Acute Toxicity of Self-Nanoemulsifying Drug Delivery System of Ipomoea reptans Poir Leaves Extract on Female Wistar Rats

a surfactant, oil phase, active pharmaceutical ingredient, and hydrophilic co-surfactant 10 . The SNEDDS is anhydrous preconcentrates of nanoemulsions with droplet sizes ranging from 20-600 nm 11 . This technology has many advantages, such as improved lipophilic drug solubilization, improved physical stability, provided greater surface area and absorption, and decreased first-pass metabolism 12,13 . However, no acute oral toxicity is observed for SNEDDS of I. reptans extracts. Therefore, this study aimed to evaluate the effect of I. reptans leaves extract SNEDDS on clinical and organ histology examination.


Preparation of extract
The Laboratory of Plant Systematic, Faculty of Biology, Universitas Gadjah Mada (01058/S.Tb./V/2017) has validated the plant. Harvesting the leaves was done on day 30 in the morning. The standardized extract of I. reptans has been acquired based on the extraction procedures by Hayati et al 14 .

Preparation of SNEDDS
The formulation of I. reptans leaf extract was obtained from Jumaryatno et al. study 9 . The compositions were Capryol 90, Tween 20, and PEG 400 with a ratio of 1 : 7 : 2 consecutively.

Observation of stability
Determination of stability of SNEDDS used centrifugation test, heating cooling cycle test, and freeze-thaw cycle test continually, as mentioned in Jumaryatno et al. study 9 .

Determination of size, polydisperse index (PDI) and zeta potential
The measurement carried out using PSA was to know the formula's size and distribution. A total of 1 mL SNEDDS was mixed with distilled water to 100 mL, stirred until nanoemulsion was obtained, and set to PSA.

Toxicity test
A limit test of Acute Toxic Class Method OECD 423 was performed to determine LD50 of SNEDDS on 2-2.5-month-old female Wistar rats 15 . The animals were supplied by the Faculty of Pharmacy, Universitas Gadjah Mada, Yogyakarta, Indonesia. The rats were kept under controlled conditions (room temperature (25±2)°C; 12 hours light/dark cycle) and allowed free access to standard food and water. The animals were acclimatized for seven days before the experiment. Ethical clearance for the experiments was obtained from the Ethical Committee Faculty of Medicine, Universitas Islam Indonesia, with number 843/KE/XII/2017. The animals were divided into the control group (vehicle/SNEDDS base) and treatment group (SNEDDS of I. reptans extract 2000 mg/kg). Each group consisted of three animals per step. There were no test animals that died unwanted during the trial period. The scheme of the limit test can be seen in Figure 1. The animals were given a dose of 2000 mg/kg for the limit test. If there were 2-3 dead animals, the dose was reduced to 300 mg/kg, and the main test should be performed. However, if 0-1 animal died, the dose administration still used 2000 mg/kg. The results (LD50) were dependent on the number of dead animals. The vehicle or extract SNEDDS was given as a single dose. For the first four hours, every 30 min after administration, rats' clinical examination (tremor, hyperactivity, eyes change, convulsion, and mortality) was observed thoroughly. The rats were examined periodically during the first 24 hours and daily after that for 14 days. Body weight was measured daily. At the end of the experiment or when the animals were found dead during the investigation, their organs (heart, liver, kidney, lung) were harvested and evaluated histopathologically.   The test result using PSA can be seen in Table I. The droplet size of nanoemulsion is varying 20-600 nm. The small size of the I. reptans extract SNEDDS droplet made the formula will not break through dilution. Therefore, it will enhance the surface area and absorption of the active ingredient 17 . As a consequence, the bioavailability of I. reptans will escalate. Measurement of PDI implies the homogeneity of particle size in SNEDDS. Higher values indicate a broader distribution and smaller droplet size uniformity 18 . Zeta potential ranging ± 30 mV suggests the stability of the nanoemulsion preserved.

RESULTS AND DISCUSSION
A negative value implies that there are free fatty acids in the emulsion 19  There are no apparent toxic effects observed during this research. In the previous study, the animal showed no harm after being administered I. reptans ethanol extract in a single high dose (>9375 mg/kg) 6 . Body weight measurement for 14 days can be found in Figure 3. There is no significant difference in weight gain between the control and treatment groups. The average daily gain in the treatment group obtained was 0.86 g/day; the control group was 1.32 g/day. The previous research exhibited 0.62±0.22 g weight gain per day in I. reptans extract at a dose of 9375 mg/kg group 6 . A histopathology organ test was run to investigate the effect of I. reptans extract SNEDDS on the organ. Cellular changes may occur, although no toxic symptoms were observed during the clinical examination. Figure 4 showed that administering I. reptans extracts SNEDDS did not make any difference in the organ. A study by Hayati et al. 8 demonstrated the same result in kidney and liver organs in female mice, although AST and ALT value rise after administration of I. reptans extracts for 14 days. Therefore, I. reptans extract SNEDDS was safe for animal and human.