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International Journal of PharmTech Research CODEN (USA): IJPRIF, ISSN: 0974-4304, ISSN(Online): 2455-9563 Vol.9, No.6, pp 424-435, 2016
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Effects of Papua Ant Nests (Myrmecodia pendens) on Level of sFlt-1, PIGF, MDA and NO in Preeclampsia-induced HUVEC Cell Line
1Department of Obstetrics and Gynecology, Faculty of Medicine, Padjadjaran University, Indonesia
2Department of Microbiology, Faculty of Dentistry, Padjadjaran University, Indonesia
3 Department of Medical Laboratory Technology, School of Health Sciences Jendral Achmad Yani, Indonesia
4The Integrated Research and Testing Laboratory, Gadjah Mada University, Indonesia
Abstract : Preeclampsia remains major cause in both maternal and perinatal mortality and morbidity. Preeclampsia causes endothelial dysfunction due to imbalance of proangiogenic and antiangiogenic indicated by increased soluble fms-like tyrosine kinase-1 (sFlt-1) and decreased placental growth factor (PlGF), also followed by oxidative stress indicated by decreased malondialdehyde (MDA) and increased nitric oxide (NO). Antioxidant is believed in preventing such mechanism. Herbal antioxidant is widely used in Indonesia, one of which is ant nests from Papua. Ant nests used is formed in water fraction and known containing antioxidant compounds such as flavonoid, tannin, terpenoid and alkaloid. This study aims to analyze therapeutic effects of ant nests on level of sFlt-1, PIGF, MDA and NO in preeclampsia-induced HUVEC cell line. Measurement of level of sFlt-1 and PlGF was performed with ELISA. Measurement of level of MDA was performed with thiobarbituric acid-reactive substances (TBARS), and level of NO was performed with NWLSSTM Nitric Oxide Assay. Data was analyzed statistically with ANOVA and Duncan test. Level of sFlt-1 and MDA in preeclampsia-induced HUVEC ATCC CRL 1730 cell line were decreased whereas level of PlGF and NO in preeclampsia-induced HUVEC ATCC CRL 1730 cell line were increased, after exposure of ant nest water fraction on concentration 31,25 ug/ml. Conclusion: Ant nest water fraction has therapeutic effects on preeclampsia. Further studies regarding development of ant nests in prevention of preeclampsia are encouraged.
Keywords: ant nests, sFlt-1, PIGF, MDA, NO.
Introduction
Preeclampsia remains major cause in both maternal and perinatal mortality and morbidity. National Vital Statistics Reports reported increased preeclampsia onset was approximately 40%.1 According to Survei Demografi Kesehatan Indonesia (SKDI) in 2013, number of maternal mortality in Indonesia was significantly increased approximately 359 per 100.000 living birth compared to SDKI in 2007. Preeclampsia contributed to maternal mortality about 23%, makes it the second cause of maternal mortality in Indonesia. 2 In Dr. Hasan Sadikin Hospital, Bandung, preeclampsia was reported in 3.5% cases and eclampsia in 2.8% cases in 2006,
while in 2008-2010 preeclampsia was reported in 4.0-10.4% and eclampsia in 2.3-4.3%.3-5 Therefore, studies regarding underlying mechanism of preeclampsia are conducted.
Endothelial dysfunction is a major point in preeclampsia and associated with alteration of angiogenics and antiangiogenics balance, placental metabolism, placental inflammation mediator, and very low density lipoprotein (VDRL) level.6 Alteration in preeclampsia occurs in presence of excess antiangiogenic, soluble fms-like tyrosine kinase-1 (sFlt-1).7 sFlt-1 is endogenic antiangiogenic protein produced by placenta which undergo ischemic and neutralize proangiogenic protein, Vascular Endothelial Growth Factor (VEGF) and Placental growth factor (PlGF).8 sFlt-1 is expressed by placenta as response of hypoxia, and increased before its clinical symptoms emerged (early trimester). The presence of sFlt-1 was further observed to diagnose preeclampsia in laboratory before hypertension and proteinuria.9,10
In preeclamptic patients, altered endothelial vessels decrease nitric oxide (NO) phosphorylation synthesis in endothelial cells due to increased sFlt-1. Decreased oxide phosphorylation synthesis affects NO bioavailability produced by endothel, that promotes alteration in cardiovascular homeostasis.11-13 NO is a free radical molecule which is reactive in a short time, but modifies the activity of trombocit in endothelial cells to undergo adhesion, aggregation, and releasing reaction. Decreased NO worsens hypoxia in preeclampsia.14,15 Hypoxia causes production of reactive oxygen species (ROS) such as superoxidation (O2), hydroxyl radical (OH) and hidrogen peroxide (H2O2) in preeclampsia following disturbance in regulation of prooxidant and antioxidant.16 Decreased enzymatic production of prooxidant and increased lipid peroxide caused by free radical malondialdehyde (MDA), are also detected. The presence of MDA is therefore used as a marker of endothelial dysfunction on molecular level. 17,18-22
Increased free radical molecules is documented in preeclamptic patients which is associated with decreased cellular antioxidant. Antioxidant is responsible in reducing damage through scavenging mechanism of radical cluster, as well as preventing oxidative stress through metal binding mechanism.23,24 It is naturally produced by cell or obtained from outside such as foods. Decreased level of Nicotinamide Adenine Dinucleotide Phospahte (NADPH) occurs in preeclampsia, and theoretically prevented by antioxidant exposure from outside that provides additional protective effects and synergistic with intracellular enzyme as internal antioxidant. 25-27 It has been reported that herbal medicine possess antioxidant properties which is known safe for patients. 28,29
Ant nests (Myrmecodia pendens) is reported to contain alkaloid, flavanoid, tanin and terpenoid. Flavanoid is water dissolved and used as antioxidant, antiangiogenic, wide spectrum antimicrobial, antithrombogenic, antiviral, decreases blood cholesterol level and inhibits cell proliferation.35 The other compound, tannin, is also hydrolyzed and has condensed protein complex and quickly reactive trapping hydroxyl radical cluster. 37,38 Previous studies show ant nests extract to possess antiangiogenic compound.36 Antiangiogenic (angiogenesis inhibitor) inhibits activity of angiogenic factors inducing extracellular matrix enzyme which attacks endothelial vessels walls and inhibits endothelial vessels cells proliferation.39
Water fraction of ant nests is considered as food alternative. However, direct exposure of compounds to preeclamptic patients has never been performed due to its clinical test which has never been done. Thus, this study aims to know effects of ant nests extract (ANE) on endothelial damage caused by angiogenesis alteration and oxidative stress in HUVEC cell line with preeclampsia by measuring level of sFlt-1, PIGF, MDA, and NO in culture.
Materials and Method
Preparation of water fraction of ant nests
Ant nests were obtained from Ayawasi village, South Sorong, West Papua. Ant nests were identified in Herbarium, Department of Biology, Faculty of Mathematics and Science, Padjadjaran University, Bandung. Tuber of ant nests was removed and cleaned, stripped into pieces and dried in oven 60°C for 5 hours. 10 g dried ant nests were boiled with 200 ml aquadest for 20 minutes. Boiled water was filtered and left until warm. Boiled water was sterilized with filtration using syringe filter millipore 0,22 μm into sterilized bottle.52
Cell culture
HUVEC ATCC CRL 1730 cell line was human endothelial cells-derived in cryopresipitat form, progenitor free, isolated from amnion blood vessels. Cell culture was done in Integrated Research and Testing Laboratory (LPPT), Gadjah Mada University. Cell culture was divided into 2 steps; thawing and passage.
Resusitation was initiated by growing HUVEC cell line into tissue culture flask containing RPMI 1640 medium, incubated to reach confluent 90%. Passage was initiated after proliferation and cell invasion 6 x 105 cell per cm2.57,58
Measurement of IC50 Water fraction of ant nests in HUVEC cell line
Measurement was performed in Integrated Research and Testing Laboratory (LPPT), Gadjah Mada University. Samples were dissolved with aquadest to obtain concentration 100 µg/ml, 1000 µg/ml and 5000 µg/ml.51 600 μl aquadest were added into cuvette and added with 3 ml sample. Solution was measured at λ=400-600 nm and absorbance was recorded at λ=517 nm. Samples with variety of serial concentration starting from 100µg/m (multiplied from 0,195 µg/ml to 100 µg/ml); 1000µg/ml (multiplied from 1,953 µg/ml to 1000 µg/ml); and 5000µg/ml (multiplied from 9,766 µg/ml to 2500 µg/ml) in HUVEC ATCC CRL 1730 cell line to measure its toxicity activity. Absorbance was measured with spectrophotometer at λ=517 nm and measured at 24 hour, to measure IC50 value from each sample. 600 μl aquadest was used as controls, added into cuvette and added with 3 ml DPPH, whereas negative control 600 μl aquadest were added into cuvette and added with 3 ml HUVEC ATCC CRL 1730 cell without ant nest water fraction. Solution was quantified using spectrophotometer at λ=400-600 nm and absorbance was recorded at λ=517 nm. Control negative value was used as standard in measurement of IC50.34
Measurement of level of sFlt-1, PlGF, MDA, and NO
Preeclampsia serum was obtained from Cibabat Hospital, Bandung. Measurement was performed in Integrated Research and Testing Laboratory (LPPT), Gadjah Mada University. Measurement of sFlt-1, PlGF, MDA and NO levels in both normal and preeclampsia-induced HUVEC ATCC CRL 1730 cell line after exposure of variety of ant nests concentration was performed. Measurement of level of sFlt-1 and PlGF was performed with ELISA. Measurement of MDA level was performed with thiobarbituric acid-reactive substances (TBARS), and level of NO was performed with NWLSSTM Nitric Oxide Assay. Data was analyzed statistically with ANOVA and Duncan test.
Compliance with Ethical Standards
Written informed consent was obtained from all participants. The ethical reviews boards of the Health Research Ethics Committee, Faculty of Medicine Padjadjaran University and Dr. Hasan Sadikin Hospital, Indonesia, approved this study.
Results
Antioxidant compounds in ant nests water fraction
Phytochemical analysis was conducted to confirm antioxindant compounds contained in water fraction of ant nests. Antioxidant compounds measured were tannin, flavanoid, alkaloid and terpenoid (Table 1 and Figure 1). Tannin, flavanoid and alkaloid compounds were present in ANE. Qualitative measurement showed color alteration to red-violet in ant nests water fraction that indicate presence of terpenoid.
Table 1 Measurement of Tannin, Flavanoid and Alkaloid contained in Ant nests
Sample |
Mass (g) |
Volume (ml) |
Concentration (ppm) |
In sample (ppm) |
Equivalent total (%b/b) |
Average |
Tannin |
0,0509 |
10 |
5090 |
4,481 |
0,088 |
0,089 |
0,0509 |
10 |
5190 |
4,609 |
0,089 |
||
Flavanoid |
0,0515 |
10 |
5150 |
42,901 |
0,833 |
0,848 |
0,0510 |
10 |
5100 |
44,030 |
0,863 |
||
Alkaloid |
0,1011 |
10 |
10110 |
73,094 |
0,723 |
0,717 |
0,1091 |
10 |
10910 |
77,542 |
0,711 |
Inhibition percentage represented by absorbance value was showed by microplate reader BIO RAD 680 XR, used as standard to measure IC50 of ANE in HUVEC ATCC CRL 1730 cell line. In this study, ant nests water fraction samples were divided into three serial dilutions, 100 μg/ml, 1000 μg/ml and 5000 μg/ml, for each group that consists of ten-fold multiplies. Measurement of ANE was performed with three replications. IC50 obtained for ant nests on 500 µg/ml was 52,57% of death percentage and on 250 µg/ml was < 20,44% of death percentage (Figure 2). Determination of ANE activity was conducted by 50% of death percentage (IC50) in HUVEC ATCC CRL 1730 to be less than 500 µg/ml indicating non-toxicity activity of cells (250µg/ml).
Figure 1 Qualitative measurement of terpenoid in ant nests. Terpenoid spot color: Red-Violet. Rf. Detected Terpenoid: 0,26 ; 0,97. Water fraction of ant nests was examined with phytochemical test to know antioxidant compounds contained. Antioxidant compounds measured were tannin, flavanoid, alkaloid and terpenoid.
Measurement of ANE effects in level of sFlt-1, PlGF, MDA, and NO was performed using ELISA for sFlt-1 and PlGF and thiobarbituric acid-reactive substances (TBARS) for MDA, and NWLSSTM Nitric Oxide Assay for NO.
Figure 2 Measurement of IC50 in Ant nests water fraction. Measurement of ant nests water fraction was performed with three replications. IC50 obtained for ant nests on 500 µg/ml was 52,57% of death percentage and on 250 µg/ml was < 20,44% of death percentage. Determination of ant nests water fraction activity was conducted by death percentage 50% (IC50) in HUVEC ATCC CRL 1730 cell line, which is less than 500 µg/ml indicating non-toxicity activity of cells (250µg/ml).
As shown in Table 2 and 3, there is difference between level of sFlt-1 based on serum used. In preeclampsia serum, sFlt-1 level was higher than that in normal. However, sFlt-1 level in preeclampsia-induced HUVEC significantly decreased following increased ant nests concentration (p<0,005). Level of sFlt-1 was significantly lower in range 29,010-31,095 pg/ml in preeclampsia-induced HUVEC ATCC CRL 1730 after exposure of ANE on concentration 62,5 µg/ml that appeared to reach its level in normal-induced HUVEC ATCC CRL 1730.
As shown in Table 4 and 5, level of PlGF were significantly different in each serum used (p<0,005). In preeclampsia serum, level of PlGF was lower than that in normal. Level of PlGF was significantly increased following increased ANE concentration approaching normal. Level of PlGF were high in range 5,005-5,647 pg/ml preeclampsia-induced HUVEC ATCC CRL 1730 after exposure of ANE in concentration 125 µg/ml approaching condition in normal serum-induced HUVEC ATCC CRL 1730.
Table 2 Effects of variety of ant nests water fraction concentration and type of serum used in sFlt-1 level
Concentration (µg/ml) |
sFlt-1 serum(pg/ml) |
|
Normal |
Preeclampsia |
|
Control 1 |
8,813 (0,565)* |
33,32 (0,764) |
Control 2 |
9,926 (0,565) |
32,845 (1,364) |
31,25 |
38,284 (1,697) |
30,202 (0,908) |
62,5 |
34,057 (1,353) |
30,053 (1,474) |
125 |
30,3185 (1,767) |
27,219 (0,608) |
250 |
26,034 (1,428) |
24,208 (2,175) |
Note: *) average and deviation standard
Control 1: serum
Control 2: serum+HUVEC
Table 3 Effects of Variety of Concentration of Ant nests water fraction on sFlt-1 level in normal and preeclampsia serum
Control Variable 2n |
Preeclampsia serum |
|||
Ant nests concentration (µg/ml) |
||||
31,25 |
62,5 |
125 |
250 |
|
sFlt-1 (pg/ml) |
|
|
|
|
X (SD) 9,92(0,56) |
30,202(0,908) |
30,053(1,474) |
27,219(0,608) |
24,208(2,175) |
Range (9,53-10,33) |
(29,560-30,844) |
(29,010-31,095) |
(26,789-27,649) |
(22,670-25,746) |
A |
B |
B |
BC |
C |
Note: Control 2n: normal serum+ HUVEC
Average followed by different alphabet showed significant difference (ρ<0,05), according to Duncan test.(A, B, C) indicates different effects of concentration on level of sFlt-1
MDA level significantly decreased in range -2,230- (-1,870)μM in preeclampsia-induced HUVEC ATCC CRL 1730 after treatment of ANE on concentration 125 µg/ml that almost reach its level in normal-induced HUVEC ATCC CRL 1730. MDA decreased following increased ant nests water fraction on concentration 125 µg/ml that close to its level in normal-induced HUVEC ATCC CRL 1730 (Table 6 and 7) .
Table 4 Effects of variety of ant nests water fraction concentration and type of serum used level of PlGF
Concentration (µg/ml) |
Serum PlGF (pg/ml) |
|
Normal |
Preeclampsia |
|
Control 1 |
6,235 (0,671)* |
1,725 (0,077) |
Control 2 |
6,357 (0,744) |
1,750 (0,283) |
31,25 |
4,191 (0,742) |
3,242 (0,629) |
62,5 |
7,674 (0,495) |
5,326 (0,454) |
125 |
8,261 (0,622) |
8,345 (0,024) |
250 |
9,822 (0,282) |
7,373 (0,733) |
Note: *) average and deviation standard
Control 1: serum
Control 2: serum+HUVEC
Table 5 Effects of variety of ant nests water fraction concentration and type of serum used level of PlGF
Control Variable2n |
Preeclampsia serum |
|||
Ant nests concentration (µg/ml) |
||||
31,25 |
62,5 |
125 |
250 |
|
PlGF (pg/ml) |
|
|
|
|
X (SD) 6,35(0,744) |
3,242(0,629) |
5,326(0,454) |
8,345(0,025) |
7,372(0,733) |
Range (5,830-6,883) |
(2,797-3,687) |
(5,005-5,647) |
(8,328-8,363) |
(6,854-7,891) |
AB |
A |
B |
C |
C |
Note: Control 2n: normal serum+ HUVEC
Average followed by different alphabet showed significant difference (ρ<0,05), according to Duncan test.(A, B, C) indicates different effects of concentration on level of PlGF
NO level significantly increased in range 0,231-0,267 ppm in preeclampsia-induced HUVEC ATCC CRL 1730 after exposure of ANE on concentration 125 µg/ml approaching condition in normal-induced HUVEC ATCC CRL 1730 (Table 8 and 9). Level of NO was increased following increased concentration approaching normal condition.
Table 6 Effects of variety of ant nests water fraction concentration and type of serum used in level of malondialdehyde (MDA)
Concentration (µg/ml) |
MDA serum (μM) |
|
Normal |
Preeclampsia |
|
Control 1 |
2,028 (0,035)* |
12,549 (1,359) |
Control 2 |
3,333 (0,396) |
13,586 (1,293) |
31,25 |
15,560 (4,313) |
8,320 (0,325) |
62,5 |
6,920 (1,131) |
5,570 (0,764) |
125 |
2,290 (0,396) |
-2,050 (0,255) |
250 |
-6,860 (1,131) |
-7,66 (1,345) |
Note: *) average and deviation standard
Control 1: serum
Control 2: serum+HUVEC
Table 7 Effects of variety of ant nests water fraction concentration and type of serum used in level of malondialdehyde (MDA)
Control Variable 2n |
Serum Preeklamsi |
|||
Ant nests concentration (µg/ml) |
||||
31,25 |
62,5 |
125 |
250 |
|
MDA (μM)
|
|
|
|
|
X (SD) |
8,320(0,325) |
5,570(0,764) |
-2,050(0,255) |
-7,66(1,343) |
3,333(0,396) |
||||
Range (3,053-3,613) |
(8,090-8,550) |
(5,030-6,110) |
(-2,230- -1,870) |
(-8,610- - 6,710) |
A |
B |
C |
D |
E |
Note: Control 2n: normal serum+ HUVEC
Average followed by different alphabet showed significant difference (ρ<0,05), according to Duncan test.(A, B, C) indicates different effects of concentration on level of MDA
Tabel 8 Effects of variety of ant nests water fraction concentration and type of serum used in level of NO
Concentration (µg/ml) |
NO Serum (ppm) |
|
Normal |
Preeclampsia |
|
Control 1 |
0,385 (0,100)* |
0,0635 (0,0615) |
COntrol 2 |
0,408 (0,125) |
0,117 (0,024) |
31,25 |
0,1845 (0,065) |
0,150 (0,052) |
62,5 |
0,264 (0,034) |
0,203 (0,015) |
125 |
0,273 (0,051) |
0,249 (0,025) |
250 |
0,314 (0,066) |
0,269 (0,045) |
Note: *) average and deviation standard
Control 1: serum
Control 2: serum+HUVEC
Table 9 Effects of variety of ant nests water fraction concentration and type of serum used in level of NO
Control Variable 2n |
Preeclampsia serum |
|||
Ant Nests concentration (µg/ml) |
||||
31,25 |
62,5 |
125 |
250 |
|
NO (ppm) |
|
|
|
|
X (SD) 0,408(0,125) |
0,150 (0,052) |
0,203 (0,015) |
0,249 (0,025) |
0,269 (0,045) |
Range (0,319-0,496) |
(0,113-0,187) |
(0,192-0,214) |
(0,231-0,267) |
(0,237-0,301) |
A |
B |
B |
AB |
AB |
Note: Control 2n: normal serum+ HUVEC
Average followed by different alphabet showed significant difference (ρ<0,05), according to Duncan test.(A, B, C) indicates different effects of concentration on level of NO
Discussion
In this study, ANE used is known to contain compounds such as tannin, alkaloid, flavanoid and terpenoid. There is difference of active compounds obtained which might be caused by different isolation method, technique, and phytochemical test sensitivity. There are no previous studies regarding secondary metabolite of ANE. Lack of information causes antioxidant and antiangiogenic mechanism on preeclampsia remains unclear.
Alteration of angiogenesis on blood vessels and extravillous trophoblast invasion occurred in preeclampsia causes absence of decreasing of placental vascular resistance. It negatively affects placental oxygen supply due to disminished blood vessels and ischemic, that promotes trophoblast villous damage.40 Ischemic causes time-dependent imbalance of prooxidant and antioxidant, in which higher free radicals are present in longer time. Free radicals produced will reach one point that exceeds its capacity, known as oxidative stress.41 Non-neutralizing free radicals in preeclamptic patients, generate cell membrane damage, disturbance in cell integrity, endothelial cells lysis, reactivity and increased vascular permeability.42
Alteration of angiogenesis balance in preeclampsia is observed through overproduction of sFlt-1.7,8,44 sFlt-1 is specific protein resulted from response to hypoxia. 43 Placental hypoxia releases free radicals into circulation and can be detected by alteration of NO and MDA level. When hypoxia occurs, placenta releases sFlt-1 expressed before clinical symptoms, hypertension and proteinuria.7,8,44 Increased sFlt-1 was followed by alteration of pro-oxidant and antioxidant balance indicated by increased MDA and decreased NO. It worsens hypoxia in preeclampsia that later causes severe hypoxia.9,10
In preeclamptic patients, increased sFlt-1 causes alteration of other angiogenics, PlGF.7,44 PlGF in preeclampsia onset is known decreased as result of increased sFlt-1.45 sFlt-1 causes alteration of endothelial blood vessels by taking place for PlGF receptor leading to decreased NO phosphorylation synthesis in endothelial cells. It changes bioavaibility of NO produced by endothel and leading to alteration of cardiovascular homeostasis which is clinically found in preeclamptic patients. 12-14 PlGF has physiology pattern in pregnancy which is consistently decreased in normal pregnancy in first and second trimester, peaking at 29 to 32 weeks and its level is found consistently low following gestational age. 11-13,43,45,46 It is thought as a result of increased sFlt-1 level starting from gestational age at 33rd week to last weeks of pregnancy. 43,45
Upregulation of sFlt-1 generate GCM1 degradation that results in decreased PlGF and metal-responsive transcription factor (MTF-1) synthesis.47,48 PlGF is a protein produced by trophoblast, endothelial cells, monosite, and erythroid cells. The presence of sFlt-1 and PlGF was frequently investigated to diagnose preeclampsia in laboratory scale before clinical symptoms.17,18 Both proteins are expressed by placenta as a response of hypoxia. 9,10
Hypoxia induces ROS release such as superoxidation (O2), hydroxyl radical (OH), and hydrogen peroxide. (H2O2). Hypoxia generates overproduction of O2, OH and H2O2 reaching its antioxidant capacity. Alteration of prooxidant and antioxidant balance is found in preeclamptic patients which is caused by increased level of MDA. Increased MDA causes its biological function disrupted, endothelial dysfunction, vasoconstriction, frozen blood process and lipid peroxidation process disrupted, biomolecule oxidative damage and DNA damage. Increased MDA level also worsens oxidative stress caused by low level of cellular NO.16-18,49-51
The result of present study showed ANE has antioxidant property as in line with previous study done by Dharsono and Soeksmanto. 34, 52 Both only used n-hexan and etyl acetate fraction instead of water. Water has higher polarity compared to n-hexan and etyl acetate as solvents. However, studies using ant nests has similarity in its antioxidant activity. Ant nests is proved for its antioxidant activity and other active compounds contained such as alkaloid, flavanoid, tanin and terpenoid. Further studies regarding difference of solvents used are encouraged.
In this study, ant nests were fully boiled in water and it is thought to dissolve antioxidant compounds of ant nests. Ant nests use is considered to dissolve more unidentified water-soluble compounds and they are thought to have better antioxidant activity. Identification of active compounds is crucial in drugs discovery and development. Moreover, interaction between dissolved compounds resulting stronger antioxidant effects might be present, although advanced studies are needed.
Concentration of ant nests water fraction used in this study starting from below 500 µg/ml which starts from 250 µg/ml and its multiple to lower concentration reaching 31,25 µg/ml. Study was continued to measure level of sFlt-1, PlGF, MDA and NO HUVEC ATCC CRL 1730 induced by normal and preeclampsia serum and treated with variety of air ant nests fraction concentration after 24 hours incubation.
Results showed level of sFlt-1 and MDA was decreased in preeclampsia-induced HUVEC ATCC CRL 1730 and its level approached sFlt-1 and MDA level in normal serum-induced HUVEC ATCC CRL 1730 without ant nests water fraction. Moreover, increased level of PlGF and NO in preeclampsia serum-induced HUVEC ATCC CRL 1730 was obtained and its level approached sFlt-1 and MDA level in normal serum-induced HUVEC ATCC CRL 1730 without ant nests water fraction exposure. Data showed significant result statistically for each parameter (sFlt-1, PlGF, MDA dan NO).
Results of ant nests water fraction in normal serum-induced HUVEC ATCC CRL 1730 was weaker than that in preeclampsia-induced HUVEC ATCC CRL 1730. As shown in Table 4.2-4.9, nests water fraction exposure with variety of concentration in normal-induced HUVEC ATCC CRL 1730 was thought increasing level of sFlt-1 and MDA and also decreasing PlGF and NO compared to controls and preeclampsia-induced HUVEC ATCC CRL 1730. It concluded that ant nests water fraction playing different role in normal cells. Ant nests water fraction is considered to have toxic activity in normal cells affecting pathophysiology of normal cells. Although, advanced studies are recommended.
Despite inadequate information in this study, results showed ANE has both antioxidant and antiangiogenic activity in normal and preeclampsia-induced HUVEC ATCC CRL 1730 cell line. It was
described that higher concentration of ant nests water fraction followed by more reduced level of sFlt-1 and MDA, and increased level of PlGF and NO as well. This is in accordance with previous studies by Hamsar dan Mizaton49 and Dharsono et al.51 that suggests that ant nests is widely consumed and believed to prevent diseases in presence of high antioxidant.31-34 It is confirmed that higher concentration of ANE resulted lower occurrence of oxidative stress and hypoxia (Table 4.3-4.6).
The result of present study suggests that ANE contains high antioxidants and antiangiogenic, one of which is flavanoid. Antioxidant in ant nests water fraction plays role as precursor to trap reactive oxygen compounds. It therefore reduces free radicals and maintains cell function including preeclampsia-induced HUVEC ATCC CRL 1730 cell line. Balance of cell function indicated by decreased occurrence of hypoxia directly affecting decreased level of sFlt-1 and MDA, followed by increased level of PlGF and NO. Thus, ant nests water fraction might be used as agent to overcome endothelial dysfunction in preeclampsia. Advanced studies regarding active compounds and optimum concentration of ant nests in embryo are encouraged and it is expected to be tested to preeclamptic patients. Studies measuring ratio average of sFlt-1/PlGF and MDA/NO as a standard in in vivo studies both in experimental animal and humans are also needed.
Conclusion
Level of sFlt-1 and MDA in preeclampsia-induced HUVEC ATCC CRL 1730 cell line was decreased whereas level of PlGF and NO was increased, both after exposure of ant nest water fraction on concentration 31,25 ug/ml.
Acknowledgement
We would like to thank The Integrated Research and Testing Laboratory (LPPT), Gadjah Mada University, Indonesia for the aid in providing research materials.
References
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