Subchronic Toxicity of Lunasin Targeted Extract (ET-Lun) from Soybean Seed (Glycine max (L.) Merr.): Perspective from Liver Histopathology, SGOT, and SGPT Levels in Sprague Dawley Rats

Indonesia is a subtropical country that is rich in natural resources and biodiversity, including various potential medicinal plants. Recently, applications of natural medicine for medications have become a trend in modern society, this then causes an increasing number of various studies in explorations and applications of plants that are believed to have medicinal properties. One of the plants that are used in traditional medicine is soybean seeds (Glycine max (L.) Merr.).1 Soybean seed has several pharmacological effects including antioxidant, estrogenic, anti-diabetes, anti-hyper cholesterol, and anti-cancer.2,3


INTRODUCTION
Indonesia is a subtropical country that is rich in natural resources and biodiversity, including various potential medicinal plants. Recently, applications of natural medicine for medications have become a trend in modern society, this then causes an increasing number of various studies in explorations and applications of plants that are believed to have medicinal properties. One of the plants that are used in traditional medicine is soybean seeds (Glycine max (L.) Merr.). 1 Soybean seed has several pharmacological effects including antioxidant, estrogenic, anti-diabetes, anti-hyper cholesterol, and anti-cancer. 2,3 Several studies to ensure the safety of natural medicine for medications should be performed. For instance, by conducting a toxicity assay. Toxicity is the potency of xenobiotics to cause damage to an organism either during use or in the environment. Toxicity assay can be divided into two types, which general toxicity (acute, sub-acute/sub-chronic, chronic) and specific (teratogenic, mutagenic, and carcinogenic). 4,5 The acute toxicity assay is an assay that detects toxicity effect, which may appear in a short time after 24 hours of single or repeated dose administrations of the test solution. 4 , 6 Subchronic toxicity is an assay that is performed to detect toxicity effect after repeated doses of oral administration in animal models for part of the life of the animal, but no more than 10 % of the entire life of the animal. 4 A previous study by Wijiasih (2017) 7 showed the extract of soybean seed contained 0.823 mg/g of lunasin. This extract significantly reduced COX-2 and iNOS expression in colon preneoplasia of mice at doses of 150 mg/kg BW and 200 mg/kg BW (p <0.05). 7 Another study proved the soybean extract was able to increase apoptosis (p = 0,001) at a dose of 150 mg/kg BW, and reduced dysplasia (p = 0,024) at a dose of 200 mg/kg BW. 8 The soybean extract have also inhibitory activity in colitis-associated colon carcinogenesis through inhibiting reduction in the number of goblet cell and microvessel density. 9 Moreover the soybean extract with targeted Lunasin (ET-Lun) could reduced tumor volume (p=0,021) and decreased EGFR expression in DMBA induced breast cancer rat model. 10 Previously, acute toxicity has been performed using doses of 500, 2000, 5000 mg/kg BW The acute toxicity showed ET-Lun has LD50 more than 5000 mg/kg BW and was practically nontoxic (unpublished data). The aim of this study was to evaluate the subchronic toxicity of ET-Lun with targeted organs and observed changes in the liver using histopathology examinations, Serum Glutamine Oxaloacetate Transaminase (SGOT), and Serum Glutamine Pyruvate Transaminase (SGPT) test.

Preparation of Simplisia, extraction and Phytochemical Screening Animal Models preparations
The study using animal models has been approved by ethical committee from Faculty of Medicine, Universitas Indonesia, Protocol ID : 20-09-1077. The animal models was weighed and underwent aclimizations for 7 days by standard feeding and drink. The animal models were placed in a cage with a husk, with 5 animals per cage. The animal was maintained in a clean room.

Determinations of Subchonic toxocity test
In this study, 40 Sprague Dawley (SD) rats aged 6 weeks (twenty female and twenty male) were divided into four groups and each group consisted of 5 male and female rats respectively. Animals were randomly assigned to a control (GN) and three treatment groups ; dose 250 mg/kg BW (G1), 500 mg/kg BW (G2), and 750 mg/kg BW (G3). The dose that used in this study was based on the previous study. 7,10 The treatment group was orally administered with ET-Lun extract for 90 days. The weight of the rat was measured one times a week for 3 months. On the 91st day, the rats were anesthetized using ketamine and xylazine, and blood samples were collected from each animal by cardiac puncture. After the collection of blood samples, the rats were sacrificed by cervical dislocation and the liver was dissected for histopathology analysis.

Determination of SGOT SGPT Level
The blood was put into a centrifuge tube and allowed to stand at room temperature for 10 minutes, then transferred to an ice bath for no more than 20 minutes and immediately centrifuged for 10 minutes at 3000 rpm. Furthermore, the serum was separated and stored in a freezer at -20 o C. The SGOT and SGPT levels were measured according to DiaSys® protocol.

Histopathology Examinations
The liver of male and female rats was immediately fixed in 10% neutral buffered formalin (NBF), fixation and, the paraffin block was made afterward. The tissue blocks were sectioned in ribbons at a thickness of 5 µm with Leica microtome (Leica DM 750, Germany). Slide preparations and Hematoxylin Eosin staining were conducted.

Data Analysis
Data were presented as mean ± SEM with 95% confidence interval and analyzed by SPSS version 24. One-way ANOVA and continued with the Tukey HSD test with a 95% confidence level to determine the differences between each group.

RESULT
In this study, defatted soybean seeds were extracted using PBS. The macerate was dried to obtain a thick extract with specific characteristics (Tables 1 and 2). Standardization of ET-Lun in the form of water value, ash value, and phytochemical screening was shown in Tables 3 and 4. Giving ET-Lun to the treatment groups for 3 months did not show a significant difference in body weight when compared to the control group ( Figure 1). Likewise, the results of the SGOT and SGPT level, and the liver weight examinations (Figure 2-4). A microscopic study of the liver was also showed no damage on the liver and the diameter central vein showed no difference between the treatment group and the control group ( Figure 5-9).

DISCUSSION
Sub-chronic ET-Lun toxicity for soybean seeds was carried out with the aim of obtaining information on the presence of symptoms of toxic effects that were not detected in the acute toxicity test. A subchronic toxicity test can be done if the plant has passed the acute toxicity test. The acute toxicity test on soybean extract obtained the results of giving soybean seed extract given in a single dose and observed death within 24 hours and continued observation for 14 days showed no death in experimental animals with doses of 500 mg/kg BW, 2000 mg/kg BW and 5000 mg/kg (unpublished data).
The results of the extraction and standardization of the extract in this study showed that ET-Lun met the quality standard of the extract. 11,12 The administration of a targeted extract of soybean seed lunasin at each different dose did not affect the bodyweight of the rats. Parameters of SGOT and SGPT levels are indicators that can indicate liver damage. 13 The presence of damage to liver cells can be characterized by increased levels of SGOT and SGPT enzymes. SGOT-SGPT are two transaminase enzymes produced by liver cells. An increase in SGOT and SGPT indicates damage to liver cells. 14 SGOT serves as an indicator and evaluation of the function of the liver and heart muscle and monitors the effects of hepatotoxic and nephrotoxic drugs, while SGPT functions as an indicator of liver function, monitoring the effects of hepatotoxic drugs. SGOT is found mainly in red blood cells, in the heart and skeletal muscle, and in the kidneys. SGPT is an enzyme found in the liver and is the most sensitive marker for liver cell damage. 13,14 The result of this study showed that there was no significant difference in the levels of SGOT and SGPT between the control group (GN) and the ET-Lun group at doses of 250 (G1), 500 (G2), and 750 mg/kg (G3). These results were supported by microscopic examination. The general architecture of the liver, the appearance of the hepatocytes, the hepatic sinusoids, portal triads, and central veins are normal as compared with controls.

CONCLUSION
The results of the sub-chronic toxicity can be concluded that ET-Lun from soybean seeds (Glycine max (L) Merr.) with a dose of 250 mg/ kg BW, 500 mg/kg BW, and a dose of 750 mg/kg BW is proven not to be toxic. There was no significant difference between control and treatment groups to SGOT and SGPT levels (p > 0.05). On microscopic observation of the central vein of the liver, there was no change or damage in each dose group. Meanwhile, the microscopic quantitative measurement of the diameter of the central vein of the rat liver showed no difference between groups (p > 0.05). The results showed that ET-Lun from soybean seeds did not cause toxicity to rats.