Diabetic Foot Ulcer (DFU) is a neurological disorder that occurs as

tissue-damaging infection by microorganisms1, that ultimately

undergoing amputations2 (± 20%)3 and affecting quality of life (QoL) 4.5.
DFU prevalence in the world is 4% -10%6, whereas in Indonesia 7,3% -
24% from Diabetes Mellitus patients7 and will continue to increase with
prevalence of risk (neuropathy and angiopathy) equal to 55.4% 8.
DFU increases the formation of free radicals that will react with nitric
oxide to be peroxinitrite, thus damaging vascular endothelial cells9 or cell
membranes characterized by elevated levels of Malondialdehyde (MDA)10.
MDA concentration affects proinflammatory and proatherogenic
processes that extends the inflammatory phase11, 12, so that there will be
delay wound healing.
Based on the above problems, a new strategy should be developed and
implemented in DFU treatment. DFU treatment is one application of
conservation model by Myra E. Levine as the structural integrity through
biological therapy that focuses on wound care4. Biological therapy acts
as an antioxidant and anti-bacterial, thus protecting DNA from oxidative
damage that eventually accelerates the epithelial phase13, 14. The ability of
antioxidant activity possessed by ionized alkaline water is caused by a very
high hydrogen content15,16,17, the activity to stimulate the synthesis of
fibroblasts and collagen18, and the ability on decreasing reactive oxygen
species production17. Based on the above description, the researchers
were motivated to conduct research on the impact of ionized alkaline
water on MDA levels in Rattus norvegicus Barkenhout, 1769 with DFU
model condition.

MATERIALS AND METHODS

Animal Test
Using Rattus norvegicus Barkenhout, 1769; 25 males, weighing 150-200
grams divided into 5 randomized treatments that were adapted 7 days in
Food and Nutrition Laboratory of Gadjah Mada University Yogyakarta.
Making Diabetic Foot Ulcer Model
The process of making hyperglycemia through induction of streptozotocin
(STZ) intraperitoneally (i.p) of rat’s abdominal peritoneal cavity . STZ
induction at 50 mg/kg with body weight 150-200 grams and monitored
after 72 hours for random blood glucose test (>200 mg/dl), after the rat
was incised to make the wound19. Making wounds in rat started by
shaving its hair on the back area, then anesthetized by ketamine (0.1
mg/kg), making DFU through a 0.5x0.5 ml biopsy wound on the skin
according to Wagner II grade wound .

Procedure
Ionized Alkaline Water (IAW) used is Kangen Water with pH 11.5 and 9.5
for 8 days. The rats were divided into three different groups. Group I
with control negative (Treatment I). Group II control positive with
induction of STZ 45 mg/Kg and 0.5 ml biopsy wound (Treatment II).
Group III were induced with STZ and 0.5 ml biopsy wound with oral
administration of 1.8 ml/200 gr (Treatment III). Group IV was induced
with STZ and 0.5 ml biopsy wound with tropical IAW administration
(Treatment IV), Group V were induced with STD administration and
0.5 ml biopsy wound by IAW administration orally 1.8ml/200 gr and
Tropical (Treatment V).

MDA Level Measurement

The examination of plasma MDA level was done in Food and Nutrition
Laboratory, PAU of Gajah Mada University Yogyakarta, measured using
TBARs method. The MDA analysis used a 0.75 ml H3PO4 reagent; 0.25
ml TBA; 0,45ml Aquabides which had been mixed with Tetra Ethoxy
Propane standards and a sample of 0.05 ml. The mixture was boiled in ±
100° C for 60 minutes and cooled and then filtered using Sep-Pak
cartridges C18. The result were read using a spectrophotometer with a
wavelength of 532 nm and interpreted by units of nmol/ml20
Data Statistics
The research method used in this research is quasy experimental. Data
were analyzed using independent t test with 95% level of confidence.

RESULT
Table 1. Characteristics of Treatment Subjects in Rattus norvegicus
Berkenhout, 1769 (Mean ± SD)
Variable Time Treatment p
I II III IV V Value
Weight Before IAW 167,4 ± 166,4 ± 171,6 ± 169,8 ± 165,2 ± 3,9 0,000
(gr) Administration 3,9 3,5 2,7 4,02
After IAW 182 ± 2,9 154 ± 3,8 157,2 ± 175,8 ± 4,6 170 ± 2,9 0,000
Administration 2,7
Glucose Before IAW 68,6 ± 1,1 262,7 ± 254,8 ± 268,4 ± 4,6 267,04 ± 7,6 0,000
Level Administration 2,9 3,4
(mg/dl) After IAW 69,4 ± 0,9 263,3 ± 255,1 ± 260,9 ± 3,2 243,2 ± 5,5 0,000
Administration 2,7 3,3

Description: Treatment I was not given streptozotocin (STZ); Treatment II – positive

control (induction STZ 45 mg / kg and 0.5 ml biopsy wound); Treatment III -
STZ induction and 0.5 ml biopsy wound by IAW orally 1.8 ml/200 gr; IV
treatment of STZ induction and 0.5 ml biopsy wound with tropical IAW
administration; V treatment of STZ induction and 0.5 ml biopsy wound with oral
administration of 1.8 ml/200 gr and tropical; N (repeat) = 5; Sig 0.05; SD
(standard deviation).

Table 1 shows that the mean value of glucose level in STZ rats after
IAW was decreasing at Group V (Treatment V) from 267,04 ± 7,6 to
243,2 ± 5,5 mg/dl, whereas in Group II (Treatment II) the glucose level
increased from 262.7 ± 2.9 to 263.3 ± 2.7 mg/dl and treatment III
increased from 254.8 ± 3.4 to 255.1 ± 3.3 mg/dl. This suggests that
IAW has an effect on decreasing glucose level in rats when compared to
rats without IAW administration. In contrary to weight, in Treatment II
and III had lost weight while on treatment V had significant weight gain.
Figure 1. Average MDA (nmol/ml) levels in Rattus norvegicus Barkenhout, 1769.
Treatment I was not given streptozotocin (STZ); Treatment II – positive control
(induction STZ 45 mg / kg and 0.5 ml biopsy wound); Treatment III - STZ
induction and 0.5 ml biopsy wound by IAW orally 1.8 ml/200 gr; IV treatment of
STZ induction and 0.5 ml biopsy wound with tropical IAW administration; V
treatment of STZ induction and 0.5 ml biopsy wound with oral administration of
1.8 ml/200 gr and tropical; N (repeat) = 5; Sig 0.05.

Figure 1 shows that the mean value of MDA (nmol/ml) in STZ rats
after being given the lowest IAW at Group V (Treatment V) is 6.72 ±
0.23 (nmol/ml) and highest level was in the second Group (Treatment
II) 8.55 ± 0.18 (nmol/ml ). This suggests that IAW has an effect on
decreasing MDA levels in rats given STZ if compared with STZ rats
without IAW administration. While in the treatment group III and IV
are not significantly different, this proves that the orally and tropically
IAW administration alone has no significant effect on MDA levels in
STZ rats.
DISCUSSION
The results of this study showed that MDA levels decreased better after
8 days of oral and tropical intervention in Rattus norvegicus Barkenhout,
1769 DFU model with p value 0.000 (6.72 ± 0.23 mg / dl). STZ
administration can cause β cell damage, resulting in ADP-ribosylation
(DNA damage) AD activation, which in turn leads to inhibition of
insulin secretion21 and disrupts antioxidant defenses22. DFU occurs due
to insulin resistance, dyslipidemia, inflammation and increased oxidative
stress 10.23 requiring antioxidants24.
The results of this study that the decreasing MDA levels caused by
inhibition of lipid peroxidation by IAW has an impact on the production
of reactive oxygen species (ROS) production. MDA is a predictor of
oxidative stress in the body, it is because there is a decrease in MDA
levels in line with decrease glucose level, thus decrease lipid peroxidation
and ultimately decrease MDA levels25. Lipid peroxide is formed due to
excess ROS that attacks cell components (lipid membranes and proteins)
by involving double fatty acid residues through the peroxidation process
into MDA26. MDA concentration affects proinflammatory and proatherogenic
processes that extend the inflammatory phase11, 12, so that there will be
delay in wound healing.

IAW as antidiabetic 17, protects cell damage β 27, insulin secretion 28,
prevents the growth of bacteria (poliformonuclear cells) 29, hydrogen-rich
molecules to decrease reactive oxygen species (ROS) and antioxidants15.
The lipid peroxidation inhibition process by capturing free radicals and
donating hydrogen atoms to form a more stable compound and stop the
chain reaction of lipid peroxidation can decrease the average MDA
levels30.
Superoxide dismutase enzymes, glutathione peroxidase and catalase
include intracellular antioxidant enzymes produced in the body which
are essential for the body to absorb free radicals that prevent cell
damage. The superoxide dismutase enzyme as one of the intracellular
antioxidant enzymes works by clearing free radicals or reactive oxygen
species (ROS) with enzymatic reactions and converting them into more
stable products. SOD catalyzes the reaction of superoxide anion free
radical (O2) superoxide into hydrogen peroxide and oxygen molecules so
they are harmless to cells31. Superoxide dismutase is an enzyme present
in intracellular fluid, which participates in the degradation of intracellular
free radical compounds. This enzyme catalyzes the disinfection of O2•
into H2O2. This enzyme inhibits the simultaneous presence of O2• and
H2O2 derived from the formation of a hydroxy radical (OH) 31. Based on
the literature and data above, the AIW mechanism to resist oxidative
stress, resulting in ROS decreasing and inhibit lipid peroxidation
resulting in decreased MDA levels and ultimately can improve the
wound healing phase.
CONCLUSION
In conclusion, the 8-day oral and tropical combination of AIW on Rattus
norvegicus Barkenhout, 1769 DFU model has an effect on decreasing
MDA levels.