International Journal of PharmTech Research
CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563
Vol.10, No.02, pp 103-108, 2017
In summary, lycopene possess high antioxidant and antiangiogenesis that plays role as precursor in scavenging reactive oxygen and reduce free radicals that recover trophoblast cells induced by preeclampsia as indicated by decrease in MDA level. Further studies regarding the optimal concentration of lycopene on embryo cell for clinical trial, are encouraged.
References
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As shown in Figure 2, MDA level reduced along with longer incubation time and higher lycopene concentration. Highest decrease after incubated for 24 hours. This might be due that trophoblast requires time to contact with compounds given that will reduce MDA level.
Figure 3. Level of MDA in serum various and incubation time.
In preeclampsia-induced trophoblast, MDA level significantly reduced (p<0,001) from 18,8923 μM to 8,5773 μM after treated with lycopene of 31,25 μg/ml incubated for 24 hours, and from 18,899 μM to 8,6671 μM after incubation for 48 hours (Figure 3).
Discussion
For our best knowledge, there is no studies regarding potency of lycopene as antioxidant and antiangiogenesis in preeclampsia. Angiogenesis occurrs in preeclampsia that leads to blood vessels derived from extravillous trophoblast invade to uterus that reduce placental vascular viability and affects placental oxygen supply due to dimished blood vessels. These events cause ischemic that promote damage in villous trophoblast (cell damage)15,16. Ischemic causes imbalance in pro-oxidant and antioxidant, release of free radicals that increase gradually in time leading to oxidative stress17. Free radicals cannot be neutralized by preeclamptic patients that causes cell damage, disturbance in cell integrity, endothelial lysis, reactivity and increase in vascular permeability13.
Oxidative stress causes increase in MDA level that causes endothelial dysfunction, vasocontriction, disturbance in blood coagulation, lipid peroxidation, biomolecule oxidative damage, and DNA damage. Increase MDA also causes nitrite oxide that worsen oxidative stress11,13,14. In preeclampsia, there is decrease in NADPH which theoritically can be prevented by antioxidant that can provide protective effect and synergistic to internal antioxidant. Antioxidant is obtained through food or drug derived from plants which has been known to possess antioxidant properties11,13.
The result of present study showed lycopene had antioxidant and antiangiogenesis activities in trophoblast induced by both preeclampsia and normal serum. Referring to study done by Wilcox (2003), Basu and Imrhan (2007), and Srinivasan (2007), tomatoes contain lycopene and is believed to prevent many diseases due to its high antioxidant content18-20. Lycopene is the most dominant antioxidant in tomatoes that possess activities such as antimicrobial, antithrombogenik, antivirus, reduce blood cholesterol and inhibit cell proliferation. Lycopene inhibits lung cancer growth in mouse21. Lycopen abundantly in cell membrane inhibits lipid peroxidase due to free radicals22.
Lycopene is a electron-rich compound and unstable which makes it more reactive to oxygen and peroxidase and free radicals11. The main characteristic of lycopene is catalytic and effectively scavenge superoxid and peroxil radical22. Agarwal and Sekhon (2010) reported that lycopene protect lipid membrane and DNA damage due to oxidative stress in vitro23. Activity of lycopene is cosidered two-folds higher than β-caroten and ten-folds higher than α-karoten24. In vitro study reported that lycopene gives protetection on lipoprotein of cell membrane, DNA and vasculer against oxidant18-20.
Underlying mechanism of preeclampsia remains unclear. Placenta and endothelial dysfunction have been proposed as the main pathophysiology of preeclampsia. Vascular disease and excessive trophoblast can promote trophoblast invasion on spiral arteries at early first and second trimester. This causes dilatation of spiral arteries that leads to reduced placental blood circulation. Incomplete spiral artery remodelling in preeclampsia causes inadequate response to increased blood supply along with development of pregnancy, which casuses reduced perfusion of utero-placenta and imbalance between pro- and antiangiogenic. These events will lead to ischemia on placental vascular6,7.
Preeclampsia is also caused by imbalance in free radicals and antioxidant in blood and placenta8. Oxidative stress occurs due to disturbance in pro-oxidant and antioxidant. Previous researches show that antioxidant decrease preeclampsia risk after exposure of vitamin E, vitamin C and lycopene in pregnant women9,10. Lycopene is a strong antioxidant commonly found in tomatoes, watermelon, guava, papaya, red winde etc. Lycopene is a electron-rich compound and unstable which makes it easily reacted to oxygen and peroxide as well as free radicals. Lycopene has been proved to possess activities in prevention of various cancers such as prostate and cataract, cardiovascular dysfunction, endometriosis, osteoporosis, etc11. This study aimed to observe lycopene on MDA level in placental trophoblast which is induced by preeclampsia in vitro.
Materials and Methods
Lycopene was isolated from tomatoes [SIGMA Aldrich] and placental trophoblast was primary culture from Laboratory of Cell Culture, Faculty of Medicine, Universitas Padjadjaran. Serum was obtained from normal pregnancy and preeclamptic women which fulfillef inclusi and exclusi criteria
Cell Culture
Trophoblast cell was cultured in media containing Amniomax and growth factor supplemented with 10% serum (normal pregnancy and preeclampsia at 34-42 gestational age), and antibiotic-antimikotic (1%Penicillin G-Streptomycin Solution Stabilised and 1% Fungizone Amphotericin B). Cells were incubated for 24 hours at 37°C 5% CO2 (v/v) untill confluent. Viability was measured with trypan blue in haemocytometer under light microscope with 400x magnification12-14.
Measurement of MDA level
Cell of 6x105 cell/ml containing 10% serum both normal and preeclampsia was replaced into 96-well microplate, and then incubated at 37°C 5% CO2 (v/v) untill confluent. Wells were washed 3-4 times with PBS 37°C. Lycopene in various concentration were distirbuted in each well, and then incubated at 24 and 48 hours 37°C 5% CO2 (v/v). Each well was washed with PBS pH 7,4 once for 5 minutes. Level of MDA was measured with TBARS (thiobarbituric acid-reactive substances) from NWLSSTM Malondialdehyde Assay Northwest (NWK-MDA01). Cells were treated with liquid containing 15% w/v trichloroacetic acid, 0,375 w/v thiobarbituric acid, 0,25 hydrichloric acid and 0,2% triton X. Furthermore, cells were carried and suspended with heating at 100°C for 15 minutes, and centrifuged at 4500 rpm for 10 min. Supernatant was measured with spectrophometer at 532 nm wavelength13.
Data Analysis
Data was analyzed with T-test if normally distributed, and Mann Whitney test if not normally distributed. Data was quantitavely analyzed with ANOVA DMRT (Duncans’s Multiple Range Test) to determine the significance among variables in each treatment with SPSS 22.
Result
Effect of lycopene on MDA level
Figure 1. Level of MDA in trophoblast cell based on serum various
Figure 1 shows there was difference in MDA level affected by serum (p<0,001). In preeclampsia-induced trophoblast exhibited higher MDA level compared to normal. Lycopene lowered the MDA level in concentration-dependent manner. Thus, MDA level in preeclampsia-induced trophoblast treated with lycopene was comparable with that in normal.
Effect of incubation time on MDA level can be seen in Figure 2. Figure 2 showed there was decrease in MDA level on trophoblast after incubated 24-48 hours.
Figure 2. Comparison of MDA level in preeclampsia-induced trophoblast treated with lycopene in various incubation time.
Effect of Lycopene on Level of Malondialdehid (MDA) in Preeclampsia-Induced Placental Trophoblast Cells
Vaulinne Basyir1*, Prima Nanda Fauziah2, Yanwirasti4, Johanes C. Mose3, Fadil Oenzil4
1Department Obstetrics and Gynecology, Faculty of Medicine, Andalas University, Padang, West Sumatra, Indonesia
2Department of Medical Laboratory Technology, School of Health Sciences Jenderal Achmad Yani Cimahi, Indonesia
3Department of Obstetrics and Gynecology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia
4Faculty of Medicine, Andalas University, Padang, West Sumatra, Indonesia
Abstract : Preeclampsia is a mjor cause in both maternal and perinatal mortality and morbidity. Underlying mechanism of preeclampsia remains unclear. It is assumed that preeclampsia is caused by imbalance in free radicals and antioxidant in blood and placenta. Lycopene, known to possess antioxidant properties, is therefore a promising agent to decrease preeclampsia risk. This study aimed to observe lycopene on MDA level in placental trophoblast which is induced by preeclampsia in vitro. Level of MDA was measured with TBARS (thiobarbituric acid-reactive substances). In preeclampsia-induced trophoblast, MDA level significantly reduced (p<0,001) from 18,8923 μM to 8,5773 μM after treated with lycopene of 31,25 μg/ml incubated for 24 hours, and from 18,899 μM to 8,6671 μM after incubation for 48 hours. Lycopene possess high antioxidant and antiangiogenesis that plays role as precursor in scavenging reactive oxygen and reduce free radicals that recover trophoblast cells induced by preeclampsia as indicated by decrease in MDA level. Further studies regarding the optimal concentration of lycopene on embryo cell for clinical trial, are encouraged.
Keywords : lycopene, MDA, preeclampsia.
Introduction
Preeclampsia is a major cause in both maternal and perinatal mortality and morbidity1. It has been reported that preeclampsia cases were 5-8% in developing countries2,3, whilst according to World Health Organization (WHO) were 0,51%-38,4% in 2005. Preeclampsia ranked second in Indonesia as the main cause in maternal mortality after hemorrhage4. Referring to Department of Obstetrics and Gynecology in Hasan Sadikin Hospital (RSHS), Bandung, number of preeclampsia occurence was approximately 4-10% in 2005. Preeclampsia contributed about 10,4% to maternal mortality in RSHS. Maternal mortality was 228/100.000 living birth compared to desired target by government in 2010 which was 125/100.000 living birth5.
International Journal of PharmTech Research, Vol.10, No.2, pp 103-108 (2017)
http://dx.doi.org/10.20902/IJPTR.2017.10115