Effect of Water Clover (Marsilea crenata) Leaf Extract on Estrogen Receptors-β in Skin Aging

Received: November, 1, 2020 Revised: December, 13, 2020 Available online: February, 2021 Extrinsic and intrinsic factors influence skin aging. Hormonal changes, especially estrogen, significantly affect intrinsic skin aging. Decreased circulating estrogen levels reduce skin collagen content and skin elasticity. Isoflavones in Marsilea crenata (MC) leaf are active substances containing compounds that mimic estrogen. This study aims to analyze MC leaf extract against the estrogen receptor (ER)-β. The sample for this research was female Wistar Rats (Rattus norwegicus). All of them were 12 months old, with their weight was between 350 to 550 grams divided into five groups. P1 P2 and P3 were grouping with MC leaf extract administration in sequential doses 20 mg/kg BW, 30 mg/kg BW, and 40 mg/kg BW. At the same time, P4 was a positive control group, and P5 was a negative control group. The independent variable was M.crenata leaf extract. The dependent variables were (ER)-β expression and dermal thickness. The data analysis utilized the one-way analysis of variance (ANOVA) showed significant differences (p< 0.05). Each group showed significant results, and group P2 showed the highest expression of (ER)-β and dermal thickness. The result showed that there were significant correlations between both variables (P<0.05). This research has proved that water clover extract could become an alternative treatment in the future for skin aging. However, further research should find a proper dose for human consumption.

Estrogen is useful in treating aging skin after six months of treatment in premenopausal women with skin aging symptoms. It improves skin elasticity and wrinkle depth is (Liu et al., 2019). Phytoestrogens bind to REα and REβ -more bound to REβ. After binding with ligands, these receptors can move from the cytoplasm to the nucleus, bind and influence the transcription control regions of small DNA or RNA, and express specific genes. Furthermore, steroids can bind to cell surface receptors, promote the formation of cytoplasmic cyclic nucleotides and related protein kinases, which in turn, through transcription factors control the expression of target genes. Therefore, phytoestrogens can influence estrogen-regulated https://doi.org/10.33086/JHS.V14.I1.1811 Winawati Eka Putri -Effect of Water Clover (Marsilea crenata) Leaf Extract on Estrogen Receptors-β in Skin Aging processes. It affects the induction of sex hormone binding globulin (SHBG) and aromatase inhibition. (Sirotkin and Harrath, 2014).
Marsilea crenata (MC) or water clover is a group of salviniales living wild in aquatic environments such as ponds, rice fields, lakes, and swamps. There are isoflavones, a part of flavonoids, in MC leaves.
Isoflavones are active substances that contain compounds that mimic estrogen. It can activate ER in mammals, so they are often called phytoestrogen isoflavones (Titisari et al., 2016). Phytoestrogens are non-steroidal organic phytochemicals. There are several types in the phytoestrogens class: lignans, stilbene, coumestans, coumarin, dihydrochalcone, triterpenoids, and flavones. In humans, phytoestrogen activity is similar to estrogen. Phytoestrogen potential is estimated to be lower than 17-β-estradiol. The action mechanism of phytoestrogens is similar to structural 17-β-estradiol. This component binds to both ER-α and ER-β (Kapuscinska, Nowak, and Mickiewicz, 2015). The background of this research because there has not been a similar study regarding MC leaf extract's effect on ER-β on skin aging. This study aims to determine MC leaf extract's effects for ER-beta on skin aging so that the research results will contribute positively to anti-aging dermatology.

RESULTS
In this study, we treated post ovariectomy female Wistar rats with MC leaf extract with three different doses, group P1 at a dose of 20 mg/kg BW, and P2 at a 30 mg/kg BW dose, and P3 at a dose of 40 mg/kg BW. Another group, P4, was given estradiol tablets 30 µg / 100 g BW as a positive control, while the last group, P0, was not given any treatment. Several rats died during the treatment in the treatment group with MC leaf extract (1 group P1, three groups P3), one rat in the treatment group with estradiol tablets, and three rats in the control group : group 0 Without treatment P2 showed the highest ER-β expression and dermal thickness in the three treatment groups with MC leaf extract. ER-β expression in the P3 group was not as high as the P2 group but was still higher than the P1 group. In contrast, the dermal thickness in the P1 group was not as high as the P2 but was still higher than the P3 group. ER-β expression and dermal thickness in the P4 group were higher than the P1 and P3 groups but lower than the P2 group. The P0 groupwithout any treatmentshowed the lowest ER-β expression and dermal thickness than other treatment groups (P1, P2, P3, and P4). The results showed that MC leaf extract affected ER-β and dermal thickness at the end of the study. There were significant differences in the ER-β expression and dermal thickness between groups with analysis using One-way ANOVA. The difference in mean ER-β expression and dermal thickness was significant when the p-value < 0,05. From the test results, the p-value was 0.000, so there was a significant difference in ER-β expression and dermal thickness between groups. Pearson correlation test analyzed a correlation between ER-β expression and dermal thickness of Wistar rats after ovariectomy.
The results of this test showed that Sig. (2-tailed) between dermal thickness and ER-β was 0.000 < 0,05, which means there was a significant correlation between both variables.

DISCUSSION
This study aims to prove the effect of MC extract on increasing ER-β expression on skin aging. In this study, the average ER-β expression and dermal thickness increased after being treated with MC leaf extract and estradiol tablets. Studies on MC leaf extract in skin aging are still rare; however, there were studies on phytoestrogens from other sources. This study's result is in line with research conducted by Laswati et al. (2016), which uses phytoestrogens from tomatoes. The ER-β of post ovariectomy rats without phytoestrogens was lower than with phytoestrogens (Laswati et al., 2016). One of the elements in M.crenata is an isoflavone. Isoflavone has a chemical structure similar to 17 -β estradiolisoflavone can bind to estrogen receptors. Isoflavones act as agonists of estrogen receptors, but isoflavones have less activity than 17 -β estradiol. However, the endogenous estrogen levels in the body influence isoflavone's effect (Pilsakova et al., 2010).
A study done by Pilkasova et al. (2010) reported that isoflavones could inhibit the activity of 5αreductase. 5α-reductase catalyzed the conversion of testosterone to 5α-dihydrotestosterone and aromatase P450. Isoflavones mediated the conversion from testosterone to estradiol. A low concentration of isoflavones inhibits aromatase activity. High isoflavone levels increase this enzyme activity (Almstrup et al., 2002). Isoflavones bind to the sex hormone binding globulin (SHBG) and stimulate its synthesis (Berrino et al. 2001). Alterations in SHBG concentration may yield changes in circulating steroid hormones (Pilsakova et al., 2010).
In another study by Mahmoud et al. (2015) reported that genistein, a component of phytoestrogens, can increase ER-β expression as the dose of genistein increases through the mechanism of phosphorylation, nuclear translocation, and ER-β transcription (Mahmoud et al., 2015). In this study, the P3 group showed ER-β expression not higher than the P2 group but higher than the P1 group. The ER-β expression in the P4 group (estradiol tablets) was higher than the P1 and P3 groups. These results align with the theory that states that phytoestrogen activity is lower than 17-β-estradiol (Pilsakova, Riecansky, and Jagla, 2010  Apart from ER-β expression, this study also examined dermal thickness. The skin is a target organ for estrogen receptor beta hormone so that when there is a decrease in estrogen with aging, it will affect the skin. Manifestations of decreased estrogen in the skin include thinning of the skin, reduced collagen, dry and thin skin, and decreased skin vascularity (Liu et al., 2019;Carneiro et al., 2020). The skin aging mechanism occurs due to an increase in free radicals / ROS, which causes activation of cytokine and growth factor receptors on the surface of keratinocytes and dermal cells and initiates downstream signal transduction pathways (Chung, Cho, and Kang, 2004;Alam and Havey, 2010). Several signaling pathways can further increase inflammatory cytokines (IL-1, IL-6, TNF-α) and collagen-degrading enzymes (MMP) (Chung, Cho, and Kang, 2004;Baumann, Saghari, and Weisberg, 2009). An increase in MMP causes a decrease in the dermis quality, including a decrease in dermal thickness due to a decrease in the amount of collagen (Kavitha and Thampan, 2008;Baumann, Saghari, and Weisberg, 2009;Mizukoshi et al., 2015). In this study, there was an increase in dermal thickness in the treatment group than in the control group that was not treated either with MC leaf extract or estradiol tablets. This result is similar to studies by Akyun, Fajariyah, and Mahriani (2019), which uses phytoestrogens in the form of black soybean ethanol extract. A study conducted by Akyun, Fajariyah, and Mahriani (2019) found that this plant extract can increase dermal thickness at a dose of 0.31 g / ml/day rather than the higher dose of 0.63 g /ml/day. The conclusion was 0.31 g / ml/day as an optimal dose (Akyun, Fajariyah, and Mahriani, 2019). Isoflavones can reduce wrinkling and skin thinning through collagen synthesis and decrease collagen degradation (Liu et al., 2019). Another study by Moraes et al. (2009) showed an increase in dermal thickness with isoflavones still lower than the topical estrogen observed for six months.

CONCLUSIONS
Administration of water clover (M.crenata) leaf extract for four weeks increases the expression of β estrogen receptors and the dermal thickness in the Wistar post ovariectomy. MC leaf extract administration at a 30 mg/kg BW dose is optimal for improving skin aging more considerably than the 40 mg/kg BW/day dose. Finally, we can conclude that phytoestrogens in water clover have positive effects as estrogen replacement therapy.