Assessment of Type 2 Anti-Diabetes on Bound Flavonoids of Barringtonia racemosa (L.) Spreng. Kernel in Glucose- Induced Diabetic Rats

Corresponding Author: Musri Musman Department of Chemistry Education, Faculty of Teacher Training and Education, Syiah Kuala University, Darussalam – Banda Aceh, Indonesia, 23111, Indonesia Tel: +6285260525207 Email: musrimusman@gmail.com Abstract: A study of type 2 anti-diabetes on bound flavonoids fraction from the kernel of Barringtonia racemosa (L.) Spreng. in glucose-induced diabetic rats was performed. This learning aimed to gather scientific information about the possibility to utilize kernel of B. racemosa as a raw material of antidiabetic drug. The antioxidant property of the bound flavonoids was determined by the DPPH scavenging method compared to the ascorbic acid. Assessment of type 2 anti-diabetes was conducted on glucose-induced diabetic Rattus norvegicus Wistar strain compared to metformin and the data were analyzed through one-way ANOVA software. Histopathological studies of the pancreas and kidney were made to get evidence of the β-cell performance and the renal tissue respectively. The DPPH testing at the wavelength of 517 nm showed the bound flavonoids and the ascorbic acid showed absorbance at 0.097 and 0.080 correlated with IC50 values of 7.51 and 6.50 ppm respectively. The results of one-way ANOVA indicated that the administration of bound flavonoids was significant (F(2,11) = 8.60, p = 0.008) to reduce blood glucose level in the tested rats. The diabetic rats treated with the extract experienced an antidiabetic effect equivalent to an antidiabetic effect of metformin. Histopathologic observations showed increasing of the granulated β-cell (F(3, 15) = 26.09, p<0.0001) and no renal tissue damage (F(3, 15) = 0.23, p = 0.873) in the tested rats. The conclusion raised from the data of this study revealed that the bound flavonoids from the kernel of B. racemosa (L.) Spreng. could be utilized as a drug source of type 2 anti-diabetes.


Introduction
The WHO report stated that diabetes is a very important public health matter to address and requires serious business and positive responses from various sectors such as government, civil society and diabetic people, food makers, pharmaceutical manufacturers and medical technology (WHO, 2016). The current findings indicate strong evidence that type 2 diabetes is nearly linked to oxidative stress (Ceriello and Motz, 2004;Pham-Huy et al., 2008;Chikezie et al., 2015;Ullah et al., 2016;Das et al., 2016) that accumulates due to the body's incapability to balance the formation of oxidants (free radicals) with the availability of reductants (antioxidants). Free radicals of Reactive Oxygen Species (ROS) can occur due to metabolic activities in the body (Wolff, 1993;Maddux et al., 2001;Devasagayam et al., 2004;Wright Junior et al., 2006), ultraviolet radiation, pesticides in food and other pollutants (Bagchi and Puri, 1998;Betteridge, 2000;Brownlee, 2001;Yoshikawa and Naito, 2002;Bansal and Bilaspuri, 2011).
ROS are formed in the nucleus and also in the cell membrane where it destroys biologically relevant molecules such as DNA, proteins, sugars and lipids (Young and Woodside, 2001). ROS have been concerned with the initiation and complications of diabetes mellitus (Martin et al., 2003;Yung et al., 2006;Iqbal et al., 2016). Excessive ROS production causes damage to cells and cell tissues. To halt the production of ROS, a compound that has the property of free radical deactivation is required. The amount of ROS (oxidants) formed in cell tissues must be balanced with the availability of antioxidants. Therefore, the administration of external sources of antioxidants can be applied in managing the ROS (Halliwell, 1995;Laight et al., 2000;Kangralkar et al., 2012;Santos-Buelga and Feliciano, 2017).
Nature has provided medicinal materials in its surroundings. Humans have and will exploit the medicinal plants to cope with the illness, i.e., Barringtonia racemosa (L.) Spreng. The plant is an evergreens mangrove association that has been used as an ethnomedicinal agent to treat a number of illnesses as shown in Table 1. Outstanding to its wide range of ethnopharmacological applications, researchers have devoted their attention to finding out the pharmacological activities of the plant as revealed in Table 2 which may be used as a source of medicinal substances. Considering the presence of secondary metabolites in B. racemosa seeds as disclosed in Table 2, the exploitation of the bound flavonoids to manage type 2 diabetes mellitus interest to be investigated. The bound flavonoids have demonstrated very strong antioxidant activity, high bioavailability and more ready absorbed in metabolism (Nijveldt et al., 2001;Kumar and Pandey, 2013). Based on data searching via the internet, information concerning to bioactive property as type 2 anti-diabetes originating from B. racemosa can not be found (Hasan et al., 2000;Sun et al., 2006;Gowri et al., 2009;Lim, 2012;Osman et al., 2015;Nazaruk and Borzym-Kluczyk, 2015;Das et al., 2016;RIRDC, 2017). Therefore, this study was the first investigation of type 2 antidiabetic property derived from the plant.  Kabir et al. (2013;Osman et al., 2015). leaves itch, chickenpox, rheumatism febrifuge Lim (2012). seeds Tumors Thomas et al. (2002). seeds, barks fish poison Manjunath (1948). seeds colic, parturition, vermifuge, febrifuge Jayaweera (1981). fruits poison wild pigs Manjunath (1948). fruits hemicrania, ophthalmia, coughs, asthma, diarrhea Nadkarni (1976). fruits coughs, asthma, diarrhea, eczema Jayaweera (1981). fruits, barks fish poison Giesen et al. (2007). barks Insecticide Manjunath (1948). barks fish poison, skin diseases Jayaweera (1981). roots deobstruent, relief in stomachache. Jayaweera (1981).   Tachibana et al. (1996). HeLa cells leaves Mackeen et al. (1997). DLA cells seeds Thomas et al. (2002).

Chemicals, Drug and Kit
All of the analytical grade chemicals, drug and the kit were procured commercially. The metformin hydrochloride (C 4 H 11 N 5 , Dexa Medica, Indonesia) was decided as a positive control of the antidiabetic drug. The tested-diabetic rats were induced by the glucose monohydrate (C 6 H 12 O 6 , Merck, Germany). The Nesco Multicheck (Gesunde Medical, Indonesia) was operated to measure the blood glucose level of the tested rats.

Sample Collection
The old fruit that has been loose from its stem was collected from Lampuuk (5° 31' 56" N 95° 24' 00" E) village of Kuta Baro SubDistrict, Aceh Besar District of Aceh Province on October 15th, 2015. The specimen was authenticated by a plant taxonomist of Syiah Kuala University under code MM-015102015.

Preparation of Fruit Sample
The collected fruits (1.50 kg, gross weight) were decorticated to pick kernels up. The kernels (0.65 kg, gross weight) were cut into thin slices and then air dried under shade for seven days. The thin slices of a dried kernel (0.20 kg, dry weight) were ground with an electric blender and sieved with 40 mm mesh sieve to get a fine powder. The powder was stored in a dark bottle at room temperature until used.

Extraction of Bound Flavonoids
The procedure of Subramanian and Nagarajan (1969) was applied in order to obtain the bound flavonoid substances. The kernel powder was Soxhlet extracted with 96% (v/v) ethanol (EtOH, 100 mL g −1 dry weight) for 24 h and then concentrated under vacuum at 45°C. The concentrated extract was further fractioned in series petroleum ether (pet ether), diethyl ether (Et 2 O) and ethyl acetate (EtOAc). The ethyl acetate fraction was hydrolyzed by refluxing with 7% sulphuric acid (H 2 SO 4 , 10 mL g −1 residue) for two hours and then the filtrate was extracted with the ethyl acetate solvent. The obtained fraction was washed with distilled water to neutrality and dried by laying in a vacuum desiccator. The bound flavonoids extract was stored in the labeled bottle for the next step.

Phytochemical Analysis of Bound Flavonoids
The secondary metabolites of the bound flavonoids extract were carried out by means of standard laboratory for phytochemical screening (Banu and Cathrine, 2015). The alkaloids were examined through the Dragendorff's tests, the Mayer's and the Wagner's (Evans, 2009), the flavonoid constituents were investigated by the Shinoda's test (Raaman, 2006), the phenolic components were evaluated by the ferric chloride test (Sangeetha et al., 2014), the saponin constituents were noticed via the frothing test (Evans, 2009), the tannins were assessed through the ferric chloride and the alkaline tests (Evans, 2009) and the terpenoids were studied over the Liebermann-Burchard's test (Harborne, 1998).

DPPH Assay
The DPPH (2,2-diphenyl-1-picrylhydrazyl, C 18 H 12 N 5 O 6 ) procedure (Huang et al., 2005) was applied to evaluate the antioxidant activity of the bounded flavonoids extract. The ascorbic acid was preferred as the standard of the antioxidant and the trial was set up in triplicate. A 10 mL of 0.1 mM methanolic DPPH solution was prepared. A control solution was made by adding 3.5 mL of 96% methanol (MeOH) to 0.5 mL of the DPPH solution. The tested extract was dissolved in the 96% methanol at five different concentrations, e.g., 2, 4, 6, 8 and 10 ppm. A three mL of each the methanolic tested extract solution was mixed with one mL of the DPPH solution. The mixture was homogenized and kept standing at room temperature for 30 min. The wavelength of 517 nm was set to measure absorbance by using UV-Vis Spectronik 20D + single-beam Spectrophotometer (Thermo Fisher Scientific, USA). The percentage inhibition of antioxidant activity was designed through the formula: Inhibition (%) = {(A 0 -A 1 )/A 0 } x 100, where A 0 was the absorbance of the ascorbic acid and A 1 was the absorbance of the extract (Hossain et al., 2016). The IC 50 value of the tested extract was calculated through the log dose inhibition curve.

In Vivo Experiment
The healthy adult Rattus norvegicus (200-250 g body weight) Wistar fatty strain (Abdul-Ghani and DeFronzo, 2010) were conditioned in a cage (Alexandru, 2011;Fawcett, 2012) for a week. After a week adaptation, a dozen rat was separated into four groups by setting: The negative control group (marked as NC group), the positive control group (marked as PC group), the dose of 100 mg kg −1 body weight group (marked as BF1 stands for the bound flavonoids extract at a dose of 100 mg kg −1 b.wt.) and the dose of 200 mg kg −1 body weight group (marked as BF2 stands for the bound flavonoids extract at a dose of 200 mg kg −1 b.wt.). Individual rat in each group was collected its blood on the 7th day and marked as a pre-treatment blood. The diabetic rat was generated by giving orally one mL of 50% (w/v) aqueous glucose monohydrate to each rat in each group (Arul et al., 2006) on the 8th and the 11th days. After a week since the glucose given, the blood was collected from individual rat to check the diabetic rat according to the value blood glucose level ≥200 mg dL −1 (ADA, 2015). This blood was noticeable as the blood obtained before treatment. After finding out the diabetic rat, all rats were given orally: The aqueous metformin of 65 mg kg −1 body weight in PC group, the aqueous tested extract of 100 mg kg −1 body weight in BF1 group and the aqueous tested extract of 200 mg kg −1 body weight in BF2 group respectively every day at 10 a.m. for seven days. Later on this point, the individual rat in each group was collected its blood. The blood was noticeable as the blood obtained after treatment. One day later, a rat in each group was selected to be sacrificed for histopathological observation on the kidney and pancreas organs. The difference of blood glucose level was stated as an antidiabetic effect. The percentage of antidiabetic effect was calculated by the formula: Antidiabetic effect (%) = {(a-b)/a} x 100, where a was blood glucose level of rat obtained before treatment and b was blood glucose level of rat obtained after treatment (Candasamy et al., 2014).

Histopathological Study
The kidney and pancreas organs were submerged in Neutral Buffered Formalin for a week and then histopathological investigations were performed (Spitalnik, 2016). The slices were tainted with Hematoxylin Eosin (HE) and studied under DP12 Olympus binocular research microscope.

Statistical Analysis
One-way ANOVA was performed using the SPSS software version 24 (IBM Corp., Armonk, New York, USA) and the SAS software version 9.1.3 (SAS Institute Inc., Cary, NC, USA) to assess the effect of bound flavonoids of B. racemosa kernel on blood glucose level of glucose-induced diabetic rats. The values were stated statistically significant difference when the p value < 0.05 following Duncan's post hoc test for comparing the treatments (Steel et al., 1997).

Phytochemical Analysis
The results of the phytochemical analysis of the bound flavonoid extract showed only flavonoid components as disclosed in Table 3.

Antioxidant Evaluation
The bounded flavonoids extract was run to antioxidant evaluation over DPPH radical scavenging method. At the wavelength of 517 nm, the absorbances of 0.080 and 0.097 for the ascorbic acid and the extract respectively were observed as shown in Table 4.

In Vivo Experiment
The in vivo experiment on the glucose-induced diabetic rats shown decreasing the blood glucose level along with increasing dose of the extract as shown in Table 5.

Histopathological Study
The histopathologic observations of the kidney and pancreas images showed results in renal damage within the normal range (Table 6) and an increase in β-cell granulation (Table 7) respectively.

Discussion
The bound flavonoids extract shown an intense red color on Shinoda's test (Raaman, 2006) based on the phytochemical analysis. In this extract, the alkaloid, phenolic, saponin, tannin and terpenoid components did not detect according to the standard procedures. This indicated that only bound flavonoids were existing in the extract. The bound flavonoids containing extract had the ability to turn deep violet color to pale yellow color in ethanolic DPPH solution. The magnitude of the reduction strength of the extract to neutralize the DPPH free radical was not much different when compared to the ability of ascorbic acid to neutralize the DPPH in terms of inhibition as shown in Table 4. The IC 50 values for the ascorbic acid and the extract were in the amount of 6.50 and 7.51 ppm respectively based on the log inhibition curve as revealed in Fig. 1. Referring to Molyneux's (2004) that activity of an antioxidant is considered as weak, moderate, strong and very strong when the IC 50 values are 150-200, 100-150, 50-100 and less than 50 ppm respectively, therefore, the bound flavonoids extract has very strong antioxidant activity (Su et al., 2014). This fact suggested the ability of the extract to reduce free radical molecule which in this case was the DPPH. Thus, the bound flavonoids contained in the extract were thought to have the ability to neutralize the DPPH molecule which has properties as a free radical and a scavenger for other free radicals (Pavithra and Vadivukkarasi, 2015). It was supposed that the flavonoids contained in the extract were able to work to neutralize free radicals produced through the metabolism process (Wang et al., 2013;Nimse and Pal, 2015;Elochukwu, 2015). Thus, the number of free radicals decreased and this reduced the oxidative stress which gave the pancreas an opportunity to secrete insulin into the blood. The result signposted by this fact was a reduction of blood glucose level in the glucose-induced diabetic rats (Bajaj and Khan, 2012;King, 2012;Czompa et al., 2017). In terms of one-way ANOVA results, there was a significant difference (p<0.05) among groups in response to antioxidants given to the tested rats. Indeed, the magnitude of the antidiabetic effect of the metformin (75.51%) and the extract (70.53 and 75.18% for BF1 and BF2 respectively) showed a very small difference in numbers as displayed in Table 5. This indicated that the bound flavonoids contained in the extract were thought to be able to reduce blood glucose level as well as the metformin as revealed in Fig. 2.
Renal histopathologic observational data were described in semi-quantitative descriptive and scores with a scale of 0 to 2 (Suhita et al., 2013). The mark 0 states no lesions in the organ. The mark 1 suggests hydropic degeneration, fatty degeneration, karyomegaly and pycnosis. The mark 2 states the occurrence of necrosa. Each individual score was then counted up and the mean of the group was determined for comparison with controls, then, a mild (score 0), moderate (score 1) and severe (score 2) lesions were identified and described.
The renal histopathologic observation was performed on proximal tubule nuclei as revealed in Table 6. The kidney is a target organ of insulin. Insulin binds to the insulin receptors via the nephron (Nakamura et al., 1983), which is essential for the proper function of the nephron, glomerulus and tubule (Hale and Coward, 2013). In insulin resistance, the insulin signaling cascade in the glomerulus seems to be impaired (Lay and Coward, 2014). In diabetic conditions, insulin stimulation in the transportation of proximal renal tubules is impaired so that glucose reabsorption decreases and glucose is excreted through urine (Horita et al., 2017). The administration of the bound flavonoids extract improved the kidney and the visible cells in the proximal tubule were the same as a normal rat (Fig. 3).  The score value of renal proximal tubule cells in diabetic rats given metformin by 0.50 expressed damage conditions in the normal range as shown in Fig. 3. The metformin could reduce hyperglycemia in the blood so it could reduce damage to proximal renal tubular cells. The one-way ANOVA results showed that the bound flavonoid extract given to diabetic rats did not cause any significant change in the histologic structure of the kidney (F(3, 15) = 0.23, p = 0.873). Indeed, the result of scores on a proximal tubular cell in diabetic rats given the extract displayed 0.25. This result indicated that the physiological functions of renal cells worked within the range of normal changes (Khoshnoud et al., 2017). This suggested that administration of the bound flavonoids extract of B. racemosa kernel in diabetic rats did not show specific damage to proximal renal tubular cells. Thus, the bound flavonoids contained in the extract did not cause damage to the kidney organs in the tested rats when applied as a controlling agent for type 2 diabetes.
The histopathologic images demonstrated that the pancreatic β-cell granulation was directly proportional to the given extract dose as shown in Table 7. The number of β-cells enhancement (F(3, 15) = 26.09, p<0.0001) for each treatment stated that the bound flavonoids extract administered to hyperglycemic rats could improve pancreatic β-cells and depresses necrosis or apoptosis of pancreatic β-cells compared to metformin as shown in Figure 4. It was assumed that the modulatory effects of bound flavonoid constituents on the blood glucose transporter by increasing insulin secretion, decreasing apoptosis and stimulating proliferation of pancreatic β-cells (Fu et al., 2012;Vinayagam and Xu, 2015;Zheng et al., 2016).

Conclusion
The bound flavonoids extract of B. racemosa kernel showed the strong antioxidant power and it displayed the type 2 anti-diabetes property. Administration of the extract with doses of 100 mg kg −1 b.wt. and 200 mg kg −1 b.wt. orally for 14 days was not causing the histopathologic disturbance on the tested rat kidney organ.

Acknowledgement
Authors are thankful to Prof. Dr. Djufri, M.Si., the plant taxonomist of Teacher Teaching and Education Faculty of Syiah Kuala University, for identification and authentication of Barringtonia racemosa (L.) Spreng.

Conflict of Interest
The authors declare that they do not have any conflict of interests.

Ethics
This original article contains unpublish material. The corresponding author states that all of the other authors have read and agreed to the manuscript and no ethical issues are involved.