1. Introduction
Preeclampsia is a multisystem disorder that complicates 3% - 8% of pregnancies in Western countries and contributes to morbidity and mortality worldwide [1]. Preeclampsia is characterized by an increased blood pressure more than 140/90 mmHg and proteinuria more than 300 mg/24 h (dipstik + 1) with or without edema. According to SKDI (2012), preeclampsia/eclampsia contributes to about 13% maternal mortality in Indonesia [2]. Its incidences in Hasan Sadikin General Hospital (RSHS) were approximately from 4 to 10% in 2017, which contributes to 10.4% maternal mortality in the hospital [3].
Underlying mechanism of preeclampsia remains unclear. Many studies suggested that it involves genetic factor, immunologic factor, vascular disease, and imbalance between free oxygen radicals and scavengers in favour of oxidants. Biochemical imbalance in preeclampsia occurs with an increase of oxidative stress and lipoperoxidation and at the same time, a deficient antioxidant protection. Lipid peroxides, as products of an altered oxidative stress, are involved in endothelial cell injury, vasoconstriction and imbalance between thromboxane and prostacyclin [4]. Levels of plasma and erythrocytic malondialdehyde (MDA), a marker of lipid peroxidation in normotensive pregnant women, have been documented in women with established preelampsia [5] [6] [7]. Thus, antioxidants are considered as an important approach to compensate lipid which is associated with preeclampsia.
The exogenous antioxidants are found in food and medicinal plants, such as fruits, vegetables, cereals, mushrooms, beverages, flowers, spices and traditional medicinal herbs. The industries processing agricultural by-products are also potentially important sources of natural antioxidants. Thus we would like to observe antioxidant activities in agricultural waste. Black rice “Sembada Hitam” is a black rice cultivar in Yogyakarta, which is planted in Sleman and Bantul region, Indonesia [8]. Biological and antioxidant activity of rice is mainly localized in its bran (the outer layer) [9] [10].
Rice bran (the outer layer) is discarded during the milling process of white rice. However, the biological and antioxidant activity of rice is mainly localized in the rice bran [8] [11]. Rice bran contains most of the biological components that include phenolic compounds, anthocyanins, phytic acid, γ-oryzanols, tocotrienols, and tocopherols, which were previously reported as antioxidants [9] [10] [12] [13]. Cempo Ireng is a black rice cultivar in Yogyakarta, which is planted in Selman and Bantul region, Indonesia [14]. The phenolic compounds in black rice bran are reported to contain more than 1 phenolic hydroxyl group, which is assumed to poss antioxidant activity [15] [16]. Black rice bran contains various phenolic acids (that is, ferulic acid, p-coumaric acid, vanillic acid, p-hydroxybenzoic, gallic acid, and protocatechuic acid), and ferulic acid has been identified as the predominant phenolic antioxidant [11] [17]. This study aimed to observe anti-angiogenesis properties of black rice bran on preeclampsia through measurement of MDA levels.
2. Materials and Method
2.1. Study Design and Sampling
This was true experimental study with posttest only control group design. Research subjects were pregnant women at 28 - 34 weeks of gestational age consisting of women with preeclampsia and normal pregnancy as controls. This study was performed in Laboratory of Molecular Genetics, Faculty of Medicine, Universitas Padjajaran, from January 2018 to January 2019. Blood samples were collected from patients admitted to Department of Obstetrics and Gynecology, Hasan Sadikin General Hospital (RSHS), and informed consent was obtained. This study was approved by Ethics Committee of Faculty of Medicine/RSHS.
2.2. Extraction of Black Rice Bran
Black rice bran was acquired from outer layer of black rice, and then filtered with 60 mm sieve. Black rice bran of 10 mg was macerated with 100 mL mixture of ethanol and HCl 1N (85:15) for 48 hours at room temperature. Extract was filtered with Whatman No.1 paper. Filtrate was remacerated with 50 mL mixture of ethanol and HCl 1N (85:15). Furthermore, extract was evaporated. Extract was stored in room temperature.
2.3. Cell Culture
HUVEC cell was cultured in RPMI 1640 containing 20% (v/v) sample (normal or preeclamptic serum) [18], 10% endothelial supplement and antibiotic-antimycotic (1% penicillin G-streptomycin Solution Stabilised and 1% Fungisone Amphotericin B) and 1% gentamisin. Cells were incubated at 37˚C 5% CO2 [19] [20] [21].
2.4. Lethal Concentration (LC50) of Black Rice Bran toward HUVEC Cell
LC50 of black rice bran was measured with Brine Shrime Lethality Test (BSLT) [20] [21]. Cells (6 × 105 cell/mL) was added with black rice bran extract in various concentrations. Absorbance was read at 517 nm wavelength. LC50 was measured with the following formula:
A0 = Absorbance control; A = absorbance of the sample (HUVEC and various concentration of black rice bran extracts).
2.5. Measurement of MDA Levels
Cells (6 × 105 cell/mL) were placed into 96-wells microplate, and incubated at 37˚C 5% CO2 (v/v). Futhermore, wells were washed with PBS 37˚C three or four times to discard remaining medium and unattached cells. Brand black rice in various concentration (0; 0.977; 1.953; 3.906; 7.813; 15.625; 31.25; 62.5; 125; 250 μg/mL) was placed into wells, and incubated for 24 to 48 hours at 37˚C 5% CO2 (v/v). Each well was washed with PBS pH 7.4 for 5 minutes, and cells were centrifuged at 3000 rpm 20 minutes. Supernatant was carried, and MDA was measured wtih TBARS (thiobarbituricacid-reactive substances) [22]. Supernatan was added with 15% w/v trichloroaceticacid, 0.375 w/v thiobarbituricacid, 0.25% hydrichloric acid and 0.2% triton X. Afterward, cells were suspended using heating at 100˚C for 15 minutes and then centrifuged at 4500 rpm for10 minutes. Supernant was read at 532 nm wavelength.
2.6. Data Analysis
Data were analyzed with two way-ANOVA, and continued with post hoc Dunnett (α = 0.05) using SPSS 21.
3. Results
LC50 of black rice bran is presented in Figure 1. Results showed LC50 of black rice bran was 500 ug/mL with mortality percentage 50.78%. This indicates LC50 less than 500 ug/mL shows non-toxicity towards HUVEC.
As shown in Table 1, MDA levels in preeclampsia-induced HUVEC were reduced after treatment of black rice bran (p < 0.05). At concentration of 62.5 μg/ml after 24 hours of incubation, MDA levels (5.0895 μM) were comparable to normal level in control group (5.7180 μM).
4. Discussion
In the present study, cytoxicity of black rice bran was determined through measurement of LC50 toward HUVEC cells (Figure 1). Results showed LC50 of black rice showed no toxicity toward HUVEC makes it appropriate for further treatment. Black rice bran exhibited very strong antioxidant properties in preeclampsia by decreasing MDA levels (Table 1). These results might be due to presence of bioactive compounds found in black rice bran. The phenolic compounds in black rice bran contain more than 1 phenolic hydroxyl group, which is known to poss antioxidant activity [15] [16]. Various phenolic acids are found such as ferulic acid, p-coumaric acid, vanillic acid, p-hydroxybenzoic, gallic acid, and protocatechuic acid [11] [17].
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Figure 1. LC50 of black rice bran towards HUVEC.
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Table 1. Level of MDA in preeclampsa-induced HUVEC treated with black rice bran.
Data are presented in mean. Different letters (a)-(c) in the same row indicates significant difference among tretament, analyzed with Duncan test (p < 0.05).
It has been reported that lipid peroxidation is the main causative factor for oxidative stress in preeclampsia. Free radicals initiate lipid peroxidation by attacking polyunsaturated fatty acids in cell membranes [23]. Uncontrolled peroxidation of fatty acids and cholesterol alter membrane fluidity and permeability as lipid peroxides are toxic compounds that damage endothelial cells, increase peripheral vasoconstriction and increase thromboxane synthesis and decrease prostacyclin synthesis [24]. Once steady state levels of blood lipid peroxides begin to rise, the stage would be set for self-perpetuating chain-reaction processes to take place. Endothelial contact with lipid peroxides would allow peroxidative damage of endothelial cell membrane lipids. Eventually, the permeability of the endothelium to plasma components, is reduced. Exposure of the vascular endothelium to lipid peroxides would begin to interfere prostacyclin synthesis, increasing the propensity for vasoconstriction and platelet aggregation [4].
5. Conclusion
Black rice bran has very strong antioxidant properties by significantly decreasing MDA levels in preeclampsia-induced HUVEC, which can be a promising strategy in preeclampsia treatment. Since this was a preliminary study to observe efficacy of black rice bran which only through measurement of MDA levels, antioxidant activiy of black rice bran is not established yet. Further antioxidant measurements using other parameters, as well as in vivo study to determine optimum concentration of black rice bran, are encouraged.