Lepidiumuridine A: A New Natural Uridine Derivative as a Phytoestrogen Isolated from the Seeds of Lepidium apetalum Willd.

There has been great interest in phytoestrogens, which are polyhydric compounds that are derived from plants and have a structure similar to that of the mammalian steroid hormone 17β-estradiol. The present study examined the estrogenic effects of a new natural uridine derivative, lepidiumuridine A (LA), that was isolated from the seeds of Lepidium apetalum. The structure was clarified and determined via analysis of extensive spectroscopic data interpretation. The activity of LA was investigated by measuring the levels of estradiol (E2), luteinizing hormone (LH), follicle stimulating hormone (FSH), and the uterus growth in mice. The proliferation experiment of MCF-7 breast cancer cells was also conducted. Western blot, in-cell western, and antagonist assays with methyl piperidino-pyrazole (MPP) were used for exploring the mechanism of the effects of LA. The results showed that LA elevated the uterine coefficient, the levels of E2, and FSH significantly. In addition, LA significantly elevated ERα expression in the uterus and MCF-7 cells. MPP inhibited the proliferation of LA-stimulated MCF-7 cell and ERα expression in MCF-7 cells. Taken together, LA had an estrogen-like effect, which was mainly mediated by the estrogen receptor ERα.


Introduction
The seeds of Lepidium apetalum Willd. have been thought as traditional Chinese herbal medicine. The use of these seeds was first chronicled in "Shennong's Herba" and described as having cold nature, an acrid and bitter taste. This herb has the ability to relieve asthma and clear away heat from the lungs, detumescence, and promoting diuresis. Furthermore, the dry mature seeds of L. apetalum and herb-Sophia (Descurainia sophia L.) Webb ex Prantl., belonging to the Cruciferae (Brassicaceae) family, were commonly called "Tinglizi"; the former was called "BeiTinglizi", and the latter was named "Nan Tinglizi" in the Chinese Pharmacopoeia 2015 [1]. Although they have been both used as "Tinglizi", distinctions have been made between their plant origin, appearance, and pharmacological activity [2,3]. Compared with the seeds of L. apetalum, the previous investigations of herb-Sophia semen were more comprehensive [4][5][6][7][8][9]. Modern pharmacology has demonstrated that the seeds of L. apetalum have a cardiotonic [10] effect, and the chemical constituents are mainly flavonoids [11][12][13][14][15].
Phytoestrogens are polyhydric compounds derived from plants and have a similar structure to mammalian steroid hormone 17 -estradiol. In our preliminary study, we found that L. apetalum exerted estrogen-like effects. In a continuing search for phytoestrogens, various chromatographic techniques were used to isolate a new natural derivative lepidiumuridine A (1) from the seeds of L. apetalum. Lepidiumuridine A was evaluated for its estrogen-like effect of in vitro and in vivo, and we explored its potential mechanisms in this study.

General Experimental
Procedure. NMR spectroscopy was performed at room temperature using a Bruker Avance III 500 MHz spectrometer with tetramethylsilane (TMS) as a standard. Optical rotations were measured using an AP-IV laboratory polarimeter, which was made by Rudolph Research Analytical. HRESIMS spectra were determined with a Bruker Maxis HD mass spectrometer. The IR spectrum was measured on a Nicolet iS10 microscope spectrometer, which was made by Thermo Scientific. UV spectra were measured using a Shimadzu UV-2401PC apparatus. P-HPLC was acquired on YMC-Pack ODS-A column (250 × 10 mm and 5 m, respectively) on a Saipuruisi LC-50 instrument with a UV200 detector. CC was performed on Diaion HP-20 adsorbent (Mitsubishi Chemical Co.), Toyopearl HW-40, MCI gel CHP-20 (Tosoh Co.), Lichroprep RP-18 gel (Merck, Darmstadt), and silica gel (Marine Chemical Industry). TLC was performed on custom silica gel G plates (Qingdao Marine Chemical Industry). The chemical reagents used for isolation were of analytical grade, and the solvents used for p-HPLC were of chromatographic grade.    and two doses of LA (25 and 50 mg/kg). The mice received LA by continuous gavage for 7 d. The animals were euthanized, and blood was collected by heart punctures, and uteri were removed and quickly weighed.

Western
Blot. Uterine proteins were extracted using a mammalian protein extraction kit (Beijing Com Win Biotech Co., Ltd.) and quantified with a Bradford protein assay kit (Pierce, Perbio Science Co.). Protein samples separated by SDS-PAGE were transferred to a PVDF membrane. The PVDF membrane was then incubated with a primary antibody (ABclonal, Boston, USA; ER 1:500, A0296; ER 1:500, A2546; GPR30 1:500, A10217) overnight at 4 ∘ C and subsequently cultivated with a secondary antibody (1:1000) for 1 h at 25 ∘ C. The proteins from the bands were quantified with a chemiluminescence gel imaging apparatus (Azure c500), and the bands were analyzed with Quantity One software.  and kept for 0.5h with LA. Other experimental steps are as described above.   (Table 1) . Compared with uridine [16], the NMR spectral data of 1 only added a glucose unit [17], and the glucose unit was connected to C-3 of ribose according to glycosidation shift ( 78.7 from 75.0). Based on the HMBC correlation of H-1 with the C-3 , the assumption was confirmed. In addition, the presence of uridine group and D-glucopyranosyl unit was determined by acid hydrolysis and TLC compared with the standards of uridine and Dglucose. Accordingly, 1 was characterized as uridine-3 -O--D-glucopyranoside [18], which is a new natural compound, namely, lepidiumuridine A (Figure 1). Table 2, as a positive control, EV increased the uterine coefficient (107.24%) and the levels of FSH, LH, and E2 in serum of mice. The different doses of LA increased the uterine coefficient (41.24% and 47.95%, respectively) and the levels of FSH and E2. See Table 2.

Effect of LA on the Uterine Expression of ER , ER ,
and GPR30. Figure 2 shows that although LA promoted the expression of ER in the uterus, it had no effect for ER and GPR30. EV promoted the expression of three proteins in the uterus. Figure 3 shows that LA (different doses) and 17 -E2 (as a positive control) promoted MCF-7 cell proliferation.

Effect of MPP on LA-Stimulated MCF-7 Cell Proliferation.
As shown in Figure 4, MPP (specific ER antagonist, 1 M) blocked the effect of LA (10 M) on MCF-7 cell proliferation.

Discussion
Phytoestrogens are polyhydric compounds obtained from plants with a structure close to that of mammalian steroid hormone 17 -estradiol [19]. Phytoestrogens act in a manner similar to that of estrogen and first bind to estrogen receptors (ERs) [20]. When the ERs bind to ligands, they induce the creation of ER homo-or heterodimers, which in turn stimulate nuclear and extracellular signaling pathways to exert estrogen-like effects [21,22]. Natural phytoestrogens have lower estrogen-like activity than synthetic estrogen, but they are associated with less risk of breast and endometrial cancers and are safer to use [23]. This is why there has been great interest in the effects and molecular mechanisms of Chinese herbal medicines containing phytoestrogens. The uterus contains a large number of ERs, and estrogen binds to this receptor, which can induce increased levels of uterine target protein (IPs) that manifests as uterine tissue hyperplasia [24] and is measurable by exogenous sources. In our current research, the uterine coefficient of young female mice was made as an index for evaluating estrogenlike activity. The experimental results displayed that LA significantly increased the uterine coefficient of immature mice, suggesting that LA has estrogen-like effects in vivo.
MCF-7 cells are estrogen receptor-positive human breast cancer cells that proliferate in reaction to estrogen or estrogen-like activated material, and they are frequently removed to identify estrogen-like activity [25]. The experimental outcomes showed that LA significantly stimulated MCF-7 cells proliferation, suggesting that LA has estrogenlike effects in vitro.
There are two types of estrogen receptors, genomic nuclear ER and ER and the nongenomic GPR30 and potentially additional nongenomic receptors [26]. The receptors mediate the physiological and pathological impacts of estrogen, and western blot and in-cell western were utilized to examine ER , Er , and GPR30 expression in the uterus. These receptors were then used to determine the molecular mechanism of the estrogen-like effect of LA. Western blot revealed that EV (0.33 mg/kg) increased ER , ER , and GPR30 protein expression in the uterus significantly. LA (25 and 50 mg/kg) significantly elevated ER expression in the uterus, indicating that LA's estrogen-like effect occurred in vivo through ER . In-cell western showed that 17 -E2 significantly boosted ER , Er , and GPR30 expression in MCF-7 cells. LA significantly elevated ER expression in MCF-7 cells, which indicated that the estrogen-like effect 6 Evidence-Based Complementary and Alternative Medicine of LA acted in vitro through ER . In addition, the ER antagonist methyl piperidino-pyrazole (MPP) inhibited LAstimulated MCF-7 cell proliferation and ER expression in MCF-7 cells.
Lepidiumuridine A, a new natural uridine derivative, was isolated from the seeds of L. apetalum. We evaluated the estrogen-like effect of LA in vitro and in vivo and explored its potential mechanisms. Previous investigations demonstrated that uridine derivatives possess significant biological properties, such as anti-HIV [27], antibacterial [28], antiviral activity [29], and anticancer [30], but no previous reports have described their estrogen-like effect. Prior evaluations have revealed that the phytoestrogens found in traditional Chinese medicine mainly consist of flavonoids, coumarins, lignans, terpenes, steroids, and other compounds [31]. The uridine derivative, lepidiumuridine A, was determined to be a new type of phytoestrogen through our study.

Conclusion
Lepidiumuridine A, as a new natural uridine derivative, was isolated from the seeds of L. apetalum. The compound has estrogen-like effect and the effect was mainly mediated by the estrogen receptor ER .

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare no conflicts of interest.