KIMIA.STUDENTJOURNAL, Vol. 1, No. 1, pp.

71-77, UNIVERSITAS BRAWIJAYA MALANG Received 6

March 2014, Accepted 17 March 2014, Published online 17 March 2014

EFFECT OF HERBAL MEDICINE FROM SPIRULINA SPRAY

BASED BIOACTIVE (SPIRULINA SP.) PROTEIN PROFILE OF
WOUNDS AND RAT PANCREATIC histology (Rattus norvegicus)
EXPOSED LOW DOSE OF MULTIPLE streptozotocin (MLD-
STZ)
Kartika Rahma, Aulanni’am, Chanif Mahdi

Department of Chemistry, Faculty of Mathematics and Natural Sciences, University of

Brawijaya
Jl. Veteran Malang 65145
* Address correspondence, Tel: +62-341-575838, Fax: +62-341-575835
Email: aulani@ub.ac.id

ABSTRACT
This research aims to determine the effect of Spirulina sp. extracts in the form of spray on
pancreatic tissue histology, and protein profiles of skin wound rats (Rattus norvegicus) induced
by Multiple Low Dose streptozotocin (MLD-STZ). The injection of MLD-STZ at a dose of 20
mg/kg BW causes Diabetes Mellitus (DM) type 1. In this study, rats were divided into 3 groups:
negative control (no STZ induction), positive control (MLD-STZ-induced), and therapy group
(MLD-STZ-induced and therapy). Therapy was given in the form of spray-based herbal
bioactive Spirulina sp. with a dose of ± 200 μL right on rat wound 3 times/day during 2 weeks
after being DM as a result of MLD-STZ induced. The method used for the analysis of protein is
SDS-PAGE and pancreatic tissue histology using hematoxylin-eosin staining methods (HE). The
results of the study showed that post-therapy causes the difference of protein band profiles of the
three treatment groups, emergence a new protein band with a molecular weight of 93 kDa in skin
wounds of diabetic rats, and this protein bands disappear in skin wound rats after treated by
herbal spray of Spirulina sp.. In addition, results of histological pancreatic tissue diabetic group
has reduction of pancreatic β cells and islet of Langerhans in the cavity width. As well, there is
improvement in the therapy group of pancreatic histology.

Keywords: diabetes mellitus, pancreatic histology, skin protein bands

INTRODUCTION
Diabetes Mellitus (DM) is a disease caused by a complex metabolic disorder, especially
in the metabolism of carbohydrates, and protein. Indirectly, this disease affects pancreatic
endocrine hormones work [1]. Hyperglycemia that is characteristic of DM can cause
complications such as microvascular abnormalities namely stroke, heart and kidney damage. Due
to these complications, many patients who suffer from DM Diabetic Foot Infection (IKD). IKD
is an infection that occurs in the lower extremities and the flood area developed into IKD which
when left untreated the infection will become gangrene [2].

Oral treatment with these drugs have side effects when consumed prolonged variety, for
example gastrointestinal [3]. Making the use of herbal medicines are believed to be safer. This
study uses algae Spirulina sp. are packaged in the form of spray for the treatment of type 1
diabetes wounds. The content of polyphenols quercetin in it can act as a scavenger to be able to
capture free radicals (superoxide radical species, ROS) and repair of pancreatic β cells to insulin
secretion can be corrected. Then the wound will close with accompaniment berlahan decrease
blood sugar levels close to normal [4].

Histopathology of islets of Langerhans in diabetic patients due to necrosis caused the

islets of Langerhans (a collection of β cells) appear to be smaller and even less compared to
normal pancreatic histology when stained with hematoxylin-eosin stain (HE) [5]. Extracts of
Spirulina sp. suspected pancreatic tissue histology can repair due to its antioxidant content.
While DM wound repair in mice will be analyzed based on different proteins with the protein
profiles in normal mice by sodium dodecyl methods Polyacrylamide Gel Electrophoresis (SDS-
PAGE). Based on the description above, the need for research on the effectiveness of the
administration of bioactive herbal based spray Spirulina sp. in its role as an adjunct therapy in
diabetes type I wound it.

RESEARCH METHODS
Materials and Equipment
This study used rats (Rattus norvegicus) Wistar males age 3 months. The use of
experimental animals in this study has received ethical No. worthy certificate. No.. KEP-176-UB
worthy of Research Ethics Commissioner UB.

The materials used in the study include Streptozotocin (STZ), PFA (Paraformaldehid) 4%,
NaCl Physiological, Alcohol-rise (70%, 80%, 90%, 95%), distilled water, Spirulina sp., Wagner
reagent, NaOH 10%, 1N HCl, CHCl (phosphate Buffer Saline) pH 7.4, xylol 1,2,3, paraffin,
hematoxylen, eosin alcohol, Entellen, citrate buffer, H3, H2SO4 98% (1.84 g / mL), NH3 0.05
M, PBS 2O2 3%, NaN3, Tris-HCl, RSB (Reducing Sample Buffer).
The equipment used in this research is a set of tools that is glasses (petri cup, pumpkin peck,
glass objects, spatula, pipette drops, 10ml measuring glass, beaker (50.100, and 250 mL), the
reaction tube, the funnel glass), mortar, micro pipette , magnetic stirrer, a set of barn rats,
scissors, tweezers, spuit, glukotes strip and glucometer (one touch), vortex (Guo-Huq),
mikrotube Eppendorf tube, polypropylene, plastic,-20oC freezer, refrigerator 40C, digital pH
meter (inolab -WTW) waterbath, knife mitokron, analytical balances, equipment Electrophoresis
(SDS-PAGE), a spray bottle, a light microscope (Olympus CX-31), LC-MS (thermo scientific),

Procedure
Observation of pancreatic histological picture
Pancreatic preparations are made using HE staining method to distinguish the degree of
insulitis was observed from a network. Observations were made with a light microscope receipts
powerful magnification 400x and 1000x.

Analysis of protein profiles of skin injury with SDS-PAGE

Profile of protein bands were characterized using SDS-PAGE method is performed according a
standard method. A total of 15 mL of protein isolates in tris-HCl in 15 mL RSB on mikrotube
tube is heated at a temperature 100Commasive Brilliant Blue 0.1% (w / v). 0C for 10 minutes.
The process of electrophoresis performed at 200 V until the lower limit of protein bands are at ±
0.5 cm from the lower limit of the gel plate. Gel staining results of running receipts Commasive
dye Brilliant Blue 0.1% (w / v).

Each skin wound protein bands was calculated using protein molecular weight marker as
a standard. Rf value of each protein band then plotted on a linear regression equation obtained
from the standard curve to obtain the BM of each protein band.

RESULTS AND DISCUSSION

Pancreatic histology with HE staining of negative control mice, mice were exposed to
MLD-STZ and spirulina extract spray treatment outcome (Spirulina sp.)
Observations on tissue histology was conducted to determine differences in the histology of the
pancreas are exposed to conditions of MLD-STZ and post-spray therapy of spirulina extract.
According to previous research there is a histopathologic changes islets of Langerhans in patients
with DM both qualitatively and quantitatively [5]. This damage as a result of the formation of
free radicals (ROS), which are potentially damaging pancreatic β cells, causing necrosis,
degeneration, and amydoloisis. This condition will affect the production of insulin in the islets of
Langerhans. Here are the histological features of each treatment are presented in picture 1.

Picture 1 Histology of the pancreas of mice by HE staining

Description:
= Island of Langerhans of the pancreas,

= Β cells of the pancreas,

Based on the observations of each treatment of pancreatic histology seen that the negative
control mice ( A1 , A2 and 400x magnification , 1000x magnification ) shows the regularity of
the arrangement of endocrine cells that spread to the islands of Langerhans cells form a uniform .
Conditions of β cells in the negative control mice was also in a state of relatively good and
normal amounts , in addition to the condition of the islets of Langerhans are relatively tight .
While in the DM group ( B1 and B2 ) showed a slightly pale color in the islets of Langerhans .
The existence of empty spaces on the network indicate damage to the pancreatic islet
degeneration followed by endocrine cells in which β -cell count decreases . The decline in β cells
is directly proportional to the level of insulin secretion resulting in the production of insulin also
decreases . It is proved that the administration of diabetogenic agent STZ as highly toxic nature
can damage cells , especially pancreatic endocrine β cells in the islets of Langerhans form of
diminution , reduction in β cell number and degranulation of these cells than that secretion of
insulin into the blood decreases [ 6 ] .

While the therapy using spirulina extract. Observations of HE staining showed that even
though the improvements have not been able to restore the pancreas under normal conditions.
Cavities are formed due to the increased free radicals (ROS) can be docked with herbal therapy
bioactive spirulina-based spray. Flavonoid compound in spirulina extract which acts as a radical
scavenger can reduce free radicals as evidenced by the increasing number of endocrine cells in
the Langerhans and islet of Langerhans in the cavity shut. Scavenging mechanisms of quercetin
when reacting with free radicals as follows [7]:
Image 2. Scavenging free radical mechanism (R •) may Kuersetin
Effect of Bioactive Spirulina herbal spray based on protein profiles of rat skin after induction of
MLD-STZ
 Induction of MLD-STZ causes the formation of type 1 diabetes is characterized by
hyperglycemia. Conditions autooksidasi hyperglycemia triggers glucose, protein glycation and
polyol pathway activation metabolism which in turn accelerates the formation of reactive oxygen
compounds (ROS). One result of the increased presence of ROS is the modification of the
protein. In general, protein modification shown by the degradation of proteins that cause changes
in protein structure. Two possible changes in the structure of the peptide chain is the
development and breakdown of proteins into smaller units without the development of
molecules.
The use of SDS-PAGE technique is one alternative to find out the changes through
pemngamatan protein electrophoresis protein profile results. Pita protein in healthy rat group,
MLD-STZ induction outcome, and there are differences in the appearances of post-therapy. In
the group of mice DM, a new protein band appeared between the same protein band visible on
the gel with a molecular weight (BM) each one 164 kDa, 145 kDa, 74 kDa, 58 kDa, and 52 kDa.
New protein appears only in kempok DM with BM 93 kDa (indicated by arrows Figure 3) that
are not available in other two groups. These results are believed to be in the DM rats with lesions
degraded as a result of the inflammatory process and the condition of DM.

Picture 3. Profiles protein band of skin from three groups

In group therapy, protein bands with 93 kDa BM is no longer appear, so this protein can
also be regarded as an antigen diabetes. This condition is believed to be due to a return to normal
blood glucose levels so that the inflammatory process is reduced. Additionally the activity of
quercetin as a scavenger of ROS that differentiation berlahan decreased proinflammatory
proteins.
When explored further, the buildup of glucose in the blood begin to decline as a result of
improvements in pancreatic β cells and increased insulin secretion, causing the flow of oxygen
and nutrients to the cells in the body, including the cells that play a role in immune increased. To
expedite the repair of the wound. Repair wound indicates that the condition of inflammation in
the body decreases. At normal blood glucose levels and there is no inflammation, agents no
longer synthesize protein synthesis BM is 93 kDa.

CONCLUSION
Herbal-based bioactive spray of Spirulina sp. given to mice at a dose of DM type 1 3
times a day for 2 weeks were able to improve the function of the pancreas and there are known
differences in protein band profiles of the three groups which have a ribbon kolompok diabetic
rats with BM 93 kDa protein that is not shared by the other two groups.

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