Study in Activity Combination of Physalis angulata and Hibiscus
sabdariffa in 70% Ethanol Extract to Decrease Blood Sugar Levels
and Histopathology of Pancreas Langerhans Island in Alloxan
Induced Diabetic Rats
Hadi Sunaryo, Ni Putu Ermi Hikmawanti, Hesty Awanis Listyaningrum
Faculty of Pharmacy and Science, Universitas Muhammadiyah Prof. Dr. Hamka
Keywords: Physalis angulata L; Hibiscus sabdariffa L; Blood sugar; Pancreatic Islet of Langerhans
Abstract: Ciplukan (Physalis angulata L) and roselle petals (Hibiscus sabdariffa L) have been shown to have blood
sugar levels decreasing properties. This study aims to determine the effect of the Ciplukan and Roselle
petals ethanol extract combination to the decrease in blood sugar levels in alloxan-induced diabetic rats
(Sprague Dawley). The diabetic rats were divided into seven groups, each consisting of four rats. Group I
(negative control), group II (positive control), group III (ciplukan 10mg/200g BW), group IV (roselle petals
60mg/200g BW), group V (ciplukan 5mg/200g BW and roselle petals 30mg/200g BW), group VI (ciplukan
5mg/200g BW and roselle petals 60mg/200g BW) and group VII (ciplukan 10mg/200g BW and rosella
petals 30mg/200g BW). All groups were induced with alloxan. Parameters observed were decreased blood
sugar levels on the 15th and 30th day. After that, surgery and histopathological observation of pancreatic
organs were conducted at the 31st day. Blood sugar percentage was analysed using one-way ANOVA with
95% significance level and continued with the Tukey test. The results showed that group number VII
combination of 70% ethanol extract ciplukan with 10mg/200g BW dose and roselle flower petal dose
30mg/200 g BW decreased blood sugar level equal to glibenclamide dose 0.052mg/200g BW (p0.05) that
is equal to 66.74% and improvement activity of pancreatic islets of Langerhans, has been shown at the
histology result.
1 INTRODUCTION
Diabetes mellitus (DM) is a disease or disorder of
fat, carbohydrate, and protein metabolism caused by
a disturbance of insulin secretion, insulin work
(sensitivity), or both characterized by
hyperglycemia. Symptoms in DM patients such as
polyuria, polydipsia, polyphagia, weight loss, and
lethargy by hyperglycemia are the most common
early symptoms (Dipiro et al., 2014).
Synthetic antidiabetic drugs have side effects of
gastrointestinal disorders, anorexia, lactic acidosis,
vitamin B12 absorption disorders, and edema. Plants
as traditional medicine are easy to obtain and
generally has lower side effects when compared to
synthetic drugs. Components in one ingredient
(usually) have mutually supportive effects, and one
plant has multiple pharmacological effects that
makes it more suitable and safer for degenerative
metabolic disease (MOH RI, 2008). The ciplukan
(Physalis angulata L.) and roselle petals (Hibiscus
sabdariffa L.) are alternatives that can be used in the
treatment of DM (Sunaryo et al., 2012; Rosemary et
al., 2014).
Sunaryo et al. (2012) reported that the fraction of
chloroform ciplukan with the dose of 2mg/20g BW
in mice has the antidiabetic effect and can improve
the number of Langerhans pancreatic cells
proportional to glibenclamide. Rosemary et al.
(2014) reported that ethanol extract of roselle petals
at 600mg/kg BW dose was able to lower blood sugar
levels comparable with glibenclamide of 77.69% in
streptozocin-induced mice.
Based on this background, this study wanted to
prove the effect of ciplukan and roselle petals
ethanol extract in combination to the lowering of
sugar levels in alloxan-induced rats. The purpose of
combining the two extracts is to obtain smaller doses
and have activities that are comparable or even
greater than the single herbal extract.
Study in Activity Combination of Physalis angulata and Hibiscus sabdariffa in 70 .
DOI: 10.5220/0008240401170122
In Proceedings of the 1st Muhammadiyah International Conference on Health and Pharmaceutical Development (MICH-PhD 2018), pages 117-122
ISBN: 978-989-758-349-0
Copyright
c
2021 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
117
2 MATERIALS AND METHODS
2.1 Plant and Preparation of Extracts
Physalis angulata L. was obtained from Merapi
Farma Herbal Yogyakarta, and Hibiscus sabdariffa
L. obtained from Balai Tanaman Obat dan Rempah
(BALITTRO) Bogor. The reference drug used was
glibenclamide. A total of 1500 g of dried herb
ciplukan and 1500 g of dried roselle petals each
made into a powder. It was then sieved with mesh
no. 40, weighed, and recorded. As many as 900 g
and 1000 g of powder were each macerated by using
ethanol 70%. They were left soaking for the first 6
hours while stirring occasionally, and then let stand
for 18 hours. The maceration was performed three
repetitions using a new liquid. The macerate was
then evaporated using a vacuum rotary evaporator
until it turns into a viscous extract (MOH RI, 2008).
2.2 Phytochemical Screening
Phytochemical screening was performed using TLC
method by preparing a 10 mg/ml test solution in
ethanol for each extract. Prepared test solutions were
bottled on a silica gel TLC plate GF254. The
separation and detection systems of the compounds
are shown in Table 1.
2.3 Preparation of Animal
The animals used for study were white male rats of
Sprague Dawley strain, aged 2-3 months with a body
weight around 150-300 g, obtained from the Bogor
Agricultural University (IPB) of the Production
Division of Working Meat and Animal Livestock.
The experiment was completed with a randomized
design, using 28 male rats divided into seven groups
consisting of four rats. Before the experimental
treatment, the animals were first acclimatized in the
cage with standard feed and drink for approximately
seven days. The protocol no. 17-05-0486 has been
approved by the Health Research Ethics Committee
of the Faculty of Medicine, Universitas Indonesia
no. 453/UN2.F1/ETIK/2017.
2.4 Measurement of Blood Sugar
Levels
The rats fasted for 12 hours before treatment. Blood
sample was collected through the eye area (sinus
orbital). Blood glucose measurements were
performed on day 15 and after administration of the
test substance on day 30 using a spectrophotometer
clinical 300.
2.5 Observation of Pancreatic
Histopathology
The pancreatic histopathological observation process
began with surgery in rats that have been
anaesthetized with ketamine. The preparation stage
of preparation included the taking of pancreatic
organs, fixation, dehydration, clearing, embedding,
cutting, staining with Haematoxylin-Eosin, closing
the dosage, and observation by microscope
(Olympus x21) with 4x10 magnification.
2.6 Data analysis
The percentage data of decreased blood sugar levels
was statistically tested using ANOVA one way with
Table 1. Separation & detection system with TLC method
Compounds Mobile Phase Detection Reagent
Alkaloid Ethyl acetate:methanol:ammonia
(4:4:1)
Dragendroff
Saponin Chloroform:methanol
(1: 4)
Vanillin-Sulfuric acid
Tanin Ethyl acetate: methanol :
Formic acid 5%
(
4: 1: 1
)
FeCl
3
Flavonoid Acetic Acid: methanol :
formic acid 5%
(
4: 1: 1
)
Cytro-borate 5%
Terpenoid n-hexane: Ethyl acetate
(4: 1)
Liebermann-Bouchard
118
95% significance level. Then proceeded with the
Tukey test to determine the significant differences
between groups.
3 RESULTS AND DISCUSSION
3.1 Physalis angulata L. and Hibiscus
sabdariffa L. extracts
Table 2 shows the results of the extract of ciplukan
herb and roselle petals. Preparation of the dried
powder was carried to enlarge its surface area so that
the solvent used can be readily absorbed into dried
powder and the active compound which is attracted
more optimally (Vitasari, 2012), while pollination
will help break the walls and cell membranes to
maximize the extraction process (Koirewoa et al.,
2016).
The dried powder was extracted by maceration
using 70% ethanol. Ethanol is considered to be a
more selective (mouldy and bacteria growing in
ethanol 20% and above), non-toxic, neutral, and
well-absorbed (Pratiwi, 2010). Using ethanol as a
universal solvent is due to its easy-to-dissolve of
polar, semi-polar, and non-polar active substances
and its ability to precipitate proteins and inhibit the
action of enzymes to avoid hydrolysis and oxidation
processes (Dharma et al., 2010). According to
Indraswari (2008), ethanol with 70% concentration
is effective in attracting active compounds. The
ethanol solvent has two sides consisting of a polar
OH-group and a non-polar CH
2
CH
3
group. The
extraction by maceration was done because this
method can be used for large quantities of samples,
the implementation is simple, does not require
special treatment and the possibility of
decomposition of the active substance by the
influence of temperature can be avoided because
there is no heating process (Dharma et al., 2010).
Percentage of extract weight to dried powder shows
the effectiveness of the extraction process. The
effectiveness of the extraction process is influenced
by the type of solvent, particle size of dried leaves
powder, method and duration of extraction
(Istiqomah, 2013). The longer the extraction time,
the higher the saturation point of the solution. The
contact between the sample and the solvent can be
increased while it was shaking, to keep the contact
between the sample and the solvent frequent,
resulting in a more perfect extraction process
(Koirewoa et al., 2016). High percentage of extract
weight to dried powder results indicate the
possibility of the chemical compounds contained in
the extract is also high (Isnawati et al., 2006)
.
3.2 Phytochemical Screening
Phytochemical screening in this study used the thin
layer chromatography (TLC) method. The purpose
of using TLC is for more accurate and better
identification of compounds contained in the extract.
TLC is a way of physical separation with elements
to be separated distributed between two phases
(stationary phase and mobile phase). Separation is
based on migration differences and distribution of
compounds or ions (Astuti, 2016). The silica gel
plate GF254 was used as stationary phase, while the
eluent was used as the mobile phase varies for each
of the secondary metabolites to be tested. A good
eluent can separate the compound in large quantities
that are marked by the appearance of stains. The
stain that formed was not tailed, and the distance
between the stains with each other was clear
(Harborne, 1987). TLC plates were activated by the
oven at temperature 115°C for 15 minutes to remove
water contained in KLT plate (Sastrohamidjojo,
2007).
The results of screening tests can be found at
Table 3. Stains obtained from the TLC were still not
Table 2. Results of Ciplukan Herb Extraction and Rosella
Flower Petals
No Name Physalis
angulata L.
herb extract
Hibiscus
sabdariffa L.
flower petals
extract
1 Extract
weight
98.80 g 234 g
2
Percentage
of extract
weight to
dried
p
owde
r
10.98 % 23.4 %
Table 3. Phytochemical Screening Results
No. Compounds Physalis
angulata L.
Hibiscus
sabdariffa L.
1 Alkaloids + -
2 Flavonoids + +
3 Saponins - -
4 Tannin + -
5 Terpenoids + +
Note: (+) = detected
(-) = not detected
Study in Activity Combination of Physalis angulata and Hibiscus sabdariffa in 70
119
completely separated because the metabolite
compounds were not yet perfectly attracted, possibly
due to unsuitable eluents, inappropriate selection of
TLC plates, and extract concentrations that were too
high.
3.3 Results Measurement of Blood
Sugar Levels
The result of blood glucose levels of each group
before and after treatment can be seen in Figure 1.
The percentage data of decreased blood sugar levels
(Table 4) were inserted into statistics to test and
determine their normality and homogeneity with
Kolmogorov and Smirnov. The data of group I is not
included in statistics because the results were
different.
The data analysis showed normal data
distribution {(p=0.819) 0.05} and homogeneity
{(p=0.418) 0.05}. The analysis was continued by
ANOVA one-way test to find out whether there was
any significant difference or not in each treatment
group with p0.05. Based on the analysis results, the
value obtained was {(p=0.044) 0.05}. These results
indicate that giving a combination of herbal extract
ciplukan and roselle petals have a significant effect
on the decrease in blood sugar levels of diabetic rats.
The data then continued with the Tukey test to
know the difference between each treatment groups.
Based on the Tukey test, there was no significant
difference (p0.05) between the positive control
group and single ciplukan group, single roselle petal
extract, combination I, combination II and
combination III. This result means that in this case,
all test groups have activities that are proportional to
Table 4. Average percentage of reduced blood glucose
levels
Treatment groups Reduced blood glucose
levels
(
%
)
± SD
Ne
g
ative 2.86 ± 0.81
Positive 63.02 ± 2.83
Ciplukan ext
act 60.86 ± 3.65
Roselle petals extract 60.12 ± 0.02
Combination I 56.77 ± 5.99
Combination II 60.50 ± 3.80
Combination III 66.74 ± 4.02
Note: The test was performed with four repetitions.
Figure 1. Graph of average blood glucose levels (mg/dl)
of each group before and after treatment. (I) Negative
control group. (II) Glibenclamid dose 0.052mg/200g BW
group. (III) Extract Ciplukan dose 10mg/200g BW
group.(IV) Extract Roselle petals dose 60mg/200g BW
group. (V) Combination I (½ dose of Ciplukan: ½ dose of
Roselle petals) group. (VI) Combination II (½ dose of
Ciplukan: 1 dose of Roselle petals) group. (VII)
Combination III (1 dose of Ciplukan: ½ dose of Roselle
petals) group.
D
C
E
F
G
B
A
Figure 2. Histology of Pancreatic Islets Diabetic Rats
with Magnification 4x10: (A) The arrows indicate the
empty spaces (necrosis) of the Langerhans pancreas
island cell in the negative control group; (B) Positive
control group, no necrosis; (C) Ciplukan Extract
group; (D) Flower Rosela extract group; (E)
Combination I group; (F) Combination II group; (G)
Combination III group
120
the positive control of glibenclamide in lowering
blood sugar levels.
3.4 Observation of Pancreatic
Histopathology
The results of pancreatic histologic preparations can
be seen in Figure 2. Necrosis is cell or tissue death
due to a reversible degeneration process. This
condition can be caused by several things including
toxins, drugs, low blood supply, no nerve
preservation, temperature, light, radioactive and
mechanical trauma (Berata et al., 2011). The alloxan
mechanism in principle occurs through several
processes that stimulant produce damage effects on
pancreatic β cells. The reactive oxygen that is
formed can cause damage to pancreatic beta cells.
The destruction of pancreatic beta cells made a
decreased insulin secretion (Szukudelski, 2001).
The results of this study have shown that the
decrease in blood sugar levels and improvement of
the pancreatic Langerhans islet cells induced the
insulin secretion to compensate for high blood
glucose levels and subsequently lowered it. The
mechanism of decreased blood sugar levels and the
improvement of the islet cells of Langerhans were
derived from the compounds of flavonoids,
anthocyanins, and terpenoids contained in ethanol
extract 70% of ciplukan and roselle petals.
Flavonoids are antioxidants that can clean up
excessive free radicals, break the chain of free
radical reactions, and binding metal ions (chelating).
Flavonoids also have an inhibitory effect on the α-
glucosidase enzyme (Taufiqurrohman, 2015).
Anthocyanin has hypoglycaemic activity by
inhibiting α-glucosidase in the intestinal lumen and
improving insulin sensitivity (Kowalczyk et al.,
2013). Terpenoids have antidiabetic activity that can
stimulate the regeneration of Langerhans island cells
so that the cell damage of Langerhans Island in
particular β cells can be reduced gradually, the
increased number of Langerhans island cells also
allows the secretion of insulin so as to compensate
for high blood sugar levels and then decrease it
(Sunaryo et al., 2012).
4 CONCLUSIONS
The combination of 70% ethanol extract of ciplukan
and roselle petals successfully decreased blood sugar
levels in alloxan-induced rats. The combination of
70% ethanol extract of ciplukan and roselle flower
petals in dose I showed lower blood sugar level,
combination dose II equal to 60.50%, and
combination dose III equal to 66.74%.
Based on the statistical study, there was no
significant difference (p0.05) between the positive
control group and single ciplukan group, single
roselle petal, combination I, combination II and
combination III. This result means that in this case
all test groups have activities that are proportional to
the positive control of glibenclamide in lowering
blood sugar levels.
The combination of 70% ethanol extract of
ciplukan and roselle petals was able to provide an
improvement of the Langerhans island cell in
alloxan-induced rats compared with the negative
control group.
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