Isolation and Characterization of New Phenolic Compounds with Estrogen Biosynthesis-Inhibiting and Antioxidation Activities from Broussonetia papyrifera Leaves

Broussonetia papyrifera leaves (BPL) as a traditional Chinese medicine are also used in livestock feed for stimulating reproduction, adipose tissue and muscle development; however, the mechanism of their action is still unknown. Through estrogen biosynthesis-guided fractionation in human ovarian granulosa-like KGN cells, five new phenolic glycosides, broussoside A–E(1–5), along with fifteen known dietary phenolic compounds, were isolated from the n-butanol extract of BPL, and their structures were elucidated on the basis of NMR spectra analysis and chemical evidence. New compounds 3, 4, 5 and the known compounds 9 and 10 were found to potently inhibit estrogen biosynthesis in KGN cells. In addition, compounds 9, 17, 18, and 20 showed strong antioxidant activity against ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) and DPPH (1, 1′-diphenyl -2-picryl-hydrazyl radical) assays. These findings suggest that BPL may improve meat quality through the regulation of estrogen biosynthesis. Furthermore, they may be useful for the discovery of potential aromatase modulators from natural products. Finally, they could be considered as a new source for natural antioxidants.


Introduction
Estrogen plays a vital role in the regulation of many biological processes, which is synthesized by aromatase in the body, the only enzyme in vertebrate that can catalyze the formation of estrogens by using androgens as substrates [1]. Synthetic aromatase inhibitors have been developed for the treatment of breast cancer; however, their clinical uses are severely limited by their side effects such as increased risk for osteoporosis and cardiovascular disease [2]. Natural products are considered a good source of aromatase inhibitors; however, only about 300 natural products have been evaluated for their effects on aromatase [3]. Estrogen is also known to affect animal reproduction, adipose tissue and muscle development [4][5][6]. In livestock husbandry, hormones such as estrogen are widely used to stimulate these processes, but they often lead to food safety issues caused by the residual hormones [5]. Thus, finding new natural aromatase modulators and feeding sources is a new option for estrogen-related diseases and livestock culturing.
Some phenolic compounds have been reported to show potent estrogen biosynthesis-inhibiting activity [7]. Besides, they have been also reported to have excellent antioxidant properties [8,9]. Natural antioxidants can protect the human body from free radicals, retard the progress of many chronic diseases, and provide a barrier to the oxidative rancidity of lipids in food, cosmetics, and pharmaceutical materials [10]. Therefore, the isolation and characterization of natural antioxidants, with little or no side effects, for use in foods or medicinal materials is still necessary for the replacement of their synthetic counterparts whose safety has been questioned for a long time.
Broussonetia papyrifera (L.) Vent., which belongs to the family of Moraceae, and is also known as paper mulberry, is a deciduous tree or shrub widely spread in Asia and Pacific countries such as China, USA and Thailand with multiple functions that are extensively applied in the papermaking, livestock feeding, medicine, etc [11][12][13]. B. papyrifera has been used for the treatment of hernias, dysentery, tinea, and oedema in traditional Chinese medicine [14]. Its leaves are often made into medicinal tea in China because their extracts have shown antihepatotoxic, antioxidant [15], antifungal, and lens aldose reductase inhibitory activities. The leaves of B. papyrifera are also widely used in China to feed livestock for improving meat quality [16]. In addition to nutritional compounds, B. papyrifera is also rich in phenolic compounds [17]. Since the first report of the use of Broussonin A as a source of phytoalexins, researchers have focused on the isolation of the bioactive constituents from B. papyrifera. Its main bioactive constituents are phenolic compounds, diterpenoids and alkaloids [12]. However, the effect of phenolic glycosides from BPL on estrogenic biosynthesis and oxidation is still unknown. In this study, B. papyrifera leaves were investigated through bioactivityguided isolation, and the effects of isolated compounds on estrogen biosynthesis and oxidation were evaluated.

Ethics Statement
The B. papyrifera leaves were collected from Chongqing, China, in June 2011 and identified by Professor Weikai Bao, Chengdu Institute of Biology, Chinese Academy of Sciences. No specific permits were required for the described field studies in Chongqing. The research sites are not privately owned or protected in any way and field studies did not involve endangered or protected species. Human ovarian granulosa-like KGN cells as described in reference [18] were kindly supplied by Prof. Yiming Mu, Chinese PLA General Hospital, Beijing, China. No specific permissions were required for these activities.
General Procedures 2,29-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,29-dipheny1-1-picrylhydrazyl (DPPH), potassium persulfate (K 2 S 2 O 8 ), and all the chromatographic solvents (methanol and acetonitrile) were purchased from Sigma (St. Louis, USA). The TSKgel HW-40C was purchased from TOSOH Corporation. Sephadex LH-20 was purchased from GE Company. HPLC-grade water was prepared from a Milli-Q system (Millipore Laboratory, Bedford, MA). Column chromatography was carried out on silica gel (200-300 mesh) supplied by Qingdao Marine Chemical Co. Thin-layer chromatography (TLC) was carried on silica gel GF 254 -precoated plates with chloroform/ methanol/water (6:4:1) and the spots were visualized by UV illumination (254 nm) and by spraying with 5% H 2 SO 4 in ethanol followed by heating. NMR spectra were recorded in DMSO-d 6 with a Bruker Avance 600 spectrometer for both 1 H-and 13 C-NMR. Coupling constants were expressed in Hertz and chemical shifts were given on a ppm scale with tetramethylsilane (TMS) as the internal standard. The spectroscopic data of 1 H-NMR, 13 C-NMR, HSQC, HMBC for broussoside A-E were shown in the Supporting Information (Figures S1-20 in File S1). Analytical high-performance liquid chromatography (HPLC) was carried out on a LabAlliance Series III with a model 201 (SSI) detector and Ultimate C 18 column (250 mm64.60 mm, 5 mm). Preparative HPLC was carried out on a P3000 with a UV3000 detector (Beijing ChuangXinTongHeng Science and Technology Co., Ltd) and Ultimate C 18 column (250 mm621.2 mm, 5 mm). Electrospray ionization mass spectrometry (ESI-MS) and High resolution electrospray ionization mass spectrometry (HR-ESI-MS) were measured on a Finnigan LCQ DECA and Bruker Apex-III mass spectrometer respectively.

Acid Hydrolysis of the New Compounds and Sugar Analysis
The new compounds 1-5 (2 mg) were added to a solution of concentrated HCl (0.5 mL), and H 2 O (1.5 mL)/dioxane (3 mL) and refluxed for 3 h. After dilution with H 2 O, the reaction mixture was extracted twice with ethyl acetate (EtOAc). The EtOAc layer of compounds 4 and 5 were then concentrated to dryness under reduced pressure. The residue was re-dissolved with methanol (2 mL) for an optical rotation measurement. The H 2 O layers of compounds 1-5 were neutralized with NaHCO 3 and then concentrated to dryness under reduced pressure. The residue was re-dissolved with H 2 O (2 mL) for optical rotation measurement and TLC analysis.

Cell Proliferation Assay
KGN cells as described in the reference [18] were seeded into 96-well plates (1.0610 3 cells/100 mL) with Dulbecco's modified Eagle's medium/nutrient mixture F-12 (DMEM/F-12) and were incubated at 37uC in a 5% CO 2 humidified atmosphere. Compounds were then added to the cells, and the plates were incubated for a further 24 h. Alamar Blue reagent (10 mL/well) was added, and the fluorescence intensities were measured using a Verioskan Flash Multimode Reader with excitation at 544 nm and emission at 590 nm. Cytotoxicity was defined as the ratio of the fluorescence intensity in the test wells to that in the solvent control wells (0.1% dimethyl sulfoxide; DMSO). The assay was conducted in triplicate.

Cell-based Estrogen Biosynthesis Assay
The cell-based estrogen biosynthesis assay was performed as previously described [19]. Briefly, KGN cells were seeded in 24well plates overnight. The following day, the cells were replaced with serum-free DMEM/F-12 medium and pretreated with the test chemicals for 24 h. Testosterone (10 nM) was then added to each well, and the cells were incubated for a further 24 h. The culture media and cell lysates were collected and stored at -20uC. The 17b-estradiol in the culture medium was quantified using a 17b-estradiol magnetic particle-based enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions. The optical density (OD) value was measured at 550 nm with the Verioskan Flash Multimode Reader (Thermo Scientific, Waltham, MA). The results, normalized to the total cellular protein content, were expressed as percentages of the control.

ABTS and DPPH Radical-scavenging Assay
The antioxidant capacity of the test samples (the extracts and phenolic compounds) were measured by their ability to scavenge the ABTS radical cation and DPPH radical using previously reported methods [20,21], with some modifications. L-Ascorbic acid was used as a positive control. Assays were performed in 96well plates. The ABTS radical was prepared by reacting 10 mL of 2 mM ABTS water solution with 100 mL of 70 mM potassium persulfate, and the mixture was allowed to stand in dark at room temperature for 12 h before use. Prior to the assay, the solution was diluted in water to give an absorbance at 734 nm of 0.7060.02 and was equilibrated at 25uC. The DPPH radical ethanol solution was obtained by dissolving 5 mg DPPH in 50 ml ethanol (EtOH) overnight in dark and then diluting to an absorbance of 0.7060.02 at 517 nm. An EtOH solution (100 mL) of each sample (10-300 mg) was added to the ABTS (DPPH) radical ethanol solution (100 mL). After reacting with the ABTS radical solution for 10 min (DPPH radical solution for 30 min), the absorbance value (Ai) of ABTS at 734 nm (or Ai of DPPH at 517 nm) was measured using a Verioskan Flash Multimode Reader (Thermo Scientific; Waltham, MA, USA). The blank absorbance (A 0 ) was measured using ethanol. The ABTS and DPPH radical solutions were prepared daily. The antioxidant activity was expressed as the percentage inhibition of the ABTS/DPPH radical and was determined by the following equation:

Statistical Analysis
All experiments were carried out in triplicate. The inhibitory concentration providing 50% inhibitory capability (IC 50 ) and the sample concentrations providing 50% scavenging capability (SC 50 ) were obtained by fitting dose-response data to a four-parametric logistic nonlinear regression model using GraphPad Prism 5.0 software (GraphPad, La Jolla, CA).

Estrogen Biosynthesis and Antioxidant Activities of the Extracts from BPL
To investigate whether the beneficial effects of BPL are because of their role in estrogen biosynthesis, we conducted a bioactivityguided isolation procedure using human ovarian granulosa-like KGN cells. Forskolin, an adenylate cyclase agonist, increases intracellular cAMP and the expression of aromatase by activating the protein kinase A/CREB pathway in ovaries; we used it here as a positive control for an estrogen biosynthesis agonist [22]. Letrozole is a potent non-steroidal aromatase inhibitor registered for clinical use, and we used it here as a positive control for an estrogen biosynthesis antagonist [23]. As shown in Figure 1A, forskolin promoted the production of 17b-estradiol in KGN cells, whereas letrozole significantly inhibited it, the same as previously reported [24]. The methanol extract (GT) and the n-butanolsoluble extract (GB) of the BPL showed the most potent estrogen biosynthesis-inhibiting activity at 50 mg/mL, whereas the CHCl 3soluble extract (GC) and the water-soluble extract (GW) had no such effect.

Effect of Isolated Compounds from BPL on Estrogen Biosynthesis in Human Ovarian Granulose-like KGN Cells
All the isolated compounds were tested for their effect on estrogen biosynthesis activity ( Figure 5). First, the cytotoxicity of the phenolic compounds (compounds 1-20) at 50 mM against human ovarian granulosa KGN cells was evaluated. The results showed that only one compound, apigenin (8), was cytotoxic, whereas the other compounds displayed no cytotoxicity on the KGN cells (data not shown). Next, the isolated compounds (except for apigenin) were examined for their effects on estrogen biosynthesis in KGN cells at 50 mM. As shown in Figure 5, compounds 2, 3, 4, 5, 9 and 10 exhibited potent estrogen biosynthesis-inhibiting activity. Compounds 2, 3, 4, 5, 9, and 10 were further subjected to IC 50 testing. As seen from Table 4, the IC 50 values of 2, 3, 4, 5, 9 and 10 were determined to be 45.02, 28.90, 20.20, 20.75, 1.32, and 11.89 mmol/L, respectively.

Discussion
In recent decades, the incidence of breast cancer has increased worldwide, and it is the main cause of cancer mortality and morbidity in women [44]. Overexposure to environmental estrogenic compounds and the western lifestyle have contributed to this increased incidence [19]. Inhibitors for the estrogen receptor and aromatase have been developed for the prevention or therapy of hormone-dependent breast cancer in postmenopausal women, and aromatase inhibitors show superior efficacy to the conventional antiestrogen receptor drugs such as tamoxifen [3]. In this study, we found that extracts from BPL were able to modulate the biosynthesis of estrogen, making it a possible beneficial health food.
Estrogen also plays an important role in the reproduction, muscle and adipose tissue formation [6][7][8]. BPL are used for feeding livestocks to improve the quality of the meat; however, the mechanism underlying this is still unknown. This study revealed that BPL extracts and the isolated natural compounds can modulate estrogen biosynthesis, suggesting that BPL may improve the meat quality by altering the endogenous estrogen levels. In addition, B. papyrifera is widely distributed in Asia and Pacific countries such as China, USA and Thailand, and is easily obtained in large quantities [13][14][15]. Thus, BPL can be developed as a natural supplement for feed in livestock husbandry to decrease the use of synthetic hormones.
In this study, we isolated several novel compounds. The new compounds 2, 3, 4, 5 and the known compounds 9, 10 exhibited estrogen biosynthesis-inhibiting activity, whereas, compounds 1, 7, 12, 18, 19 and 20 showed estrogen biosynthesis-promoting activity. Apigenin (8) and luteolin (9) were previously reported to potently inhibit aromatase activity [19]. Compounds 2 and 9 also inhibited estrogen biosynthesis and were obtained in large quantities. These two compounds may, therefore, make a large contribution to the strong inhibition of estrogen biosynthesis by the n-butanol extract of BPL, despite some compounds having an estrogen biosynthesis-promoting effect. To the best of our knowledge, this is the first time that compounds 3, 4, 5, and 10 have been reported to inhibit estrogen biosynthesis, making them suitable for further investigation as new estrogen biosynthesis inhibitors for the prevention of breast cancer.
Free radicals are responsible for food decay and cause oxidative damages to biological systems [45]. According to the source and substrate attack mechanism of free radicals, antioxidants are divided into three categories: enzyme inhibitors, metal chelators, and radical scavengers. The former two prevent the generation of radicals indirectly, while the latter one scavenges radicals directly. Owing to their direct nature, the last category of radical- Table 3. 1H-NMR spectral data of the newly isolated compounds 4 and 5 (600 MHz in DMSO-d6, d in ppm, J in Hz).  scavenging antioxidants has received much attention [46]. Therefore, the antioxidant capability of the extracts, fractions, and isolated compounds from the BPL was examined by ABTS and DPPH free radical assays. The compounds luteolin (9),  luteoloside (17), orientin (18), and isoorientin (20) showed very potent ABTS/DPPH radical-scavenging activity. From a structural viewpoint, the four natural antioxidants were luteolin and luteolin derivatives with two vicinal phenolic hydroxyls on the B ring. These results suggest that antioxidant activity is determined not only by the number of phenolic hydroxyl groups but also by the position on the aromatic rings, which is consistent with the findings of a previous study [43]. Oxidative stress is responsible for many chronic diseases such as cancer and cardiovascular disorders [47]. The finding in this study that luteolin (9), luteoloside (17), orientin (18), and isoorientin (20) exhibit potent antioxidative activity not only explains the beneficial effects of BPL on human health, but also validates them as potential new food antioxidant supplements.
In conclusion, through estrogen biosynthesis-guided fractionation in human ovarian granulosa-like KGN cells, five new phenolic glycosides, together with fifteen known dietary phenolic compounds, were isolated from the n-butanol extract of BPL. BPL are used in livestock feed for stimulating reproduction, adipose tissue and muscle development; however, the mechanism is still unknown. The finding that the newly identified compounds 3, 4, and 5 and the known compounds 9 and 10 inhibit estrogen biosynthesis in KGN cells indicated that BPL may improve meat quality through the modulation of the endogenous estrogen levels. The finding that compounds 9, 17, 18, and 20 showed potent antioxidant activity also contributes to the explanation of the beneficial effects of BPL on human health and validates them as potential new food antioxidant supplements.