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 (500 mg/kg BW) and CS (250 mg/kg BW) in this study
151 were based on previous studies by Fitrianda et al. (2017) Sakir and Kim (2019)
152 that showed a significant and same effect. The combination doses based on trial.
153 The treatment was administered by using oral gavage for rats on 21 days. The
154 Animal handling during treatment was same as acclimatization.
155 Blood was drawn through the eye vein (orbital sinus) using the retro-
156 orbital plexus method 4 times: before injecting STZ and NA, before treatment (0
157 day), 14 days and 21 days after the treatment. Microhaematocrit was scrapped on
158 the medial canthus (under the eyeball towards the foramen poticus) and rotated 4
159 times to injure the plexus. Then, the blood was centrifuged at 1000 rpm ± 10
160 minutes at 40oC to obtain the supernatant/serum.
161

162 Fasting Blood Glucose (FBG) Levels Measurement

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

167 Insulin Levels Measurement

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

173 Insulin Resistance Measurement

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

179 Malondialdehyde (MDA) Levels Measurement

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

184 Data Analysis

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

190 RESULTS AND DISCUSSION

191 The results of this study indicate that there is a significant effect of the
192 combination treatment of sappan wood extract and gotu kola on decreasing fasting
193 blood glucose levels, insulin resistance based on HOMA-IR index, and MDA
194 levels as well as an increase in insulin levels in T2DM rats.
195 1. Fasting Blood Glucose Levels
196 Table 1 shows that fasting blood glucose levels in all groups before STZ
197 NA induction were normal. Fasting blood glucose (FBG) levels experienced a
198 significant increase in all groups after STZ NA induction (before each group was
199 treated). This proves the success after 72 hours of STZ (65 mg/kg bw) and NA
200 (230 mg/kg bw) induction. STZ and NA cause delayed onset of diabetes through
201 β cell damage and immunologic reactions. STZ is a diabetagonic agent that causes
202 damage to pancreatic β cells while NA aims to protect the cytotoxic effects of
203 STZ (Szkudelski et al., 2013). NA is a derivative of vitamin B3 (niacin) which
204 functions to increase the concentration of NAD+ or partially inhibit PARP-1
205 (Kaur, 2017). Moreover, the data between groups either before or after STZ NA
206 induction were not significantly so that success of randomization and the results
207 are homogeneous.
208 Table 1. The Effect of Combined Extracts of Sappan Wood and Gotu Kola on
209 Fasting Blood Glucose Levels
Group Mean ± SD (mg/dl)
Before STZ-NA 0th day 14th day 21st day
Control 70.81 ± 1.04 267.92 ± 3.33 271.01 ± 3.91 d
273.39 ± 3.51e
Glibenclamid 72.46 ± 2.24 267.26 ± 5.01 103.89 ± 3.73 a
84.04 ± 4.29a
CS 70.22 ± 2.62 265.22 ± 4.36 137.03 ± 3.44 b
114.76 ± 3.79c
CA 70.22 ± 2.88 267.86 ± 5.30 152.39 ± 4.09 c
127.50 ± 3.63d
CSCA1 69.23 ± 2.84 264.98 ± 4.17 146.82 ± 3.69 c
122.44 ± 2.74d
CSCA2 70.42 ± 3.24 264.80 ± 3.88 135.60 ± 3.76b 101.36 ± 2.63b
CSCA3 70.81 ± 2.54 264.98 ± 4.78 103.82 ± 3.10a 82.73 ± 2.06a
p 0.529 0.698 0.000 0.000
210 CS: Sappan wood extract 250 mg/kg BW; CA : Gotu kola extract 500 mg/kg BW; CSCA1 :
211 Combination of sappan wood extract 125 mg/kg BW and gotu kola extract 750 mg/kg BW;
212 CSCA2: Combination of sappan wood extract 250 mg/kg BW and gotu kola extract 500 mg/kg
213 BW; CSCA3: Combination of sappan wood extract 375 mg/kg BW and gotu kola extract 250
214 mg/kg BW. Mean values with different superscript letters (a, b, c, d, e) within a column are
215 significantly different (p<0.05) based on ANOVA and Tukey’s post-hoc test.

216 The results showed that all treatments groups experienced a significant
217 decrease in FBG levels after 14 and 21 days of treatment, except for the control
218 group. After 21 days of treatment, there was a much greater decrease in FBG
219 levels closely to normal FBG levels (before STZ NA induction) than the decrease
220 14 days after treatment, although there was still a decrease in FBG levels. Blood
221 glucose levels did not decrease in the control group because STZ inhibited the
222 Krebs cycle so that ATP production in the mitochondria was limited and
223 continuously reduced pancreatic β cell nucleotides as reported by (Szkudelski et
224 al., 2013).
225 The combination treatment showed that the CSCA1 treatment was not
226 significantly different from the CA treatment. CSCA2 treatment was also not
227 significantly different from CS in reducing FBG levels after 14 days of treatment,
228 but after 21 days, CSCA2 treatment significantly reduced FBG levels compared to
229 CS, CA, or CSCA1, while CSCA1 treatment still showed no difference with CA,
230 even though it was given treatment for 21 days. This shows that the proportion of
231 CSCA1 is not better in reducing FBG levels than the treatment without the
232 combination (CA or CS). CSCA3 treatment showed the most reduction in FBG
233 levels among other treatments. CSCA3 treatment for 21 days of treatment
234 (69.73%) decreased FBG levels more than 14 days of treatment (61.69%) in the
235 control group. CSCA3 treatment was not significantly different from
236 Glibenclamide after 14 and 21 days of treatment. This shows that CSCA3
237 treatment is the best combination proportion with the same ability as
238 Glibenclamide in reducing FBG levels in T2DM rats.
239 This decrease in FBG levels occurs because the bioactive compound of the
240 two ingredients that are thought to be effective as antidiabetic substances which
241 are caused by the positive effect of antioxidants in both ingredients such as
242 flavonoids. Flavonoids are the most popular compounds as antioxidants because
243 of their ability to breakdown free radicals and modulate signals to several cells.
244 Flavonoids work by activating the Phosphoinositide 3-kinase (PI3K/AKT)
245 pathway, inhibiting gluconeogenesis, and stimulating glycogen synthesis.
246 Flavonoids work by activating the synthesis and translocation of Glucose
247 transporter type 4 (GLUT4), increasing hexokinase activity in the liver, reducing
248 the occurrence of pancreatic β cell apoptosis, activating PPARϒ expression to
249 improve glucose uptake, activating the AMPK pathway, inhibiting tyrosine kinase
250 activity, and activating NF-kB (Al-Ishaq et al., 2019).
251 Gotu kola also has main compounds that have potential as antioxidants,
252 especially its asiaticoside and asiatic acid. Thipkaew et al. (2012) reported that
253 asiaticoside has antioxidant effect on neuropathy diabetic rats. Another result of
254 study reported that siatic acid also has potential as an antidiabetic agent through
255 increased glycolysis by restoring the activity of enzymes such as hexokinase,
256 glucose-6-phosphate dehydrogenase (G6PDH), and pyruvate kinase and found a
257 decrease in glycogen in the liver in STZ-induced diabetic rats (Ramachandran and
258 Saravanan, 2013).
259

260 2. Insulin Levels

261 All treatment groups experienced a significant increase in insulin secretion
262 both 14 and 21 days after treatment, except the control group (Table 2). The
263 control group actually experienced an insignificant decrease 0 to 21 days after
264 treatment, presumably due to the inability of insulin to mobilize blood glucose
265 into cells caused by insulin receptor resistance that occurs continuously. Then, the
266 function of pancreatic β-cells is disrupted so that they are unable to produce
267 enough insulin to compensate for insulin resistance (Saisho, 2015).
268 Table 2. The Effect of Combined Extracts of Sappan Wood and Gotu Kola on
269 Insulin Levels
Group Mean ± SD (pg/ml)
0th day 14th day 21st day
Control 421.74 ± 6.89 417.38 ± 6.09a 413.92 ± 5.66a
Glibenclamide 426.47 ± 5.50 515.38 ± 5.91e 547.01 ± 5.74d
CS 425.56 ± 8.21 484.65 ± 8.12cd 514.65 ± 8.40c
CA 428.47 ± 7.75 476.29 ± 7.68bc 499.01 ± 8.92b
CSCA1 421.20 ± 4.91 469.01 ± 5.84b 488.83 ± 7.12b
CSCA2 421.20 ± 4.40 492.11 ± 6.38d 524.65 ± 4.29c
CSCA3 424.65 ± 6.04 510.65 ± 6.42e 542.65 ± 5.70d
p 0.323 0.000 0.000
270 CS: Sappan wood extract 250 mg/kg BW; CA : Gotu kola extract 500
271 mg/kg BW; CSCA1 : Combination of sappan wood extract 125 mg/kg BW
272 and gotu kola extract 750 mg/kg BW; CSCA2: Combination of sappan
273 wood extract 250 mg/kg BW and gotu kola extract 500 mg/kg BW;
274 CSCA3: Combination of sappan wood extract 375 mg/kg BW and gotu kola
275 extract 250 mg/kg BW. Mean values with different superscript letters (a, b,
276 c, d, e) within a column are significantly different (p<0.05) based on
277 ANOVA and Tukey’s post-hoc test.

278 The combination treatment showed that CSCA1 was not significantly
279 different from CA after 14 and 21 days of treatment. CSCA2 treatment also was
280 not different significantly from CS in terms of increasing insulin level. This
281 showed that CSCA1 treatment was not better than CA and CSCA2 treatment was
282 not better than CS treatment. CSCA3 treatment was increasing insulin levels the
283 most after 14 days (22.34%) and 21 days of treatment (31.09%) compared to the
284 control group. CSCA3 was not significantly different from Glibenclamide
285 treatment. This means CSCA3 treatment is the best proportion of combination of
286 both and has the same ability as Glibenclamide to increase insulin levels.
287 Impaired IRS production, decreased GLUT-4 translocation, and glucose
288 oxidation cause a decrease in insulin levels, so glucose does not enter cells and
289 remains in circulation (hyperglycemia) (Lee, 2006) in (Nurhidajah and
290 Nurrahman, 2017). Increased insulin levels are thought to be because of a positive
291 effect on insulin sensitivity, thereby increasing secretion of insulin after treatment.
292 Compounds that play a role in insulin secretion, especially through the Calcium
293 (Ca) pathway, are flavonoids (Al-Ishaq et al., 2019). The increase in Ca ions in
294 the cytoplasm of pancreatic β-cells will cause insulin to be secreted.
295 Brazilin is the main ingredient in sappan wood, including the flavonoid
296 group and works specifically in inhibiting protein kinase C and insulin receptor
297 serine kinase which plays a role in the regulation of insulin signaling. Brazilin
298 induces GLUT4 from intracellular storage areas to plasma membrane via PI3K
299 activation without affecting protein and GLUT4 synthesis (Nirmal et al., 2015).
300 Asiatic acid has also been shown to increase insulin secretion by enhancing the
301 PI3KT/Akt signaling pathway. Moreover, these compounds also improve glucose
302 response by increasing muscle protein GLUT-4, IRS (Insulin Receptor Substrate),
303 IRS-1, and IRS-2 as reported by Ramachandran and Saravanan (2015, 2013).
304

305 3. Insulin Resistance

306 Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) is an
307 insulin resistance biomarker that uses fasting plasma glucose and insulin
308 concentration to determine insulin sensitivity. The HOMA-IR is simple, efficient,
309 and validated for evaluating insulin resistance against DM. The results of this
310 study showed that all groups, except the control group, experienced a significant
311 decrease after 14 and 21 days of treatment,. The results of the control group are
312 not different from the result in a study conducted by Wang et al., (2020) which
313 reported that there was no significant difference in the HOMA-IR index in
314 patients with duration of T2DM of less than 1 year and patients with duration of
315 T2DM of 30 years.
316 Table 3. The Effect of Combined Extracts of Sappan Wood and Gotu Kola on
317 HOMA-IR index
Group Mean ± SD (nmol/ml)
0th day 14th day 21st day
Control 8.37 ± 0.23 8.38 ± 0.24d 8.38 ± 0.21d
Glibenclamide 8.44 ± 0.24 3.96 ± 0.14a 3.40 ± 0.16a
CS 8.36 ± 0.24 4.92 ± 0.19b 4.37 ± 0.20bc
CA 8.50 ± 0.30 5.37 ± 0.21c 4.71 ± 0.18c
CSCA1 8.26 ± 0.17 5.10 ± 0.12bc 4.43 ± 0.08c
CSCA2 8.26 ± 0.11 4.94 ± 0.08b 3.94 ± 0.12b
CSCA3 8.33 ± 0.10 3.92 ± 0.08a 3.32 ± 0.05a
p 0.423 0.000 0.000
318 CS: Sappan wood extract 250 mg/kg BW; CA : Gotu kola extract 500
319 mg/kg BW; CSCA1 : Combination of sappan wood extract 125 mg/kg BW
320 and gotu kola extract 750 mg/kg BW; CSCA2: Combination of sappan
321 wood extract 250 mg/kg BW and gotu kola extract 500 mg/kg BW;
322 CSCA3: Combination of sappan wood extract 375 mg/kg BW and gotu kola
323 extract 250 mg/kg BW. Mean values with different superscript letters (a, b,
324 c, d, e) within a column are significantly different (p<0.05) based on
325 ANOVA and Tukey’s (14th day) and Games Howell (21th day) post-hoc
326 test.

327 The combination treatment showed that the CSCA1 and CSCA2
328 treatments were not significantly different from the CA and CS treatments after 14
329 days and 21 days of treatment. This shows that the proportion of the combination
330 of CSCA1 and CSCA2 is not better in reducing the HOMA-IR index than the
331 treatment without the combination (CA and CS treatment). The treatment that
332 showed the ability to reduce the HOMA-IR index the most and was not
333 significantly different from Glibenclamide treatment after 14 and 21 days of
334 treatment was CSCA3. CSCA3 treatment, compared to the control group, was
335 able to reduce HOMA-IR as much as 61.69% after 14 days of treatment and
336 69.73% after 21 days of treatment.
337 If the HOMA-IR index is higher, the uptake and use of glucose by body
338 cells will be disrupted, resulting in an increase in blood glucose levels. In this
339 study, the increase in insulin ability was thought to be caused by the repair
340 response of target cells (muscle, adipose, and liver) to activate the use of glucose
341 in cells as indicated by resistance values based on the HOMA-IR index which
342 decreased after treatment. CSCA3 treatment showed the most optimal decrease in
343 insulin resistance values and was not significantly different from the
344 Glibenclamide.
345

346 4. MDA Levels

347 In table 4, it is known that the MDA levels of all groups showed a
348 significant decrease after 14 days and 21 days of treatment, except the control
349 group. The control group after 14 days of treatment did not show a significant
350 difference, in fact, there was a significant increase after 21 days of treatment. This
351 is thought to be due to the occurrence of excessive metabolic stress as a result of
352 the development of T2DM conditions through several metabolic pathways;
353 polyols, hexosamines, Advanced Glycation End Products (AGEs), and Protein
354 Kinase Activation (PKC). Moreover, an increase in MDA levels proves that STZ-
355 NA induction increases in ROS levels through a shift in the balance of redox
356 reactions due to changes in carbohydrate and lipid metabolism which will increase
357 the formation of ROS from glycation reactions and lipid oxidation, thereby
358 reducing the antioxidant defense system (Halliwell and Gutteridge, 2015). STZ
359 has the potential as a source of free radicals, damaging DNA and causing cell
360 death. A study reported a significant increase in MDA levels and decreased
361 endogenous antioxidant enzyme activity after STZ induction (Husna et al., 2019).
362 Table 4. The Effect of Combined Extracts of Sappan Wood and Gotu Kola on
363 MDA levels
Group Mean ± SD (nmol/ml)
0th day 14th day 21st day
Control 9.61 ± 0.30 9.84 ± 0.28e 10.06 ± 0.27e
Glibenclamide 9.28 ± 0.34 3.63 ± 0.44a 2.32 ± 0.24a
CS 9.04 ± 0.43 5.48 ± 0.53bc 3.91 ± 0.16c
CA 8.51 ± 0.38 6.84 ± 0.23d 4.70 ± 0.20d
CSCA1 8.88 ± 0.25 6.36 ± 0.13c 3.90 ± 0.18c
CSCA2 9.26 ± 0.53 5.18 ± 0.15b 3.25 ± 0.46b
CSCA3 9.49 ± 0.30 4.45 ± 0.22a 2.04 ± 0.37a
p 0.082 0.003 0.000
364 CS: Sappan wood extract 250 mg/kg BW; CA : Gotu kola extract 500
365 mg/kg BW; CSCA1 : Combination of sappan wood extract 125 mg/kg BW
366 and gotu kola extract 750 mg/kg BW; CSCA2: Combination of sappan
367 wood extract 250 mg/kg BW and gotu kola extract 500 mg/kg BW;
368 CSCA3: Combination of sappan wood extract 375 mg/kg BW and gotu kola
369 extract 250 mg/kg BW. Mean values with different superscript letters (a, b,
370 c, d, e) within a column are significantly different (p<0.05) based on
371 ANOVA and Games Howell (14th day) and Tukey’s (21th day) post-hoc
372 test.

373 The results of this study showed that the CSCA1 and CSCA2 treatments
374 were not significantly different from the treatment without the combination,
375 namely, CS treatment (after 14 days of treatment), but after 21 days of treatment,
376 CSCA2 significantly reduced MDA levels compared to CS and CSCA1. These
377 results indicate that the proportion of CSCA1 combination is not better at
378 reducing MDA levels than the treatment without the combination, namely, CS.
379 The CSCA3 treatment showed the most reduction in MDA levels among the other
380 treatments and the significance was the same as the Glibenclamide treatment
381 either 14 or 21 days after treatment. This indicates that the proportion of CSCA3
382 treatment is the best combination of sappan wood extract and gotu kola for
383 lowering MDA levels, and is similar to Glibenclamide. CSCA3 treatment for 21
384 days (60.38 %) suppressed MDA levels more than the control group's 14 days
385 treatment (53.22 %).
386 Bioactive components of CSCA3 treatment are thought to be more
387 effective in lowering the production of free radicals like ROS and other oxidants,
388 inhibiting lipid peroxidation, and improving pancreatic cell injury in diabetic rats
389 than other treatments. Brazilin has anti-inflammatory properties that prevent the
390 formation of NO and iNOS (Nirmal et al., 2015). According to the study in
391 metabolic syndrome rats by Pakdeechote et al. (2014), increased bioavailability of
392 asiatic acid helped reduce ROS and other proinflammatory cytokines.
393 Furthermore, it is claimed to be able to inhibit lipid peroxidation and a number of
394 proinflammatory cytokines due to an increase in FFA in T2DM patients. Lower
395 MDA levels indicate inhibition of lipid peroxidation. Muchtaromah et al. (2016)
396 found that antioxidant overload increased MDA levels in diabetic rats, suggesting
397 that an effective dose and duration of administration is required.
398

399 CONCLUSION
400 The combination of secang wood extract and gotu kola had a significant
401 effect in reducing insulin resistance and MDA levels. The proportion of CSCA3
402 which was a combination of sappan wood extract 375 mg/kg BW and gotu kola
403 extract 250 mg/kg BW was the optimal dose to improve the condition of diabetic
404 rats. The decrease in insulin resistance with the HOMA-IR index of 61.69% and a
405 significant decrease in MDA levels of 53.22% occurred after 14 days of CSCA3
406 treatment, but more significant decrease occurred on day 21 (69.73 % for HOMA-
407 IR and 60.38% for MDA level). This study shows that combination of sappan
408 wood and gotu kola has potential to decrease oxidative stress and, increase insulin
409 secretion and insulin sensitivity in conditions of T2DM.
410

411 ACKNOWLEDGEMENT
412 We would like to express our gratitude to the parties who have helped us a
413 lot in conducting the research.
414

415 CONFLICT OF INTERESTS STATEMENT

416 The authors declare that there is no conflict of interest with the parties
417 involved in this research.
418

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