Erythrina subumbrans leaves extracts improved Diabetic condition by reducing MDA and 8-OHDG on Rat model

 

Anton Bahtiar*, Anggi Aprilia Prawidi, Syifa Amalia, An’nisa Safitri, Babay Asih Suliasih

Department of Pharmacology and Toxicology, Faculty of Pharmacy, Universitas Indonesia,

Kampus UI Depok, 16424, West Java, Indonesia.

*Corresponding Author E-mail: anton.bahtiar@farmasi.ui.ac.id

 

ABSTRACT:

Erythrina subumbrans, commonly known as DadapDuri, have been used to treat diabetes by people in the West Sumatra region. This study was designed to determine  E.subumbransextract on blood glucose and MDA levels in diabetic Wistar rats induced by low-dose Streptozotocin and a high-fat diet. Type II Diabetes Mellitus rats induced by High-fat diet (HFD) and followed by two times injection of a combination of Nicotinamide (110mg/kg BW) and low-dose Streptozotocin (40mg/kg BW). The rat was randomized and then divided into six groups (n=4). Diabetic rats were treated with E. subumbransextract orally in doses of 50, 100, and 200mg/200gBW once daily for three weeks. Metformin (90mg/200gBW, orally) was used as the reference drug. Blood glucose levels were measured every 7th day using a glucometer for three weeks of treatment. After treatment, the serum MDA and 8-OHdG were calculated. Intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test were performed on the last day of treatment. E. subumbransextract at a dose of 200mg/200gBW administered orally significantly (P < 0.05) could lower and normalize blood glucose levels compared to the Negative control group. The decrease in serum MDA and 8-OHdG levels during E. subumbransextract treatment at dose 3 was significantly different (P 0.05) than the HFD/STZ-NA control group. In this study, it can be concluded that E. subumbransleaves show promising hypoglycemic action and antioxidant effects starting at doses of 200mg/200gBW.

 

KEYWORDS: Diabetes Mellitus, High Fat Diet, Streptozotocin, Erythrina subumbrans, Hypoglycemic, Oxidative Stress, MDA, 8-OHdG.

 

 


INTRODUCTION:

Background:

Diabetes mellitus (DM) is a chronic metabolic disease characterized by increased blood glucose levels due to decreased insulin secretion, and insulin cannot be used effectively1. Hyperglycemia is a fundamental biochemical feature of type 2 diabetes mellitus. Hyperglycemia can stimulate the production of free radicals or reactive oxygen species (ROS). When the body's defense system is weak and becomes unable to counteract the increased ROS, the result is an imbalance between ROS and their protection, resulting in oxidative stress conditions2.

 

 

Lipids are the main target of ROS. Lipid peroxidation will produce highly reactive aldehydes, including malondialdehyde (MDA). High levels of MDA in the serum or plasma of DM patients have been widely reported2.

 

Management of Type 2 DM can be done by implementing a healthy lifestyle, diet, exercise, and pharmacological therapy3. In addition to conventional medicine, one of the diabetes control efforts carried out by the people is the use of herbal as alternatives medicines4.

 

E. subumbrans ans have been used to treat diabetes by peoplein the West Sumatra region5. Several studies have been carried out on plants belonging to the genus Erythrina that show pharmacological effects on diabetic rats6,7. In vitro evaluation of E. subumbrans could inhibit the activity of α-glucosidase8.

The animal model of DMT2 was prepared bydieting high-fat and streptozotocin injection. Animal models with a high-fat diet and low-dose streptozotocin induction (HFD/STZ) are animal models that are similar to humans with type 2 diabetes mellitus. Administration of HFD will cause insulin resistance, and the addition of low-dose streptozotocin injection will cause mild dysfunction of β-cells without completely interfering withinsulin secretion9. Nicotinamide administration protects cells against Streptozotocin cytotoxicity by preventing cell damage10,11.

 

Based on the description above, the content of the E. subumbrans plant can be used in the treatment of diabetes. However, in vivo testing of E. subumbrans leaf extract has not been carried out in rats. Therefore, this study was aimed to examine the effect of E. subumbrans leaves on animal models of type 2 diabetes mellitus induced by low dose HFD and STZ. The effect of E.subumbrans leaf ethanol extract with three variations of doses was evaluated through the parameters of blood glucose levels and MDA levels.

 

MATERIAL AND METHODS:

This research has fulfilled the ethical review of the Faculty of Medicine Universitas Indonesia Ethics Committee with certificate no. KET-68/UN2.F1/ETIK/PPM. 00.02/2021. This study used six treatment groups (N=4). The group consisted of a normal control group, a negative control, positive control, and three groups with variations of the dose of the extract 50mg, 100mg, and 200mg/200grBB rats, respectively.

 

Diabetes Mellitus Rat Model Induction Method:

Wistar male ratsweighed 180-200g were obtained from the Laboratory of Pharmacology and Therapeutics Faculty of Medicine, Padjadjaran University.

 

A high-fat diet (HFD) with the composition of 50% standard diet, 20% tallow, 10% butter, and 20% sucrose was carried out for four weeks12, followed by two injections of the streptozotocin-nicotinamide (STZ-NA). The second STZ-NA combination injection was performed three days after the first injection. Blood glucose levels in rats were measured for three days after the second injection. If the blood glucose level is stable at 280mg/dL for three days, the rat is declared diabetes and can be used for extract testing.The dose of Streptozotocin used in the experiment was 40 mg/kgBW, and the dose of Nicotinamide was 110 mg/kgBW13.

 

Chemical material:

The materials used in this research are Metformin (Hubei Vanz Pharm Co Ltd., China), Streptozotocin (Chem Cruz, USA), CMC- Na, ether, Citric acid (Merck, Germany), Sodium citrate (Merck, Germany), Nicotinamide (Aneka chemical, Indonesia), Insulin (Humulin, Indonesia), Anhydrous glucose (Pudak scientific, Indonesia), NaCl 0.9% (Ecosol Braun, Malaysia), Rat MDA (Malonaldehyde) ELISA Kit (FineTest, China).

 

Instruments:

The instruments used in this study were animal scales (Mettle Toledo, Ohio, USA), analytical scales (Ohaus, New Jersey, USA), glassware (Pyrex, Indonesia), syringes (Terumo, Tokyo, Japan), rat probes (Cadence Science, Rhode Island, USA), microtube (Nesco, Indonesia), yellow tip (Nesco, Indonesia), blue tip (Nesco, Indonesia), microcentrifuge ( Neuve NF 048, Turkey), glucometer and strip (Accu-Chek and Autocheck), micropipette (Socorex, Switzerland), microplate reader (GloMax), 10mL vial; 20mL and 50mL, refrigerator (GEA, Indonesia), deep freezer (Polytron, Indonesia), pH meter (HANNA, Metter Toledo, Ohio, USA), microhematocrit (Marienfield, Germany) and porcelain cup (Jangkar, Indonesia).

 

Extraction Method:

E. subumbrans leaves, with a plant age of one year. E. subumbrans leaves were obtained from the Research Institute for Spices and Medicines (Balitro; Bogor, Indonesia). E. subumbrans leaves were extracted by maceration twice and replicated using 70% ethanol as solvent (8).

 

Measurement of blood glucose levels:

Blood glucose levels were measured before administering the extract and every seven days after the Extract administration. Glucose levels in blood were evaluated using a glucometer (Autocheck)14.

 

Lipid Profile Measurement:

Analysis of triglyceride, cholesterol, HDL-C, and LDL-C levels was carried out at four sampling points (before being given a high-fat diet, after being given a high-fat diet, after STZ-NA induction, and after being given extract). Blood was collected then centrifuged at 5000 rpm for 10 minutes. The resulting supernatant was aliquoted into microtubes. Lipid profile measurements were carried out with Cholesterol FS Diasys, Triglyceride FS Diasys, and HDL-C Precipitant Diasys reagent kits15.

 

Measurement of Urea Serum:

Mice were blood drawn from the orbital sinus vein for analysis of serum urea. The collected blood was stored in a 1.5mL microtube without anticoagulant. Then the blood was centrifuged at 5000rpm for 5 min. Serum was then transferred to another microtube before being analyzed16. Measurements were made using the Urea FS Kit (DiaSys)17.

 

Measurement of Creatinine Serum:

Serum was then transferred to another microtube before being analyzed16. Measurements were made using the Creatinine FS Kit (DiaSys).

 

MDA Serum Level Measurement:

After giving the extract for three weeks, an analysis of the MDA levels in each test group was carried out. Blood from the orbital sinus was collected, and MDA measurements were carried out using the ELISA KIT (Fine Test)18.

 

Measurement of Serum 8-OHdG:

Before taking measurements, several steps were taken, namely removing the reagent from the freezer and letting it sit, making wash buffer, standard, biotin-labeled antibody working solution, and SABC working solution. The 8-OHdG measurement procedure can be carried out using the ELISA Kit when all these steps have been carried out19.

 

Intraperitoneal Glucose Tolerance Test (IpGTT)

On the 21st day after administration of the extract, the rats fasted for 12-16 hours. Furthermore, the rats were injected with 40% glucose solution (2g/kgBW) (20). A glucometer measured blood glucose levels through rats at 0, 30, 60, 90, and 120 minutes. The area under the curve (AUC) represent glucose tolerance in the rat. The AUC value was obtained using the Graph Pad Prism 9 application.

 

Insulin Tolerance Test (ITT):

On day 22, after administration of the extract, the rats were injected with insulin intraperitoneally at a dose of 0.75IU/kg20. A glucometer measured blood glucose levels through rats at 0, 30, 60, 90, and 120 minutes. The area under the curve (AUC) represent the sensitivity of rat tissue to insulin. The AUC value was obtained using the Graph Pad Prism 9 application by entering the blood glucose levels of each rat five times, and then it would produce a graph and the total area of ​​the curve.

 

Data analysis:

The data from this research will be measured and analyzed statistically using the SPSS 24.0 application. Data on changes in blood glucose levels and MDA levels obtained were checked for normality and data distribution. Suppose the data is normally distributed and homogeneous. In that case, the data from each group will be compared with One-way ANOVA, followed by the Least Significant Difference Test (BNT) to see differences between treatment groups. Suppose the data are not normally distributed or homogeneous. In that case, statistical analysis will be carried out using the Kruskal-Wallis test followed by the Mann-Whitney test to see differences between treatment groups. An independent T-test was performed if the data were normally distributed and homogeneous to see a significant difference between the two treatment groups that are not related. All values ​​will be expressed as Mean±SEM. The results of the analysis with a p-value <0.05 will be considered statistically significant

 

RESULT AND DISCUSSION:

A. Identification E. subumbrans Leaf Ethanolic Extract by Thin Layer Chromatography.   

Thin Layer Chromatography of E. subumbrans leaf extract was separated using eluent ethyl acetate: methanol: water (6:4:2). The combination of eluent is used to separate alkaloid compounds in the extracts. The alkaloids were then identified under 366 nm UV light and showed a blue fluorescence spot21.

 

Figure 1: Thin Layer Chromatography of E. subumbrans leaf extract. The eluent was separated for alkaloids contained Ethyl acetate: methanol: water (6: 4: 2) and identified under 366 nm UV light21. Black arrow indicated alkaloids.

 

The alkaloids are shown by the black arrow that blue fluorescence spot under 366nm UV light. This result was similar to the research conducted by Abraham (2014) that the separation of alkaloid compounds with eluent variations of ethyl acetate: methanol: water (6: 4: 2) under a 366 nm UV lamp, the alkaloids fluorescence blue, blue-green, or purple21. In previous studies, E. subumbrans contain four erythrinan alkaloids: erythramine, hypaphorine, erysodine, and erysopine22.

 

B. Effects of E. subumbrans Leaf Extract on Blood Glucose Levels:

The first step in this experiment wase stablishing animal models; administering a high-fat diet (HFD) did not increase blood glucose levels compared to the normal group administered with a standard diet. However, after administrated STZ-NA injection to HFD rats, the average blood glucose level could increase more than 280 mg/dL, as shown in Table 1.


Table 1. Measurement of Blood Glucose Levels During Administration of E. subumbrans Leaf Extract

 Group

Average Blood Glucose Level (mg/dl ± SEM)

After STZ (D0)

D7

D14

D21

D21-D0

Normal

97±4.1

109.7 ± 1.6 c

98.7 ± 4.1c

113.2 ± 5.2 c

16.0 ±5.1

Negative

362.7 ± 46.8a

430.5 ± 32.1 ab

465.0 ± 44.9 ab

426.0 ± 63.3 ab

68.0 ±127.1

Positive

373.0 ± 17.7a

190.0 ± 63.7 c

194.2 ± 46.3 c

194.7 ± 22.5 ac

-178.2 ± 16.8

E. subumbransextract dose1

372.0 ±4.6a

383.0 ± 3.1

391.0 ± 5.2

453.0 ± 6.3

35.0 ± 4.3

E. subumbransextract dose2

343.0 ±13.5a

463.2 ± 21.6 b

395.2 ± 34.7 ab

365.6 ± 6.7 ab

16.3±21.6

E. subumbransextract dose3

357.7 ± 10.1a

264.2 ±43.9 ac

213.4 ± 42.1 ac

186.2 ± 48.6 c

-171.7 ± 43.9

Note: normal = 0.5% CMC administration; negative = 0.5% CMC; positive = metformin 90mg/200gBW rats; E. subumbrans extract dose 1= 50mg/200gBB rats; E. subumbrans extract dose 2= 1000mg/200gBB rats; E. subumbrans extract dose 3= 2000mg/200gBB rats.  (a) = p <0.05 compared to the normal group; (b) = p<0.05 compared to the positive group; (c) = p<0.05 compared to the negative group.  (#) = p < 0.05 (vs negative group); (*) = p > 0.05 (vs positive group).

 


At the first week to third weeks after administration of either the extract or metformin, Metformin group and dose 3 have significantly decreased blood glucose levels.  However, the blood glucose level in Metformin and Dose III treatment has not been recovered to the normal group value (p > 0.05).

 

The formation of animal models of type 2 diabetes mellitus was carried out by providing a high-fat diet (HFD) and two injections of the combination of Nicotinamide 110mg/kg and low dose streptozotocin 40 mg/kg. Differences in blood glucose levels of normal rats and rats treated with HFD and Streptozotocin and Nicotinamide showed increased blood glucose levels. These results are the same as research conducted by other researchersusing the STZ-NA induction method with a combination of HFD20. Giving HFD to rats can cause insulin resistance, although it can't cause hyperglycemia or diabetes. Induction of insulin resistance and glucose intolerance was easier in HFD-treated. HFD aims to initiate insulin resistance, an important feature of type 2 diabetes mellitus20.

 

STZ-NA combination injection can cause hyperglycemia, and this is due to the partial destruction of pancreatic cells, which will reduce insulin levels in the blood and increase blood glucose levels (11). STZ induces diabetes by transporting cells through the glucose transporter 2(GLUT2) and causing DNA damage, and increasing the activity of poly(ADP-ribose) polymerase (PARP-1), which functions to repair DNA. However, overactivity of this enzyme can reduce intracellular NAD+ and ATP, resulting in necrosis of insulin-producing cells. Thus, the combination of STZ and HFD can induce diabetes mellitus conditions, namely insulin resistance and stable blood glucose levels, which resemble the syndrome in humans. Nicotinamide administration can protect by inhibiting PARP-1 activity, thereby preventing the reduction of NAD+ and ATP in cells10,11.

 

The ethanolic extract of E. subumbrans leaves was given for three weeks after the rats were established to have diabetes. Based on this study, the ethanolic extract of E. subumbrans leaves had the effect of lowering glucose levels starting at a dose of 200mg/200gr body weight of rats. Giving E. subumbrans extract on blood glucose levels in diabetic rats can be caused by phytochemical compounds in the extract. Based on research conducted by Phukhatmuen et al., pterocarpan, flavanones, and phenolic derivatives contained in the twigs and roots of E.subumbrans have antidiabetic effects. Pterocarpan compounds such as Erythrina, 1-methoxyerythrabyssin II, erythrabyssin II; and flavanone compounds such as 5-hydroxysophoranone isolated from the twigs and roots of E. subumbrans showed the ability to inhibit the a-glucosidase enzyme (8). The enzyme a-glucosidase causes the degradation of oligosaccharides, which is categorized as a cause of hyperglycemia because it can release glucose. With inhibition of a-glucosidase in the intestine, the rate of hydrolytic breakdown of oligosaccharides will decrease, thereby reducing carbohydrate digestion. Consequently, the degree of hyperglycemia will decrease23.

 

C. Effects of E. subumbrans Leaf Extract on Lipid profile:

Excess lipid intake and increased lipogenesis can cause atopic lipids to accumulate in metabolic organs resulting in increased adipose lipolysis in obese and T2DM patients, increasing the release of glycerol and fatty acids24.

 

Obesity and Type 2 Diabetes Mellitus are associated with increased lipolysis in adipose tissue, thereby increasing the production of cytokines such as TNF-a and IL-6 by macrophages present in adipose tissue. The release of TNF-a and IL-6 from macrophages is due to the secretion of adipocytokines such as RBP4 (Retinol-Binding Protein 4) from adipocytes. Fatty acids, including LCFAs (Long Chain Fatty Acids), are released by lipolysis and then taken up by muscles and liver via the Scavenger Receptor Class B member 1 (SRB1) fatty acid transporter. In muscle, LCFA thioester (LCFA-CoA) is imported into the mitochondria for β-oxidation via the carnitine shuttle to convert LCFA-CoA to LCACs (Long-Chain Acylcarnitines). Incomplete β-oxidation leads to the accumulation of acylcarnitine, which is associated with insulin resistance and hyperglycemia. In the liver, LCFA is imported into the mitochondria and oxidized to produce Acetyl-CoA, which activates pyruvate carboxylation, increasing PEP production (phosphoenolpyruvate) from pyruvate. Glycerol produced from the lipolysis process is converted to G6P (Glucose-6-phosphate), resulting in glucose production. An increased metabolic substrate to the liver causes insulin resistance and hyperglycemia25.

Type 2 diabetes is linked to many interconnected lipid and lipoprotein abnormalities in the blood, including low HDL cholesterol, a high prevalence of small dense LDL particles, and high triglycerides. Despite normal LDL cholesterol levels, many people develop these problems. These changes are also part of the insulin resistance syndrome (also known as the metabolic syndrome), linked to type 2 diabetes in many cases.In reality, pre-diabetic people often have an atherogenic pattern of risk factors, such as greater total cholesterol, LDL cholesterol, triglycerides, and lower HDL cholesterol, than people who don't acquire diabetes. Insulin resistance significantly impacts lipoprotein particle size and concentrations in the VLDL, LDL, and HDL subclasses26.

 

The ethanolic Extract of E. subumbrans leaves has improved the lipid profiles of animal models of type II diabetes mellitus, as shown in Table 2. The increase in HDL in the dose 3 group could be due to the flavonoids contained in the extract. The extraction and isolation of twigs and roots E. subumbrans plants showed positive results for flavonoids. The isolation results from this study showed that the roots and twigs of the E. subumbrans plant contained compounds from the pterocarpan group, flavanones, flavones, isoflavones, and phenol derivatives8. In addition to flavonoid compounds, ethanolicExtract of E. subumbrans also showed positive results for alkaloids, as shown in Figure 1. E. subumbrans contains erythrinan alkaloids in erythramine, hypaporine, erysodine, and erysopine22. Based on the research results by Kumar et al. (2011), erythramine contained in the same genus can reduce blood glucose levels and lipid profiles in streptozotocin-induced rats. The presence of secondary metabolites such as flavonoids and alkaloids can increase Lecithin Cholesterol Acyltransferase (LCAT). LCAT activity can increase cholesterol ester transport from peripheral tissues to the liver and stimulate circulating HDL production and secretion6. There was a decrease in adipose tissue weight and hepatic glycogen content in the animals model induced by Streptozotocin27. In theanimal model given the ethanolic Extract of E. subumbrans leaves, there was an increase in body weight. The ethanol extract of E. subumbrans leaves has the effect of lowering blood glucose levels. Thus, suppression of hepatic gluconeogenesis and hepatic blood glucose release is associated with inhibiting lipolysis in adipose tissue28.

 

D. Effects of E. subumbrans Leaf Extract on Creatinine and Ureum serum

Diabetic nephropathy, commonly known as diabetic kidney disease, is a condition caused by diabetes usually occurs in patients with T1DM and T2DM. In DM, glycemic control is not maintained for a long time, causing diabetic nephropathy. In DKD, changes in renal structure occur in the form of mesangial expansion, thickening of the glomerular and tubular basement membranes, and glomerular sclerosis. DKD usually causes clinical signs such as albuminuria, increased blood pressure, decreased GFR, increased cardiovascular events, and mortality from cardiovascular causes29.


 

 


Table 2. The Lipid profile levels of the treated and untreated rat.

 

Kelompok

The difference in Lipid Profile Levels Before and After Administration of Extract of E. subumbrans leaves (mg/dL) (Rata-rata ± SEM)

TG

TC

HDL-C

LDL-C

Normal

-0.71± 0.84

0.63 ± 0.16

2.01 ± 1.97

-6.31 ± 2.96

Negative

86.68 ± 7.25

322.53 ± 9.48

-5.74 ± 3.79

274.67 ± 7.17

Positive

-85.83 ± 6.21*

-16.00 ± 1.86*

18.50 ± 5.69*

-8.89 ± 2.92*

E. subumbrans extract dose1

-38.81 ± 7.62*

21.69 ± 5.16*

-3.30 ± 1.05

26.50 ± 7.67*

E. subumbrans extract dose2

-57.65 ± 7.90*

44.07 ± 4.90*

-3.01 ± 7.21

16.91 ± 1.14*

E. subumbrans extract dose3

-63.46 ± 7.57*

-12.00 ± 1.71*

16.73 ± 2.49*

-5.86 ± 2.52*

Note: TG: Triglycerides; TC: Total Cholesterol; HDL-C: High-Density Lipoprotein -C; LDL-C: Low-Density Lipoprotein-C; * = (p<0.05)compared to the negative group.

 

Table 3. Serum Creatinine Levels After and Before Administration of Extract

Group

Before Administration of Extract (mg/dl)

After Administration of Extract (mg/dl)

Difference (mg/dl)

Normal

0.44 ± 0.11

0.49 ± 0.19

0.05 ± 0.14

Negative

1.01 ± 0.34*

1.54 ± 0.52*

0.53 ± 0.44*

Positive

0.92 ± 0.20*

0.38 ± 0.11#

-0.54 ± 0.10#

E. subumbrans extract dose 1

1.07 ± 0.00*

0.94 ±0.00

-0.13 ±0.00

E. subumbrans extract dose 2

1.19 ± 0.09*

1.06 ± 0.42

-0.13 ± 0.37

E. subumbrans extract dose 3

1.06 ± 0.34*

0.52 ± 0.06#

-0.54 ± 0.29#

Note: (*) p<0.05 compared to the normal group, (#) p<0.05 compared to the negative group


Inflammation, oxidative stress, and renal apoptosis can be linked directly or indirectly to the pathogenesis of diabetic nephropathy. Excessive ROS generation due to hyperglycemia causes an imbalance in the body to counteract ROS production due to decreased antioxidant defenses that cause kidney dysfunction.

 

Table 3 showed a significant increase in serum creatinine after 21 days since the injection of STZ at a dose of 40 milligrams per kilogram of body weight and Nicotinamide and also showed a significant difference between the positive and normal groups (p<0.05). These indicate a significant decrease in serum creatinine after 21 days of usingE. Subumbrans leaf ethanol extract.

 

Table 4. UreumSerum Levels Before and After Administration of Extract

Group

Before Administration of Extract (mg/dl)

After Administration of Extract (mg/dl)

Difference

(mg/dl)

Normal

26.95 ±

3.24

24.06 ±

3.49

-2.89 ± 3.08

Negative

40.09 ±

4.18*

50.94 ± 12.63*

10.85 ± 15.68*

Positive

45.23 ± 10.89*

28.77 ±

6.91#

-16.45 ± 13.04 #

E. subumbrans extract dose 1

35.85 ±

0.00*

42.16 ±

0.00

6.32 ±

0.00

E. subumbrans extract dose 2

47.19 ±

9.43*

46.37 ±

7.43

-0.82 ± 10.34

E. subumbrans extract dose 3

47.22 ± 10.36*

32.06 ±

6.66#

-15.16 ± 10.00 #

Note: (*) p<0.05 compared to the normal group, (#) p<0.05 compared to the negative group

 

Table 4 showed a significant difference between the normal group and the positive and negative groups (p<0.05). These indicate a significant increase in serum urea compared to the normal group in the negative group. In addition, it showed a significant decrease in serum urea compared to the normal group in the positive group.

 

Table 5. Changes in MDA Levels during Administration of Extract

Group

Changes in MDA Levels during Extract Administration (ng/ml) (Mean ± SEM)

Normal

-145.55 ± 108.39

Negative

556.67 ± 168.34*

Positive

-141.11 ± 88.41#

E. subumbrans extract dose 1

191.67 ± 258.34

E. subumbrans extract dose 2

-121.67 ± 95#

E. subumbrans extract dose 3

-248.89 ± 79.73#

Note: (*) p<0.05 compared to the normal group, (#) p<0.05 compared to the negative group

 

Figure 5. shows an increase in MDA levels in rats induced by HFD and STZ/NA combination. Accumulation of free radicals such as superoxide anion during HFD/STZ induction can lead to loss or reduction of antioxidants, causing oxidative stress and lipid peroxidation conditions. These are characterized by increasing levels of MDA. In a diabetic state, the electron transport chain in the mitochondria will be activated and will cause more Reactive Oxygen Species (ROS) production. Excessive ROS production due to increased glucose and FFA oxidation will also lead to progressive metabolic and mitochondrial dysfunction. These will result in a state of oxidative stress, which can potentially reduce insulin secretion from the pancreas and inhibit insulin signaling in tissues.

 

ROS and oxidative stress can react and cause the peroxidation of polyunsaturated fatty acids (PUFA) and produce the final product, namely malondialdehyde (MDA). STZ injection, a cytotoxic drug, exerts its cytotoxic effect through free radical-mediated mechanisms. The hyperglycemia condition caused by HFD/STZ induction increased ROS and oxidation products such as lipid peroxidation product: MDA. The mechanism that causes oxidative stress in DM can be caused by overproduction of superoxide anion radicals (O2•−) in the mitochondrial electron transfer chain, glucose autoxidation, as well as activation of the polyol pathway, and activation of protein kinase C (PKC) that the administration of extracts at doses 1 and 2 did not significantly reduce MDA levels. While in the positive group and dose 3, there was a significant difference in changes in MDA levels during the extract administration compared to the negative group (p 0.05). These illustrate that administering metformin and ethanol extract of Dadap thorn leaves at a dose 3 can reduce MDA levels in diabetes mellitus.

 

Table 6. 8-OHdG Serum Level Difference Before and After Administration of Extract

Group

Difference (ng/ml)

Normal

-54.50 ± 0.71

Negative

48.25 ± 39.24*

Positive

18.50 ± 0.00#

E. subumbrans extract dose 1

-13.25 ± 4.60#

E. subumbrans extract dose 2

34 ± 12.73

E. subumbrans extract dose 3

-2.25 ± 8.84#

Description: basedontheAnovatest, (*)p<0.05for the normal group and (#) p>0.05 for the positive group

 

In addition to serum creatinine and urea levels, serum 8-OHdG levels were also analyzed because it was known that serum 8-OHdG levels increased with decreased kidney function(30). The difference in serum levels of 8-OHdG for all treatment groups as shown in Table 6.8-Hydroxydeoxyguanosine (8-OHdG) results from oxidative DNA damage caused by particular enzymatic cleavage following ROS-induced DNA damage. In mitochondria and nuclear DNA, the guanine base is 8-hydroxylated.Another study conducted on STZ-induced diabetic rats showed that serum levels of 8-OHdG in the diabetes control group were higher than in other groups. In addition, 8-OHdG levels are elevated in tissues and body fluids in diabetic subjects31-33.

 

Flavonoids are reported to be contained in the E. subumbrans, including flavanones, isoflavones, and pterocarpans34. Research conducted by Phukhatmuen et al. (2021) carried out the extraction and isolation of the twigs and roots of the E. subumbrans showed pterocarpan compounds, flavanones, flavones, isoflavones, and phenol derivatives. Some of these compounds showed -glucosidase inhibitor activity, namely eryvarin D, 1-methoxyerythrabyssin I, erythrabyssin II, and 5-hydroxysophoranone8.

 

CONCLUSION:

E. subumbrans extracts could improve diabetesin model rats by reducing lipid peroxidation and 8-OHDG produced during treatment. Therefore improve the kidney function and lipid profiles of serum and blood glucose of diabetic model rats. It is very promising to be developed as a diabetes drug in the future.

 

CONFLICTS OF INTEREST:

There are no conflicts of interest.

 

ACKNOWLEDGMENT:

This research was supported by The Ministry of Research and Technology/BRIN Republic of Indonesia 2021. NKB 057/UN2.RST/HKP.05.00/2021

 

REFERENCE:

1.     Skyler JS, Bakris GL, Bonifacio E, Darsow T, Eckel RH, Groop L, et al. Differentiation of Diabetes by Pathophysiology, Natural History, and Prognosis. Diabetes [Internet]. 2016/12/15. 2017 Feb;66(2):241–55. Available from: https://pubmed.ncbi.nlm.nih.gov/27980006

2.     Tiwari BK, Pandey KB, Abidi AB, Rizvi SI. Markers of Oxidative Stress during Diabetes Mellitus. Dusinska M, editor. J Biomarkers [Internet]. 2013;2013:378790. Available from: https://doi.org/10.1155/2013/378790

3.     Chong S, Ding D, Byun R, Comino E, Bauman A, Jalaludin B. Lifestyle Changes After a Diagnosis of Type 2 Diabetes. Diabetes Spectr [Internet]. 2017 Feb;30(1):43–50. Available from: https://pubmed.ncbi.nlm.nih.gov/28270714

4.     Pang G-M, Li F-X, Yan Y, Zhang Y, Kong L-L, Zhu P, et al. Herbal medicine in the treatment of patients with type 2 diabetes mellitus. Chin Med J (Engl) [Internet]. 2019 Jan 5;132(1):78–85. Available from: https://pubmed.ncbi.nlm.nih.gov/30628962

5.     Bahtiar A, Vichitphan K, Han J. Leguminous plants in the Indonesian archipelago: Traditional uses and secondary metabolites. Nat Prod Commun. 2017;12(3).

6.     Yashwant Kumar A, Nandakumar K, Handral M, Talwar S, Dhayabaran D. Hypoglycaemic and anti-diabetic activity of stem bark extracts Erythrina indica in normal and alloxan-induced diabetic rats. Saudi Pharm J  SPJ  Off Publ Saudi Pharm Soc [Internet]. 2010/10/31. 2011 Jan;19(1):35–42. Available from: https://pubmed.ncbi.nlm.nih.gov/23960740

7.     Kumar A, Lingadurai S, Shrivastava TP, Bhattacharya S, Haldar PK. Hypoglycemic activity of Erythrina variegata leaf in streptozotocin-induced diabetic rats. Pharm Biol [Internet]. 2011 Jun 1;49(6):577–82. Available from: https://doi.org/10.3109/13880209.2010.529615

8.     Phukhatmuen P, Meesakul P, Suthiphasilp V, Charoensup R, Maneerat T, Cheenpracha S, et al. Antidiabetic and antimicrobial flavonoids from the twigs and roots of Erythrina subumbrans (Hassk.) Merr. Heliyon [Internet]. 2021;7(4):e06904. Available from: https://www.sciencedirect.com/science/article/pii/S2405844021010070

9.     Guo X-X, Wang Y, Wang K, Ji B-P, Zhou F. Stability of a type 2 diabetes rat model induced by high-fat diet feeding with low-dose streptozotocin injection. J Zhejiang Univ Sci B [Internet]. 2018 Jul;19(7):559–69. Available from: https://pubmed.ncbi.nlm.nih.gov/29971994

10.  Ghasemi A, Khalifi S, Jedi S. Streptozotocin-nicotinamide-induced rat model of type 2 diabetes (review). Acta Physiol Hung [Internet]. 2014;101(4):408–20. Available from: https://akjournals.com/view/journals/036/101/4/article-p408.xml

11.  Szkudelski T. Streptozotocin–nicotinamide-induced diabetes in the rat. Characteristics of the experimental model. Exp Biol Med [Internet]. 2012 May 1;237(5):481–90. Available from: https://doi.org/10.1258/ebm.2012.011372

12.  Dwitiyanti, Harahap Y, Elya B, Bahtiar A. Study of Molecular Docking of Vitexin in Binahong (Anredera cordifolia (Ten.) Steenis) Leaves Extract on Glibenclamide-CYP3A4 Interaction. Pharmacogn J. 2019 Nov;11:1471–6.

13.  Srinivasan K, Viswanad B, Asrat L, Kaul CL, Ramarao P. Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: A model for type 2 diabetes and pharmacological screening. Pharmacol Res [Internet]. 2005;52(4):313–20. Available from: https://www.sciencedirect.com/science/article/pii/S1043661805001039

14.  Morley LA, Gomez TH, Goldman JL, Flores R, Robinson MA. Accuracy of 5 Point-of-Care Glucometers in C57BL/6J Mice. J Am Assoc Lab Anim Sci [Internet]. 2018 Jan 1;57(1):44–50. Available from: https://pubmed.ncbi.nlm.nih.gov/29402351

15.  Bahtiar A, Amandasari F. The effect of 70% Ethanolic extract of Dayak Onion Bulbs and Cowpea on Cardiovascular Parameters of Hypoestrogen Model Rats. J Nat Remedies. 2019;19(2).

16.  Widyastuti DA, Ristianti MA, Sari IM. The Study of Blood Creatinin and Urea Concentration of Wistar Rats (Rattus norvegicus) due to Sodium Nitrite Induction. J ILMU KEFARMASIAN Indones Vol 17 No 1 JIFIDO  - 1035814/jifi.v17i1560  [Internet]. 2019 Apr 24; Available from: http://jifi.farmasi.univpancasila.ac.id/index.php/jifi/article/view/560

17.  Bahtiar A, Miranda AJ, Arsianti A. The Effect of Artocarpus altilis (Parkinson) Fosberg Extract Supplementation on Kidney Ischemia-Reperfusion Injury Rat. Pharmacogn J. 2021;13(1).

18.  Bahtiar A, Utami PS, Noor MR. The Antioxidant Effects of the Ethanolic Extract of Binahong Leaves Unilateral Ureteral Obstruction Rat Model. Pharmacogn J. 2021;13(1).

19.  Kim JY, Prouty LA, Fang SC, Rodrigues EG, Magari SR, Modest GA, et al. Association between fine particulate matter and oxidative DNA damage may be modified in individuals with hypertension. J Occup Environ Med [Internet]. 2009 Oct;51(10):1158–66. Available from: https://pubmed.ncbi.nlm.nih.gov/19786898

20.  Zhang M, Lv X-Y, Li J, Xu Z-G, Chen L. The characterization of high-fat diet and multiple low-dose streptozotocin induced type 2 diabetes rat model. Exp Diabetes Res [Internet]. 2009/01/04. 2008;2008:704045. Available from: https://pubmed.ncbi.nlm.nih.gov/19132099

21.  Abraham A, Fasya AG, Fauziyah B, Adi TK. Uji Antitoksoplasma Ekstrak Kasar Alkaloid Daun Pulai (Alstonia scholaris, (L.) R. BR) Terhadap Mencit (Mus musculus) Balb/C Yang Terinfeksi Toxoplasma Gondii Strain Rh. Alchemy; ALCHEMY (Vol3, No1; 03-2014) [Internet]. 2014; Available from: https://ejournal.uin-malang.ac.id/index.php/Kimia/article/view/2896

22.  Folkers K, Shavel J, Koniuszy F. Erythrina Alkaloids. X. Isolation and Characterization of Erysonine and Other Liberated Alkaloids. J Am Chem Soc [Internet]. 1941 Jun 1;63(6):1544–9. Available from: https://doi.org/10.1021/ja01851a016

23.  Younus M, Hasan MM ul, Ahmad K, Sharif A, Asif HM, Aslam MR, et al. α-Glucosidase Inhibitory, Anti-Oxidant, and Anti-Hyperglycemic Effects of Euphorbia nivulia–Ham. in STZ-Induced Diabetic Rats. Dose-Response [Internet]. 2020 Jul 1;18(3):1559325820939429. Available from: https://doi.org/10.1177/1559325820939429

24.  Perry RJ, Camporez J-PG, Kursawe R, Titchenell PM, Zhang D, Perry CJ, et al. Hepatic Acetyl CoA Links Adipose Tissue Inflammation to Hepatic Insulin Resistance and Type 2 Diabetes. Cell [Internet]. 2015 Feb 12;160(4):745–58. Available from: https://doi.org/10.1016/j.cell.2015.01.012

25.  Yang S, Han Y, Liu J, Song P, Xu X, Zhao L, et al. Mitochondria: A Novel Therapeutic Target in Diabetic Nephropathy [Internet]. Vol. 24, Current Medicinal Chemistry. 2017. p. 3185–202. Available from: http://www.eurekaselect.com/node/152279/article

26.  Krauss RM. Lipids and Lipoproteins in Patients With Type 2 Diabetes. Diabetes Care [Internet]. 2004 Jun 1;27(6):1496 LP – 1504. Available from: http://care.diabetesjournals.org/content/27/6/1496.abstract

27.  Jack BU, Malherbe CJ, Mamushi M, Muller CJF, Joubert E, Louw J, et al. Adipose tissue as a possible therapeutic target for polyphenols: A case for Cyclopia extracts as anti-obesity nutraceuticals. Biomed Pharmacother [Internet]. 2019;120:109439. Available from: https://www.sciencedirect.com/science/article/pii/S0753332219330562

28.  Hatting M, Tavares CDJ, Sharabi K, Rines AK, Puigserver P. Insulin regulation of gluconeogenesis. Ann N Y Acad Sci [Internet]. 2017/09/03. 2018 Jan;1411(1):21–35. Available from: https://pubmed.ncbi.nlm.nih.gov/28868790

29.  Lin Y-C, Chang Y-H, Yang S-Y, Wu K-D, Chu T-S. Update of pathophysiology and management of diabetic kidney disease. J Formos Med Assoc [Internet]. 2018;117(8):662–75. Available from: https://www.sciencedirect.com/science/article/pii/S0929664617308033

29.Dai L, Watanabe M, Qureshi AR, Mukai H, Machowska A, Heimbürger O, et al. Serum 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, is associated with mortality independent of inflammation in chronic kidney disease. Eur J Intern Med [Internet]. 2019 Oct 1;68:60–5. Available from: https://doi.org/10.1016/j.ejim.2019.07.035

30.  Giugliano D, Ceriello A, Paolisso G. Oxidative Stress and Diabetic Vascular Complications. Diabetes Care [Internet]. 1996 Mar 1;19(3):257 LP – 267. Available from: http://care.diabetesjournals.org/content/19/3/257.abstract

31.  Park KS, Kim JH, Kim MS, Kim JM, Kim SK, Choi JY, et al. Effects of Insulin and Antioxidant on Plasma 8-Hydroxyguanine and Tissue 8-Hydroxydeoxyguanosine in Streptozotocin-Induced Diabetic Rats. Diabetes [Internet]. 2001 Dec 1;50(12):2837 LP – 2841. Available from: http://diabetes.diabetesjournals.org/content/50/12/2837.abstract

32.  Andican G, Burçak G. Oxidative Damage To Nuclear Dna In Streptozotocin-Diabetic Rat Liver. Clin Exp Pharmacol Physiol [Internet]. 2005 Aug 1;32(8):663–6. Available from: https://doi.org/10.1111/j.0305-1870.2005.04247.x

33.  Rukachaisirikul T, Chokchaisiri S, Suksamrarn A. Chemical Constituents of the Roots of Erythrina subumbrans. Chem Nat Compd [Internet]. 2014;49(6):1127–8. Available from: https://doi.org/10.1007/s10600-014-0838-7

 

 

 

 

 

Received on 12.10.2021             Modified on 21.03.2022

Accepted on 15.06.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(12):5651-5658.

DOI: 10.52711/0974-360X.2022.00953