1 The Effect of Combined Extracts of Sappan Wood (Caesalpinia sappan L.

)
2 and Gotu Kola (Centella asiatica L.) in Improving Diabetic Condition in Rats
3 Binti Maulina Putri1, Brian Wasita2, Ratih Puspita Febrinasari3
4 1
Postgraduate Program of Nutrition Sciences, Universitas Sebelas Maret,
5 Surakarta 57126, Indonesia
6 2
Department of Anatomical Pathology, Faculty of Medicine, Universitas Sebelas
7 Maret, Surakarta 57126, Indonesia
8 3
Department of Pharmacology, Faculty of Medicine, Universitas Sebelas Maret,
9 Surakarta 57126, Indonesia
10
11 ABSTRACT
12 This study aimed to determine the efficacy of combination of sappan wood and
13 gotu kola extracts in reducing insulin resistance and MDA levels in diabetic rats
14 with induction of streptozotocin 65 mg/kg body weight (BW) and nicotinamide
15 230 mg/kg BW. Forty-two male Sprague Dawley rats weighing ±200 grams
16 were divided into 7 groups: 1) control, 2) glibenclamide 0.45 mg/kg BW, 3)
17 sappan wood extract (CS) 250 mg/kg BW, 4) gotu kola extract (CA) 500 mg/kg
18 BW, 5) combination of extracts of sappan wood and gotu kola 1 (CSCA1) 125
19 mg/kg BW + 750 mg/kg BW, 6) combination 2 (CSCA2) 250 mg/kg BW + 500
20 mg/kg BW, 7) Combination 3 (CSCA3) 375 mg/kg BW + 250 mg/kg BW. Then,
21 measurement using the HOMA-IR index as insulin resistance levels based on
22 fasting blood glucose and insulin levels was conducted. The Thiobarbituric Acid
23 Reactive Substance (TBARs) method was used to measure MDA levels. All
24 measurements were taken before treatment, 14 days after treatment, and 21 days
25 after treatment. CSCA3 is a significant group (p<0.05) to reduce insulin
26 resistance (-3.32±0.05) and MDA levels (-2.04± 0.37 nmol/ml) on day 21 after
27 treatment. The treatment of CSCA3 was not statistically different (p>0.05) from
28 glibenclamide treatment. CSCA3 is the best proportion of sappan wood and gotu
29 kola extracts, and it has the same ability as glibenclamide. The combination of
30 sappan wood and gotu kola extracts has the potential to be a functional drink for
31 people with diabetes.
32 Keywords: centella asiatica, combined extracts, diabetes mellitus, MDA, sappan
33 wood
34 *Corresponding author: Binti Maulina Putri. +628 2157 5789 97.
35 bintimaulina.bm@gmail.com
36 INTRODUCTION
37 Diabetes mellitus (DM) is the most common metabolic condition in
38 society. An increase in blood glucose levels caused by impaired insulin
39 production, insulin resistance, or both, as well as carbohydrate, lipid, and protein
40 metabolism abnormalities, is the most prevalent symptom (World Health
41 Organization, 2019). A survey by the International Diabetes Federation revealed
42 that type 2 diabetes mellitus (T2DM) accounts for 90% of all cases of diabetes.
43 DM causes death for approximately 4.2 million individuals aged 20 to 79 in 2019.
44 The global prevalence of diabetes is expected to be around 463 million people
45 (9.3%) in 2019 and will rise to 700.2 million people (10.9%) in 2045, which is a
46 51% increase (IDF, 2019).
47 Diabetes impacts individual health and the economy’s a country. The
48 impact of diabetes on individual health is macrovascular problems that cause
49 morbidity and mortality, such as cardiovascular disease, peripheral vascular
50 disease, and cerebrovascular disease, as well as microvascular problems (which
51 frequently occur), such as retinopathy, nephropathy, neuropathy, chronic disease,
52 and diabetic ulcers (Silva et al., 2017). All of these health issues reduce the
53 quality of human resources, therefore increasing the burden of health costs in a
54 country.
55 Insulin resistance (IR) is expected to be the main pathogenesis of T2DM
56 because it is a diabetic condition that is frequently undiagnosed when it arises. A
57 cohort study by Wang et al. (2020) found a stronger association of diabetes
58 incidence among adults in China with insulin resistance than with pancreatic beta
59 cell dysfunction. The body is unable to utilize the insulin in hyperglycemia
60 condition caused by receptors of insulin have been disrupted. The pancreas then
61 compensates for the loss of insulin synthesis, resulting in hyperinsulinemia.
62 T2DM development results in pancreatic beta-cell dysfunction and decreased
63 insulin production (Saisho, 2015). IR is important to be analyzed as a marker of
64 T2DM.
65 The development of T2DM is also affected by oxidative stress.
66 Hyperglycemia caused an increase in free radicals, particularly the type of
67 Reactive Oxygen Species (ROS) in all body tissues. The presence of high free
68 radicals in the body can cause lipid peroxidation in cell membranes, resulting in
69 the formation of malondialdehyde (MDA). MDA is a carcinogenic secondary
70 product that is more persistent than other aldehydes, so it is the ideal indicator for
71 oxidative stress on lipids (Ayala et al., 2014).
72 Metformin and sulfonylureas (glibenclamide, glycuidone, glicazide, and
73 glimepiride) are two oral anti-diabetic medications that T2DM patients in
74 Indonesia regularly take. However, they cannot avoid adverse effects such as
75 nausea and hypoglycemia (Putra et al., 2017). So, treatments for T2DM that have
 76 fewer side effects and, are less toxic, and inexpensive. Antioxidant activity at a
77 high level has been found to have a therapeutic impact. Antioxidants serve as the
78 body's defense system, trapping oxidants, producing inflammatory mediators,
79 repairing damaged molecules, and beginning and enhancing endogenous
80 antioxidant production (Adwas et al., 2019). Antioxidants are required to improve
81 insulin sensitivity and oxidative stress conditions.
82 Sappan wood (Caesalpinia sappan L.) and gotu kola (Centella asiatica L.)
83 are known as natural ingredients that have been proven to be effective in treating
84 T2DM due to their strong antioxidants. Both of the plants have been used as
85 functional drinks for people with T2DM (BPOM, 2016; Fitriyanti et al., 2020).
86 There has been no research reported on the effect of combination of these two
87 natural components on DM. Many people combine plants or other ingredients that
88 have a synergistic effect or are more potent than a single ingredient in traditional
89 drink recipes to reduce side effects. Therefore, researcher analyzed the effect of
90 combined extracts of sappan wood on fasting blood glucose, insulin levels, insulin
91 resistance, and MDA levels in diabetic rats induced with STZ NA. The
92 researchers expected that this study could show the potentials of combination of
93 sappan wood and gotu kola as a functional drink which is more effective in
94 improving T2DM conditions than functional drink without combination.
95

96 METHODS
97 Design, Location, and Time
98 The design of this study was a randomized control group pretest-posttest
99 design. The material origin and extraction process location Materia Medika Batu,
100 Malang City, Indonesia. The research on experimental animal was carried out at
101 The House of Experimental Rats, Center for Food and Nutrition Studies, UGM,
102 DIY Yogyakarta. The research was carried out from June to July 2021 with
103 ethical clearance by The Research Ethics Committee of Faculty Medicine,
104 Universitas Sebelas Maret No 36/ UN27.06.6.1/KEP/EC/2021 and ID:
105 01/02/04/38.
106

107 Materials and Tools

108 The materials for the treatment were sappan wood and gotu kola extracted
109 with 96% ethanol as solvent. Modeling of hyperglycemia was done by inducing
110 streptozotocin (STZ) dissolved in citrate buffer and nicotinamide (NA) dissolved
111 in saline. FBG was measured by the Glucose GOD FS Kit (DiaSys, Germany).
112 Insulin levels was measured by mouse insulin ELISA Kit (FineTest, China).
113 H3PO4, TBA, methanol, distilled water, and 1,1,3,3-tetraethoxypropane (TEP)
114 were used for the measurement of MDA levels.
115 The tools used in this study were rotary evaporator, reciprocating shaker,
116 syringe, oral gavage, microhematocrit, waterbath, cooling bath, centrifuge,
117 microplate reader, cuvette and Uv-Vis spectrophotometer.
118

119 Extraction of Sappan Wood and Gotu Kola

120 Sappan wood and gotu kola were washed and dried before being mashed
121 and being processed into simplicia. The extraction method used was maceration.
122 Sappan wood was diluted with 96% ethanol as solvent in a ratio of 1:9 for 120
123 hours, while gotu kola was diluted with a ratio of 1:5 for 96 hours and stirred at
124 room temperature. The amount of solvent is adjusted to the simplicia and must be
125 completely submerged. Then, each was filtered with filter paper. The filtrate was
126 evaporated at speed of 100 rpm and temperature of 60 oC. The evaporation of
127 sappan wood and gotu kola took 4 hours and 2 hours, respectively. The
128 evaporation was stopped when the solvent was no longer dripping and the extract
129 has thickened. The result of extracts was thick liquid with a yield of 10.71% from
130 700 grams of sappan wood simplicia, while the extract of gotu kola leaves
131 obtained a yield of 15.71% from 700 grams of gotu kola simplicia.
132

133 Animal Study

134 Total number of samples in this study was 42 male white rats (Rattus
135 novergicus), Sprague Dawley strain. The rats were 8-10 weeks old and weighed
136 ±200 grams. The rats were acclimatized for 7 days under standard animal housing
137 conditions (with temperature being controlled at 25±2°C and maintained with 12
138 light-dark cycle) and given standard AD II feed of 10-20 grams and ad libitum
139 water. T2DM modeling for all rats was carried out by intraperitoneal injection in
140 rats using 230 mg/kg BW of NA dissolved in 2 mg/200 gr BW of saline. After 15
141 minutes, 65 mg/kg BW of STZ dissolved in 2 mg/200 gr BW of citrate buffer was
142 injected (Muhlishoh et al., 2019). Hyperglycemic conditions (> 150 mg/dL) were
143 obtained within 72 hours (Ghasemi et al., 2014). Then, randomization was done
144 and the rats were divided into 7 groups; each group was given different
145 treatments: distilled water (Control), Glibenclamide 0.45 mg/kg BW
146 (Glibenclamide), sappan wood extract (CS) 250 mg/kg BW, gotu kola extract
147 (CA) 500 mg/kg BW, combination of sappan wood and gotu kola extracts 1
148 (CSCA1) 125 mg/kg BW + 750 mg/kg BW, combination 2 (CSCA2) 250 mg/kg
149 BW + 500 mg/kg BW, and combination 3 (CSCA3) 375 mg/kg BW + 250 mg/kg
150 BW. The doses of CA and CS in this study were based on previous studies by
151 Fitrianda et al. (2017) Sakir and Kim (2019) that showed a significant effect.
152 Futhermore, antioxidant activity of CS (34.172 ppm) showed more stronger than
153 CA (160.236 ppm) (Putri et al., 2021). The treatment was administered by using
154 oral gavage for rats on 21 days. The Animal handling during treatment was same
155 as acclimatization.
156 Blood was drawn through the eye vein (orbital sinus) using the retro-
157 orbital plexus method 4 times: before injecting STZ and NA, before treatment (0
158 day), 14 days and 21 days after the treatment. Microhaematocrit was scrapped on
159 the medial canthus (under the eyeball towards the foramen poticus) and rotated 4
160 times to injure the plexus. Then, the blood was centrifuged at 1000 rpm ± 10
161 minutes at 40oC to obtain the supernatant/serum.
162

163 Fasting Blood Glucose (FBG) Levels Measurement

164 FBG levels were measured by the GOD-PAP (Enzymatic Calorimetric
165 Test of Glucose Oxidase Phenol 4-Aminophenazone). The method was applied
166 according to the method used by Subiyono et al., (2016).
167

168 Insulin Levels Measurement

169 Insulin levels were checked by reacting serum with monoclonal anti-
170 mouse insulin (antibodies) that had been coated in microplate wells and the
171 reagents provided in the mouse insulin ELISA kit. The procedure for analysis was
172 performed following the protocol specified for the kit (FineTest, China).
173

174 Insulin Resistance Measurement

175 The assessment of insulin resistance was based on the FBG levels and
176 insulin levels. Assessment of insulin resistance used a simple method with the
177 HOMA-IR calculation formula (Fitriyanto et al., 2020).
fasting blood sugar level ( mg/dL ) x fasting insulin level ( μg / L )
178 HOMA-IR =
405
179

180 Malondialdehyde (MDA) Levels Measurement

181 MDA levels were measured with serum as a sample, standard, and blank
182 using the Thiobarbituric Acid Reactive Substance (TBARs) method according to
183 the method used by (Zainuddin et al., 2019).
184

185 Data Analysis

186 All data obtained were presented as mean and standard deviation. Analysis
187 was done by using SPSS (IBM, version 23) and one-way ANOVA statistical test,
188 as well as Post Hoc Tukey HSD test and Games-Howell test which has a
189 significant value <0.05.
190

191 RESULTS AND DISCUSSION

192 The results of this study indicate that there is a significant effect of the
193 combination treatment of sappan wood extract and gotu kola on decreasing fasting
194 blood glucose levels, insulin resistance based on HOMA-IR index, and MDA
195 levels as well as an increase in insulin levels in T2DM rats.
196 1. Fasting Blood Glucose Levels
197 Table 1 shows that fasting blood glucose levels in all groups before STZ
198 NA induction were normal. Fasting blood glucose (FBG) levels experienced a
199 significant increase in all groups after STZ NA induction (before each group was
200 treated). This proves the success after 72 hours of STZ (65 mg/kg bw) and NA
201 (230 mg/kg bw) induction. STZ and NA cause delayed onset of diabetes through
202 β cell damage and immunologic reactions. STZ is a diabetagonic agent that causes
203 damage to pancreatic β cells while NA aims to protect the cytotoxic effects of
204 STZ (Szkudelski et al., 2013). NA is a derivative of vitamin B3 (niacin) which
205 functions to increase the concentration of NAD+ or partially inhibit PARP-1
206 (Kaur, 2017). Moreover, the data between groups either before or after STZ NA
207 induction were not significantly so that success of randomization and the results
208 are homogeneous.
209 Table 1. The Effect of Combined Extracts of Sappan Wood and Gotu Kola on
210 Fasting Blood Glucose Levels
Group Mean ± SD (mg/dl)
Before STZ-NA 0th Day 14th Day 21st Day
Control 70.81 ± 1.04 a
267.92 ± 3.33 a
271.01 ± 3.91 d
273.39 ± 3.51e
Glibenclamid 72.46 ± 2.24 a
267.26 ± 5.01 a
103.89 ± 3.73 a
84.04 ± 4.29a
CS 70.22 ± 2.62 a
265.22 ± 4.36 a
137.03 ± 3.44 b
114.76 ± 3.79c
CA 70.22 ± 2.88 a
267.86 ± 5.30 a
152.39 ± 4.09 c
127.50 ± 3.63d
CSCA1 69.23 ± 2.84 a
264.98 ± 4.17 a
146.82 ± 3.69 c
122.44 ± 2.74d
CSCA2 70.42 ± 3.24 a
264.80 ± 3.88 a
135.60 ± 3.76 b
101.36 ± 2.63b
CSCA3 70.81 ± 2.54 a
264.98 ± 4.78 a
103.82 ± 3.10 a
82.73 ± 2.06a
p 0.529 0.698 0.000 0.000
211 Mean values with different superscript letters (a, b, c, d, e) within a column are significantly
212 different (p<0.05) based on ANOVA and Tukey’s post-hoc test
213 p : One-Way ANOVA test

214 The results showed that all treatments groups experienced a significant
215 decrease in FBG levels after 14 and 21 days of treatment, except for the control
216 group. After 21 days of treatment, there was a much greater decrease in FBG
217 levels closely to normal FBG levels (before STZ NA induction) than the decrease
218 14 days after treatment, although there was still a decrease in FBG levels. Blood
219 glucose levels did not decrease in the control group because STZ inhibited the
220 Krebs cycle so that ATP production in the mitochondria was limited and
221 continuously reduced pancreatic β cell nucleotides as reported by (Szkudelski et
222 al., 2013).
223 The combination treatment showed that the CSCA1 treatment was not
224 significantly different from the CA treatment. CSCA2 treatment was also not
225 significantly different from CS in reducing FBG levels after 14 days of treatment,
226 but after 21 days, CSCA2 treatment significantly reduced FBG levels compared to
227 CS, CA, or CSCA1, while CSCA1 treatment still showed no difference with CA,
228 even though it was given treatment for 21 days. This shows that the proportion of
229 CSCA1 is not better in reducing FBG levels than the treatment without the
230 combination (CA or CS). CSCA3 treatment showed the most reduction in FBG
231 levels among other treatments. CSCA3 treatment for 21 days of treatment
232 (69.73%) decreased FBG levels more than 14 days of treatment (61.69%) in the
233 control group. CSCA3 treatment was not significantly different from
234 Glibenclamide after 14 and 21 days of treatment. This shows that CSCA3
235 treatment is the best combination proportion with the same ability as
236 Glibenclamide in reducing FBG levels in T2DM rats.
237 This decrease in FBG levels occurs because the bioactive compound of the
238 two ingredients that are thought to be effective as antidiabetic substances which
239 are caused by the positive effect of antioxidants in both ingredients such as
240 flavonoids. Flavonoids are the most popular compounds as antioxidants because
241 of their ability to breakdown free radicals and modulate signals to several cells.
242 Flavonoids work by activating the Phosphoinositide 3-kinase (PI3K/AKT)
243 pathway, inhibiting gluconeogenesis, and stimulating glycogen synthesis.
244 Flavonoids work by activating the synthesis and translocation of Glucose
245 transporter type 4 (GLUT4), increasing hexokinase activity in the liver, reducing
246 the occurrence of pancreatic β cell apoptosis, activating PPARϒ expression to
247 improve glucose uptake, activating the AMPK pathway, inhibiting tyrosine kinase
248 activity, and activating NF-kB (Al-Ishaq et al., 2019).
249 Gotu kola also has main compounds that have potential as antioxidants,
250 especially its asiaticoside and asiatic acid. Thipkaew et al. (2012) reported that
251 asiaticoside has antioxidant effect on neuropathy diabetic rats. Another result of
252 study reported that siatic acid also has potential as an antidiabetic agent through
253 increased glycolysis by restoring the activity of enzymes such as hexokinase,
254 glucose-6-phosphate dehydrogenase (G6PDH), and pyruvate kinase and found a
255 decrease in glycogen in the liver in STZ-induced diabetic rats (Ramachandran and
256 Saravanan, 2013).
257

258 2. Insulin Levels

259 All treatment groups experienced a significant increase in insulin secretion
260 both 14 and 21 days after treatment, except the control group (Table 2). The
261 control group actually experienced an insignificant decrease 0 to 21 days after
262 treatment, presumably due to the inability of insulin to mobilize blood glucose
263 into cells caused by insulin receptor resistance that occurs continuously. Then, the
264 function of pancreatic β-cells is disrupted so that they are unable to produce
265 enough insulin to compensate for insulin resistance (Saisho, 2015).
266 Table 2. The Effect of Combined Extracts of Sappan Wood and Gotu Kola on
267 Insulin Levels
Group Mean ± SD (pg/ml)
0th Day 14th Day 21st Day
Control 421.74 ± 6.89a 417.38 ± 6.09a 413.92 ± 5.66a
Glibenclamide 426.47 ± 5.50a 515.38 ± 5.91e 547.01 ± 5.74d
CS 425.56 ± 8.21a 484.65 ± 8.12cd 514.65 ± 8.40c
CA 428.47 ± 7.75a 476.29 ± 7.68bc 499.01 ± 8.92b
CSCA1 421.20 ± 4.91a 469.01 ± 5.84b 488.83 ± 7.12b
CSCA2 421.20 ± 4.40a 492.11 ± 6.38d 524.65 ± 4.29c
CSCA3 424.65 ± 6.04a 510.65 ± 6.42e 542.65 ± 5.70d
p 0.323 0.000 0.000
268 Mean values with different superscript letters (a, b, c) within a column are significantly
269 different (p<0.05) based on ANOVA and Tukey’s post-hoc test
270 p : One-Way ANOVA test

271 The combination treatment showed that CSCA1 was not significantly
272 different from CA after 14 and 21 days of treatment. CSCA2 treatment also was
273 not different significantly from CS in terms of increasing insulin level. This
274 showed that CSCA1 treatment was not better than CA and CSCA2 treatment was
275 not better than CS treatment. CSCA3 treatment was increasing insulin levels the
276 most after 14 days (22.34%) and 21 days of treatment (31.09%) compared to the
277 control group. CSCA3 was not significantly different from Glibenclamide
278 treatment. This means CSCA3 treatment is the best proportion of combination of
279 both and has the same ability as Glibenclamide to increase insulin levels.
280 Impaired IRS production, decreased GLUT-4 translocation, and glucose
281 oxidation cause a decrease in insulin levels, so glucose does not enter cells and
282 remains in circulation (hyperglycemia) (Lee, 2006) in (Nurhidajah and
283 Nurrahman, 2017). Increased insulin levels are thought to be because of a positive
284 effect on insulin sensitivity, thereby increasing secretion of insulin after treatment.
285 Compounds that play a role in insulin secretion, especially through the Calcium
286 (Ca) pathway, are flavonoids (Al-Ishaq et al., 2019). The increase in Ca ions in
287 the cytoplasm of pancreatic β-cells will cause insulin to be secreted.
288 Brazilin is the main ingredient in sappan wood, including the flavonoid
289 group and works specifically in inhibiting protein kinase C and insulin receptor
290 serine kinase which plays a role in the regulation of insulin signaling. Brazilin
291 induces GLUT4 from intracellular storage areas to plasma membrane via PI3K
292 activation without affecting protein and GLUT4 synthesis (Nirmal et al., 2015).
293 Asiatic acid has also been shown to increase insulin secretion by enhancing the
294 PI3KT/Akt signaling pathway. Moreover, these compounds also improve glucose
295 response by increasing muscle protein GLUT-4, IRS (Insulin Receptor Substrate),
296 IRS-1, and IRS-2 as reported by Ramachandran and Saravanan (2015, 2013).
297

298 3. Insulin Resistance

299 Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) is an
300 insulin resistance biomarker that uses fasting plasma glucose and insulin
301 concentration to determine insulin sensitivity. The HOMA-IR is simple, efficient,
302 and validated for evaluating insulin resistance against DM. The results of this
303 study showed that all groups, except the control group, experienced a significant
304 decrease after 14 and 21 days of treatment,. The results of the control group are
305 not different from the result in a study conducted by Wang et al., (2020) which
306 reported that there was no significant difference in the HOMA-IR index in
307 patients with duration of T2DM of less than 1 year and patients with duration of
308 T2DM of 30 years.
309 Table 3. The Effect of Combined Extracts of Sappan Wood and Gotu Kola on
310 HOMA-IR index
Group Mean ± SD (nmol/ml)
0th Day 14th Day 21st Day
Control 8.37 ± 0.23a 8.38 ± 0.24 d
8.38 ± 0.21d
Glibenclamide 8.44 ± 0.24a 3.96 ± 0.14 a
3.40 ± 0.16a
CS 8.36 ± 0.24a 4.92 ± 0.19b 4.37 ± 0.20bc
CA 8.50 ± 0.30a 5.37 ± 0.21 c
4.71 ± 0.18c
CSCA1 8.26 ± 0.17a 5.10 ± 0.12bc 4.43 ± 0.08c
CSCA2 8.26 ± 0.11a 4.94 ± 0.08 b
3.94 ± 0.12b
CSCA3 8.33 ± 0.10a 3.92 ± 0.08a 3.32 ± 0.05a
p 0.423 0.000 0.000
311 Mean values with different superscript letters (a, b, c) within a column are significantly
312 different (p<0.05) based on ANOVA. Tukey (14 th day) and Games Howell (21 th day)
313 are post-hoc test
314 p : One-Way ANOVA test
315 The combination treatment showed that the CSCA1 and CSCA2
316 treatments were not significantly different from the CA and CS treatments after 14
317 days and 21 days of treatment. This shows that the proportion of the combination
318 of CSCA1 and CSCA2 is not better in reducing the HOMA-IR index than the
319 treatment without the combination (CA and CS treatment). The treatment that
320 showed the ability to reduce the HOMA-IR index the most and was not
321 significantly different from Glibenclamide treatment after 14 and 21 days of
322 treatment was CSCA3. CSCA3 treatment, compared to the control group, was
323 able to reduce HOMA-IR as much as 61.69% after 14 days of treatment and
324 69.73% after 21 days of treatment.
325 If the HOMA-IR index is higher, the uptake and use of glucose by body
326 cells will be disrupted, resulting in an increase in blood glucose levels. In this
327 study, the increase in insulin ability was thought to be caused by the repair
328 response of target cells (muscle, adipose, and liver) to activate the use of glucose
329 in cells as indicated by resistance values based on the HOMA-IR index which
330 decreased after treatment. CSCA3 treatment showed the most optimal decrease in
331 insulin resistance values and was not significantly different from the
332 Glibenclamide.
333

334 4. MDA Levels

335 In table 4, it is known that the MDA levels of all groups showed a
336 significant decrease after 14 days and 21 days of treatment, except the control
337 group. The control group after 14 days of treatment did not show a significant
338 difference, in fact, there was a significant increase after 21 days of treatment. This
339 is thought to be due to the occurrence of excessive metabolic stress as a result of
340 the development of T2DM conditions through several metabolic pathways;
341 polyols, hexosamines, Advanced Glycation End Products (AGEs), and Protein
342 Kinase Activation (PKC). Moreover, an increase in MDA levels proves that STZ-
343 NA induction increases in ROS levels through a shift in the balance of redox
344 reactions due to changes in carbohydrate and lipid metabolism which will increase
345 the formation of ROS from glycation reactions and lipid oxidation, thereby
346 reducing the antioxidant defense system (Halliwell and Gutteridge, 2015). STZ
347 has the potential as a source of free radicals, damaging DNA and causing cell
348 death. A study reported a significant increase in MDA levels and decreased
349 endogenous antioxidant enzyme activity after STZ induction (Husna et al., 2019).
350 Table 4. The Effect of Combined Extracts of Sappan Wood and Gotu Kola on
351 MDA levels
Group Mean ± SD (nmol/ml)
0th Day 14th Day 21st Day
Control 9.61 ± 0.30a 9.84 ± 0.28 e
10.06 ± 0.27e
Glibenclamide 9.28 ± 0.34a 3.63 ± 0. 44a
2.32 ± 0.24a
CS 9.04 ± 0.43a 5.48 ± 0.53bc 3.91 ± 0.16c
CA 8.51 ± 0.38a 6.84 ± 0.23 d
4.70 ± 0.20d
CSCA1 8.88 ± 0.25a 6.36 ± 0.13c 3.90 ± 0.18c
CSCA2 9.26 ± 0.53a 5.18 ± 0.15 b
3.25 ± 0.46b
CSCA3 9.49 ± 0.30a 4.45 ± 0.22a 2.04 ± 0.37a
p 0.082 0.003 0.000
352 Mean values with different superscript letters (a, b, c) within a column are significantly
353 different (p<0.05) based on ANOVA and Tukey’s post-hoc test
354 p : One-Way ANOVA test

355 The results of this study showed that the CSCA1 and CSCA2 treatments
356 were not significantly different from the treatment without the combination,
357 namely, CS treatment, after 14 days of treatment, but after 21 days of treatment,
358 CSCA2 significantly reduced MDA levels compared to CS and CSCA1. These
359 results indicate that the proportion of CSCA1 combination is not better at
360 reducing MDA levels than the treatment without the combination, namely, CS.
361 The CSCA3 treatment showed the most reduction in MDA levels among the other
362 treatments and the significance was the same as the Glibenclamide treatment
363 either 14 or 21 days after treatment. This indicates that the proportion of CSCA3
364 treatment is the best combination of sappan wood extract and gotu kola for
365 lowering MDA levels, and is similar to Glibenclamide. CSCA3 treatment for 21
366 days (60.38 %) suppressed MDA levels more than the control group's 14 days
367 treatment (53.22 %).
368 Bioactive components of CSCA3 treatment are thought to be more
369 effective in lowering the production of free radicals like ROS and other oxidants,
370 inhibiting lipid peroxidation, and improving pancreatic cell injury in diabetic rats
371 than other treatments. Brazilin has anti-inflammatory properties that prevent the
372 formation of NO and iNOS (Nirmal et al., 2015). According to the study in
373 metabolic syndrome rats by Pakdeechote et al. (2014), increased bioavailability of
374 asiatic acid helped reduce ROS and other proinflammatory cytokines.
375 Furthermore, it is claimed to be able to inhibit lipid peroxidation and a number of
376 proinflammatory cytokines due to an increase in FFA in T2DM patients. Lower
377 MDA levels indicate inhibition of lipid peroxidation. Muchtaromah et al. (2016)
378 found that antioxidant overload increased MDA levels in diabetic rats, suggesting
379 that an effective dose and duration of administration is required.
380

381 CONCLUSION
382 The combination of secang wood extract and gotu kola had a significant
383 effect in reducing insulin resistance and MDA levels. The proportion of CSCA3
384 which was a combination of sappan wood extract 375 mg/kg BW and gotu kola
385 extract 250 mg/kg BW was the optimal dose to improve the condition of diabetic
386 rats. The decrease in insulin resistance with the HOMA-IR index of 61.69% and a
387 significant decrease in MDA levels of 53.22% occurred after 14 days of CSCA3
388 treatment, but more significant decrease occurred on day 21 (69.73 % for HOMA-
389 IR and 60.38% for MDA level). This study shows that combination of sappan
390 wood and gotu kola has potential to decrease oxidative stress and, increase insulin
391 secretion and insulin sensitivity in conditions of T2DM.
392

393 ACKNOWLEDGEMENT
394 We would like to express our gratitude to the parties who have helped us a
395 lot in conducting the research.
396

397 CONFLICT OF INTERESTS STATEMENT

398 The authors declare that there is no conflict of interest with the parties
399 involved in this research.
400

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