Teratogenic Effects of Durian Fruit (Durio Zibethinus Linn) on White Mice Fetus (Mus Musculus L)

Pharmauho: Jurnal Farmasi, Sains, dan Kesehatan 2021, 7(1); 5-10 p-ISSN: 2442-9791, e-ISSN: 2715-4181

Teratogenic Effects of Durian Fruit (Durio Zibethinus Linn) on White Mice Fetus (Mus musculus L)

Mesa Sukmadani Rusdi*, M. Rifqi Efendi, Hafizhatul Hilma

Department of Pharmacy, Universitas Dharma Andalas, Padang, West Sumatra 25127

Abstract Article Info: Durian (Durio zibethinus L.) is one of the most consumed and liked fruit in Indonesia, Received: 13 August 2020 but there was no evidence about the safety of consuming durian during pregnancy. This Accepted: 16 March 2021 DOI: 10.33772/pharmauho. study was determine the level of safety of durian fruit consumption during pregnancy v7i1.13489 by conducted teratogenicity test of durian fruit (Durio zibethinus Linn) on fetuses of white mice (Mus musculus L). Teratogenicity test was conducted in vivo, by step: preparation of experimental animal, observation during administration, laparotomy, fixation and observation of morphological defects. Data were analyzed using Analysis of Variance (ANOVA). If the results were significant (P < 0.05), the analysis was continued using Duncan Multiple Range Test. Administration durian during pregnancy did not affects the bodyweight of the mice (P > 0,05), but significantly influences the number of fetuses (P < 0,05) and its body weight (P < 0,05). Consumption of durian during pregnancy is safe in 32,5 mg / KgBW, but it has the potential teratogenic in 65 mg / KgBW and 97,5 mg/KgBW groups. Teratogen effects that were found in several fetuses were underweight, dead, hemorrhage, maternal death and there were resorption sites.

Keywords: teratogenicity, durian, mice, Durio zibethinus

1. Introduction During pregnancy, women’s nutritional needs for durian consumption during pregnancy. Thus, this study energy, protein and micronutrients significantly increase. aimed to determine the level of safety of durian Pregnant women require an additional 285 kcals/day. Good consumption during pregnancy, and its teratogenic effects nutritional for pregnant women will prevent malnutrition in on fetuses of white mice (Mus musculus L) the mother and fetus. While, the consequences of poor nutritional status and inadequate nutritional intake for 2. Methods women during pregnancy and lactation not only directly affect the women’s health status but may have a negative Research protocol had been approved by the Ethics impact on infant birth weight and early development [1]. Committee of the Faculty of Medicine, Universitas Andalas Therefore, to meet the additional requirements of (Number: 429/KEP/FK/2019). Teratogenicity test was pregnancy, pregnant women were suggested to consume conducted in vivo, performed by preparation of various types of fruits and vegetables. experimental animal, observation during administration, Based on Central Statistics Agency (BPS) regarding laparotomy, fixation, and observation of morphological the most consumed fruit per week in 2015, durian was defects. ranked 4th [2]. It was stated that durian fruit liked by most of the Indonesian population and it can be liked by pregnant 2.1 Material women. Durian contains protein, fat, carbohydrate, beta- Flesh of durian were obtained from the center of carotene, vitamin B2, and vitamin C. It is also reported to durian, Tarandam, Padang. This research also used have flavonoids, phenolic acids, and sugars. One of the aquadest, gabapentin, red alizarin solution, and bouin's dominant compounds that influence the taste and aroma of solution. Twenty female white mice (Mus musculus Linn) durian flesh is esters, sulfur compounds, ketone aged ± 2-3 months with a bodyweight of ± 20-30 grams, compounds, and various other compounds which are healthy, had never been in experiments and showed normal mostly alcohols such as methanol and ethanol [3,4]. In behavior were used in this study. Otherwise, 5 healthy male some studies, durian has been shown to have several mice aged ± 2-3 months were also needed for mating. bioactivities, such as antioxidants, reacts to increase Bouin's solution consists of formaldehyde, glacial acetic immune responses, reduce cholesterol levels [5], anti- acid, and saturated picric acid [11]. Alizarin red solution is inflammatory [6], antibacterial and antifungal [7]. made by adding 6 mg of alizarin red in one liter of 1% Although durian has good properties for health, but aqueous KOH [12]. there was not enough evidence that stated the safety of

Pharmauho: Jurnal Farmasi, Sains, dan Kesehatan 2021, 7(1); 5-10 Rusdi, et al

2.2 Preparation laparotomy was done to remove the fetus from uterus. Mice were dissected in the upper part of the abdomen until uterus The durian dose variations in this study were based on was seen. The fetus was removed by cutting from the uterus the research of Kumolosasi [8], it was stated that an average and placenta. Furthermore, it was observed whether there consumption of durian fruit in adult healthy humans was 5 were resorption sites that were marked by the presence of pieces or equivalent to 250 g, and the maximum red lumps as the place where the fetus was implanted. The consumption of durian fruit was 10 pieces or equivalent to number of fetuses were counted, whether it were still alive 500 g. This dose was then converted to mice doses. Durian or have died. Then the fetus was dried with a tissue and flesh was separated from the seeds, then weighed as much weighed to find out the average birth weight. Then, observe as 32,5 mg/ KgBW, 65 mg/ KgBW, 97,5 mg/ KgBW for the presence or absence of visual abnormalities such as the treatment groups, aquadest for negative control groups and tail, earlobe, eyelid, number of fronts and rear fingers [9]. gabapentin 50 mg/KgBW for positive control group. For preparation of the test solution, the converted doses was 2.7 Fixation and Observation of Morphological Defects made in 50 ml aqua dest. The test preparation was carried out in 10 days in a row starting on 6th to 15th day of After being observed visually, one-third of the number pregnancy [9]. of fetuses from one parent were fixed with Bouin's solution in 14 days until it was yellow and hard. Furthermore, the Table 1. Grouping of Experimental Animals fetus was removed and dried. The outside of the fetus Groups Treatment examined was the ear, eyes, feet, and tail. After that, Negative Control (P-) Only given aqua dest observed cleft palate by slipping a scalpel slowly [13]. Positive Control (P+) Given gabapentin 50mg/KgBW Other two-thirds then were soaked with red alizarin I (P1) Given durian 32,5 mg/KgBW solution, leave it for 2-3 days, while occasionally shaken II (P2) Given durian 65 mg/KgBW III (P3) Given durian 97,5 mg/KgBW until the fetus becomes transparent and the bones look red. Then, observe bone abnormalities, such as the size and the 2.3 Animal Preparation and Estrous Cycle Determination distance between the ribs, bone abnormalities of the skull, and coccyx. Then compare all the observations with Acclimatization was carried out for 7 days to adapt the controls [13]. animal with the experimental environment. Food and drink were given, body weight weighed daily and behavior was 2.8 Data Analysis observed. During the acclimatization, the estrous cycle was determined by visual observation of the vagina. Estrous The body weight of Pregnant mice were analyzed cycle characterized by a reddish and gummy texture of using the two-way analysis of variance (ANOVA). Number vagina. The experimental animal considered healthy if the of fetuses and fetal body weight, were analyzed using the change of body weight were no more than 10%, visually one-way analysis of variance (ANOVA). If the results were showed normal behavior and had an estrous cycle of 4-5 significant (P < 0.05), the analysis was continued using days [10]. Duncan Multiple Range Test with 95% confidence interval The fetal defect was measure after they were fixed in 2.4 Mating of Experimental Animal Bouin’s and red alizarin solutions. During the estrous period, the experimental animals 3. Result and Discussion were mated. Ratio of male and female was 1: 4. Male mice were put into female mice cages at 16.00 in the afternoon This research was to determine impact of and separated again tomorrow morning. In the morning, consumption of flesh of durian fruit (Durio zibethinus vaginal plug was examined. Vaginal plugs indicated mice Linn) in mice during pregnancy. Before the treatment, mice have experienced copulation and signed as the 1st day of were acclimatized for 7 days to familiarize and adapt the pregnancy. Mice that have been pregnant were separated experimental condition. and those who have not were mixed back with male mice The test preparation were given to the pregnant mice th th [9]. for 10 days, started on the 6 day until the 15 day of pregnancy [14,15]. In gestation period, there is 2.5 Observations during the Administration of Test organogenesis. It is signed by cell differentiation to form organs, so the fetus is susceptible to incoming teratogenic Female mice were weighed daily. If there was a drastic substances. This period is referred to as the critical period weight loss and accompanied by bleeding around the of pregnancy [16]. The treatment was not given on first vagina, the mice had miscarriage or spontanous abortion trimester (1st - 5th days of pregnancy). According to Sadler then the animal must be killed and examined. The sick mice, [17], during the first trimester of pregnancy, zygote is whether because of treatment or disease, were no longer continuously divided and produce two groups of cells. One included [9]. cell group will become embryo’s body, and the other becomes complementary cells, which include the 2.6 Laparotomy trophoblast, periblast, and auxiliary cells. These cells Laparotomy performed on the 18th day of pregnancy. protect the embryo and make contact between the embryo Pregnant mice were killed by neck dislocation, then and the parent. Many teratogens have the ability to inhibit this division and kill embryo, which was involved in

Pharmauho: Jurnal Farmasi, Sains, dan Kesehatan 2021, 7(1); 5-10 Rusdi, et al blastocyst formation. However, most of the time the Result showed on the 6th to 10th days of pregnancy embryo survives; its subsequent development does not there had not been a significant increasing in body weight. generally seem to be compromised [18,19]. It is also said The body weight of pregnant mice began to increase on 11th the first trimester of pregnancy has totipotency properties. to 18th days. This was because of fetal development, Totipotency is a condition where the embryo is not increased amniotic fluid volume, placenta and amniotic susceptible to teratogenic substances and can repair itself membrane [20]. Besides, the number of fetuses also affects from damaged tissue. Whereas on the 16th day onwards, the body weight of pregnant mice. During the observation, teratogen compounds did not cause morphological defects, it was found that 1 pregnant mice of P3 group died on the but could cause functional abnormalities. Morphological 15th day of pregnancy. This may caused by spontaneous defects can generally be detected easily at birth or shortly abortion. Spontaneous abortion could occur because of after birth. However, functional disorders such as: CNS genetic susceptibility due to high dose exposure in mice disorders, was difficult to be detected quickly after birth individuals even though they originate from the same breed [13]. [21]. The results of observation of bodyweight of pregnant Treatment of Durian (Durio zibethinus Linn) and mice during pregnancy can be seen from Table 2 and Figure duration of pregnancy significantly influence the average 1. weight of pregnant mice during pregnancy (p<0,05), but not the interaction of these factors (p > 0.05). The slower Table 2. Average pregnant mice body weight during pregnancy body weight gain was seen in postive control group, and P3 Pregnant Mice Body Weight (g) treatment, but not statistically different to another treatment Days P-b P+a P1c P2b P3a groups during pregnancy. It might because of the number 1 26,10 25,50 27,98 25,90 24,20 of fetuses in the uterus. Pregnant mice's body weight was 2 25,63 25,75 27,85 26,00 25,13 depended by the number of fetuses. The increasing number 3 27,05 25,70 29,38 27,05 25,75 of fetuses will gain the bodyweight of the mother [9] 4 27,03 26,15 29,35 28,03 26,45 On the 18th day was performed laparotomy. 5 27,23 25,85 29,40 28,55 26,98 Pregnancy should be terminated not more than one day 6 27,33 26,93 29,78 29,00 27,35 before the expected time of delivery. For mice, it was 18th 7 27,88 27,15 30,23 28,98 27,78 day - 19th day [21]. It aimed to avoid spontaneous birthing. 8 28,28 26,98 30,75 30,00 28,33 The mice which gave birth spontaneously, tend to eat the 9 28,48 27,05 32,13 30,20 29,35 defective, dead and nearly die offspring (Cannibalism 10 29,25 27,20 33,75 30,93 30,15 Rodentia). Thus, it could affect the results of study. 11 30,85 28,00 34,10 32,40 30,85 12 31,90 27,78 34,60 32,73 31,78 Table 3. Fetal body weight of durian (D. zibethinus Linn) treated 13 32,50 28,43 34,78 33,43 32,33 mice 14 33,40 28,80 37,20 34,80 33,83 Treatment Average Fetal Body Weight of Mice (g) 15 33,33 30,10 37,00 36,00 33,23 P- 0,86±0,08d 16 33,83 30,85 37,08 35,63 35,07 P+ 0,25±0,17a 17 34,68 31,70 37,90 36,28 35,53 P1 0,92±0,16d 18 35,13 33,00 38,95 35,78 36,30 P2 0,72±0,10c Data are expressed as mean; Average data with different superscript b are significantly diffrerent (p<0,05); P- = negative control group; P + P3 0,58±0,32 = positive control group; P1 = 32.5mg / KgBW group; P2 = 65mg / Data are expressed as mean±SD; Average data with different KgBW group; P3 = 95.7mg / KgBW superscript are significantly diffrerent (p<0,05); P- = negative control group; P + = positive control group; P1 = 32,5mg / KgBW group; P2 = 65mg / KgBW group; P3 = 95,7mg / KgBW group

40.00 Besides, laparotomy was performed to observe the resorption sites [9]. Furthermore, the fetus was removed 35.00 and separated from the uterus and placenta. Fetuses that 30.00 have been cleaned were counted and weighed. The results of observations on fetal weight can be seen in Table 3. 25.00 Based on the results of one-way ANOVA, the

Bodyweight (gram) Bodyweight administration of durian fruit significantly affected fetal 20.00 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 body weight (p < 0.05). Day The test was continued by Duncan Multiple Range Test. Negative control group and P1 group were in the same P- P+ P1 P2 P3 coloumn, by the meaning these were not statistically different. But, in P2 and P3 groups were proven to reduce Figure 1. Graph of Average Weight in Pregnant Mice During fetal weight. In this study, the average fetal body weight Pregnancy, P- = negative control group; P + = positive control decreased in line with the higher dosage of treatment. Fetal group; P1 = 32.5mg / KgBW group; P2 = 65mg / KgBW group; body weight is an important parameter to determine the P3 = 95.7mg / KgBW group effect of foreign compounds on the fetus. Fetal weight loss

is the lightest form of the effects of teratogenic agents.

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Impaired fetal development causes abnormalities including imbalance. Under normal circumstances, the embryo fetal growth restriction (intrauterine growth retardation) develops in an isotonic amniotic fluid. Foreign substances and mild errors of morphogenesis (abnormalities of in the tissue could make osmotic imbalance. Thus, can phenogenesis), such as epicanthic folds, clinodactyly, and cause disturbances in pressure and viscosity of fluids in others [23]. embryos, and lead to a ruptured blood vessels and hemorrhage [25]. Table 4. Fetal number of mice treated with durian (D. zibethinus According to Polifka and Friedman [24], if a toxic Linn) effect cannot be tolerated by an embryo, the embryo will Treatment Average Fetal Number of Mice die. The effect of death can be caused by genetic factors P- 5,0±3,56a (sensitivity) of the fetus and teratogen compounds found in P+ 12,5±3,11b the preparation. Any chemical and physical agents with P1 6,2±3,86a small molecular weights can easily enter the embryo across a,b P2 9,0±3,37 the placenta. The fetus that is not yet fully developed cannot a P3 6,2±2,63 metabolize foreign substances properly, resulting fetal Data are expressed as mean±SD;Average data with different superscript are significantly diffrerent (p<0,05); P- = negative control disorders. The higher the foreign substances dose level get group; P + = positive control group; P1 = 32.5mg / KgBW group; P2 in the embryo, will lead to a higher probability of growth = 65mg / KgBW group; P3 = 95.7mg / KgBW group retardation, malformation, fetal death and resorption. Spontaneous hemorrhage occured because of a disruption Table 4 showed the impact number of fetuses on of platelet and caused impaired blood circulation. administration of durian (D. zibethinus). It was found that Hemorrhage is blood loss from the cardiovascular system, the administration of durian significantly affected the accompanied by accumulation in the body tissue. In this number of live fetuses (p < 0,05). In this study, it was found study, there were 2 fetuses in P2 group experienced that the number of dead fetuses increased in line with tha hemorrhage (Figure 3). This may be happened because of higher dosage treatment. In P1 group, all fetuses was live repeatedly administration of high dose of durian fruit, as well as negative controls. In P2 group, there were 2 dead which causing high concentrations in the blood and osmotic fetuses, in different parents. In P3 group, there were 10 imbalance. Under normal circumstances, the embryo dead fetuses in one parent. While in positive control groups, develops in an isotonic amniotic fluid. Foreign substances there were 4 dead fetuses, 10 dead fetuses, and 11 dead in the tissue could make osmotic imbalance. Thus, can fetuses for parent 1, 2 and 3 respectively (Figure 2). cause disturbances in pressure and viscosity of fluids in embryos, and lead to a ruptured blood vessels and hemorrhage [25]

a b

Figure 2. Dead fetus compared to normal fetus; (a) Dead fetus; Figure 3. Hemorrhage fetus (b) normal fetus Observations on the pregnant mice body weight and According to Polifka and Friedman [24], if a toxic fetal body weight were less able to show the effects of effect cannot be tolerated by an embryo, the embryo will disability because fetal body weight was influenced by the die. The effect of death can be caused by genetic factors number of fetuses from one breed. The more number of (sensitivity) of the fetus and teratogen compounds found in fetuses in the uterus, the lower the weight of each fetus [26]. the preparation. Any chemical and physical agents with Observation on the number of live fetuses, dead fetuses and small molecular weights can easily enter the embryo across resorption sites can be observed after the fetus had been the placenta. The fetus that is not yet fully developed cannot removed from the uterus. It was seen in Table 5 metabolize foreign substances properly, resulting fetal The fetal defect was measured after they were fixed disorders. The higher the foreign substances dose level get in Bouin's solution and red alizarin. One-third of the total in the embryo, will lead to a higher probability of growth fetus was soaked in Bouin's solution for fourteen days. retardation, malformation, fetal death and resorption. Fetuses that were immersed in Bouin's solution become Spontaneous hemorrhage occured because of a disruption hard, yellow, and easily observed the external and visceral of platelet and caused impaired blood circulation. bodies. Parameters observed were eyelids, ears, tails, toes, Hemorrhage is blood loss from the cardiovascular system, and cleft palate [11]. In P2 group, there was 1 resorption accompanied by accumulation in the body tissue. In this site. It was signed by the presence of red lumps as the place study, there were 2 fetuses in P2 group experienced where the fetus was implanted (Figure 4). In P3 group, hemorrhage (Figure 3). This may be happened because of there was 1 fetus had a shorter tail compared to the fetus in repeatedly administration of high dose of durian fruit, P- (Figure 5). According to Lu [13], disabilities which which causing high concentrations in the blood and osmotic include tails, straight legs, malrotation of limbs or claws are

Pharmauho: Jurnal Farmasi, Sains, dan Kesehatan 2021, 7(1); 5-10 Rusdi, et al

Table 5. Observation of defects in each treatment group Groups Observations P- P+ P1 P2 P3 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 Feet n n n n n n n n n n n n n n n n n - n n Fingers n n n n n n n n n n n n n n n n n - n n Skeletal n n n n n n n n n n n n n n n n n - n n Tail n n n n n n n n n n n n n n n n n - n 1 Cleft Palate n n n n n n n n n n n n n n n n n - n n Ear n n n n n n n n n n n n n n n n n - n n Eyelid n n n n n n n n n n n n n n n n n - n n Resorption Site ------1 - - - - - Delayed Growth ------Dead - - - - 4* 10* 11* ------2* - 2* - 10* - - Hemorrhage ------1* - 1* - - - - Information : P- = negative control; P + = positive control; P1 = 32.5mg/KgBW group; P2 = 65mg/KgBW group; P3 = 95.7mg/KgBW group; n = normal; - = None; * = number of fetuses (according to the data in the column)

classified as minor anomalies. Minor anomaly is a type of transparent and made bones become dark red. Thus, the disability that will not interfere the survival, growth, shape of fetal bone can be observed more easily[12]. After development, fertility, and longevity of animals. Also, there comparing with negative control group, there was not found was not find fetus with cleft palate abnormalities (Figure bone abnormalities in all treatment groups including the 6). Cleft palate is a condition where there is an abnormal positive control group. gap in the roof of the mouth because some parts fail to join during pregnancy. Cleft palate is one of the most common congenital malformations worldwide. Environmental factors, such as smoking, alcohol consumption, intake of drugs during pregnancy, advanced maternal age, have been demonstrated to be a risk of cleft palate [27].

a b c d

Figure 6. Comparison of horizontal slit in the mouth to observed cleft palate abnormality; (a) Control negative group; (b) Control Positive Group; (c) Treatment Group P1; (d) Treatment Group 2 a b (P2); (e) Treatment Group 3 (P3)

Figure 4. Resorpsion site compared to normal fetus; (a) Resorpsion site; (b) normal fetus Many chemical agents have the ability to penetrate animal tissue and developing fetus, negatively impact the reproductive health of human [28,29]. According to several authors [18,29,30], fetuses are susceptible to teratogenic substances during the second trimester. During this period (organogenesis phase), the eyes, brain, heart, skeletal, urogenital, and other organs are formed. If the fetus defects, this will lead to fetal death and form a red mass (fetal resorption) because of no more totipotential properties of a b the cells [18]. When fetal damage occurred during this period, it could not be repaired and neither further Figure 5. Normal fetus compared to shorter tailed fetus; (a) developed. This condition is one of teratogenic indication normal tailed fetus; (b) Shorter tailed fetus of an agent [16,18,28], as also seen this studybecause of no

more totipotential properties of the cells [18]. When fetal The two-third of fetuses were soaked with red alizarin damage occurred during this period, it could not be repaired solution for two to three days. This solution is used for and neither further developed. This condition is one of coloring mice skeletal system, made the fetus become

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