(PPAR-γ) expression in adipocytes, which is relevant to its function to
regulate lipid deposition [13]. In the current study, we attempted to
study in vivo antidiabetic, anti-inflammatory, and antioxidative activ­
ities of white saffron in STZ-induced diabetic rats. Low and high dosages
of WSP were administered to diabetic and non-diabetic rats for a month
to compare their effects. H&E staining of pancreatic tissues was also
conducted to compare β-cells integrity.

2. Materials and Methods

2.1. Materials

White saffron was planted in manure-enriched soil in a 4000-meter

square private garden located in Sedayu, Bantul, Yogyakarta. Ready-
to-harvest white saffron (mature, aged 10 months) is characterized by
its fallen leaves, bright yellow-flesh rhizomes, and mango-like smell.
After harvesting, white saffron rhizomes (all parts: main rhizome, first
branch, and second branch) were washed, peeled, and blanched with hot
water containing 0.05% citric acid for 5 min. Then, they were cut into
small pieces, dried in a cabinet dryer, and ground to obtain WSP.

2.2. Measurement of curcumin content in white saffron

Curcumin content of white saffron was measured from different parts

of the rhizomes, including the main rhizome, first branch, and second
branch (Fig. 1). Initially, fresh white saffron was ground and extracted
with ethanol and its total curcuminoid content was quantified spectro­
photometrically at the wavelength of 425 nm. For quantifying the cur­
cumin content, 5 µL of the filtrate was introduced into thin-layer
chromatography (silica gel 60 F254) and put into a chamber containing
chloroform:methanol mobile phase (98:2 v/v). The plate was taken and
dried once it was thoroughly developed with the eluent. The absorbance
of the formed spots was measured by a densitometer at 425 nm, and the
curcumin content was determined from the total curcuminoid.
2.3. Animal study
Twenty four white rats (Rattus norvegicus L.) (male, aged 8 weeks,
bodyweight: 180–210 g) were divided into four groups, namely PC, NC,
WSP 1.5, and WSP 4.5 (6 rats for each group). Adaptation feeding
(standard diet) was given for one week, following STZ induction for NC,
WSP 1.5, and WSP 4.5 groups by a single intraperitoneal (IP) injection
(45 mg/kg) in citrate buffer (pH 4.5). Meanwhile, the PC group was
normal rats without STZ induction. After 3 days of STZ induction, rats
were measured their blood glucose levels for confirmation of diabetes
status (higher than 250 mg/dL is considered diabetes), stated as week
0 (W0) [14–16]. WSP was given orally using a blunt microsyringe, and
the study was conducted for one month (Fig. 2). To assess the antidia­
betic, anti-inflammatory, and antioxidative activities of WSP in diabetic
rats, the representative variables, including blood glucose and insulin,
IL-6, IL-8, and TNFα, and SOD and MDA were measured at different time
intervals (week 1, 2, and 4: W1, W2, and W4, respectively). The animal
study was conducted ethically in the Center for Food and Nutrition
Studies (PSPG UGM), Gadjah Mada University, Indonesia, under the
Fig. 1. White saffron rhizome and its parts: main rhizome, first branch, and
second branch (before and after peeling and slicing).
approval of the Health Research Ethics Committee, Faculty of Health
Science, Yogyakarta Respati University (UNRIYO), Yogyakarta,
Indonesia (ethical clearance number: 180.3/FIKES/PL/VII/2019).
2.4. Red blood cells and blood plasma sample preparation
Rats were anesthetized intramuscularly with ketamine (60 mg/kg)
and then their blood were withdrawn from the orbital sinus route using
EDTA as the anticoagulant and the obtained blood was transferred into
an eppendorf tube following centrifugation at 3000 rpm for 10 min to
isolate red blood cells (RBCs) and blood plasma for further assays.
2.5. Determination of blood glucose level
Glucose level was quantified using the commercially available Dia­
Sys kit (DiaSys Diagnostic System, Germany). The kit has a sensitive
determination of glucose concentrations from 1 to 400 mg/dL. Glucose
determination was done after enzymatic oxidation by glucose oxidase.
Quinoneimine was used as the colorimetric indicator, which was
generated via hydrogen peroxide-mediated oxidation of 4-aminoanti­
pyrine and phenol (peroxidase was used as the catalyst, Trinder’s re­
action). Briefly, freshly isolated blood plasma (10 µL) or standard
(100 mg/dL, 10 µL) was mixed with 1.0 mL of reagent containing
phosphate buffer (pH 7.5), phenol, 4-aminoantipyrine, glucose oxidase,

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Fig. 2. A schematic diagram of the animal study. Twenty four white rats (Rattus norvegicus L.) were divided into four groups: PC, NC, WSP 1.5, and WSP 4.5 (6 rats
for each group). Adaptation feeding (standard diet) was given for one week, following STZ induction (blue lines) for NC, WSP 1.5, and WSP 4.5 groups. After 3 days
of STZ induction, rats were measured their blood glucose levels (red lines) for confirmation of diabetes status, stated as week 0 (W0). Then, daily oral WSP was given
for WSP 1.5 and WSP 4.5 groups until W4 (purple lines). At W1, W2, and W4, rats were analyzed their blood glucose, insulin, IL-6, IL-8, TNFα, and MDA levels as well
as SOD activity. At the end of the study, all rats were sacrificed for obtaining their pancreatic tissues. (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)

and peroxidase. Then, the mixture was incubated at 25 ºC for 10 min,
following the absorbance measurement at a 500-nm wavelength. The
glucose concentration was measured by comparing the absorbance of
samples with the standard, which was expressed in mg/dL.
2.6. Determination of insulin level
reaction, each well was added with 3,3′ ,5,5′ -tetramethylbenzidine
(TMB) substrates (90 µL) following the final incubation for 10–20 min in
dark conditions. To stop the reaction, stop solution (50 µL) was added
into each well, yielding a yellow color. The plate was then placed into a
microplate reader to measure its absorbance at 450 nm and then the
insulin concentration was calculated based on the standard curve.

An enzyme-linked immunosorbent assay (ELISA) kit (Fine-Test,
China) was employed to measure the insulin level following the given
instruction. Briefly, 100 µL of standard curve solutions, known diluted
blood plasma samples, and blank (dilution buffer) were placed into a 96-
well plate, sealed, and incubated at 37 ºC for 1.5 h. Then, the solution
was discarded and washed twice with washing buffer before adding
100 µL of biotin-labeled antibody. The plate was then incubated again
for another 1 h. After incubation finished, solutions were removed and
washed thrice with washing buffer (1–2 min gentle shaking for each
wash). Immediately, 100 µL of horseradish peroxidase (HRP)-strepta­
vidin conjugate (SABC) solution was added into each well following
incubation at 37 ºC for 30 min. Then, solutions were again removed and
washed five times with washing buffer. To visualize HRP enzymatic
2.7. Determination of IL-6, TNFα, and IL-8 levels
IL-6, TNFα, and IL-8 levels were also measured using an ELISA kit
(Fine-Test, China), following the same procedure as the kit for
measuring insulin level. However, every kit used a different standard
with different ranges of a standard curve (the detection sensitivity is
different). The procedure of measurement was as follows. Briefly, 100 µL
of standard curve solutions, known diluted blood plasma samples, and
blank (dilution buffer) were placed into a 96-well plate, sealed, and
incubated at 37 ºC for 1.5 h. Then, the solution was discarded and
washed twice with washing buffer before adding 100 µL of biotin-
labeled antibody. The plate was then incubated again for another 1 h.
After incubation finished, solutions were removed and washed thrice

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D. Pujimulyani et al.
with washing buffer (1–2 min gentle shaking for each wash). Immedi­
ately, 100 µL of SABC solution was added into each well following in­
cubation at 37 ºC for 30 min. Then, solutions were again removed and
washed five times with washing buffer. To visualize HRP enzymatic
reaction, each well was added with 90 µL of TMB substrates following
the last incubation for 10–20 min in dark conditions. To stop the reac­
tion, stop solution (50 µL) was added into each well, yielding a yellow
color. The plate was then placed into a microplate reader to measure its
absorbance at 450 nm and then the IL-6, TNFα, and IL-8 concentrations
were calculated based on the standard curve.
2.8. Determination of SOD activity
SOD activity was measured using the SOD activity kit (GenWay
Biotech Inc., USA), following the procedure from the manufacturer.
Briefly, 500 µL of isolated RBCs were diluted into 2.5 mL (5 ×) using
cold distilled water, mixed, and centrifuged at 10000g for 10 min to
pellet the RBC membranes. Then, the obtained supernatant was
collected and diluted 100 × prior to SOD activity assay. To conduct the
assay, three different blanks were prepared for each sample in different
wells in a 96-well plate. Firstly, blank 1 consisted of distilled water,
water-soluble tetrazolium salts (WST) working solution, and enzyme
working solution (20, 200, and 20 µL, respectively). Secondly, blank 2
consisted of sample solution, WST working solution, and dilution buffer
(20, 200, and 20 µL, respectively). Thirdly, blank 3 consisted of distilled
water, WST working solution, and dilution buffer (20, 200, and 20 µL,
respectively). Finally, each sample solution (20 µL) was mixed with WST
working solution (200 µL) and enzyme working solution (20 µL). Then,
the plate was incubated at 37 ºC for 20 min, following an immediate
measurement of absorbance in a microplate reader (450 nm wave­
length). SOD activity was expressed as % inhibition rate, by using this
equation:
(Abs blank 1 − Abs blank 3) − (Abs sample − Abs blank 2)
SOD activity(%inhibition rate) =
(Abs blank 1 − Abs blank 3)
2.9. Determination of MDA level
A thiobarbituric acid reactive substance (TBARS) method was
employed to estimate the MDA level. Briefly, a solution of phosphoric
acid:blood plasma (15:1 v/v, 800 µL) was freshly prepared in 15-mL
polypropylene tube and mixed with thiobarbituric acid solution
(250 µL, 40 mM). The obtained mixture was diluted with distilled water
(450 µL), gently vortexed, and heated for 1 h (the lid was closed tightly
during heating). Then the solution was cooled down in an ice bath,
mixed gently, and applied to a prewashed Sep-Pak C18 column. Meth­
anol (4 mL) was used to elute TBARS from the column. Finally, the MDA
level was measured from a generated standard curve of tetraethox­
ypropane (TEP) in a spectrophotometric method (wavelength: 532 nm),
where the level of lipid peroxides was indicated as nmol of MDA.
2.10. H&E staining
The euthanasia procedure was conducted by a single injection of
ketamine solution (100 mg/kg). Then, rats were dissected and harvested
their pancreatic tissues, following fixation in Bouin’s solution. After
fixation, the tissues were dehydrated, embedded in paraffin, and
sectioned at 5 µm. The selected tissue sections were then stained with
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Toxicology Reports 9 (2022) 1213–1221
H&E for comparing the histological characteristics between samples,
especially islets of Langerhans and β-cells integrity.
2.11. Statistical analysis
The obtained data were expressed as mean ± standard deviation
(SD) of six repetitions (n= 6). Statistical analysis was conducted to
measure any significant differences using one-way analysis of variance
(ANOVA, first stage) and Duncan’s Multiple Range Test (DMRT, post hoc
test) with a 95% confidence interval (p < 0.05). In addition, to deter­
mine a significant difference between two selected groups, unpaired
two-tail Student’s t-test with significant levels set to nsp > 0.05,
* p < 0.005, * * p < 0.01 * ** p < 0.001, and * ** * p < 0.0001 was
included.
3. Results and Discussions
3.1. Curcumin content in white saffron
Curcuma mangga Val. contains many bioactive compounds including
curcumin, gallic acid (GA), catechin (CA), epicatechin (EP), epi­
gallocatechin (EPG), epigallocatechin gallate (EPGG), and gallocatechin
gallate (GG). In our previous study, we have characterized the GA, CA,
EP, EPG, EPGG, and GG contents in fresh Curcuma mangga Val. extract,
namely 12.4, 13.4, 44.2, 11.3, 3.7, and 15.9 mg/100 g dried extract,
respectively [17]. In the present study, we measured the curcumin
content of Curcuma mangga Val. from different parts of the rhizomes. The
main rhizome had more than double the curcumin content of the first
and second branches, which were 88.6 vs 37.5 and 31.4 mg/100 g dried
extract, respectively (Table 1).
3.2. White saffron maintains rats bodyweight
× 100
As seen from Fig. 3, the bodyweight of diabetic rats without WSP
intervention gradually declined from 203.2 to 183.2 g. Meanwhile, the
PC group showed increased bodyweight from 203.3 to 239.2 g. After
treatment with WSP 1.5 and 4.5, the bodyweight of rats indicated a
steady increase, in which WSP 4.5 showed a significant increase of the
bodyweight at the end of study. Similar results were reported on STZ-
induced diabetic rats [18]. STZ caused a significant reduction in the
bodyweight of albino rats as observed after 2, 4, 6, and 8 weeks
post-induction [18].
3.3. White saffron reduces glucose level and improves insulin level
In general, diabetic rats had blood glucose levels nearly four times
higher than non-diabetic rats, as shown in Fig. 4A, while the figures for
WSP 1.5 and 4.5 exhibited a visible drop in blood glucose levels from W1
to W4. In the WSP 1.5 group, blood glucose level decreased from
270.39 mg/dL (W1) to 186.33 and 125.10 mg/dL (W2 and W4,
Table 1
Curcumin contents of fresh Curcuma mangga Val. extract from different parts of
the rhizomes.
Rhizome parts Curcumin content (mg/100 g dried extract)
Main rhizome 88.6
First branch 37.5
Second branch 31.4
D. Pujimulyani et al.
Fig. 3. Measurement of rats bodyweight from the beginning of study until W4
of WSP treatment. Data are presented as mean ± SD (n = 6).
respectively). Similarly, in the WSP 4.5 group, blood glucose level
decreased from 264.77 mg/dL (W1) to 144.54 and 87.66 mg/dL (W2
and W4, respectively). These results indicated that the reduction in the
glucose level was dose- and time-dependent with the optimal treatment
was WSP 4.5, in which circulating glucose was almost reaching the
normal level after one month of treatment.
According to Widowati (2008), the mechanism of action of various
plants as antidiabetic agents can be divided into three parts [19]. (1)
They can agglomerate intestinal-mucosa membrane protein and build a
protective layer that can inhibit glucose intake and normalize blood
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Toxicology Reports 9 (2022) 1213–1221
glucose rate. (2) They can accelerate blood circulation and kidney
filtration and excretion that enhance urine production-mediated glucose
excretion through the kidneys so that the glucose level in the blood will
decrease. (3) They can accelerate glucose excretion through increased
metabolism or fat storage mediated by the pancreas to produce insulin
[19].
White saffron contains an abundance of phenolic compounds [10,
17]. Phenolic compounds such as flavonoids, phenols, flavonols, and
proanthocyanidins in plants have potential as antioxidants and antidi­
abetic agents [20]. Flavonoids that are beneficial to diabetic patients
include interradiain, quercetin, rutin, diosmin, luteolin, CA, and cin­
namic acid [20,21]. Flavonoids appear as aglycones and glycosylated
and methylated derivatives [22]. Flavonoid compounds are believed to
have antidiabetic effects due to their antioxidant properties that can
protect our bodies against free radicals and other oxidizers. A recent
study showed that Cistus laurifolius L. contained GA, CA, EP like Curcuma
mangga Val. and other bioactive compounds (e.g., rutin, p-coumaric
acid, and resveratrol) and its treatment (250 mg/kg C. laurifolius L.
extract) in STZ-induced diabetic rats could reduce blood glucose level
from 355 to 288 mg/dL and increase insulin level from 9.5 to 14.8
μU/mL [23].
On the other hand, Fig. 4B shows that the insulin level was the lowest
in diabetic rats, while rats given WSP 1.5 and 4.5 for four weeks showed
relatively high insulin levels close to the normal rats, which were 491.02
and 516.52 pg/mL, respectively. White saffron contains curcumin,
which acts as antioxidant. A recent study showed that curcumin could
maintain β-cell function in vivo by inhibiting phosphodiesterase activity
[24]. As a result, the production of insulin could be regulated due to the
normal function of β-cells.
Fig. 4. (A) Blood glucose levels of diabetic rats
after treatment with WSP 1.5 and WSP 4.5 for
one week (W1), two weeks (W2), and four
weeks (W4). Adaptation feeding was given
before STZ induction and confirmation of dia­
betes status, stated as week 0 (W0). (B) Blood
insulin levels of diabetic rats after treatment
with WSP 1.5 and WSP 4.5 for four weeks. PC:
non-diabetic rats, NC: diabetic rats. Data are
presented as mean ± SD (n = 6). Different no­
tations with the same color (green, pink, blue,
red, or black) indicate a significant difference
among groups. (For interpretation of the refer­
ences to colour in this figure legend, the reader
is referred to the web version of this article.)
D. Pujimulyani et al. Toxicology Reports 9 (2022) 1213–1221

Fig. 5. (A) IL-6, (B) TNF-α, and (C) IL-8 levels of diabetic rats after treatment with, WSP 1.5 and WSP 4.5 for one week (W1), two weeks (W2), and four weeks (W4).
PC: non-diabetic rats, NC: diabetic rats. Data are presented as mean ± SD (n = 6). Different notations with the same color (blue, red, or black) indicate a significant
difference among groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. White saffron reduces IL-6, TNF-α, and IL-8 levels
The anti-inflammatory effects of WSP against diabetes are shown in
Fig. 5A-C. It is clear that diabetic rats had much higher IL-6, TNF-α, and
IL-8 levels than the normal rats. Meanwhile, diabetic rats treated with
WSP 1.5 and 4.5 showed a significant decrease in IL-6, TNF-α, and IL-8
levels from W1 to W4. In the WSP 1.5 group, IL-6, TNF-α, and IL-8 levels
of diabetic rats decreased from 190.25, 13.49, and 326.68 pg/mL (W1)
to 137.17, 11.66, 170.48 pg/mL (W2) and 122.10, 9.94, and 161.68 pg/
mL (W4), respectively. Similarly, in the WSP 4.5 group, IL-6, TNF-α, and
IL-8 levels of diabetic rats dropped from 187.42, 12.84, and 322.10 pg/
mL (W1) to 140.10, 9.94, and 134.02 pg/mL (W2) and 97.39, 8.11, and
89.09 pg/mL (W4), respectively.
The phenolic and flavonoid compounds from white saffron might
play a key role in reducing those pro-inflammatory cytokines [25]. IL-6
is mainly secreted by white blood cells and its overproduction might
induce insulin resistance [26]. Meanwhile, TNF-α, one of the critical
inflammatory cytokines, is secreted by macrophages/monocytes during
severe inflammation and plays a primary role in various cellular
signaling pathways [27,28]. The high level of TNF-α in the bloodstream
is associated with multiple chronic diseases, including cardiovascular
diseases, inflammatory bowel disease, and diabetes [27,28]. On the
other hand, IL-8 is a chemokine that directly contributes to macrophage
infiltration and activation in adipose tissue and has been studied to get
involved in type 2 diabetes and atherosclerosis [29]. WSP intervention
for one month could reduce the IL-6, TNF-α, and IL-8 levels in diabetic
rats.
3.5. White saffron improves SOD activity and reduces MDA level
According to the data in Fig. 6 A,B, it is clear that, as increased dose
and prolonged time of WSP treatment, the SOD activity of diabetic rats

Fig. 6. (A) SOD activity and (B) MDA level of diabetic rats after treatment with WSP 1.5 and WSP 4.5 for one week (W1), two weeks (W2), and four weeks (W4). PC:
non-diabetic rats, NC: diabetic rats. Data are presented as mean ± SD (n = 6). Different notations with the same color (blue, red, or black) indicate a significant
difference among groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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D. Pujimulyani et al.
increased considerably, while the MDA level of diabetic rats declined
significantly compared to the NC group. In the WSP 1.5 group, the SOD
activity of diabetic rats increased from 23.53% (W1) to 50.63% and
57.05% (W2 and W4, respectively), while the MDA level decreased from
9.08 nmol/mL (W1) to 6.59 and 4.28 nmol/mL (W2 and W4, respec­
tively). Similarly, in the WSP 4.5 group, the SOD activity of diabetic rats
increased from 24.84% (W1) to 63.52% and 70.83% (W2 and W4,
respectively), while the MDA level decreased from 8.62 nmol/mL (W1)
to 5.66 and 3.25 nmol/mL (W2 and W4, respectively). After treatment
with WSP 1.5 and 4.5 for one month, both SOD activity and MDA level of
diabetic rats were closely related to the PC group, while the NC group
showed relatively low SOD activity and high MDA level, as seen in Fig. 6.
White saffron antioxidants in the form of curcumin and polyphenols
could increase SOD levels as the forefront of defense against free radicals
[10,30]. Recently, Pujimulyani et al. (2020) [31] have studied an oral
administration of WSP to the oxidized peanut oil-treated wistar rats. The
results showed that WSP treatment could increase the SOD and vitamin
E levels and reduce the MDA levels [31]. MDA is the final product of
lipid oxidation. High MDA levels are influenced by lipid peroxide levels,
which also indirectly indicate a high amount of free radicals [32]. In the
previous study, white saffron extract and fractions showed the ability to
capture free radicals, such as nitric oxide (NO) and hydrogen peroxide
(H2O2) [25].
3.6. White saffron maintains β-cells integrity
Pancreatic β-cells play a primary role in producing and releasing
insulin in a strictly controlled manner in order to keep the circulation of
glucose at a normal range [33]. It is crucial to preserve β-cell structure
and function and prevent damages that can cause cell death-induced
disrupted cell integrity. According to Anděl et al. (2014) [34], various
factors could damage or destroy β-cells, including metabolic factors (e.
g., hyperglycemia, lipotoxicity, and reactive oxygen species), pharma­
cological factors (e.g., antimicrobial medication and antidepressants),
Fig. 7. H&E staining of pancreatic tissues, showing the islets of Langerhans and β-cell integrity after treatment with (C) WSP 1.5 and (D) WSP 4.5 for four weeks. (A)
PC: non-diabetic rats, (B) NC: diabetic rats. Scale bar: 50 µm.
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cystic fibrosis (e.g., infections, inflammation, and autoimmunity),
environmental toxic factors (e.g., STZ and rat poison), impaired insulin
secretion, exocrine disorders, and viruses [34].
From Fig. 7A-D, it is clear that the NC group showed disrupted β-cells
structure and integrity compared to the PC group, which was caused by
diabetes. β-cells and the islets of Langerhans in the PC group were in
normal condition. Meanwhile, WSP 1.5 and WSP 4.5 groups indicated a
significant improvement in β-cells regeneration compared to the NC
group. There are three different types of impaired functions on the
destruction of β-cells, namely decreased sensitivity to glucose, impaired
insulin secretion (biphasic profile and pulsatility), and decreased β-cell
mass-induced uncontrolled glucose homeostasis [35,36].
Natural compounds in various forms, such as berberine, curcumin,
and mangiferin as well as extracts of medicinal plants have been found
to have protective and regenerative effects on β-cells [36]. For instance,
a recent study showed that curcumin had anti-diabetic effects as it could
help to regenerate β-cells in vivo by the immunomodulatory effect on T
helper1-related cytokines (IL-2) as well as the immunosuppressive ac­
tion on IFN-γ, IL-6, and IL-1β [37]. Other study showed that intraperi­
toneal injections of turmeric (Curcuma longa) extract, which is the main
source of curcumin, could significantly reduce blood glucose level from
around 400–100 mg/dL and showed protective effects on pancreatic and
renal structure and function [38]. In addition to that, extract of
Sargassum oligocystum algae, which is rich in phytochemical compounds
such as tannins, alkaloids, saponins, and flavonoids, could regenerate
β-cells of diabetic rats as well as maintain their function as measured by
calculating the homeostasis model assessment of β-cells (HOMA-B) [39].
4. Conclusions
According to the results of the in vivo study, WSP exhibited antidi­
abetic, anti-inflammatory, and antioxidative effects on diabetic rats.
Rats given WSP 1.5 and WSP 4.5 had blood glucose, insulin, IL-6, TNF-α,
IL-8, and MDA levels as well as SOD activity that were close to the PC
D. Pujimulyani et al.
group and the WSP treatment showed a dose- and time-dependence
during the normalization process. The higher the WSP dose given to
diabetic rats, the lower the blood glucose, IL-6, TNF-α, IL-8, and MDA
levels and the higher the SOD activity and the insulin level of diabetic
rats. Similarly, the longer the WSP intervention in diabetic rats, the
lower their blood glucose, IL-6, TNF-α, IL-8, and MDA levels and the
higher the SOD activity and the insulin level of diabetic rats. In addition,
WSP treatments could significantly improve β-cell regeneration as
observed by H&E staining in the end of the study. Taken together, white
saffron is a promising aromatic spice having potential antidiabetic
effects.
CRediT authorship contribution statement
Dwiyati Pujimulyani: Conceptualization, Methodology, Validation,
Resources, Data curation, Writing – original draft, Writing – review &
editing, Supervision, Project administration, Funding acquisition.
Wisnu Afi Yulianto: Conceptualization, Methodology, Validation,
Writing – review & editing, Supervision. Astuti Setyowati: Conceptu­
alization, Methodology, Writing – review & editing. Prastyo Prastyo:
Formal analysis, Investigation, Data curation, Writing – original draft.
Sulkhan Windrayahya: Software, Data curation, Writing – original
draft, Writing – review & editing, Visualization. Ali Maruf: Software,
Data curation, Writing – original draft, Writing – review & editing,
Visualization
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
We gratefully acknowledge the funding from Excellence Higher Ed­
ucation Fundamental Research [Grant No.: 111/SP2H/LT/DRPM/2019]
by The Indonesian Ministry of Research, Technology and Higher Edu­
cation (RISTEK DIKTI, Indonesia).
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