Preeclampsia (PE) is a severe pregnancy disorder posing significant health risks to both the mother and the fetus without available preventive measures or treatments. More specifically, the main pathological feature of early-onset PE (EO-PE) is the incomplete transformation of the spiral artery. Meanwhile, aspirin has proven effective for the treatment and prevention of PE. Previous studies indicated a relationship between the phytochemical compound of kopyor coconut water (KCW) and nutritional treatment for PE. Therefore, the objective of this study was to determine the efficacy of KCW as a potential preventive treatment for PE. An experimental laboratory method was used with pre-test and post-test control group designs. The samples comprised 35 pregnant Wistar rats, divided into five groups with seven members each. Rats were then randomized into control and groups exposed to L-NAME, L-NAME and aspirin, L-NAME and KCW 2 ml/200 gBW, as well as L-Name and KCW 2 ml/200 gBW for GD4 to 19. The results showed that after administering L-NAME to induce EO-PE, mean arterial pressure (MAP), proteinuria, placental hypoxia, oxidative stress, and endothelial dysfunction decreased due to KCW nutritional treatment. Specifically, KCW nutrition prevented the uterine spiral artery from expanding and reduced the number of neutrophils. The decreased survival rate caused by L-NAME-induced PE was reversed by providing KCW nutrition. Moreover, results indicated that KCW was a potential alternative for the prevention and treatment of EO-PE even at doses of 2 or 3 ml/200 gBW, offering insights for the community and clinical practitioners in treating PE as a therapeutic option.

artery spiralis uteri, early onset, eNOS, HbF, HIF1α, MAP, L-NAME, preeclampsia, proteinuria

Preeclampsia (PE) is one of the main causes of morbidity and mortality in mothers and fetuses (Bouter and Duvekot 2020). According to reports, 500,000 fetuses and 76,000 pregnant women pass away from PE annually (Poon et al. 2021). This condition is characterized by systolic blood pressure ≥140 mmHg and diastolic blood pressure ≥90 mmHg on at least two measurements taken 4 hours apart in previously normotensive women, which begin 20 weeks into pregnancy (ACOG 2020). PE is classified into two categories, early-onset (EO) and late-onset (LO), which have unique effects on the fetus and neonate. Perinatal mortality increases approximately 10-fold higher in EO and twice in LO (Poon et al. 2021).

Early onset Preeclampsia (EO-PE) is a condition that can endanger the lives of the mother and the fetus (Staff et al. 2022). The onset of the condition is characterized by trophoblast invasion disorders, uterine spiral artery transformation, immune maladaptation, and elevated markers of endothelial dysfunction (Staff 2019). Patients with PE, especially those with EO, have significantly higher levels of HIF-1α in their serum due to placental ischemia and trophoblast hypoxia (Tianthong and Phupong 2021). Moreover, excessive systemic inflammation can increase local oxidative stress responses, leading to decreased antioxidants. Furthermore, the liver and lymphocytes produce alpha-1 microglobulin (A1M), which aids cells in fending off damage from oxidative stress and fetal hemoglobin (HbF) (Kristiansson et al. 2020). Additionally, an imbalance between VEGF and PlGF causes decreased NO and increased eNOS, which in turn causes endothelial damage and vascular dysfunction, ultimately leading to endothelial dysfunction (Pereira et al. 2021). Consequently, endothelial dysfunction leads to increased vascular resistance, heightened glomerular permeability, and other symptoms, including cerebral edema and liver dysfunction, ultimately resulting in PE (Jung et al. 2022).

The management of PE using aspirin has proven to be a cost-effective strategy compared to aspirin prophylaxis for high-risk women. Aspirin, an anti-inflammatory drug, is a non-steroidal molecule that acts as a cyclooxygenase (COX) inhibitor (Shanmugalingam et al. 2020). However, other potential side effects, such as bleeding, are a serious concern (Rolnik et al. 2022). Prevention and treatment interventions for PE are more effective in the early phase before spiral artery remodeling progresses towards worsening effects, leading to hypertension, proteinuria, tissue edema, and multiple systemic organ damage (Lecarpentier et al. 2016). The standard treatment for PE has not been established. Even though, recently, daily low-dose aspirin (150 mg/day) has been reported to reduce the risk of EO-PE (before 34 weeks of pregnancy) if it is initiated before the first trimester of pregnancy. However, aspirin does not reduce the risk of LO-PE (at or after the 34^th week of pregnancy). Data reported that there were approximately 90% of cases of LO-PE in developed countries and 40% to 70% in developing countries (Kalafat et al. 2020). In addition, long-term treatment with aspirin has negative impacts, including bleeding at the last stage of pregnancy (Hastie et al. 2021).

Herbal remedies, such as Extra Virgin Olive Oil (EVOO), which has high levels of monounsaturated fatty acids that can inhibit the angiotensin-converting enzyme (ACE), control blood pressure, and lower urine levels of nitric oxide and 8-isoprostane, are another treatment option for PE (Alcaide-Hidalgo et al. 2020). However, consuming high doses of olive oil may lead to weight gain and nausea in some individuals (Lakshmanan et al. 2020). Previous studies reported that coconut water attenuated blood pressure and mitigated inflammation (Bhagya et al. 2012; Airaodion et al. 2020; Lakshmanan et al. 2020). In addition, it reduces oxidative stress induced by isoproterenol-induced myocardial infarction (Prathapan and Rajamohan 2011).

Several bioactive natural products have been used both as prophylaxis and treatment to prevent or alleviate disorders (Cragg and Pezzuto 2016). The advantage of using natural products is their high tolerability and minimal side effects. The coconut tree, commonly referred to as the “tree of life,” is a tropical plant whose entire part of the plant is beneficial to human life (Rao and Najam 2016). Typically, one coconut has 300 milliliters of water, ranging in pH from 3.5 to 6.1 based on the type, maturity, and climate (Lopez 2023). Young coconut water is rich in minerals and amino acids, and its composition is similar to that of an isotonic fluid, making it ideal for replacing bodily fluids (Yong et al. 2009). L-arginine (Arg), a functional amino acid (AA), is an essential substrate for the production of NO, creatine, polyamines, homoarginine, and agmatine in mammals, including humans, acting as protein building blocks (Wu et al. 2021). Nitric oxide (NO) increases blood flow to tissues, while arginine and its by-products are important for physiology and metabolism (Tuyekar et al. 2021).

Kopyor Coconut Water (KCW) (Cocos nucifera L. var. Kopyor), commonly known as Kelapa Kopyor in Indonesia, is a member of the Cocos nucifera family and a new substitute for PE due to the high arginine content. A previous study reported that KCW consists of vitamins C and E. The prominent amino acids are glutamic acid, alanine, and arginine. In addition, KCW contains high levels of Mn, Zn, and Mg but low levels of vitamins B1, B2, and C, as well as sucrose (Santoso et al. 1996).

A previous study revealed that tender coconut water (TCW) could lower blood pressure in hypertensive rats by reducing oxidative stress (Bhagya et al. 2012). In addition, another previous study also reported that TCW affects free radicals due to mercury exposure (Zulaikhah and Wibowo 2018). However, the mechanism behind anti-inflammatory properties has not been determined (Radenahmad et al. 2012). TCW regulates the expression of inflammatory mediators and NO production mediated by cytokines, Nos2 mRNA, and iNOS protein expression in the primary hepatocytes of rats (Lakshmanan et al. 2020). This study proposed that KCW would prevent the primary cause of EO-PE, uterine spiral artery remodeling, due to its anti-inflammatory properties.

According to the results, the administration of 75 mg/kg BW/day of L-nitro-arginine methyl ester inhibited NO synthesis in the endothelium, which mimics the effects of PE and functions in vascular relaxation, followed by the failure of syncytiotrophoblasts to transform from a proliferative epithelial to an invasive endothelial subtype. This resulted in the spiral artery’s imperfect remodeling, which is thought to be the cause of EO-PE and is characterized by a narrowing diameter. Failure causes the fetal placenta to become hypoxic, which is indicated by elevated HIF1 levels in placental tissue. Subsequently, placental hypoxia led to increased lipid peroxidation in L-nitro-arginine methyl ester-administered rats, causing elevated free radical production.

KCW is a natural plant product with antioxidant properties in several experimental models and protects the liver from chemical toxin injury (Bhagya et al. 2012). Previous studies reported that oral kopyor administration reduced oxidative stress caused by isoproterenol myocardial infarction (Prathapan and Rajamohan 2011) and decreased inflammation (Rao and Najam 2016) in rats. However, the mechanism of the vasoconstriction effect is unknown. This study found that KCW activates NO synthesis through eNOS levels and simultaneously affects USAD.

The administration of KCW, both at 2 ml/200 g BW and 3 ml/200 g BW, prevented vasoconstriction in the uterine spiral artery, reduced placental hypoxia, and lowered serum HbF levels and lipid peroxidation. In addition, KCW prevented endothelial dysfunction by reducing eNOS levels, decreasing MAP and urine protein levels, and preventing a decrease in the number of live fetuses induced by L-nitro-arginine methyl ester. An important pathophysiologic theory pertaining to EO-PE is the partial transformation of the spiral artery, which is vital for the placenta and fetus’s nutrition supply (Staff 2019).

The results showed that there was a significant difference between the PE+LDA group, PE 2 ml/200 g BW, and PE 3 ml/200 g BW compared to only the PE group. This was consistent with a study that was conducted by Lalita (2019). The findings demonstrated a significant effect on USAD when 60 Mus Musculus were given anti-Qa at a dose of 10 ng from the first to the fourth day of pregnancy. KCW’s high arginine content can promote the synthesis of NO. Meanwhile, it has been established that NO, an endothelial vasodilator, is flawed in PE (Sari 2019).

Hypoxia-inducible factor 1-alpha, also known as HIF1α, is a major transcriptional regulator of the adaptive response to hypoxia (Tal 2012). Based on the results, KCW mitigated hypoxia in the placenta of PE rats. There was a significant difference (p < 0.05) between the PC and PE groups. On the other hand, there was no significant difference (p > 0.05) between the PE+2 ml/200 g BW and 3 ml/200 g BW groups compared to the PE group. Likewise, a study conducted at the Department of Obstetrics and Gynecology, Safdarjung Hospital, New Delhi, India, on ninety women with EO-PE discovered a significant difference in HIF1α expression between the PC and PE groups (Rath et al. 2016). The high content of arginine in coconut water is a precursor for the powerful endothelial vasodilator, namely nitric oxide (NO), which plays a role in increasing blood vessel diameter. Moreover, L-arginine decreases malondialdehyde levels and HIF-1α expression to effectively repair oxidative damage caused by hypoxia (Al-Rasheed et al. 2016).

Hemoglobin fetus (HbF) is the primary oxygen-carrying protein in the human fetus (Perutz et al. 1987). The results showed that KCW reduced oxidative stress levels in serum HbF. On the fourth day of pregnancy, there were no significant differences in any of the groups. However, on day 20, the HbF levels in the PC and PE groups differed significantly. Additionally, compared to PC, there were significant differences between the PC+LDA, PC 2 ml/200 g BW, and PC 3 ml/200 g BW groups. The findings were in line with a study that found that 86 pregnant patients with PE who visited the obstetric unit at St. George Hospital had noticeably higher serum HbF concentrations and went on to develop PE (Anderson et al. 2016). Increased Hb-F leakage into the mother›s blood may act as a mediator for systemic effects. Moreover, HbF can also raise blood pressure and improve blood vessel contractility.

Endothelial nitric oxide synthase is an enzyme that regulates the availability of NO (Gambardalle et al. 2020). According to the results, KCW mitigated endothelial dysfunction. On the fourth day of pregnancy, there were no significant differences found in any of the groups. However, the HbF levels of the PC and PE groups differed significantly on day 20. In comparison to PE, significant differences were also seen between the PE+LDA and PE 3 ml/200 g BW groups. The findings are in line with a study that found that, compared to 143 pregnant women without PE, 63 pregnant women with PE at Bharati Hospital and Gupte Hospital in Pune, Maharashtra, India, had comparatively lower levels of eNOS (Dsouza et al. 2016). In general, eNOS is a crucial mediator for cardiovascular homeostasis, controlling blood vessel diameter and preserving normal levels of cell division and apoptosis. (Heiss et al. 2015). As previously reported, 48 male Sprague-Dawley rats given oral l-arginine supplementation (100 mg/kg/day) for 12 weeks had reduced eNOS gene expression in the abdominal aorta (Adejare et al. 2020). This suggests that the high arginine content in KCW may increase eNOS levels. It might be due to arginine serving as a substrate for eNOS to produce nitric oxide, which in turn plays a crucial role in regulating vascular tone and blood pressure through vasodilation (Elms et al. 2013).

Mean arterial pressure and proteinuria are the primary indicators for diagnosing PE. Results revealed that the administration of KCW reduced MAP and proteinuria levels. There was no significant difference in MAP or proteinuria on the fourth day of pregnancy across any group. However, significant differences between the PE and PC groups as well as the PE, KCW 2 ml/200 g BW, and KCW 3 ml/200 g BW groups were observed on days 13 and 19 (Fig. 6a). The results were consistent with a study conducted by Bhagya et al. (2012), in which six male Sprague-Dawley rats were exposed to 4 mL/100 g BW high-variety TCW for 42 days, then reduced blood pressure from (145±2) mmHg to (126±3) mmHg. It might be due to the high content of minerals, amino acids, and vitamins in KCW. Those minerals help to counteract the effects of sodium and regulate fluid balance in the body (Zarean and Tarjan 2017). Moreover, it plays a role in relaxing blood vessels and modulating blood pressure (Xie et al. 2020) by vasodilating and attenuating blood pressure (Li et al. 2022). In addition, an antioxidant may help to improve endothelial function and reduce oxidative stress (Benachi et al. 2020).

Fetal death or distress is another effect of EO-PE, and the number of live fetuses is affected by KCW administration but not placental or fetal body weight. This study may be considered the first report of KCW effects on the number of viable fetuses in rats suffering from PE. The total number of alive and dead offspring at birth is the number of normal fetuses, based on litter size (Eisen 1989). According to Inglis (1980), mice typically have litter sizes of eight to eleven, but Smith and Mangkoewidjojo (1988) reported that litter sizes could reach as high as fifteen. The age of the parents, the time of birth, the food, and the surroundings all affect the number of children (Rosenfeld et al. 2003). The quantity and quality of food provided to the parents, the time of mating, the number of eggs laid, and the degree of embryo mortality are among the environmental factors that have a significant impact on the number of fetuses in a litter (Otto et al. 2015). The number of live fetuses has a strong influence on placental and fetal body weight, although it is not significant.

The results of this study showed that pregnancy hypertension, L-nitro-arginine methyl ester-induced proteinuria, and adverse pregnancy outcomes like placental weight and fetal growth restriction were all exacerbated by uterine spiral artery narrowing. Followed by the uterine spiral artery constriction, and the attenuation of live pup numbers might be due to placental hypoxia. In addition, results showed the effects of KCW on pregnancy outcomes in L-nitro-arginine methyl ester-induced EO-PE rats. The increased survival ratio was enhanced by KCW nutritional treatment but did not affect placental weight in EO-PE rats for either of the two doses or aspirin treatment. The findings indicate that KCW significantly affects MAP, proteinuria, lipid oxidation, and autophagy, among other PE symptoms. However, placental diameter in EO-PE was not observed. It was established that lesions in the villous and vascular placenta and aberrant trophoblast invasion were closely related to EO-PE. Histopathological characteristics of the placenta of rats given L-nitroarginine methyl ester to induce EO-PE need to be noted in order to further validate the effects of KCW.

In conclusion, KCW improved uterine spiral artery remodeling, prevented placental hypoxia, and raised levels of the antioxidant HbF in plasma in PE Wistar rats, all of which prevented endothelial dysfunction. KCW stimulated the HIF1α and eNOS pathways, which led to a decrease in HbF and eNOS levels. Additionally, this study showed that in rats induced with L-nitroarginine methyl ester, KCW nutritional treatment could improve the preventive and therapeutic effects of EO-PE. The findings provided a novel strategy for the management and avoidance of PE in people.

Conceptualization: F.F, S.S, D.I, S.S, I.N, V.W; Validation: D.I, S.S, S.S, F.F; Methodology: V.W, D.I, F.F; Investigation: S.S, D.I, S.S; Writing original draft preparation: F.F, D.I; Data Curation: D.I, V.W, F.F; Writing-Review and Editing: F.F, S.S, D.I, S.S, I.N, V.W; Visualization: D.I, V.W, F.F; Supervision: S.S, S.S, I.N; Project Administration: D.I, F.F; Funding Acquisition: F.F. All authors have read and agreed to the published version of the manuscript.

There is no financial support. This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

The Research Ethics Committee approved the protocol of this research study, Faculty of Medicine, Universitas Sebelas Maret (UNS), with the 301/UNS/27.06.9.1/TU.00/2022 number and 01 November 2022.

The corresponding author can provide the datasets created and/or analyzed during this study upon request.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.