INTRODUCTION:

During pregnancy, some common infections can cause serious illness to the mother and their baby. A study has been shown that 15% of pregnancies undergo a miscarriage because of infection¹. Depending on the data source, 10–25% of patients undergo a miscarriage in developed countries². Antiphospholipid antibody syndrome, hypertension, and overweight maternal are maternal comorbidities that increase the risk of miscarriage³.

Expectant management, medical, and surgical are types of treatment for women without comorbid conditions. These treatments have been reported to be effective and safe to decrease miscarriage. Although many pharmaceutical drugs are available, the potential of drugs to cause harm, exemplified by thalidomide in 1958 cannot be ignored. A study reported that several pharmaceutical drugs are not recommended for pregnant women. To overcome this problem, herbal medicine can be an alternative as a potential drug for pregnant women both in developing and developed countries. The advantages of herbal medicine are relative safety with minimal side effects⁴. Additionally, herbal medicine can be used to stimulate immunity and reduce the cost of therapy in pregnant women⁵. Apium graveolens or celery has a potential candidate and has several active compounds, such as flavonoids and apigenin. Several studies demonstrated that A. graveolens extracts have function antioxidant⁶, hepatoprotective⁷, and anti-inflammatory activities⁸.

Progesterone-induced blocking factor (PIBF) has a function in the maintenance of human pregnancy and the progesterone-dependent immunomodulation by the mother. Additionally, progesterone plays a crucial role in regulating the maternal immune response and preventing rejection of the fetal semi-allograft, which is mediated through PIBF⁹. Lymphocytes are expressed progesterone receptors that stimulate PIBF expression during pregnancy¹⁰. Therefore, PIBF is crucial to support and maintenance healthy pregnancies. Recently, PIBF has been found in premature pregnancy termination and preterm births¹¹. Besides, the role of Th2 cytokine also plays a critical role in maintaining pregnancy. It is indicated by impairment of Th1/Th2 ratio in women with complications during pregnancy¹².

Understanding the roles of A. graveolens in PIBF level during pregnancy may help in pregnancy management to prevent inflammation and infection. Therefore, this study’s aim was to investigate the effect of A. graveolens extract on PIBF level on mice models and pregnant women.

MATERIALS AND METHOD:

Study design:

This study was approved by the Development Planning Board Bappeda (Research Division, No: 070/0816/VIII/2020). Preclinical research was true experimental research using 32 pregnant mice, divided into four groups randomly. The 1st group was the control group (P1). While, P2, P3, and P4 groups were pregnant mice given with 1mg/g, 2mg/g, and 4mg/g of Apium graveolens extract orally for 14 days. The inclusion criteria for the mice experiment were pregnant mice, aged 3 months, and had body weights around 20-25grams (n= 40). The exclusions criteria were illness or physical injury to mice during treatment. Mice were injected with 5 IU pregnant mare serum gonadotropin (PMSG), then injected with 5 IU human chorionic gonadotropin (hCG) after 48 hours. The female and male mice were placed in one cage. Then, female mice were observed to have a vaginal plug as an indicator of pregnancy.

Clinical research was completely randomized design. Thirty pregnant women (6 weeks-12 weeks of gestation) at Mrs. Sri Lumintu Clinic, Surakarta, Indonesia divided into two groups. Treatment groups were given A. graveolens capsule 534mg/50kg BW, Fe, and calcium, while the control group was given placebo, Fe, and calcium orally for 14 days. The inclusion criteria of the human experiment were pregnant women (n=30), aged 21-35 years old, and all trimester gestation, while the exclusion criteria were pregnant women with comorbidities such as hypertension, diabetes mellitus, and others.

Preparation of Apium graveolens:

Fresh celery (A. graveolens) was purchased from Ledoksari Market, Surakarta, Indonesia. The celery herbs were rinsed with water and dried under the sun for 24 hours then incubated for 4 hours at 45°C.

Determination of Apium graveolens or celery dose:

The effective dose of A. graveolens was determined using pregnant mice experiment before A. graveolens supplementation was given in pregnant women. There were four groups in this study (P1 = negative control; P2 = pregnant mice injected with 1mg/g BW dose of A. graveolens extract; P3 = pregnant mice injected with 2 mg/g BW dose of A. graveolens extract; P4 = pregnant mice injected with 4mg/g BW dose of A. graveolens extract) during 14 days. E. coli (5 cc) were injected in pregnant mice on day 2 and day 9 to decrease their immune system.

Sample collection:

A total of 3ml blood sample was collected from pregnant women (6 weeks-12 weeks of gestation) at Mrs. Sri Lumintu Clinic, Surakarta, Indonesia. Moreover, 1mL of mice blood was obtained from pregnant mice. Blood samples were placed into a 5 mL sterile centrifuge tube and immediately centrifuged at 3000rpm, 4°C for 15 minutes. The supernatant was aspirated and placed into 1.5mL centrifuge tubes. All samples were labeled and stored at -80°C until further analysis.

Determination of PIBF level:

The level of PIBF in serum was measured by ELISA. The kits of human and mice PIBF (code E3714Hu) produced by BT Lab, China were used according to the manufacturer’s instructions. Plates were developed using tetramethylbenzidine substrate (Thermo Fisher Scientific, USA), stopped using 2 N H₂SO₄, and read at a dual-wavelength of 450 nm in a microplate reader (BIO-RAD, USA) to obtain the optical density (OD) and concentration values.

Statistical analysis:

All statistical calculations were performed using SPSS statistical software. The data distribution was assessed by the independent samples test. P-value <0.05 was considered statistically significant.

RESULT:

Characteristics of pregnant women:

The women’s demographic data are presented in Table 1. The median maternal and gestational ages were 26 years and 12 weeks, respectively of 30 women, 17 (56.7%) were primigravida (first time pregnant) and 13 (43.3%) were multigravida (have many pregnancies). The median PlBF was 266.13 (95% of confidence interval) and there were 10% of women have an abortion history.

Table 1. Sample characteristics of pregnant women.

Characteristics	Value
Maternal ages (yr)	26±13.5
Gravida (%):
1	56.7
2	43.3
Gestational age (week)	12±12.4
Parity (number of live births) (%)	100
History of abortion (%)	10
PIBF	266.13

PIBF level in mice:

We first examined the effective dose of A. graveolens extracts against the PIBF level in pregnant mice. The mean values of the PIBF level in each group of pregnant mice are presented in Figure 1.

Figure 1. The difference of Mice mean PIBF level in all groups.

As shown in figure 1, the third group given by 2mg/g BW (P3) dose of A. graveolens extract significantly increased PIBF level compared to all groups (P1, P2, and P4). The statistical significance of differences was analyzed using one-way ANOVA by SPSS. The post hoc test was conducted to determine the differences among all groups. The PIBF level in mice is presented in Table 2.

Table 2. The mean differences of PIBF level between-group (mice).

Group	Group
Group	P1	P2	P3	P4
P1	-	0.260	0.000^*	0.773
P2	0.260	-	0.002^*	0.393
P3	0.000^*	0.002^*	-	0.000^*
P4	0.773	0.393	0.000^*

Note: P1 = mice were given distilled water. P2, P3, and P4 groups were injected with Apium graveolens extract for 14 days. Each group consisted of 8 pregnant mice.

We observed a significant difference in PIBF level between P3 and P1, P3 and P2, P3 and P4 based on the post hoc test. This finding suggested that the effective dose of A. graveolens was 2mg/g BW of pregnant mice. Therefore, this dose was used for supplementation of pregnant women in this study.

PIBF level in pregnant women:

To understand the profile of PIBF in pregnant women, we measured PIBF levels in the serum of pregnant women using the ELISA method as presented in Figure 2.

Figure 2. The differences of mean PIBF level between-group (human).

Note: Control group was treated with pregnant placebo, Fe, and Calcium for 14 days orally. Treatment group was supplemented with A. graveolens L. capsule 534 mg/50 kg BW, Fe, and calcium for 14 days orally. Each group consisted of 15 pregnant women.

The treatment group significantly increased PIBF level compared to the control group. This finding revealed that A. graveolens supplementation induced PIBF level in pregnant women. A study reported that PIBF concentrations are found in the serum of pregnant women and have a potential biomarker to support pregnancy outcomes¹³. PIBF level is found to be increased in pregnant women and followed by advancing trimesters and increasing gestational age¹⁴. This study is in accordance with previous studies, suggesting a crucial role of PIBF in pregnancy. PIBF also regulates cytokine secretion and cytotoxic cell activity^15,16. Besides, PIBF has function as a molecule with inhibitory effects on cell-mediated immune reactions¹⁷.

DISCUSSION:

Pregnancy is a complex immunological condition. PIBF is found in lymphocytes during pregnancy and plays a crucial role in supporting and maintaining pregnancy in humans and mice. It is helpful in feto-maternal communication by mediating the immunological action of progesterone¹⁸. PIBF has several functions, such as regulating the cell cycle. Impairment of PIBF isoforms reduces the immune regulatory function, resulting in miscarriage¹⁹. A study reported decreased implantation, and increased resorption rate of pregnant mice occur in pregnant mice during the peri-implantation period after being treated with anti-PIBF²⁰. In addition, PIBF may exacerbate placental inflammation. This condition leads to the release of reactive oxygen species (ROS) and proinflammatory cytokines such as TNF-α and IL-6 and the activation of cytotoxic T cells²¹. Besides, PIBF contributes to the altered maternal immune system by decreasing NK activity and regulating Th2-type cytokine production. A Th2-dominant cytokine balance regulates normal pregnancy. PIBF protects pregnancy in mice by controlling NK activity¹⁹. Decidual NK cells are functionally different from their circulating counterparts. Additionally, decidual NK cells provide a favourable environment for embryo development, implantation, and placentation in normal pregnancy, but they are equipped with cytotoxic molecules to fight intrauterine infection²². Therefore, Apium graveolens supplementation is needed to induce robust immune responses against pathogens in pregnant women.

A. graveolens (celery) belongs to the Apiaceae family and is widely grown in Europe, Africa's tropical and subtropical regions, and Asia²³. Celery has several bioactive compounds, such apigenin, coumarin glycosides, flavonoids, limonene, selinene, and vitamins A and C. The numerous bioactive compounds are a major reason for celery being a potential candidate for traditional medicine. Several bioactive celery compounds with chemical structures are shown in Figure 3^19,24.

Figure 3: Chemical structure of A. graveolens flavonoid compounds.

The most beneficial effect of medicinal herbs is due to numerous bioactive compounds¹⁹. According to in vivo and in vitro studies, A. graveolens can be used to treat cardiovascular disorders, rheumatic disorders, and urinary tract obstruction for a long time²⁵. Flavonoids and other phenolic compounds are abundant in plants. They have numerous biological activities, including antioxidant properties with minimal side effects. Moreover, A. graveolens plays an important role in the immune system²⁶, reproductive system, hormonal disorders, liver disorders, kidney disease, neurological and mental disorders²⁷.

In preclinical research, we investigated the effective dose of A. graveolens in pregnant mice towards PIBF level. It is recognized as a potential therapeutic agent or marker in lymphocyte immunotherapy. We found that at 2mg/g BW dose of A. graveolens extract was the effective dose to increase PIBF level. A. graveolens contains apigenin, kaempferol, limonene, and trans-anethole that are useful to protect the female reproductive system from dangerous properties and teratogenic effects of radiation²⁸. Progesterone levels in serum increase significantly in groups given 50 and 100 mg/kg of A. graveolens extract compared to control in female rats²⁹. Properly balanced doses determine the potential effects of individual herbs. Proper doses have positive and protective effects, but the higher doses and long-term exposure may harm normal reproductive functions. A. graveolens extract also increases progesterone production at a 75μg/mL dose, while 37.5 μg/mL and 300μg/mL have no significant effect³⁰. Therefore, we used our proper dose (2mg/g BW) to continue our clinical research in pregnant women as a supplement. Under normal pregnancy conditions, the concentration of PIBF steadily rises during pregnancy. A study reported that oxidative stress effect PIBF binding to PIBF-receptors in lymphocytes during pregnancy. Over the last 50 years, oxidative stress biomarkers have been found in the blood of preeclampsia patients³¹. Therefore, antioxidant supplementation may reduce the ROS effect and repair PIBF binding in normal pregnancy to prevent miscarriage.

In our clinical research, A. graveolens supplementation in pregnant women significantly increased PIBF levels compared to the control group. In a previous study, the protective effect of celery was attributed to the antioxidant properties of celery¹⁹. Flavonoids are abundant in celery with antioxidant properties to increase the level of sex hormones and the ovaries' activity and remove free radicals³². The findings of Hussein et al. study demonstrated that phytoestrogen on celery supplementation improved the number of successful conception and fertilization due to its chemical structure that might affect estrogen and progesterone levels in serum³³. Progesterone leads the local expression of immunomodulatory molecules such as PIBF and others³⁴. Therefore, it has an essential role to establish and maintaining pregnancy³⁵. Besides, previous studies have shown the immunological effects of progesterone, such as cytokine balance and NK activity mediated by PIBF¹⁹. Progesterone and PIBF levels increase in the later trimesters, emphasizing the importance of progesterone and PIBF in healthy pregnancies³⁶. A study reported that aqueous celery extracts at a dose of 100 mg/kg increased the average percentage childbirth ratio³⁷. To our knowledge, our study is the first study on serum PIBF levels related to celery supplementation in pregnant women.

On the other hand, Apigenin, a polyphenolic flavonoid, is found in celery³⁸. Apigenin can suppress both pro-inflammatory and cytotoxic T cell responses³⁹. Apigenin can also cross the blood-brain barrier (BBB) and has been shown to exert anti-inflammatory effects on BV-2 and primary microglial cells through inhibition of p38 and JNK. In addition, flavonoids exert an anti-inflammatory effect via interfering with the development of inflammatory mediators such as IL-6, TNF-α, and IL-1β in several cell lines through the MAPK signaling pathway. Furthermore, Apigenin prevents apoptosis by protecting the cell against inflammatory stresses^38,40. In this case, we show that flavonoids increase the Th2 cell by inducing the PIBF level. Furthermore, PIBF mediates the immunomodulatory effects of progesterone in cells with a high proliferation rate⁴¹. PIBF plays a crucial role in the maternal immune system during healthy pregnancies²⁷. In addition, PIBF induces asymmetric antibodies and inhibits NK cell degradation, and plays an essential immune regulator for successful pregnancy^42,43,44.

CONCLUSION:

A higher PIBF level of serum was found in pregnant women with A. graveolens supplementation after day 14. A. graveolens in PIBF level during pregnancy can be recognized in the pregnancy to prevent inflammation and infection.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest regarding the publication of this article.

ACKNOWLEDGMENTS:

This work was supported by grant research from the Ministry of Research and Technology, Republic of Indonesia (16/FI/PKS-KCOVID-19.A/VI/2020).

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Received on 22.09.2021 Modified on 18.12.2021

Research J. Pharm. and Tech 2022; 15(10):4463-4468.

DOI: 10.52711/0974-360X.2022.00748