Effect Of Mangosteen Peel Extract On Increasing The Number Of Endocrine Cells In Pancreas Langerhans Islands And Decreasing Tumor Necrosis Factor ...
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
Effect Of Mangosteen Peel Extract On Increasing
The Number Of Endocrine Cells In Pancreas
Langerhans Islands And Decreasing Tumor
Necrosis Factor Alfa In
Streptozotocin-Induced Male Rats
Novera Herdiani1*, Bambang Wirjatmadi2 , Kuntoro3, Edza Aria Wikurendra4
1
Doctoral Program of Public Health, Faculty of Public Health, Airlangga University, Surabaya, Indonesia and
Department of Public Health, Faculty of Health, Universitas Nahdlatul Ulama Surabaya, Indonesia
2
Faculty of Public Health, Airlangga University, Surabaya, Indonesia
3
Faculty of Public Health, Airlangga University, Surabaya, Indonesia
4
Department of Public Health, Faculty of Health, Universitas Nahdlatul Ulama Surabaya, Indonesia
Email novera.herdiani-2019@fkm.unair.ac.id
DOI: 10.47750/pnr.2023.14.03.439
This study aims to analyze the effect of mangosteen peel on increasing the number of pancreatic islets in Langerhans endocrine
cells and decreasing TNF-α in male white rats induced by streptozotocin. The research conducted was laboratory experimental in
nature. The research design used is a Randomized Post Test Only Control Group Design. The samples were 24 male white rats of
the Wistar strain. Rats were grouped into six groups: negative control group (KN), positive control STZ (K1), positive control
metformin synthetic drug therapy (K2), mangosteen rind extract therapy dose of 200 mg/kgbb (K3), a dose of 400 mg/kgbb (K4),
a dose of 600 mg/kgbb (K5). Administration of therapy for 14 days orally. Pancreatic organs were examined by HE and IHC staining.
Pancreatic histopathological observation and TNF-α expression were carried out microscopically. Statistical analysis using the
Kolmogorov-Smirnov, ANOVA test, and Pearson's correlation. The results of the effect of mangosteen peel extract can increase the
proliferation of endocrine cells in the islets of Langerhans, at a dose of 600 mg/kg BW/day. While the decrease in TNF-α expression
was shown by all doses of mangosteen peel extract and appeared to be significantly different when compared to the K1 group,
namely the STZ-induced group without any therapy. Pearson's correlation results show that there is an inverse relationship between
the number of endocrine cells and TNF-α expression. The conclusion is that administration of mangosteen peel extract can increase
the number of pancreatic islets of Langerhans endocrine cells and decrease Tumor Necrosis Factor Alpha in streptozotocin-induced
male white rats. Suggestions need to do research on anti-inflammatory cytokines that occur in endocrine cells of the islets of
Langerhans due to streptozotocin-induced mangosteen peel extract in rats so that there is a balance with pro-inflammatory
cytokines.
Keywords : mangosteen peel, islets of langerhans, streptozotocin, TNF-α
1. Introduction
Diabetes Mellitus (DM) is a major health problem worldwide. DM is a group of metabolic diseases characterized by
hyperglycemia resulting from impaired insulin secretion, insulin activity, or both (Kangralkar et al., 2010).
Hyperglycemia is a clinical indication of DM, a state of continuous hyperglycemia triggers dysfunction and decreased
mass of pancreatic β cells as insulin producers. Dysfunction and decreased mass of pancreatic β-cells indicate a
histological change in the islets of the Langerhans pancreas and trigger an inflammatory process that occurs in the
pancreas (Mandarim-de-Lacerda, 2019). DM is related to the occurrence of an inflammatory process. Destructive pro-
inflammatory cytokines can affect insulin sensitivity and the function of pancreatic beta cells. The pathogenesis of
Journal of Pharmaceutical Negative Results ¦ Volume 14 ¦ Regular Issue 03 ¦ 2023 3539type 2 diabetes mellitus is characterized by metabolic disorders, namely a decrease in peripheral tissue response in
response to insulin (insulin resistance) (Kumawat et al., 2009). Damage to peripheral tissues is thought to result from
an increase in free radicals in the body, which damage insulin receptors or glucose transporters found in cell
membranes (Ueda-Wakagi et al., 2019). Based on the International Diabetes Federation (IDF) stated that in 2005 in
the world there were 200 million (5.1%) people with diabetes (diabetes) and it is suspected that 20 years later, namely
in 2025 it will increase to 333 million (6.3%) people (Atlas, 2015). In 2030, there will be 21.3 million people with
DM (diabetes) in Indonesia (Kesehatan & RI, 2013). The 2018 Riskesdas results in Indonesia showed that there was
an increase in the prevalence of DM from 6.9% in 2015 to 8.5% in 2018. DM with poor control of blood glucose
levels can eventually cause pancreatic β-cell disorders, including a decrease in cell mass and function(Ma et al., 2012).
Damage to pancreatic β cells in type 2 DM can be caused by genetic factors, and other factors such as an increase in
inflammatory cytokines, namely Tumor Necrosis Factor alpha (TNF-α) ) (Lampropoulou et al., 2020).
Cytokines are a glycoprotein inferred from aide T cells, natural killer (NK) cells, and macrophages, which play
a vital part in the body's reaction. Type 1 helper T cells (Th-1) produce IFN-γ (interferon gamma), IL-2 (interleukin-
2), and TNF-α (Tumor Necrosis Factor alpha). These cytokines enact macrophages, to make proinflammatory
cytokines such as TNF-α and actuate cytotoxic effector immune mechanisms from macrophages (Ban et al., 2020).
TNF-α is a cytokine that acts as a pyrogen. At low levels, it activates the cellular immune system. The double part of
cytokines, particularly TNF-α, which at the proper levels will give assurance and mending. Be that as it may,
intemperate levels will cause very severe and fatal tissue damage(Zhang et al., 2016). Physiologically, the body can
carry out defense reactions, maintain homeostasis, and eliminate irritants, resulting in recovery, but if immune cells
are unable to repair through homeostatic mechanisms, the damage spreads, continues to be chronic, reversible or
irreversible, and cell death (Becker et al., 2007). Streptozotocin (STZ) is a group of N-nitroso compounds that are
sources of NO free radicals, used to induce hyperglycemia in experimental animals, because they are specifically toxic
to pancreatic beta cells (Akbarzadeh et al., 2007). Damage to pancreatic beta cells in rats as a result of streptozotocin
induction is associated with the formation of peroxynitrite in the pancreas. Damage to pancreatic beta cells results in
decreased insulin production resulting in impaired glucose metabolism in the body which results in DM(Roh et al.,
2016). Control of hyperglycemia has been suggested as an important step in the management of DM(Volpe et al.,
2018). Management of DM requires multidisciplinary treatment which includes non-drug therapy and drug therapy.
Synthetic drugs have a short mechanism of action and are often accompanied by adverse side effects, which is why
plant compounds are used for the treatment of diabetes.
Based on research by Widowati et al., (2016) stated that giving mangosteen rind extract with a concentration
of 1% w/v, 2% w/v, and 3% w/v can reduce edema volume or have anti-inflammatory properties. The mangosteen
rind extract concentration of 3% w/v can provide an optimal anti-inflammatory effect because there are compounds
that are larger than the mangosteen peel extract concentrations of 1% and 2% w/v. The procedure of flavonoids in
restraining the method of irritation happens in two ways, to be specific by repressing capillary penetrability and
repressing the digestion system of arachidonic acid and the emission of lysosomal enzymes from neutrophil cells and
endothelial cells
Flavonoids play an important role in maintaining permeability and increasing capillary resistance. Therefore,
flavonoids are used in pathological conditions such as impaired permeability of the blood vessel walls. Flavonoids
primarily act on the microvascular endothelium to reduce hyperpermeability and inflammation. Several flavonoid
compounds can inhibit the release of arachidonic acid and the secretion of lysosomal enzymes from the membrane by
blocking the cyclooxygenase and lipoxygenase pathways, thereby reducing levels of prostaglandins and leukotrienes
(mediators of inflammation) (Astuti et al., 2019).
Flavonoids can function as anti-inflammatories because they can inhibit the formation of proinflammatory
cytokines such as TNF-α (Akhlaghi & Bandy, 2009). Flavonoid compounds in mangosteen peel extract (Garcinia
mangostana L.) are phytochemicals that play a part in accelerating the activation of T lymphocyte cells so that
activated T lymphocyte cells can produce various mediators including IFN-γ, a major stimulatory cytokine to activate
monocytes and macrophages. Activated macrophages then release cytokines, namely IL-1 and TNF, which then
activate T and B lymphocyte cells. T lymphocyte cells develop into CD4+ T cells and CD8+ T cells. Activated
macrophages will release cytokines, namely IL-1 and TNF which activate lymphocyte cells. Activated lymphocyte
cells will then produce IFN-γ which will stimulate monocytes to the tissue. The alpha-mangosteen and gamma-
mangostin compounds significantly have the potential as anti-inflammatories by reducing PGE2 production. Gamma-
mangosteen inhibits the conversion of arachidonic acid to PGE2 in microsomal, this causes inhibition of the
cyclooxygenase pathway(Zayyan et al., 2016)
Journal of Pharmaceutical Negative Results ¦ Volume 14 ¦ Regular Issue 03 ¦ 2023 3540Based on previous research, it was shown that the use of herbal medicine from mangosteen rind in diabetes
mellitus patients was able to control blood sugar levels. Another study conducted by Wulandaria & Ersamb, (2011)
stated that mangosteen rind proved effective in lowering blood glucose levels. Most studies examine blood glucose,
but the problem is that it is not yet clear whether mangosteen peel can inhibit TNF-α expression through a mechanism
of decreasing pro-inflammatory cytokines that occur together in the body, therefore further research is still needed.
Traditional medicine is not a single active compound, the reaction process is slow in the body, besides that safety and
cleanliness are not guaranteed (Xie et al., 2015). So we need an active compound from extracts of natural ingredients
that have gone through a separation and purification process and whose dose has been determined through pre-clinical
trials (tests on animals) to determine the level of safety, so research is still needed on rats. In connection with the
problems above, it is necessary to look for a supporting therapy that can accelerate recovery and reduce the number
of complications in people with Diabetes Mellitus. This study looked at the effect of mangosteen peel on increasing
the number of pancreatic islets of Langerhans endocrine cells and decreasing TNF-α. Research on the use of
mangosteen peel for the treatment of Diabetes Mellitus in humans is still very limited, so as an initial stage of an
experiment to find out the benefits of giving mangosteen peel extract to increasing the number of pancreatic
Langerhans islet endocrine cells and decreasing TNF-α induced by streptozotocin which will first be developed in
animals. try a male white rat.
2. Materials and Methods
2.1 Materials
The tools used at this stage were a vortex (Guo Huq Touch Meyer), 29.9 G1 ml disposable syringe (Terumo syringe),
staining jar, paraffin block, microtome, cover glass, object glass, light microscope (Olympus). The materials used in
the study included: male white rats (Rattus norvegicus) Wistar strain aged 60-70 days, body weight 150-200 grams,
mangosteen fruit, metformin 45 mg/kg, TNF-α Abclonal reagent 100 μL (Catalog No. A11534), Streptozotocin (STZ)
(MP Biomedicals, Inc. Ohio), citrate buffer pH 4.5, Mayer hematoxylin dye (Pierce), eosin, DAB chromogen
(Santacruz biotechnology), PBS (Phosphate Buffered Salinexylol), paraffin, NaCl 0.9%, ether.
2.2 Data collection procedures
This investigation is a true experiment using a completely randomized design with a randomized posttest-only control
group design approach. The research was conducted at the Airlangga University Surabaya Biochemistry Laboratory
with Ethical Approval no. 655/HRECC.FODM/VIII/2022. White rats as experimental animals were adapted for seven
days in the laboratory, then fasted for one day. Therapy was given for 14 days. The mice were divided into six groups:
(1) The negative control group that did not receive any treatment (KN), (2) The group of rats induced by STZ without
being given mangosteen peel extract as a positive control (K1), (3) The group of rats induced by STZ which given the
synthetic drug metformin as a positive control (K2), (4) the group of STZ-induced rats that were given mangosteen
peel extract at a dose of 200 mg/kg BW/day (K3), (5) the group of STZ-induced rats that were given mangosteen peel
extract at a dose of 400 mg/kg BW/day (K4). The histopathology of the islets of Langerhans is a number obtained
from counting the number of endocrine cells of the islets of Langerhans. Mangosteen peel extract is mangosteen rind
extracted using 96% ethanol. Streptozotocin (STZ) is a simple pyrimidine derivative and is a substance commonly
used to induce diabetes mellitus in experimental animals because it can cause damage to pancreatic β cells.
2.3 Addition of Mangosteen Peel Extract
Mangosteen peel extract was only given to groups of K3, K4, and K5 rats according to a predetermined dose for each
group of hyperglycemic rats. Each hyperglycemic rat was given orally 1-2 ml of the extract every morning for 14
days. The dose is calculated based on the use of Simplicia by humans, adult humans consume as much as 5 grams to
7 grams of Simplicia and converted to mice multiplied by 0.018 for a rat body weight of 200 grams(Lukiati et al.,
2019)
2.4 STZ Injection in Male White Rats
The dose of Streptozotocin (STZ) used in this study was a single dose of 45 mg/kg BW which was used to induce
intraperitoneal male white rats (Rattus norvegicus) to obtain treatment animals whose blood glucose had shown
hyperglycemia (Zafar & Naqvi, 2010). STZ injection in mice was carried out intraperitoneally (i.p) in an acidic
environment through the abdominal peritoneal cavity. Mice were held by the nape of the neck, anterior posterior
extremities, and tail. The abdomen is sprayed with alcohol and then pinched until pink or almost white skin is visible.
Journal of Pharmaceutical Negative Results ¦ Volume 14 ¦ Regular Issue 03 ¦ 2023 3541The needle is injected and then STZ is injected into that part. Injections were made once during the study after
adaptation was made. The part of the abdomen that was bulging as a result of the injection was then rubbed briefly
until it was slightly normal
2.5 Hematoxylin Eosin (HE) Staining Procedure
HE staining was carried out using the Harris method, in which the dried tissue was put into xylol I for 3 minutes in a
special place, xylol II, absolute alcohol I and II, alcohol 96%, 80%, 70%, tap water respectively for 1 minute, dye for
5-10 minutes, tap water for 2-5 minutes, 3-10 dyes of acid alcohol, 4-7 drops of tap water, 6 drops of ammonia, enough
distilled water, eosin dye for 15 minutes, distilled water for 1-2 minutes, 70% and 80% alcohol for 1-2 minutes, then
aerated to remove the remaining staining.
2.6. Immunohistochemistry Staining Procedure (IHC)
Tissue samples were soaked sequentially, namely xylol for 3-5 minutes, absolute ethanol for 1-3 minutes, and 70%
ethanol for 1-3 minutes. At that point wash 3 times with aquabidest and clean the edges of the slide with a tissue. Drop
3% H2O2 at room temperature brooding for 10 minutes, at that point wash 3x PBS, and clean the edges of the slide
with a tissue. Drop Trypsin 0.025%, and brood at 370C for 6 minutes, at that point wash 3x PBS and clean the edges
of the slide with a tissue. Drop Ultra V Square, hatch at room temperature for 5 minutes, at that point clean the edges
of the slide (no ought to wash). The following process is by dribbling the parameters to be measured, within the shape
of TNF-α which has been weakened (1:100) hatching at room temperature ± 25 - 30 minutes. At that point wash the
PBS 3x, and clean the edges of the slides with a tissue. Drop Biotinilated, and brood at room temperature for 10
minutes, at that point wash the PBS 3x and clean the edges of the slide with a tissue. Drop HRP Polymer, and hatch
at room temperature for 10 minutes. At that point wash the PBS 3x, and clean the edges of the slides with a tissue.
Drop Touch chromogen (20μl/1 ml substrate), hatch at room temperature for 5-15 minutes (dim room), at that point
wash with aquabidest 3x and clean. The final process was carried out by painting with Meyer Hematoxylin, incubating
at room temperature for six until fifteen minutes, and washing with running water 3 times. The final washing is done
by soaking it in water for 10 minutes. Then drying and mounting are carried out
2.7 Procedure for counting cells in the islets of Langerhans
The counting of the number of cells in the islets of Langerhans was carried out microscopically using the quantitative
method, by observing all the islets of Langerhans in one preparation and then counting the number of all normal
endocrine cells in the islets of Langerhans at 400x magnification.
The counting of the number of cells in the islets of Langerhans was carried out microscopically using the
quantitative method, by observing all the islets of Langerhans in one preparation and then counting the number of all
normal endocrine cells in the islets of Langerhans at 400x magnification
2.8 Procedure for Counting the Number of TNF-α Expressing Cells
TNF-α expressed by macrophages. Expression was seen by a positive reaction to an anti-TNF-α anti-rat monoclonal
antibody. Calculation per 10 high power (HPF) viewed with a light microscope with a magnification of 400 times and
counting the number of TNF-α positive cells when they produce a brownish color in the cytoplasm
2.9 Data analysis
The data obtained from the counting of the number of endocrine cells of the islets of Langerhans were tested for
normality with the Kolmogorov-Smirnov test. If the data is normally distributed, parametric analysis is performed
using one-way ANOVA and if it is significantly different (pSmirnov test on the normal esteem of the number of endocrine cells within the islets of Langerhans which shows that the data is normally distributed (p>0.05), then proceed with parametric analysis using the Analysis of Variant (ANOVA) test. Analysis using the ANOVA test showed significantly different results (p
in each treatment group (400x magnification, HE staining) Information: yellow arrow ( )
indicates endocrine cell death, and the white arrow ( ) shows normal endocrine cells.
The results in Figure 1. show the changes seen in K1 induced with STZ without therapy, that is, many endocrine cells
in the islets of Langerhans experience cell death so that they appear to be non-uniform. Meanwhile, group K2 was a
positive control group that was treated with a chemical synthesis drug for diabetes mellitus, namely metformin. The
figure shows that many cells are in a normal state compared to group K1. The diameter of the islets of Langerhans in
the figure does not appear to be too large but has more and more evenly distributed endocrine cells. The
histopathological picture also showed the presence of endocrine cells that experienced death, although the number
was not as high as in the K1 group.
In the K3 group, several endocrine cells were still found to have died, but the severity and number were not
more than those in K1. The histopathological picture of the pancreas on K4 shows that the distribution of endocrine
cells in the islets of Langerhans is more uniform, although there are still cells that experience death, the number of
cells that experience death is not as much as in K3, while the histopathological picture on K5 shows the diameter of
the islets of Langerhans in the picture looks very large with a higher number of normal endocrine cells and evenly
distributed.
3.2 Exspression of TNF-α
Based on the Kolmogorov-Smirnov test on the mean (mean) percentage of positive cells expressing TNF-α in the
islets of Langerhans, it showed that the data were normally distributed (p>0.05), so a parametric analysis was
performed using the Analysis of Variant (ANOVA) test. Analysis using the ANOVA test showed significantly
different results (pKN K1 K2
K3 K4 K5
Figure 2. Immunohistochemical test results on pancreatic TNF-α expression of male white rats (Rattus norvegicus)
in each treatment group (400x magnification).
Information: red arrows ( ) indicate positive cells that express TNF-α, and white arrows ( )
show cells that did not express TNF-α.
3.3 Relationship of Endocrine Cell Number and TNF-α Expression
Based on the data obtained from counting the number of endocrine cells and expression of TNF-α in the islets of
Langerhans, Pearson's correlation test was then performed to determine whether there was a relationship between the
two. The results of the Pearson correlation test that has been carried out show that the correlation between the number
of endocrine cells and the expression of TNF-α in the islets of Langerhans has a correlation coefficient of -0.795 and
a significance of 0.000. This shows that there is an inverse relationship between the number of endocrine cells that are
still normal and the expression of TNF-α in the islets of Langerhans, as shown in Table 3
Tabel 3. Relationship of Endocrine Cell Number and TNF-α Expression in Islets of Langerhans
Variable Correlation value P value
Number of endocrine cells -0.795 0.000
TNF-α Expression
4. Discussion
4.1 Discussion of Number of Endocrine Cells in Islets of Langerhans
The results of statistical analysis of the number of islets of Langerhans endocrine cells in this study showed that the
K1 group as a positive control was only induced by STZ without giving therapy, showing that the number of islets of
Langerhans endocrine cells was the least and significantly different from all other groups. The condition of diabetes
mellitus that occurred in white rats in this study was caused by STZ induction. Research by Hanchang et al., (2019)
said that STZ can increase the concentration of free Ca2+ ions in pancreatic beta cells which in turn opens Ca2+ ion
channels thereby increasing the influx of Ca2+ ions into pancreatic beta cells. The excessive influx of Ca2+ ions can
cause the activation of a number of enzymes such as ATPase, phospholipase, and endonuclease. ATPase enzymes can
cause a decrease in ATP, phospholipases can cause a decrease in phospholipids, proteases cause damage to the
Journal of Pharmaceutical Negative Results ¦ Volume 14 ¦ Regular Issue 03 ¦ 2023 3545pancreatic beta cell membrane and endonucleases can cause damage to chromatin which will ultimately cause damage
to pancreatic beta cells Hanchang et al., (2019). So based on the results of the study, it appears that K1 has the least
number of normal endocrine cells because there are so many cells that have been damaged by the administration of
STZ. Damage to the pancreatic beta cells triggers a decrease in insulin secretion, which will result in high glucose
levels in the blood or commonly called hyperglycemia(Rom et al., 2019). Hyperglycemia that occurs continuously
can cause hyperglycemic stress conditions which are associated with increased oxidative stress(Luc et al., 2019).
In the STZ-induced K3 group and treated with mangosteen rind extract at 200 mg/kg BW/day the results were
not significantly different from the STZ-induced K1 group and without any therapy. This is probably due to the fact
that at a dose of 200 mg/kg BW/day, the active compounds contained in that dose are still very small to be able to
repair the condition of damaged endocrine cells in the islets of Langerhans. Meanwhile, the K5 group which was
induced by STZ was then treated with the highest dose of mangosteen peel extract, which was 600 mg/kg BW/day,
which showed good results, which were not significantly different from KN as the negative control group. with
mangosteen peel extract dose of 200 or 400 mg/kg BW/day. Based on research by Hasan et al., (2016) conducted a
phytochemical analysis of mangosteen peel extract, from these results the phytochemical compounds that were
prominent in the mangosteen peel extract were flavonoids and triterpenoids. Flavanoids and triterpenoids are known
to have antioxidant activity, especially flavonoids. The effectiveness of flavonoids in counteracting free radicals
largely depends on their structure, hydrophobicity, biological activity, and also oxidative activity. The capability and
termination of radical chain reactions by flavonoids mainly depend on the presence of o-hydroxyl groups in ring B.
This allows the formation of intramolecular hydrogen bonds between hydroxyl groups which increases the stability
of phenoxyl radicals (Majewska et al., 2011).
The large number of endocrine cells in the K5 group also indicated better endocrine cell proliferation in the
administration of mangosteen rind extract at a dose of 600 mg/kg BW/day. Pancreatic beta cell proliferation can occur
if the normal pancreatic beta cells that are still in the islets of Langerhans undergo replication. The number of
pancreatic beta cells can also increase if neogenesis occurs. Neogenesis occurs when acinar cells in the pancreas
undergo transdifferentiation into pancreatic beta cells or can also be produced from pancreatic progenitor cells in the
pancreatic ductal epithelium(Moulle et al., 2017). Teta et al., (2007) stated that research conducted on rats showed
that beta cell replication had a greater role in beta cell proliferation, compared to progenitor cell differentiation
(neogenesis), both in normal physiological situations and in certain pathological conditions
The increased number of pancreatic beta cells due to proliferation will also increase insulin secretion. Insulin
itself can stimulate pancreatic beta-cell mitosis in vitro (Davis et al., 2020). Hsiao et al., (2019) stated that in newborn
rats that were directly injected with streptozotocin (STZ) as a diabetes induction agent on the first day of birth and
then treated with insulin, pancreatic beta cell regeneration appeared to occur in these mice. So it was concluded that
mangosteen peel extract increased the number of islets of Langerhans endocrine cells in male white rats given
streptozotocin.
4.2 Discussion of TNF-α Expression
Based on a statistical analysis of TNF-α expression, it can be seen that group K1, which was the STZ positive control
group without any therapy, showed relatively high results of TNF-α expression and was significantly different from
all other groups, both the diabetic group that was given therapy and the group non-diabetic control. This shows that
TNF-α is a cytokine that has a role in the pathogenesis of diabetes mellitus. The results of this study are in line with
several studies linking the condition of diabetes mellitus with TNF-α, Fathy, et al., (2019) stated that type 2 diabetes
mellitus sufferers show high concentrations of TNF-α in serum, this is not only a predictive factor type 2 diabetes
mellitus but can also exacerbate this condition. TNF-α also has an important role in research on type 1 diabetes mellitus
and TNF-α concentrations are known to be increased in type 1 diabetes patients(Jorns et al., 2020). Patients with type
1 diabetes mellitus not only have higher concentrations of TNF-α in serum but also in arterial walls when compared
to non-diabetics (Demine et al., 2020). According to Oh et al., (2021), expression of TNF-α in pancreatic beta cells
increases the degree of inflammation in non-obese diabetic (NOD) mice and TNF-α is required for the development
of diabetes in NOD mice.
Many studies have examined the role of TNF-α in the pathogenesis of diabetes mellitus, both type 1 diabetes
mellitus and type 2 diabetes mellitus. Alzamil, (2020) conducted research on the pathogenesis of TNF-α in type 2
diabetes mellitus, the results of this study showed that there is a relationship between hyperglycemia and insulin
resistance. Xu et al., (2021) based on the results of their research stated that high TNF-α production in pancreatic beta
cells can cause hyperglycemia and can affect GLUT-2 expression. Pancreatic beta cells from the study mice did not
express GLUT-2, which is a transmembrane protein that is normally expressed at high levels in pancreatic beta cells
Journal of Pharmaceutical Negative Results ¦ Volume 14 ¦ Regular Issue 03 ¦ 2023 3546and is important in glucose uptake and metabolism at the cellular level. Xu et al., (2021) also added that local
expression of TNF-α might lead to the production of inflammatory factors such as prostaglandins which can ultimately
increase the severity of diabetes mellitus and affect insulin or GLUT2 expression. The presence of inflammation
indicates a protective response against infection and tissue repair, but in fact, inflammation can also contribute to local
damage in tissues (Wellen and Wotamisgil, 2005). The inflammatory response is associated with a variety of plasma
proteins and pro-inflammatory cytokines. Pro-inflammatory cytokines such as TNF-α are produced during acute and
chronic inflammatory processes and are commonly detected in diabetes mellitus. TNF-α appears in the early phase of
the inflammatory response and has an important role in the pathophysiology of pancreatic damage(Wellen, 2005).
Meanwhile, the expression of TNF-α in the treated diabetic group showed decreasing results wherein the K5
group which was treated with mangosteen peel extract at a dose of 600 mg/kg BW/day was not significantly different
compared to the K2 group, namely the diabetic group which was treated with metformin. This indicates that the ability
of metformin and mangosteen peel extract is almost the same in reducing the expression of TNF-α as a pro-
inflammatory cytokine which plays a role in increasing the severity of diabetes mellitus.
Mangosteen peel extract is known to contain flavonoids and saponins saponin (Hasan et al., 2016). Saponins
are known to play a role in increasing membrane permeability and increasing anti-inflammatory activity by inhibiting
the release of inflammatory mediators (Barbosa, 2014). Meanwhile, based on research conducted by Kartasasmita et
al., (2009), flavanoids have anti-inflammatory activity through inhibition of the cyclooxygenase-2 (COX-2) enzyme
which is induced and resides in inflamed tissues. So it was concluded that mangosteen peel extract could reduce the
expression of TNF-α in male white rats that were given streptozotocin.
4.3 Relationship Between Number of Endocrine Cells and Expression
The results of the Pearson correlation test that has been carried out show that the correlation between the number of
endocrine cells and the expression of TNF-α in the islets of Langerhans has a correlation coefficient of -0.795 and a
significance of 0.000. This shows that there is an inverse relationship between the number of endocrine cells that are
still normal and the expression of TNF-α in the islets of Langerhans. The fewer endocrine cells of the islets of
Langerhans that are still normal, the higher the expression of TNF-α in the islets of Langerhans, and vice versa. In the
K1 group, which was a group of STZ-induced white mice without any therapy (diabetic control), TNF-α expression
was very high and the number of normal endocrine cells was very small compared to the other groups. The high
expression of TNF-α in the K1 group was possible because the small number of normal endocrine cells in this group
indicated that there were many cells that died as a result of STZ administration without therapy. According to
Bergsbaken et al., (2009), the presence of cell death will stimulate an immune response because cells that experience
death will be considered a "danger signal". Based on the research of Maeda & Fadeel, (2014), cells that experience
death then trigger the secretion of TNF-α from macrophages, so the more cells that experience death, the expression
of TNF-α will also be higher.
Oxidative stress due to high blood glucose levels in diabetes mellitus causes intensive production of pro-
inflammatory cytokines such as TNF-α in cells. In line with the development of diabetes mellitus and the damage that
occurs in the pancreas, especially in the islets of Langerhans, macrophages will infiltrate in or around the islets of
Langerhans in the pancreas and also become a source of proinflammatory cytokines (Hotamisligil, 2005). The essence
of the disorder in diabetes mellitus is glucose metabolism and transport, which is related to insulin secretion so this
disorder causes hyperglycemia, the release of free fatty acids (FFA), and proinflammatory cytokines such as TNF-α.
In type 1 DM, decreased insulin synthesis occurs as a result of the pancreatic beta cell damage caused by cell damage
resulting from autoimmune disorders and TNF-α is also involved in the process (Zhao et al., 2019). Whereas in type
2 DM there is a disturbance in the activity of pancreatic beta cells where the number of pancreatic beta cells is
associated with increased concentrations of proinflammatory cytokines, chemokines, and free fatty acids (FFA), as
well as chronic hyperglycemia (Wang et al., 2010). In humans, FFAs induce the release of TNF-α as a
proinflammatory cytokine (Silva et al., 2019).
5. Conclusion
Mangosteen peel extract contains active antioxidant compounds that can reduce free radicals in pancreatic tissue.
Oxidative stress can be prevented by reducing free radicals in pancreatic tissue, thereby repairing pancreatic beta cell
damage in diabetes mellitus.
Acknowledgment
Journal of Pharmaceutical Negative Results ¦ Volume 14 ¦ Regular Issue 03 ¦ 2023 3547The authors would like to express their deepest gratitude to Lembaga Pengelola Dana Pendidikan
(LPDP/Indonesian Endowment Fund for Education) under the Ministry of Finance of the Republic of Indonesia as the
sponsor for their doctoral studies and the support of this publication. We also thank Universitas Airlangga Surabaya
and Universitas Nahdlatul Ulama Surabaya for their permission and research support.
Acknowledgments to the Promoter and Co-promoter who have provided advice and guidance for this
investigation and to all lecturers in the Public Health Doctoral Program, Faculty of Public Health, Airlangga University
Surabaya
Funding
This work was supported by the Indonesian Endowment Fund for Education (LPDP)
Conflict of interest
The authors declare no competing interests
References
1. Akbarzadeh, A., Norouzian, D., Mehrabi, M. R., Jamshidi, S., Farhangi, A., Verdi, A. A., Mofidian, S. M. A., & Rad, B. L. (2007). Induction
of diabetes by Streptozotocin in rats. Indian Journal of Clinical Biochemistry, 22(2), 60–64. https://doi.org/10.1007/BF02913315
2. Akhlaghi, M., & Bandy, B. (2009). Mechanisms of flavonoid protection against myocardial ischemia–reperfusion injury. Journal of
Molecular and Cellular Cardiology, 46(3), 309–317.
3. Alzamil, H. (2020). Elevated serum TNF-α is related to obesity in type 2 diabetes mellitus and is associated with glycemic control and insulin
resistance. Journal of Obesity, 2020.
4. Astuti, K. W., Wijayanti, N. P. A. D., Yustiantara, P. S., Laksana, K. P., & Putra, P. S. A. (2019). Anti-inflammatory activity of mangosteen
(Garcinia Mangostana Linn.) rind extract nanoemulgel and gel dosage forms. Biomedical and Pharmacology Journal, 12(04), 1767–1774.
5. Atlas, D. (2015). International diabetes federation. IDF Diabetes Atlas, 7th Edn. Brussels, Belgium: International Diabetes Federation, 33.
6. Ban, E., Jeong, S., Park, M., Kwon, H., Park, J., Song, E. J., & Kim, A. (2020). Accelerated wound healing in diabetic mice by miRNA-497
and its anti-inflammatory activity. Biomedicine & Pharmacotherapy, 121, 109613.
7. Barbosa, A. D. P. (2014). An overview of the biological and pharmacological activities of saponins. Int J Pharm Pharm Sci, 6(8), 47–50.
8. Becker, D., Banerjee, R., Dickman, M., Gladyshev, V., & Ragsdale, S. (2007). Redox biochemistry. John Wiley & Sons.
9. Bergsbaken, T., Fink, S. L., & Cookson, B. T. (2009). Pyroptosis: host cell death and inflammation. Nature Reviews Microbiology, 7(2),
99–109.
10. Davis, J. C., Alves, T. C., Helman, A., Chen, J. C., Kenty, J. H., Cardone, R. L., Liu, D. R., Kibbey, R. G., & Melton, D. A. (2020). Glucose
response by stem cell-derived β cells in vitro is inhibited by a bottleneck in glycolysis. Cell Reports, 31(6), 107623.
11. Demine, S., Schiavo, A. A., Marín-Cañas, S., Marchetti, P., Cnop, M., & Eizirik, D. L. (2020). Pro-inflammatory cytokines induce cell death,
inflammatory responses, and endoplasmic reticulum stress in human iPSC-derived beta cells. Stem Cell Research & Therapy, 11(1), 1–15.
12. Fathy, S. A., Mohamed, M. R., Ali, M. A. M., El-Helaly, A. E., & Alattar, A. T. (2019). Influence of IL-6, IL-10, IFN-γ and TNF-α genetic
variants on susceptibility to diabetic kidney disease in type 2 diabetes mellitus patients. Biomarkers, 24(1), 43–55.
13. Hanchang, W., Khamchan, A., Wongmanee, N., & Seedadee, C. (2019). Hesperidin ameliorates pancreatic β-cell dysfunction and apoptosis
in a streptozotocin-induced diabetic rat model. Life Sciences, 235, 116858.
14. Hasan, A. E. Z., Nashrianto, H., Juhaeni, R. N., & Artika, I. M. (2016). Optimization of conditions for flavonoid extraction from mangosteen
(Garcinia mangostana L.). Der. Pharmacia Lettre, 8(18), 114–120.
15. Hotamisligil, G. S. (2005). Role of endoplasmic reticulum stress and c-Jun NH2-terminal kinase pathways in inflammation and origin of
obesity and diabetes. Diabetes, 54(suppl_2), S73–S78.
16. Hsiao, C.-C., Lin, C.-C., Hou, Y.-S., Ko, J.-Y., & Wang, C.-J. (2019). Low-energy extracorporeal shock wave ameliorates streptozotocin-
induced diabetes and promotes pancreatic beta cell regeneration in a rat model. International Journal of Molecular Sciences, 20(19), 4934.
17. Jorns, A., Wedekind, D., Jähne, J., & Lenzen, S. (2020). Pancreas pathology of latent autoimmune diabetes in adults (LADA) in patients and
in a LADA rat model compared with type 1 diabetes. Diabetes, 69(4), 624–633.
18. Kangralkar, V. A., Patil, S. D., & Bandivadekar, R. M. (2010). Oxidative stress and diabetes: a review. Int J Pharm Appl, 1(1), 38–45.
19. Kartasasmita, R. E., Herowati, R., Harmastuti, N., & Gusdinar, T. (2009). Quercetin derivatives docking based on the study of flavonoids'
interaction with cyclooxygenase-2. Indonesian Journal of Chemistry, 9(2), 297–302.
20. Kesehatan, K., & RI, K. K. (2013). Riset kesehatan dasar. Jakarta: Badan Penelitian Dan Pengembangan Kesehatan Departemen Kesehatan
Republik Indonesia.
21. Kumawat, M., Pahwa, M. B., Gahlaut, V. S., & Singh, N. (2009). Status of antioxidant enzymes and lipid peroxidation in type 2 diabetes
mellitus with microvascular complications. Open Endocrinol J, 3(1), 12–15.
22. Lampropoulou, I. T., Stangou, Μ., Sarafidis, P., Gouliovaki, A., Giamalis, P., Tsouchnikas, I., Didangelos, T., & Papagianni, Α. (2020).
TNF-α pathway and T-cell immunity are activated early during the development of diabetic nephropathy in Type II Diabetes Mellitus.
Clinical Immunology, 215, 108423.
23. Luc, K., Schramm-Luc, A., Guzik, T. J., & Mikolajczyk, T. P. (2019). Oxidative stress and inflammatory markers in prediabetes and diabetes.
J. Physiol. Pharmacol, 70(6), 111–113.
24. Lukiati, B., Nugrahaningsih, N., & Arifah, S. N. (2019). The Role of Sechium edule Fruits Ethanolic Extract in Insulin Production and
Malondialdehyde Level in Stz-Induced Diabetic Rat. Journal of Tropical Biodiversity and Biotechnology, 4(1), 11–17.
25. Ma, Z. A., Zhao, Z., & Turk, J. (2012). Mitochondrial dysfunction and β-cell failure in type 2 diabetes mellitus. Experimental Diabetes
Research, 2012.
26. Maeda, A., & Fadeel, B. (2014). Mitochondria released by cells undergoing TNF-α-induced necroptosis act as danger signals. Cell Death &
Disease, 5(7), e1312–e1312.
Journal of Pharmaceutical Negative Results ¦ Volume 14 ¦ Regular Issue 03 ¦ 2023 354827. Majewska, M., Skrzycki, M., Podsiad, M., & Czeczot, H. (2011). Evaluation of the antioxidant potential of flavonoids: an in vitro study.
Acta Poloniae Pharmaceutica, 68(4), 611–615.
28. Mandarim-de-Lacerda, C. A. (2019). Pancreatic islet (of Langerhans) revisited. Histology and Histopathology, 34(9), 985–993.
29. Moulle, V. S., Ghislain, J., & Poitout, V. (2017). Nutrient regulation of pancreatic β-cell proliferation. Biochimie, 143, 10–17.
30. Oh, E., McCown, E. M., Ahn, M., Garcia, P. A., Branciamore, S., Tang, S., Zeng, D.-F., Roep, B. O., & Thurmond, D. C. (2021). Syntaxin
4 enrichment in β-cells prevents conversion to autoimmune diabetes in non-obese diabetic (NOD) mice. Diabetes, 70(12), 2837–2849.
31. Roh, S.-S., Kwon, O. J., Yang, J. H., Kim, Y. S., Lee, S. H., Jin, J.-S., Jeon, Y.-D., Yokozawa, T., & Kim, H. J. (2016). Allium hookeri root
protects oxidative stress-induced inflammatory responses and β-cell damage in the pancreas of streptozotocin-induced diabetic rats. BMC
Complementary and Alternative Medicine, 16(1), 1–10.
32. Rom, S., Zuluaga-Ramirez, V., Gajghate, S., Seliga, A., Winfield, M., Heldt, N. A., Kolpakov, M. A., Bashkirova, Y. V, Sabri, A. K., &
Persidsky, Y. (2019). Hyperglycemia-driven neuroinflammation compromises BBB leading to memory loss in both diabetes mellitus (DM)
type 1 and type 2 mouse models. Molecular Neurobiology, 56(3), 1883–1896.
33. Silva, L. B., dos Santos Neto, A. P., Maia, S. M. A. S., dos Santos Guimarães, C., Quidute, I. L., Carvalho, A. de A. T., Júnior, S. A., &
Leão, J. C. (2019). The role of TNF-α as a proinflammatory cytokine in pathological processes. The Open Dentistry Journal, 13(1).
34. Teta, M., Rankin, M. M., Long, S. Y., Stein, G. M., & Kushner, J. A. (2007). The growth and regeneration of adult β cells do not involve
specialized progenitors. Developmental Cell, 12(5), 817–826.
35. Ueda-Wakagi, M., Nagayasu, H., Yamashita, Y., & Ashida, H. (2019). Green tea ameliorates hyperglycemia by promoting the translocation
of glucose transporter 4 in the skeletal muscle of diabetic rodents. International Journal of Molecular Sciences, 20(10), 2436.
36. Volpe, C. M. O., Villar-Delfino, P. H., Dos Anjos, P. M. F., & Nogueira-Machado, J. A. (2018). Cellular death, reactive oxygen species
(ROS), and diabetic complications. Cell Death & Disease, 9(2), 1–9.
37. Wang, C., Guan, Y., & Yang, J. (2010). Cytokines in the progression of pancreatic β-cell dysfunction. International Journal of Endocrinology,
2010.
38. Wellen, K. E. (2005). hotamisligil GS. Inflammation, Stress, Diabetes. J Clin Invest, 115, 1111–1119.
39. Widowati, W., Darsono, L., Suherman, J., Fauziah, N., Maesaroh, M., & Erawijantari, P. P. (2016). Anti-inflammatory effect of mangosteen
(Garcinia mangostana L.) peel extract and its compounds in LPS-induced RAW264. 7 cells. Natural Product Sciences, 22(3), 147–153.
40. Wulandaria, D. D., & Ersamb, T. (2011). Study Of α-Mangostin Compound And Antidiabetic Assay From Fruit Hull Of Garcinia
Mangostana Linn.
41. Xie, Z., Sintara, M., Chang, T., & Ou, B. (2015). Daily consumption of a mangosteen‐based drink improves in vivo antioxidant and anti‐
inflammatory biomarkers in healthy adults: a randomized, double‐blind, placebo‐controlled clinical trial. Food Science & Nutrition, 3(4),
342–348.
42. Xu, P., Xiao, J., & Chi, S. (2021). Piperlongumine attenuates oxidative stress, inflammation, and apoptosis by modulating the GLUT‐2/4
and AKT signaling pathways in streptozotocin‐induced diabetic rats. Journal of Biochemical and Molecular Toxicology, 35(6), 1–12.
43. Zafar, M., & Naqvi, S. N.-H. (2010). Effects of STZ-induced diabetes on the relative weights of kidney, liver, and pancreas in albino rats: a
comparative study. International Journal of Morphology, 28(1).
44. Zayyan, A. B., Nahzi, M. Y., & Kustiyah, I. (2016). Effect of mangosteen peel extract (Garcinia mangostana L.) on the number of lymphocyte
cells in pulp inflammation. Dentino, 1(2), 140–145.
45. Zhang, C., Xi, L., Zhao, S., Wei, R., Huang, Y., Yang, R., Su, L., & Liu, X. (2016). Interleukin-1β and tumor necrosis factor-α levels in the
conjunctiva of diabetic patients with symptomatic moderate dry eye: case–control study. BMJ Open, 6(8), e010979.
46. Zhao, C.-N., Wang, P., Mao, Y.-M., Dan, Y.-L., Wu, Q., Li, X.-M., Wang, D.-G., Davis, C., Hu, W., & Pan, H.-F. (2019). Potential role of
melatonin in autoimmune diseases. Cytokine & Growth Factor Reviews, 48, 1–10.
Journal of Pharmaceutical Negative Results ¦ Volume 14 ¦ Regular Issue 03 ¦ 2023 3549You can also read
