Evaluation of Propolis and Milk Administration on Caffein-Induced Mus Musculus Fetus Skeletal

40 PharmOriginal Sci Res, Article Vol 5 No 1, 2018 Pharmaceutical Sciences and Research, 5(1), 2018, 40-48 Evaluation of Propolis and Milk Administration on Caffein-Induced Mus musculus Fetus Skeletal Dwisari Dillasamola1*, Almahdy A1, Novita Purnama Sari1, Biomechy Oktomalio Putri2, Noverial2, Skunda Diliarosta3 1Faculty of Pharmacy Andalas University, West Sumatra 2Medical of Faculty Andalas University, West Sumatra 3Faculty of Mathematics and Natural Sciences, Padang State University, West Sumatra ABSTRACT Caffeine consumption by pregnant women at doses above 300 mg/day was suggested to cause skeletal damage. Propolis with high flavonoids concentration could increase the number of osteoblasts. This research aims to evaluate the effect of propolis and milk administration on fetal skeletal of caffeine-induced female mice (Mus musculus). Mice were divided into six groups: negative group, positive group of caffeine (a dose of 75 mg/kg BW), positive group of propolis ARTICLE HISTORY (a dose of 1400 mg/kg BW), positive group of milk (200 ml), group D1 (caffeine 75 mg/kg BW and propolis 1400 mg/kg BW) and group D2 (caffeine 75 mg/kg BW and milk 200 ml). Data were Received:October 2017 processed using one-way ANOVA. The results showed that administration of propolis and milk Revised : February 2018 on caffeine-induced mice during pregnancy does not affect the mice body weight, the number Accepted: April 2018 of fetuses and fetal weight significantly (P> 0.05). No skeletal defects detected in group D1 and D2 (observation with Alizarin solution) compared to the negative group. In conclusion, the admnistration of propolis at the dose of 1400 mg/kg BW and 200 ml of milk can repair skeletal damage caused by caffeine induction. Keywords: caffeine; fetus; osteoblasts; propolis; skeletal *corresponding author Email: [email protected] INTRODUCTION Although it has not been ascertained fetal defects with caffeine consumption in humans, but it was the fact that In general, pregnant women still consume caffeine every caffeine can come across the placenta to amniotic fluid, day (FDA, 2010). Caffeine can be consumed through umbilical cord blood, plasma and urine of newborns, coffee, colas, chocolate, tea, soft drinks, food, and and across breastmilk barrier, both in mice and humans. medicine (Nawrot et al., 2002). Numerous studies in Those facts about caffeine can potentially interfere animals have shown that caffeine can cause birth defects, with fetal development (Mose et al., 2008). Caffeine premature birth, reduced fertility, and increase the risk of consumption limit in pregnant women is less than 300 birth with low weight that leads to spontaneous abortion mg/day (OTIS, 2015). Pregnant women who consume (Committee on Toxicity, 2001; Weng et al., 2008). caffeine more than 300 mg/day are increasing the risk of fetal to suffer growth retardation, skeletal abnormalities, Caffeine (1,3,7-trimethylxanthine) is an active compound and miscarriage (Committee on Toxicity, 2001). This which has a variety of pharmacological effects (Nawrot is evidenced by research on the teratogenic effects of et al., 2002). Caffeine has beneficial effects as a stimulant caffeine on fetal mice that had been done previously. for the central nervous system and respiratory system. Caffeine is bound to cause skeletal system abnormalities The effect is characterized by reduced fatigue and and cleft palate (Nishimura & Nakai, 1960). weak bronchodilation effect (Welsh et al., 2001; Burns et al., 2014). On the other hand, the adverse effects of Caffeine is able to intervene mitosis which decreases the caffeine can vary such as insomnia, anxiety, increased activity of DNA polymerase enzyme, induces mitotic blood pressure and pulse rate as well as a risk factor for cells mammal prior to synthesis of DNA replication osteoporosis (Mesas et al., 2011; Drake et al., 2013; phase, inhibit the enzyme activity phosphodiesterase, Marquinaa et al., 2013). inhibit osteogenesis process and potentially cause abnormal development of the embryo (Beck & Urbano, 1991). The negative influence of caffeine on fetal ISSN 2407-2354 Evaluation of Propolis and Milk Administration Pharm Sci Res, Vol 5 No 1, 2018 41 exacerbated by the extension of the half-time of caffeine Canadian Medical Association Journal in 2007, pregnant in pregnant women from 3.5 hours to 16 hours. The women who consume milk gave birth to babies whose extension of half-time period will result in a substantial weight increases as much as 41 g (Mehta, 2007). accumulation of caffeine potentially disrupt the fetus and placenta (European Food and Safety Authority, 2015). Reproductive toxicity is one of the toxicity tests for the preparations of herbs and chemicals that will be One solution to reduce the negative effects caused by consumed by humans. Reproductive toxicity test which caffeine is the use of herbal preparations in pregnant is frequently used is the teratogenicity test. Teratology is women. Most people assume that herbal preparations the study of the causes, mechanisms and manifestations are safer than modern chemical drugs (Broussard et al., of embryonic defect (abnormal) (Wilson & Fraser, 2010). At the moment, one of the most popular herbal 1973). Teratogen is a chemical that can significantly preparations is propolis. Propolis is a substance produced affect fetal development and the effects of changes by honey bees which contains flavonoids chrysin start from lethality until deformity (malformations) and (5,7-dihidroxy-2-phenyl-4H-chromen-4-one) [16], growth retardation (Wilson & Fraser, 1973). Teratology caffeic phenetyl acid ester (50%), wax (30% ), essential principle is the provision of test compounds in animals oils (10%), pollen (5%), and other organic components during pregnancy and sees its effect on fetal development (5%) (Franz, 2008). Propolis is sticky pollen collected so that the ability or potential toxicity of compounds from plants, especially from flower, leaves, and shoots. against a developing fetus cells is known (Almahdy, Then, pollen is mixed with bees’ saliva (Marcucci et al., 2012). 2001). Therefore, researchers wanted to prove that propolis can Propolis works to improve the pathological condition reduce the negative effects of caffeine on the fetal skeletal of the sick body, works as an antioxidant and boost the system during the period of organogenesis. Researchers immune system both humoral and cellular (El Sohaimy also want to compare the provision of milk that has & Masry, 2014). In an experiment conducted before proven good effect on bone and is safe for consumption (Bereket et al., 2014), propolis can improve skeletal by pregnant women. abnormalities in rats by accelerating osteogenesis. That occurrence may also apply to the fetus. The fetus METHOD may encounter osteogenesis process due to exposure to caffeine. Based on previous research (Sartika et The tools used are micropipette (Eppendorf®), vortex al., 2013), it is also proven that provision of propolis (Etech®), analytical balance, refrigerator, rubber gloves, extracts have an influence on bone repairment time of masks, treatment cage, cage maintenance, surgical orthodontic tooth movement. The mechanism was by scissors, a petri dish, toilet roll, watch glass, tweezers, increasing significantly the number of osteoblasts. The pipette, measuring cups, beaker glass. results are consistent with research conducted before (Elwakkad et al., 2008) which showed propolis effect Materials used are pure caffeine powder (Brataco), on bone formation. Then, the latest research (Jeong et propolis (Melia Propolis®) from PT. Melia Sehat al., 2016) showed that the provision of propolis can Sejahtera, sterilized milk (Nestle® Bear Brand), distilled significantly lower the maximum concentration of water, and the solution containing Alizarin Red (1% caffeine in plasma. KOH and alizarin red 6 mg / L). Another solution to prevent the adverse effects of Experimental animals were white mice (Webster) females caffeine for pregnant women is consuming milk aged less than two months with a weight of 20-30 grams, (Winarno, 1993). Milk is a nutritious liquid produced by healthy and had never experienced treatment of the drug. the mammary glands of female mammals. Milk contains Also, the male mice also required for mating. Male mice fat and fat-soluble vitamins such as vitamin A, vitamin that used to be healthier and live approximately three D and vitamin E (Burger et al., 2007). Milk is a source months. of calcium and phosphorus which is very good for growing bones and teeth (Weinsier & Krumdieck, 2000). Preparation of the Test Solution Additionally, milk is safe for consumption by pregnant The test substance used for this study was caffeine, women (Mehta, 2007) and is an antidote to the toxic propolis and milk. Caffeine stock solution was made by substances that can harm pregnant women (Guharaj & dissolving 300 mg of caffeine in 60 ml of distilled water, Chandran, 2003). According to the American Academy propolis stock solution was made by dissolving 4 ml of of Neurology’s Annual Meeting in 2010, the risk of propolis (1 ml propolis = 900 mg) in 40 ml of distilled multiple sclerosis in the infant is lower whose mothers water and milk was pack in 10 mL solution). consumed milk (Paul, 2010). Then, according to the ISSN 2407-2354 42 Pharm Sci Res, Vol 5 No 1, 2018 Dwisari Dillasamola et al Acclimatization of Experimental Animal and Laparotomy Determination Estrous Cycle Laparotomy performed on day 17 of pregnancy. Acclimatization conducted for ten days to familiarize the Mice were killed by cervical dislocation, then do the animals with the experimental environment. They were laparotomy to remove the fetuses of mice. How mice given adequate food and drink, bodies were weighed were dissected at the abdomen upward until it looks daily, and behavior was being observed. During the uterus containing a fetus. Fetus removed by cutting acclimatization, estrous cycle is determined by mice the uterus and placenta. Furthermore, the presence or vagina visual observation. Mice in estrous period showed absence of resorption site marked by a red blob as the vagina with red-colored and gummy texture. implantation point of the fetus was observed.

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