Hypoglicemic and Antioxidant Activity of Petiveria alliacea in Diabetic Rat Models

Article history: Received 13 May 2020 Received in revised form 29 May 2020 Accepted 03 May 2020


Introduction
Diabetes mellitus (DM) is a degenerative disease that is experienced by most people in the world. According to WHO 1 , 347 million people worldwide suffer from diabetes mellitus and is expected in 2030, diabetes mellitus will be the seventh leading cause of death in the world. The deaths allegedly due to complications of diabetes affect various organs of the body. In Indonesia, the estimated prevalence will increase to 21.3 million in 2030. 2 DM is a condition of hyperglycemia in the body. DM can be caused due to the disruption of the synthesis and secretion of insulin, and also can be caused by the resistance of tissue sensitivity to insulin. Various complications of diabetes are caused by the imbalance condition of oxidants and antioxidants known as oxidative stress. Highly reactive free radicals can cause tissue damages. 3 This leads to complications in many organs. Oxidative stress in the body can be determined by measuring the levels of malondialdehyde (MDA) in the blood which is the result of lipid peroxidation. MDA levels of blood increase along with oxidative stress in the body.
Petiveria alliacea is a plant often used to treat various diseases. This plant is very useful in the health fields, such as cancer, diabetes, muscular pain, skin diseases, various central nervous system disorders, respiratory and pulmonary infections, and malaria. 4 In addition, P. alliacea also showed a protective effect against L. monocytogenes and immunomodulatory effect on hematopoiesis. 5 The contents of P. alliacea leaf extract are flavonoids types astilbi, engeletin, and leridal-chalcone; flavanone types leridal, leridol, and leridol-5-methyl ether; flavonol types myricitrin; benzyl-2-hydroxyethylsulfide; isoarborinol; isoarborinol astetat; isoarborinol cinnamate; alkaloid types of allantoin; triterpene species ilexgenin; senfol, tannins, and polyphenols. 6,7 P. alliacea leaves are also known to contain lipids such as lignoceryl lignocerate, linoleic acid, nonadecanoic acid, palmitic acid, stearic acid, as well as inorganic compounds such as KNO 3 . 7 Tannins in P. alliacea could provide a hypoglycemic effect. Linoleic acid and allantoin can stimulate insulin secretion. 8 In addition, tannins and flavonoids also have antioxidant effects through capture free radicals, enhance the activity of endogenous antioxidants, and suppress oxidative stress. P. alliacea has an antioxidant capacity by 54%. 10 With its ability as an antioxidant, P. alliacea can suppress oxidative stress and reduce the complications of diabetes.
This study aims to determine the hypoglycemic and antioxidant activity of the ethanolic extract of P. alliacea in diabetic rats.

Preparation of extract
The extract used is the result of maceration using 96% ethanol at the Laboratory of Pharmacology, Faculty of Medicine, Universitas Airlangga. Preparation of extract followed protocol implemented by Pharmacology Department of Universitas Airlangga. Dried powder of P. alliacea leaves (500 g) were soaked in 96% aquaeus etanol within 3x24 hours (2 l, 1.5 l, 1.5 l) at room temperature. The leaves extract was filtered and concentrated by heating in a waterbath at temperature of 45°C in order to obtain a thick leaf extract of P. alliacea.
The extract was suspended in 1% CMC-Na at concentration of 9 mg/ml, 18 mg/ml, and 36 mg/ml. To make this, sequentially, 900 mg, 1,800 mg, and 3,600 mg extracts were dissolved in 100 ml of 1% CMC-Na. Different volume is given depends on the bodyweight of rats. The new suspension made every 6 days.

Animals
Experimental animals used were healthy males Wistar strain albino rats (Rattus norvegicus L.) aged 2-3 months with bodyweight ± 200 grams. Rats were obtained from the Laboratory of Pharmacology, Faculty of Medicine, Universitas Airlangga. Selection of male sex aims to reduce hormonal influences. All animals were accustomed for approximately 1 week in clean cage with ad libitum water and food. The animals were treated in accordance with the standard guideline. The research work was approved by the Institutional Ethics Committee of Medical Research, Faculty of Medicine, Universitas Airlangga (174/EC/KEPK/FKUA/2015).

Induction of experimental diabetes and experimental design
Streptozotocin (STZ) used was purchased from Department of Physiology, Faculty of Medicine, Universitas Airlangga. Induction of diabetes followed induction protocol implemented by Purwanto and Liben.11. Rats were fasted for 4 hours and received single intraperitoneal injection of 50 mg/kg STZ which was freshly dissolved in 0.01 mol/L citrate buffer (pH 4.5). After injection, rats were given 10% w/v dextrose solution to prevent sudden hypoglycemic post-injection. Two days after injection, rats were fasted for 6 hours and blood glucose levels were measured. Rats with blood glucose levels ≥200 mg/dl were used.
On third day, rats were divided randomly into 6 groups of 6 rats each. The first group (N) was normal rats treated with 1% CMC-Na. Negative control group (NC), also treated with 1% CMC-Na was used as negative control. Positive control group (PC) were given metformin at doses 150 mg/kg. Metformin dose was determined based on Reagan-Shaw et al. Formula.12 Groups Pal90, Pal180, and Pal360, served as P.alliacea-treated groups and received extract at doses 90, 180, and 360 mg/kg respectively. Metformin and P.alliacea were dissolved in a 1% CMC-Na. Therapy was given intragastrically every morning for 14 days.

Blood glucose level measurement
After 14 days of treatments, rats were fasted for 6 hours and blood samples were taken from the lateral tail vein. Blood glucose level was measured using EasyTouch GCU (Bioptik Technology Inc.).

Blood malondialdehyde (MDA) level measurement
At the end of the study (day 15) levelof MDA was measured. The rats were sacrificed and blood was drawn from the heart. Measurement of MDA using Esterbauer and Cheeseman13 technique modified by the researcher and conducted at the Laboratory of Biochemistry, Faculty of Medicine, Universitas Airlangga. 500 μl sample taken and added 4.5 ml of cold PBS (phosphate buffered saline). 4 ml of the supernatant is then taken and added to 1 ml of 15% w/v Trichloroacetic acid (TCA). Furthermore, given 1 ml of 0.37% w/v Thiobarbituric acid (TBA) solution in 0.25 N HCl and heated in a waterbath at temperature 80°C for 15 minutes. Then cooled at room temperature for 60 minutes, and centrifuged at speed of 3,000 rpm for 15 minutes. Supernatant absorbance is then measured on a spectrophotometer at λ = 532 nm.

Statistical analysis
All raw datas were analyzed using one-way ANOVA with Welch Test F continued with Games-Howell Post Hoc test, Paired t-test, and Wilcoxon Signed Rank test. Statistical analysis is using SPSS 17.0 statistical package for Windows. All values were displayed as mean±SD. P values < 0.05 were considered as significant.

Results
There was no difference in blood glucose level between groups before induction of diabetes. After 48 hours of injection, blood glucose level significantly increased and persisted until the end of the study ( Figure 1).
As shown in table 1, blood glucose level was decreased in P. alliacea leaf extracts and metformin treated-groups. Unlike metformin, a significant reduction in MDA level was found in the group treated with P. alliacea at dose 360mg/kg compared to negative control group (p=0.021).

P. alliacea shows hypoglicemic activity with chronic treatment of diabetic rats
After administration of P. alliacea leaf extracts for 14 days, blood glucose level was found to decrease significantly from 374.8 mg/dl to 222.5 mg/dl (90 mg/kg) and 420 mg/dl to 209.2 mg/dl (360 mg/kg). Metformin as an oral antidiabetic used in this experiment was also showed a significant reduction of blood glucose level ( Figure 2). P. alliacea shows antioxidant activity with chronic treatment of diabetic rats MDA level in the diabetic group (7.048 nmol/ml) was increased compared with non-diabetic (6.747 nmol/ml) although statistically not found a significant increase (p = 0.098). A significant reduction in MDA level was found in the group treated with P. alliacea at dose 360mg/kg compared to negative control group (p = 0.021). . As shown in Figure 3, the higher dose of the extract, the lower the blood level of MDA. This showed that higher antioxidant level was achieved while increasing the dose. Figure 1 Comparison blood glucose level before and after STZ induction.

Discussion
DM is a degenerative disease characterized by chronic hyperglycemia in the body affecting millions of people worldwide. The deaths allegedly due to complications of diabetes which affect various organs of the body. New treatment using plants with fewer side effects began to be utilized and studied for many years. In this study, antidiabetic effect of P. alliacea leaves in STZ-induced type 2 DM rats was studied. STZ has selective toxic effects on pancreatic β cells that reach 90% success rate. 11 STZ causes the death of pancreatic β-cells through a process of alkylation on the DNA and the formation of ROS that lead into DNA fragmentation. 14,15 As a result, a significant increase in blood glucose level was obtained 48 hours post-injection ( Figure 1). When P. alliacea ethanolic leaves extract was administered for 14 days, it showed valuable hypoglycemic effect and significantly decreased blood glucose level almost same level as metformin-treated group especially at dose 90mg/kg and 360mg/kg (Figure 2). Lores and Cires (1990) found that extract from leaf and stem powder of P. alliacea was able to lower blood glucose level more than 60% after one-hour oral administration. 16 This effect may due to P. alliacea containing flavonoids that increase the activity of endogenous antioxidant enzymes and regenerate damaged pancreatic β cells. 17 It has been found that antioxidants can improve insulin sensitivity. One of the oldest oral antidiabetic and commonly used as first line treatment in DM is metformin, which also works as insulin sensitizer. Few studies showed that metformin can decrease free radicals and restore antioxidant status which explained its protective role in preventing cardiovascular complications. 18,19 Besides its antioxidant activity, P. alliacea leaves is thought to have a stimulating effect on insulin secretion. P. alliacea contain an unsaturated free fatty acids named linoleic acid which can stimulate insulin secretion and increase glucose-induced insulin secretion in rats in a dose-dependent manner. 8 In addition, in a dose-dependent manner, allantoin compounds are also known can increase insulin secretion and decrease basal blood glucose level. 9 Tannins have also been observed to increase insulin activity and enhance the glucose uptake through mediators of the insulin-signalling pathways, especially in adipocyte. These effects are proven by similarity of doseresponse curve of tannin-induced glucose transport with insulin. 20,21,22 Phenolic compounds have been attributed in induction of β cell regeneration and direct action in adipose tissue that enhance the insulin activity. 22 Interestingly, as seen in Figure 2, it was found the doseresponse curves of P. alliacea was non-monotonic. Nonmonotonic dose-response (NMDR) may presents as a bellshaped or U-shaped profile with the highest responses at low and high dose as seen in our study. 23 Several studies have described the effects of chemical compounds that can generate NMDR curves such as endocrine disruptor chemicals (EDC). 24 These compounds affect the hormone system in the synthesis, secretion, transport, binding, and metabolism of hormones. These compounds can also mimic or block the effects of hormones and cause effect on sensitive tissues, including endocrine tissues, through several mode of actions. 23,25 This finding may suggests that P. alliacea leaf extracts contain chemical compounds that can work as hormones or affect the endogenous hormone.
Hyperglycemic conditions trigger oxidative stress that can be measured by MDA level. Many experimental studies have shown the potential of plant derived antioxidant as treatment of DM. In this study, P. alliacea leaves extract decreased blood MDA levels. These results is in line with previous study showed P. alliacea had antioxidant effect by 54%. 10 P. alliacea leaves extract contains flavonoids and tannins that can work as an antioxidant and reduce oxidative stress condition. Flavonoids can work directly as an antioxidant which protect the body from free radicals and also increase the activity of antioxidant enzymes. 17 Tannins are not only enhanced the activity of endogenous antioxidant, but also increased the antioxidant levels. Tannins also decreased the MDA concentrations in liver, heart, and kidney. 26 Figure 3 shows that P. alliacea leaves extract decreased blood MDA levels and its relationship was dose-dependent. This means that the higher dose of the extract, the lower the blood level of MDA.

Conclusion
P. alliacea has a direct hypoglycemic effect and is thought to have a stimulating effect on insulin secretion and enhancing insulin activity. Moreover, P. alliacea can also work as an antioxidant. This antioxidant effect is not obtained on metformin.