The antagonistic activity of Padina arborescens extracts on mPRα

Abstract The current study attempted to evaluate the antagonistic activity of compounds isolated and purified from the marine algae Padina arborescens during cultivation. The compounds were collected on a filter, concentrated on ODS columns and separated by HPLC. Two peaks that showed competitive progesterone binding activity with membrane progesterone receptor α (mPRα) were purified. Their physiological activity was further uncovered by in vitro and in vivo oocyte maturation and ovulation-inducing assays using zebrafish. The compounds inhibited the induction of oocyte maturation and ovulation. Moreover, the results showed that the compounds have antagonistic activity against mPRα. The purified compounds with antagonistic activity against mPRα would be considered as new pharmaceutical candidate. Graphical Abstract


Introduction
Reproductive hormones play vital roles in the reproductive systems of both males and females during pubertal development as well as their growth and sexual characteristics. These reproductive hormones include both steroid and peptide hormones (Manocha et al. 2018). Among them, steroids such as progesterone is key regulators that control reproduction. Progesterone occurs naturally in the body and plays key roles in controlling the meiotic cell division of oocytes and ovulation (Manocha et al. 2018). In addition to the well-established actions of progesterone through the nuclear progesterone receptors (nPRs), a new concept of steroid action through membrane progestin receptors (mPRs) was raised after the discovery of the mPRs (Zhu, Bond, et al. 2003;Zhu, Rice, et al. 2003). mPRs are 7-transmembrane receptors that were renamed as members of the progestin and adipoQ receptor (PAQR) family after a genome-wide phylogenetic study. The first identified mPR, mPRa (also referred to as PAQR7), is the seventh gene in the family and one of the five mPR genes implicated in physiological functions in reproductive tissues (Pang et al. 2013).
A number of researchers have reported that mPRs can be found in a wide range of organisms, and progestin binding activity has been discovered in numerous species, from fish to humans (Tokumoto et al. 2006;Ben-Yehoshua et al. 2007;Smith et al. 2008;Thomas 2008;Tubbs and Thomas 2009;Tokumoto et al. 2012). Various rapid nongenomic actions have been shown to occur through mPRs, such as mammalian sex behavior, breast tumor progression, human T cell attack on fetuses, and the inflammatory response (Chien et al. 2009;Dressing et al. 2012;Xie et al. 2012;Frye et al. 2013Frye et al. , 2014Lu et al. 2015;Vares et al. 2015). Currently, mPRs have been proven to be potential cellular mediators of a variety of progesterone responses. Consequently, scientists are now extremely interested in identifying new mPR ligands. Org OD 02-0 was previously established in early research (Polikarpova et al. 2017;Rezanujjaman et al. 2020;Levina et al. 2021). Thus, the identification of new hormonal compounds from natural resources that can interact with mPR as novel pharmaceutical candidates would be a great breakthrough in reproductive or hormone research.
Marine algae-derived bioactive compounds have been considered potential pharmaceutical agents due to their various activities, including antifungal, antibacterial, antiviral, antioxidant and cytotoxic properties (Carroll et al. 2021). Currently, researchers have been attracted to the identification of a relevant group of water-soluble natural product secondary metabolites known to present notable, potent biological activities (Berlinck et al. 2021). Compounds with pharmacological potential and antimicrobial, cytotoxic, cardioprotective, hepatoprotective and immunostimulatory activities have already been reported in Padina species (Rushdi et al. 2021;Antony et al. 2022Antony et al. , 2021. Previously, we detected mPR-interacting compounds secreted from marine organisms in the seawater of coral reefs in Mauritius (Tokumoto et al. 2017). We were interested in the marine algae Padina, as these organisms secrete mPR-interacting compounds; additionally, the sampling point is the major habitat of these species. In the present study, we concentrated the secreted compounds from Padina arborescens using a fish cultivation filter and followed by compound purification. The mPRa-interacting ability of the purified compounds was then demonstrated.

Results and discussion
Thalli of Padina arborescens were collected at the marine field of University of Tsukuba or Shizuoka University in Shizuoka prefecture and were cultured in aquariums ( Figure  S1). Compounds were collected on polyester filters during the cultivation of Padina arborescens in a glass aquarium and then eluted from the filter with ethanol and concentrated on ODS columns. Then, the compounds were separated by 4 HPLC steps. Among the four fractions from the first HPLC separation, the third and fourth fractions showed binding activity. In this study, the fourth fraction was further purified, and two compounds (peak 1 and peak 2) were separated ( Figure S2). Both peaks showed mPRa binding activity ( Figure S3).
The physiological activity of the purified compounds was further analyzed by in vitro and in vivo assays using zebrafish. In the in vitro assays using oocytes, the activity on mPR was analyzed through the induction of oocyte maturation (Tokumoto et al. 2004). In vivo, agonistic or antagonistic activity of the compounds against mPR and nPR was analyzed by oocyte maturation and ovulation (Tokumoto et al. 2011). The compounds showed no effect on the induction of fish oocyte maturation and ovulation. Thus, we demonstrated antagonistic activity against induction with the natural hormone 17,20b-DHP. Inhibition of oocyte maturation induced by 17,20b-DHP was demonstrated for both compounds in an in vitro assay ( Figure S4). Additionally, in vivo, potent inhibition of ovulation was shown for both compounds. This inhibitory activity was also observed against oocyte maturation after treatment with the compound in peak 1, but not the compound in peak 2. These results suggested that the compounds purified from Padina arborescens have antagonistic activity against mPRa.
Only a small number of ligands for newly identified mPRa have been demonstrated thus far, and they are currently under investigation. The mPR-interacting chemicals as ligands acting on mPRa from marine algae would provide new insight into reproductive biology, pharmacology, physiology and the medical sector.

Conclusion
In this study, two secreted compounds from Padina arborescens were purified. The purified compounds possess mPRa-interacting activity. A physiological assay demonstrated that the compounds act as antagonists against fish oocyte maturation and ovulation. Consequently, the newly purified compounds from Padina arborescens would be the novel pharmaceutical candidates as antagonists of mPRa.

Funding
This work was supported by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan; JSPS KAKENHI grant number 20K06719 (to TT).