#TITLE_ALTERNATIVE#

• Main Author: WIDIYA LESTARI NIM: 30713005, METI
• Format: Dissertations
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/28873
• Institution: Institut Teknologi Bandung

World Health Organization estimates that around 303,000 newborns worldwide died due to congenital anomalies. One of the resolution of congenital abnormalities is to strengthen research and studies of aetiology, diagnosis and prevention, by developing research on developmental toxicology or teratogenic test. Somen of teratogenic test has been developed. Rapid, reliable and cost-effective testing method is the most effective method and limit the use of animals in the laboratory are the main demand. <br /> <br /> <br /> <br /> <br /> <br /> <br /> The Brine Shrimp Test (BST) is appropriate method to fulfil the demand. The method is fast, simple, cost-effective, and can reduce the use of higher level vertebrate animal, so it can be used as teratogenic screening method for plant extract. Some plants are often used by women for health care, including pomegranate (Punica granatum L.), coriander (Coriandrum sativum L.), liman (Elephantopus scaber L.), fennel (Foeniculum vulgare M.), and kaemferia (Kaemferia galanga L.) The usage of five plants by women especially during the conception and pregnancy period are feared to affect growth and development of conception result. Therefore teratogenic testing is required on the five plants, both BST and conventional methods to provide safety information during pregnancy, and apply BST method as a teratogenic screening test on plant extracts. <br /> <br /> <br /> <br /> <br /> <br /> <br /> The goals of this research were to analyze teratogenic screening test using BST on some teratogenic substances, analyze teratogenic effect of pomegranate pericarp, coriander fruit, liman leaves, fennel fruit, and kaemferia rhizome extracts by BST, compare the result of teratogenic screening test of plant extracts using BST and Wistar rat. <br /> <br /> <br /> <br /> <br /> <br /> <br /> The research was performed through several testing steps, beginning with brine shrimp environmental optimization including temperature, light, acidity, salinity of medium, and ratio of sea water volume and number of cysts.Then BST testing was done as teratogenic screening using 6 teratogenic potent substances (methotrexate, captopril, sodium diclofenac, phenytoin, warfarin and valproic acid) as followed: artemia cysts were hatched into 1st stage nauplii, then taken and put into seawater medium which contain test substance and kept alive until 2nd stage, 3rd stage and 4th stage. Number of deaths, morphological abnormalities, body length, and retarded of development were observed for each stage. Number which was obtained from each parameter then analyzed and teratogenic potential index was calculated to measure teratogenic potential of the test substance.Then BST was used for teratogenic screening of 5 plant extracts.The potential teratogenic plant extracts were then observedfor teratogenic test in rat to compare the results of the two teratogenic tests. <br /> <br /> <br /> <br /> <br /> <br /> Teratogenic testing in rat began with a teratogenic dose-seeking process, therefore acute toxicity was carried out using fixed dose method. LD50 value which was obtained from acute toxicity test then used to determine Maternal Tolerance Dose (MTD), which is tolerance dose by mother but may cause abnormalities in fetus. Pregnant rats were given plant extract at 11 days gestation, then at 19 days gestation rats dissected to see the condition of the uterus, implantation site, corpus luteum, dead fetus, living fetus and fetus with external abnormalities. The dosage from MTD test result was continued for teratogenic testing by giving in different time, to see affect the extract on fetus development, and to look for the most susceptible period on fetus development that can cause abnormalities after giving extract to pregnant rats. Pregnant rats were given by plant extracts into 3 different periods, ie 9-11 days gestation, 12-14 days gestation and 15-17 days gestation. At 19 days gestation rats were dissected to evaluate in uterine condition, implantation site, corpus luteum, dead fetus, living fetus and fetus with abnormalities. <br /> <br /> <br /> <br /> <br /> <br /> <br /> Based on optimization results were obtained that brine shrimp can live until naupli stage 4 in temperature 20 ± 5 0 C, standard light grow lite (10 Watt), salinity 9 ppt and density 10 nauplii/5 mL. The BST result showed hatch ability of cysts in methotrexate 0.015 mg/mL, captopril 0.25 mg/mL, sodium diclofenac 0.075 mg/mL, phenytoin 1.56 mg/mL were significantly different compared to control (p <0.05). Nauplii survival in methotrexate 0.015 mg/mL, captopril 0.25 mg/mL, sodium diclofenac 0.075 mg/mL, phenytoin 1.56 mg/mL and valproic acid 2.5 mg/mL were significantly different to control (p <0.05). The morphological abnormalities were found in methotrexate 0.015 mg/mL, captopril 0.25 mg/mL. Nauplii with retarded development were expressed in methotrexate 0.015 mg/mL, captopril 0.25 mg/mL, sodium diclofenac 0.075 mg/mL, phenytoin 1.56 mg/mL and valproic acid 2.5 mg/mL. Significant difference in body length was presented in captopril 0.25 mg/mL, and phenytoin 1.56 mg/mL compared to control (p <0.05). Potential teratogenicity index value of methotrexate was0.53, captopril 0.80, sodium diclofenac 0.57, phenytoin 0.70, warfarin, 0.21, and valproic acid 1.00. Pearson’s correlation between potential teratogenicity index on BST method and mammalia method showed significantly positive correlation (r = 0.930, p<0.01). It revealed that both methods gave linear results and proven that BST sensitive for screening teratogenic test. <br /> <br /> <br /> <br /> <br /> <br /> The BST screening results on the extract showed hatch ability of cysts in fennel fruits extracts 1 mg/mL and 0.5 mg/mL, coriander fruits extract 1 mg/mL, pomegranate pericarp extract 6 mg/mL, and liman leaves extracts 6 mg/mL, 3 mg/mL and 1.5 mg/mL were significantly different to control (p <0.05). Survival nauplii in fennel fruits extracts 1 mg/mL and 0.5 mg/mL, liman leaves extracts 6 mg/mL and 3 mg/mL were significantly different to control (p <0.05). The morphological abnormalities were exposed on coriander fruits extract 1 mg/mL, liman leaves extract 6 mg/mL, and pomegranate pericarp extract 3 mg/mL. Nauplii with retarded development were presented on fennel fruits extracts 1 mg/mL and 0.5 mg/mL, coriander fruits extracts 1 mg/mL and 0.25 mg/mL, pomegranate pericarp extracts 6 mg/mL, 3 mg/mL and 1.5 mg/mL, liman leaves extracts 3 mg/mL and 1.5 mg/mL. Nauplii body length on plants extract showed no significantly difference compared to control. Based on five parameters observation then was calculated potential teratogenicity index.Liman leaves extract had the largest potential teratogenicity index, which was 0.27 at 6 mg/mL,0.11 at 3 mg/mL, and 0.19 at 0.5 mg/mL, followed by coriander fruit extract with potential teratogenicity index value 0.14 at 1 mg/mL. Based on BST test, liman leaves extract and coriander fruit extract were potential teratogenic. <br /> <br /> <br /> <br /> <br /> <br /> <br /> Based on acute toxicity test was obtained LD50 of extract of liman leaves extract and coriander fruit extractwas > 5000 mg/kg bb.The result of MTD test of liman leaves extracts showed that the percentage of preimplantation loss in the 1185 mg/kg bw liman group significantly increased (12.80%) compared to the control group (1.59%), total intra uterine death in the liman group at dose of 3750 mg/kg bw showed the largest percentage (19.41%), live fetal percentage at dose of 3750 mg/kg bb showed the most decrease (80.59%), average of body weight at dose of 3750 mg/kg bw tended the most decrease (2.04%) compared to the others, the percentage of fetus with the most external abnormalities at dose of 375 mg/kg bw (12.91%). <br /> <br /> <br /> <br /> <br /> <br /> <br /> The result of MTD test coriander fruits expressed that the percentage of preimplantation loss in coriander group dose 375 mg/kg bw significantly increased (12.39%) compared to control group (1.59%), total intra uterine death in coriander group at dose of 3750 mg/kg bw showed the largest percentage (20.94%), the percentage of live fetus at dose of 3750 mg/kg bw reported the greatest decrease (79.06%), the percentage of fetus with the most external abnormalities was dose of 1185 mg/kg bb (9.94%). Generally the result of MTD test exposed that liman leaves dose of 375 mg/kg bb was the group that most affect the development of rat fetus. <br /> <br /> <br /> <br /> <br /> <br /> <br /> The results of the exposure period test showed that period C dose of 375 mg/kg bw had the lowest of weight increase (86.78 g), the largest preimplantation loss was B period dose of 375 mg/kg bw (5.17%), the greatest percentage of total mortality in period B dose of 270 mg/kg bw (16.33%), the lowest live fetus percentage in period B dose 270 mg/kg bw (82.96%), period B showed real decrease in average of body weight of fetus compared to the control (2.24 g) especially for dose of 270 mg/kg bw (2.02 g), the most percentage of malformation of fetus in period B was 14.55%. <br /> <br /> <br /> <br /> <br /> <br /> <br /> The conclusion of this study Brine Shrimp Test can be used as an alternative method for teratogenic screening based on test results on five parameters of developmental disorder and teratogenic potential index, teratogenic screening test showed fennel fruits extract and pomegranate pericarp extract were not teratogenic, coriander fruits extract and liman leaves extract have teratogenic properties, whereas kaemferia rhizome extract was larvacida in naupli or embriolethal in fetus. Similar to BST, the result teratogenic test using Wistra rats, showed liman leaves extract had teratogenic properties although only mild teratogenic. Gestational age 12-14 days was the most susceptible period to rat fetal development caused by administering of liman leaves extract. <br />