Antioxidant Total and HOMA-IR of Diabetic Rats Given Crocatum piper and Andrographis paniculata Leaf Extracts

Background: type-2 diabetes mellitus (T2DM) is a silent killer which the prevalence continues to increase in every year. Increased oxidative stress occurs in T2DM. Red betel and bitter herb leaf extracts (RBBH) contain a lot of antioxidants. This combination is expected to provide better safety than if used singly because the content of andrographolid in bitter herb has effect such as nausea, vomiting, loss of appetite, and antifertility if consumed in high doses. Objective: the study aimed to prove the effect of red betel and bitter herb leaf extracts on antioxidant total and Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) in T2DM rats given high-fat diet and Streptozotocin (STZ) induction. Methods: experimental randomized study with pre-post-test control group design using 25 Sprague Dawley male rats. T2DM model was conducted by providing high-fat feed for 14 days and induction of Streptozotocin-Nicotinamide , then given a combination of red betel and bitter herb leaf extracts at doses of 237.5 mg/kg BW, 225 mg/kg BW, and 212.5 mg/kg BW for 21 days. The measurement of antioxidant total used 2,2-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) method. HOMA-IR measured by ELISA (Enzyme-linked immunosorbent assay) method using insulin level equation and fasting blood glucose level measured by glucose oxidase-peroxidase aminoantrypirin (GOD-PAP). Data analysis used paired t-test, wilcoxon test, and ANOVA test to analyze differences in antioxidant total and HOMA-IR value among groups and followed by Bonferroni post-hoc test. Results: all treatments could reduce HOMA-IR and significantly increase antioxidant total (p<0.05). The most decrease in HOMA-IR and increase in antioxidant total at dose 237.5 mg/kg BW of red betel and bitter herb leaf extracts. Conclusion: the combination of red betel and bitter herb leaf extracts with dose 237.5 mg/kg BW, 225 mg/kg BW, and 212.5 mg/kg BW can improve blood glucose, insulin, and HOMA-IR levels in type-2 diabetes mellitus rats.


INTRODUCTION
Diabetes mellitus (DM) is one of the health problems with high mortality and morbidity rates in the world including in Indonesia.It is classified in degenerative disease as silent killer and most people do not realize they suffer until complications occur. 1 The International Diabetes Federation (IDF) reported that in 2017 there were 425 million people in the world have diabetes.Indonesia ranks the sixth highest after China, India, America, Brazil, and Mexico.That number is expected growing up to 629 million people in 2045 and 90% of these cases are type-2 diabetes mellitus (T2DM). 2 Insulin resistance is the failure of tissues to respond the insulin activity, as well as that is a strong predictor of T2DM and hyperinsulinemia. 3Insulin resistance can be determined by examining the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) of fasting blood glucose and fasting insulin levels. 4The increasing of reactive oxygen species (ROS) formation in hyperglycemia or diabetes mellitus results in oxidative stress. 5High levels of ROS can reduce antioxidant total.Prediabetic patients have an increase in oxidative stress characterized by a decrease in antioxidant total compared with healthy patients. 6Antioxidants can protect body from negative effects of oxidants, free radicals, and inflammation by counteracting ROS and preventing the formation of ROS. 7 Therapy for DM like insulin injection and oral antidiabetic which have side effects such as dizziness, nausea, and anorexia make patients switch it to natural treatment from herbal plants. 80][11] Red betel leaf extract 200 mg/kg BW can reduce blood glucose levels in diabetic rats.Red betel leaf extract contains high antioxidant like flavonoid which acts as antihyperglycemic agents. 9Bitter herb leaf extract with a dose of 200 mg/kg BW for 10 days shows a significantly decrease in fasting blood glucose. 10This combination is expected to provide better safety than if used singly because the content of andrographolid in bitter herb has effect such as nausea, vomiting, loss of appetite, and antifertility if consumed in high doses. 12A combination of two ingredients like bitter herb and red betel is carried out to provide safety from the effects of consuming bitter leaves without reducing its benefits.This study aimed to determine the combination of red betel and bitter herb leaf extracts against antioxidants total and HOMA-IR in type-2 diabetes mellitus rats..

Plant material
Red betel (Piper crocatum) leaf obtained from Tawangmangu, Karang Anyar Regency, Central Java on October 2018.The extracts of bitter herb (Andrigraphis paniculata) obtained from PT. Industri Borobudur Herbal Medicine, Central Java.Red betel leaf was tested for substance identification at the Plant Systematics Laboratory, Faculty of Biology, University of Gadjah Mada, Yogyakarta.

Chemicals and reagents
Streptozotocin (STZ) and nicotinamide (NA) obtained from Nacalai Tesque, Japan.The Antioxidant Total Kit purchased from Randox Laboratory, England.The Rat INS kit (Insulin) brand "Fine Test" and blood glucose kit brand "Diasys" obtained from Holzheim, Germany.

Preparation of Piper crocatum leaf extract
Extracts made by maceration method using 70% ethanol solvent.The leaves were thinly sliced and dried with good air circulation and were not directly exposed to sunlight.After dried, the leaves were mashed then weighed and put into a container for maceration.Maceration using 70% ethanol solvent with ratio 1:10 for two days protected from sunlight, while stirring, filtered with filter paper to get macerata.The pulp was macerated using 70% ethanol by same procedure.All maceratas were combined and evaporated with vacuum rotary evaporator at 40°C until thick ethanol extract obtained and dried with freeze dryer to get dry extract (powder). 13,14

Animal laboratory
Sprague Dawley male rats aged 8-10 weeks, weight 150-200 gram were obtained from the Center for Food and Nutrition Studies Laboratory of Gadjah Mada University, Yogyakarta and placed in individual stainless-steel cages.Room temperature was around 24-27°C and humidity 60-65% with adequate ventilation and lighting using 12 hours cycle of light and dark.Rats fed 20 g of comfeed II per day and given water by ad-libitium.Caring procedures and research treatments were adjusted to the ethics of experimental animal research at the Center for Food and Nutrition Studies Laboratory of Gadjah Mada University, Yogyakarta.

Design experiments
The study used true experimental study with randomized pre-post-test with control group design.Twenty five animals (rats) were divided into 5 groups randomly, such as negative control group (healthy rats) (K1), positive control group (T2DM conditioning without treatment) (K2), combined intervention of red betel and bitter herb leaf extracts 237.5 mg/kg BW (P1), combined intervention of red betel and bitter herb leaf extracts 225 mg/kg BW (P2), combined intervention of red betel and bitter herb leaf extracts 212.5 mg/kg BW (P3). 15The distribution of RBBH dose combinations based on modification from Thirumalai et al. (2014)  showed that effective dose of red betel leaf extract on reducing cholesterol level and Akhtar et al. (2016)  showed that effective dose of bitter herb on restoring metabolic profile of obesity with diabetes.The Comparisons of red betel and bitter herb doses were respectively 187.5 mg/kg BW and 50 mg/kg BW (75%:25%) on dose 1, 125 mg/kg BW and 100 mg/kg BW (50%:50%) on dose 2, and 62.5 mg/kg BW and 150 mg/kg BW (25%:75%) on dose 3.
Rats were acclimatized for a week and then given high-fat feed for 14 days, then induced by Streptozotocin 55 mg/kg BW and nicotinamide 100 mg/kg BW intraperitoneally.Experimental animals were categorized as diabetes mellitus if their blood glucose level more than 200 mg/dl. 16Technical of extracts administration used feeding tube for 21 days in a row.The study has obtained Ethical Clearance approval from the Health Research Ethics Commission of Faculty of Medicine Universitas Diponegoro (Dr.Kariadi Hospital Semarang) with registered number 114/EC/H/FK-RSDK/X/2018.

Blood sampling
Blood sampling was conducted after 3 days of STZ induction and 21 days after intervention.Rats were fasted overnight before blood was drawn through orbital plexus.Blood samples were centrifuged at 4000 rpm for 15 minutes and then serum was taken to analyze antioxidant total, insulin, and glucose levels.

HOMA-IR value
HOMA-IR value was obtained from analysis of insulin and blood glucose levels with the following formula, HOMA-IR = {fasting glucose (mg/dl) x fasting insulin (µU/ml)}/405.

Statistical analysis
Data were analyzed computerization and displayed in mean±SD.The number of sample was less than 50, so normality test using Shapiro-Wilk.The different test of antioxidant total and HOMA-IR value before and after intervention used paired t-test, while different test of antioxidant total and HOMA-IR value among groups used one-way ANOVA continued with Bonferroni post-hoc test.Significant value was stated if p<0.05.

Characteristics of experimental animal
The study used 25 Sprague Dawley male rats.There were no sick or died rats in this study.The mean of feed intake in all groups did not increase before or after the administration of intervention, while all intervention groups showed that there was an increase in body weight.The analysis results of intervention effect on feed intake showed in Table 1 and intervention effect on body weight could be seen in Table 2.

Blood glucose level
All intervention groups showed significantly decreased in blood glucose level compared to control group (Table 3).The combined intervention of red betel and bitter herb leaf extracts 237.5 mg/kg BW (P1) showed the highest decreased in blood glucose level.Paired t-test showed that there were differences in blood glucose levels before and after intervention (p<0.05).

Insulin level
All intervention groups showed significantly decreased in insulin level.The combined intervention of red betel and bitter herb leaf extracts 237.5 mg/kg BW (P1) showed the highest decreased in insulin level.Paired t-test showed that there were differences in insulin levels before and after intervention (p<0.05).

HOMA-IR value
All intervention groups showed significantly decreased in HOMA-IR value.The combined intervention of red betel and bitter herb leaf extracts 237.5 mg/kg BW (P1) showed the highest decreased in HOMA-IR value.Paired t-test showed that there were differences in HOMA-IR value before and after intervention (p<0.05).

Antioxidant total level
All intervention groups showed significantly increased in antioxidant total level mean.The highest increased in antioxidant total level mean occurred in group with combined intervention of red betel and bitter herb leaf extracts 237.5 mg/kg BW (P1).Paired t-test showed that there were differences in antioxidant total levels before and after intervention (p<0.05).

DISCUSSION
High-fat feed is given to every rat as much as 20 grams for 14 days.High-fat feed for 2 weeks showed an increase in body weight. 17High-fat feed was also increase percentage of body fat and risk of insulin resistance. 18The induction of STZ-NA in experimental animals was conducted on day-15.Streptozotocin induction caused damage to pancreatic beta cells.Nicotinamide was given to slowing down damage, preventing damage, and preventing total damage to pancreatic beta cells. 19High-fat feed and STZ-NA induction in T2DM conditioning could form obesity and insulin resistance. 16,17uring the study, weight of rats was weighed every week.The weight of treated rats increased and approached to healthy control group body weight.While, rats in control group had weight loss.This has happened because control group (K2) was in T2DM conditioning group which one of its characteristics was have drastically weight loss due to glycogenolysis and lipolysis which caused decrease in muscle mass and fat tissue.Whereas, the weight of treatment groups has increased because it was able to repair pancreatic beta cells so that it can increase insulin production.Improved insulin availability in tissue triggers an increase in fat tissue and muscle mass, thereby affecting weight gain. 9,20he administration of red betel leaf extract for 21 days could increase body weight. 20igh-fat feed and STZ induction have impact on insulin resistance and pancreatic beta cell damage, causing increase in blood glucose level or hyperglycemia. 18,19In contrast to this study, control group (K2) had decrease in fasting blood glucose levels by -90±57.04(19.5%), although it was still in diabetic category (≥135 mg/dl).This could be in control group (K2), after the administration of high-fat feed and STZ induction, it continued with standard feeding so that changed in eating patterns in rats resulted in a decrease in blood glucose levels even though the decrease was not as good as compared to the intervention group.
The decrease in blood glucose levels in intervention group was caused by presence of bioactive compounds contained in extract of red betel leaf including flavonoid, alkaloid, tannin, and saponin, while bitter herb extracts contained andrographolid, flavonoid, saponin, phenolic total, and anthraquinone. 21,22,23As antidiabetic agent, flavonoid and alkaloid are able to regenerate damaged pancreatic beta cells. 8Red betel leaf extract could reduce blood glucose level in diabetic rats. 24Alkaloid works by stimulating hypothalamus to increase the secretion of Growth Hormone Releasing Hormone (GHRH), so that the secretion of Growth Hormone (GH) in pituitary increased.High GH level will stimulate liver to secrete insulin-like growth factor-1 (IGF-1).IGF-1 has effect in inducing hypoglycemia and decreasing gluconeogenesis so that blood glucose level and insulin requirements decreased.IGF-1 through negative feedback system will normalize GH level again. 8itter herb is not only contained flavonoid, but also andrographolid which can inhibit gluconeogenesis and alpha glukosidase in the intestine. 24,25ndrographolid is the main component of lactone compound in bitter her which belongs to diterpenoid group. 26Bitter herb extract could reduce blood glucose level in diabetic rats within 10 days. 10It can be said that bitter herb extract is as good as red betel leaf extract to lower blood glucose level.
The highest increase in insulin level was found in combined intervention of red betel and bitter herb leaf extract with dose 237.5 mg/kg BW (P1), while the lowest increase in insulin level was at dose 212.5 mg/kg BW (P3).Red betel leaf extract could increase blood insulin level up to 41.50% compared with diabetic group. 27Study on red betel leaf extract has a good effect, but the other hand bitter herb extract is also has effect as good as red betel leaf extract.The administration of bitter herb extract for 10 days could increase insulin level in diabetic rats. 10The content of alkaloid in red betel leaf extract and bitter herb has hypoglycemic activity by increasing insulin level through regenerate damaged pancreatic beta cells, protect from damage, and stimulate insulin release.Increased insulin secretion is due to stimulation of sympathomimetic nerves by alkaloid compound. 28,29lavonoid compounds also have hypoglycemic activity which have a role in increasing the activity of antioxidant enzymes and improving sensitivity of insulin receptors. 30Tannin can form a layer that protects intestine and causes intestinal epithelial membrane to constrict, thereby reducing the absorption of nutrients that can inhibit carbohydrate intake and rate of increase in blood sugar is not too high. 8,30Flavonoid compounds contained in red betel extract and bitter herb are thought to improve sensitivity of insulin receptors.Flavonoids are also protective against damage to pancreatic beta cells and restore insulin sensitivity to cells and can even increase insulin sensitivity.Flavonoids in improving insulin sensitivity act as ROS-trapping antioxidant.The excess ROS can activate several kinases which can disrupt phosphorylation of insulin receptor substrate 1 (IRS-1) and insulin receptor substrate 2 (IRS-2) which play a role in insulin signaling intracelullar. 31ncreasing level of antioxidant total occurred in all intervention groups was due to red betel and bitter herb leaf extract which had quite high antioxidant activity content.Antioxidant activity contained in red betel and bitter herb leaf extracts were 84.474% and 87.135%, respectively.Antioxidants have property to protect pancreatic cells from free radicals (superoxide, hydrogen peroxide, nitric oxide, and hydroxyl radicals) and prevent damage to pancreatic beta cells due to oxidation. 24xidative stress in diabetes mellitus occurs due to changes in carbohydrate and lipid metabolism so that it can reduce antioxidant defense system.The content of flavonoid and tannin in red betel and bitter herb leaf extract acts as an antioxidant that can protect and capture free radicals, besides that alkaloid compound has ability to stop free radical chain reactions. 30The main content of bitter herb is andrographolid which can also gradually reduce ROS so that it can improve insulin sensitivity. 32

CONCLUSION
The combination of red betel and bitter herb leaf extract with dose 237.5 mg/kg BW, 225 mg/kg BW, and 212.5 mg/kg BW can increase antioxidant total and improve blood glucose, insulin, and HOMA-IR levels in type-2 diabetes mellitus rats.

Table 1 .
Mean of feed intake changes before and after RBBH intervention intervention of red betel and bitter herb leaf extracts.Values are shown as mean±SD (n = 5 rats/group).%∆: the percentage of changes between pre and post-intervention.p: paired t-test.p': one-way ANOVA test followed by Bonferroni post-hoc test.*: significant value at p<0.05

Table 2 .
Mean of body weight changes before and after RBBH intervention RBBH: combined intervention of red betel and bitter herb leaf extracts.Values are shown as median (minimum-maximum) (n = 5 rats/group).%∆: the percentage of changes between pre and post-intervention.p: paired t-test.p': one-way ANOVA test followed by Bonferroni post-hoc test.*: significant value at p<0.05

Table 3 .
Mean of blood glucose level changes before and after RBBH intervention RBBH: combined intervention of red betel and bitter herb leaf extracts.Values are shown as mean±SD (n = 5 rats/group).%∆: the percentage of changes between pre and post-intervention.p: paired t-test.p': one-way ANOVA test followed by Bonferroni post-hoc test.*: significant value at p<0.05

Table 4 .
Mean of insulin level changes before and after RBBH intervention intervention of red betel and bitter herb leaf extracts.Values are shown as mean±SD (n = 5 rats/group).%∆: the percentage of changes between pre and post-intervention.p: paired t-test.p': one-way ANOVA test followed by Bonferroni post-hoc test.*: significant value at p<0.05

Table 5 .
Mean of HOMA-IR value changes before and after RBBH intervention RBBH: combined intervention of red betel and bitter herb leaf extracts.Values are shown as mean±SD (n = 5 rats/group).%∆: the percentage of changes between pre and post-intervention.p: paired t-test.p': one-way ANOVA test followed by Bonferroni post-hoc test.*: significant value at p<0.05

Table 6 .
Mean of antioxidant total level changes before and after RBBH intervention