Effects of Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) Hull Extracts on the Secretion of and Estradiol In Vivo and In Vitro

Shih-Min Hsia1, Chih-Lan Yeh1, Yueh-Hsiung Kuo2 , Paulus S. Wang3* and

Wenchang Chiang1*

1Graduate Institute of Food Science and Technology, National Taiwan University,

Taipei 106, 2Department of Chemistry, National Taiwan University, Taipei 106, and

3Department of Physiology, School of Medicine, National Yang-Ming University,

Taipei 112, Taiwan, Republic of China

Keywords: adlay hulls; progesterone; estradiol; aromatase; P450scc; StAR

Short Title: ADLAY SEED EXTRACTS ON E2, P4 IN VIVO AND IN VITRO

*Both authors are corresponding authers because of their equal contributions.

*Corresponding authors:

1Wenchang Chiang Ph.D. Institute of Food Science and Technology, National Taiwan University Taipei 106, Taiwan, R.O.C. Tel: 886-2-33664115 E-mail: [email protected]

3Paulus S. Wang, Ph.D. Department of Physiology, School of Medicine, National Yang-Ming University Shih-Pai, Taipei 11221, Taiwan, R.O.C. Tel: 886-2-28267082 FAX: 886-2-28264049 E-mail: [email protected]

1 Abstract

Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf.) has been used as a traditional

Chinese medicine for the dysfunction of endocrine system. However, the effect of adlay seed on the endocrine system has not yet been reported. In the present study, both in vivo and in vitro effects of methanolic extract of adlay hull (AHM) on progesterone synthesis were studied. AHM was partitioned with different solvents including water, 1-butanol, ethyl acetate and n-hexane. Four subfractions named

AHM-Wa (water fraction), AHM-Bu (1-butanol fraction), AHM-EA (ethyl acetate fraction) and AHM-Hex (n-hexane fraction) were obtained. Granulosa cells (GCs) were prepared from pregnant mare serum gonadotropin-primed immature female rats and challenged with different reagents including human chorionic gonadotropin (hCG,

0.5 IU/ml), 8-bromo-adenosine-3’,5’-cyclic monophosphate (8-Br-cAMP, 0.1 mM), forskolin (10 μM), 25-OH-cholesterol (10 μM) and (10 μM) in the presence or absence of AHM (100 μg/ml). The functions of steroidogenic enzyme including protein expression of the steroidogenic acute regulatory protein (StAR), cytochrome P450 side chain cleavage enzyme (P450scc) and aromatase were investigated. The expressions of StAR mRNA have also been explored by real-time reverse transcription polymerase chain reaction (real time RT-PCR). Accumulation of cAMP was measured by the radioimmunoassay. The enzyme activities of P450scc and

2 3β-hydroxysteroid dehydrogenase (3β-HSD) were analyzed by measurement of the accumulation of pregnenolone and progesterone, respectively. The aromatase activity

3 3 was analyzed by measurement of H20 in response to 1 H-andostenedione. In an in vivo study, AHM decreased plasma progesterone and estradiol levels after an intravenous injection of AHM (2 mg/ml/kg). In the in vitro studies, AHM decreased progesterone and estradiol via an inhibition on (1) the cAMP-PKA signal transduction pathway, (2) the cAMP accumulation, (3) P450scc and 3β-HSD enzyme activities, (4)

P450scc and StAR protein expressions and StAR mRNA expressioins, and (5) the aromatase activity in rat granulosa cells. These results suggest that AHM decreased the production of progesterone via mechanisms involving the inhibition of cAMP pathway, enzyme activities and protein expressions of P450scc and StAR in GCs.

3 Introduction

Coix lachryma-jobi L. var. ma-yuen Stapf (a Chinese medicine named Yi-yi-lan), commonly called adlay (Job’s tears), is an annual crop. It has long been consumed as both an herbal medicine and a food supplement. It has been described by the ancient

Chinese medical book Pen-Taso Kang Mu [1] as an efficient remedy for number of maladies and particularly beneficial to the digestive system. It is widely planted in

Taiwan, China, and Japan, and considered as a healthy food supplement.

Recent studies have shown some physiological effects of adlay extracts. The adlay extracts inhibit growth of Ehrlich ascites sarcoma and their active components have been identified as coixenolides [2]. A number of benzoxazinones were isolated from adlay seed and showed anti-inflammatory activity [3]. The coixans A, B, and C isolated from adlay seed express hypoglycemic activity in rats [4]. The lipid components in plasma and feces decrease in rats fed with adlay seed [5]. Ingestion of coix seed tablets increases the activities of cytotoxic T-lympocytes and natural killer cells [6]. A decrease in fibrinolytic activities in plasma has been observed in rats on a adlay seed mixed diet [7]. Numerous reports have indicated that the consumption of adlay seed is beneficial to human health [8-14]. Recently, we found that some traditional Chinese medicines, such as bufalin, Chansu, digoxin, ginsenoside-Rb1 and evodiamine, have inhibitory effects on peripheral hormone production [15-19]. The inhibition by these Chinese medicines might occur though different specific sites in rat granulosa cells, Leydig cells and anterior pituitary. Although adlay has many

4 biological functions, the relationship between its action and endocrine function has not yet been reported. Only one study indicated that adlay seed had an ovulatory-active substance [20]. Neverthlesss, some medical reports and study have also suggested that adlay seeds should not be used during pregnancy although no scientific evidence was given [21] Therefore, it is conceivable that adlay plays an important role in regulating endocrine functions.

Progesterone and estradiol are ovarian hormones of pregnancy and are responsible for preparing the reproductive tract for zygote implantation and the subsequent maintenance of the pregnant state. Whether adlay affect the secretion of progesterone and estradiol in granulosa cells is still unknown. The purpose of this study is to examine the direct effect of adlay extracts on the production of hormones (P4 or E2) in vivo or in vitro. In the present study, we examine whether adlay extract exerts direct on the production of progesterone and estradiol in vivo. We also examine whether AHM exerts direct on the production on cAMP-PKA pathway, or on the function of P450scc, 3β-HSD or aromatase in GCs. Cells were treated with human chorionic gonadotropin (hCG), forskolin (adenylate cyclase activator),

8-bromo-cAMP (8-Br-cAMP, cAMP analogue) with or without AHM to examine whether ABM inhibited cAMP-PKA signal transduction pathway. Both

25-OH-cholesterol (substrate for P450scc) and pregnenolone (substrate for 3β-HSD) were added to cells with or without AHM to determine whether AHM affected enzyme activity. The expressions of StAR and P450scc mRNA have also been explored by real-time reverse transcription polymerase chain reaction (real time

5 3 RT-PCR). The aromatase activity was analyzed by measurement of H20 in response to 13H-andostenedione. In an in vivo study, AHM decreased plasma progesterone and estradiol levels after an intravenous injection of AHM.

6 Materials and methods

Reagents

Pregnant mares serum gonadotropin (PMSG), Dulbecco's modified Eagle medium

(DMEM)/F12, fatty acid-free bovine serum albumin (BSA), penicillin-G, sodium bicarbonate, streptomycin sulphate, human chorionic gonadotropin (hCG), insulin, medium-199 (M199), L-glutamine, N-2-hydroxyethylpiperazine-N'-2-ethanesulpho nic acid (HEPES), progesterone (P4), dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The cAMP enzyme immunoassay

(EIA) system was obtained from Assay Designs, Inc. (Ann Arbor, MI, USA).

Plant materials

Adlay was purchased from a local farmer who planted Taichung Shuenyu No.4

(TSC4) of Coix lachryma-jobi L. var. ma-yuen Stapf. in Taichung, Taiwan in March

2000 and harvested it in July of the same year. The air-dried adlay seed was separated into three different parts including adlay hull, adlay testa, and polished adlay. All the materials were blended in powder form, and screened through a 20-mesh sieve

(aperture 0.94 mm).

Methanol extractions of adlay seeds

Each sample powder (100 g) was extracted with 1 liter of methanol stirred on

7 Thermolyn Nuova stirring/heating plate (Dubuque, IA) at room temperature for 24 h.

Contents were filtered through #1 filter paper (Whatman Inc., Hillsboro, OR, USA).

The filtrate was concentrated to dryness in vacuum condition to obtain dried methanolic extract form and stored at -20 ℃. The methanolic extracts from different parts of adlay seeds were named as AHM (hulls), ATM (testa), and PAM (polished adlay), respectively.

Partition and fractionation of methanol extract of adlay hull (AHM)

The AHM was further partitioned with different solvents including water, 1-butanol, ethyl acetate and n-hexane. Four subfractions, named AHM-Wa (water fraction),

AHM-Bu (1-butanol fraction), AHM-EA (ethyl acetate fraction) and AHM-Hex

(n-hexane fraction) were obtained. The solvent was evaporated under reduced pressure to give dried extract stocks and stored at -20 ℃. The active AHM-EA part was further fractionated with normal phase silica gel column chromatography using ethyl acetate-hexane mobile phase into thirteen fractions (A to M). The effects of fractions of AHM on progesterone and estradiol release by rat granulosa cells were further studied (Fig. 1A). For in vitro treated rat granulosa cells, powder of adlay extract was dissolved in DMSO to prepare a stock solution. The final concentration of

DMSO was less than 0.1%.

HPLC analysis of AHM-EA fractions F and G

8 The most active fractions F and G were subjected to HPLC analysis using a

KH2PO4:CH3CN mobile system. Cosmosil 5C18-MS (5μm, 25 cm x 4.6 mm) was used to quantify components in AHM-EA-F and AHM-EA-G (Fig. 1B).

Isolation and culture of rat granulosa cells (GCs)

Immature female Sprague Dawley rats 25~27 days of age were housed in a temperature-controlled room (22 ± 1 ℃) with 14 h of artifical illumination daily

(06:00 to 20:00) and were given food and water ad libitum. The preparation of GCs was modified from the method described elsewhere [22;23]. The immature female rats were injected subcutaneously with PMSG (15 IU/rat). Forty-eight hours later, rats were killed by cervical dislocation. Ovaries were excised and transferred into the sterile DMEM/F12 (1:1) medium, containing 0.1% BSA, 20 mM HEPES, 100 IU/ml penicillin-G, 50 μg/ml streptomycin sulphate. The 5- 10 ovaries were assigned as one dispersion. After trimming free fat and connective tissues, the follicles were punctured with a 26-gauge needle to release GCs. The harvested cells were pelleted and resuspended in growth medium (DMEM/F12 containing 10% fetal calf serum, 2

μg/ml insulin, 100 IU/ml penicillin-G, and 100 μg/ml streptomycin sulphate). Cell viability was greater than 90% as determined using a haemocytometer and trypan blue method. GCs were aliquoted in 24-well plates at approximately 1×105 cells per well and incubated at 37 ℃ with 5% CO2 -95% air for 2 days. Generally, at least 6 dispersions (n=6) of GCs were incubated in each group. Morphologically the cultured granulosa cells maintained a characteristic round (or polygonal) shape, throughout our

9 culture conditions.

Gel electrophoresis and Western blotting

The western blotting method has been reported previously [24-26]. The granulosa cells (2×106 cells) were incubated with medium containing AHM-EA-G (0, 100 μg/ml) for 2 h. At end of incubation, cells were washed twice with ice-cold saline and detached by trypsinization (1.25 mg/ml). The cells were collected and extracted in homogenization buffer (pH 8.0) containing 1.5% Na-lauroylsarcosine, 1 x 10-3 M

EDTA, 2.5 x 10-3 M Tris-base, 0.68% PMSF, and 2% proteinase inhibitor cocktail, and then disrupted by ultrasonic sonicator in ice-bath. Cell extracts were centrifuged at 13,500 × g for 10 min [26]. The supernatant fluid was collected and the protein concentration was determined by colorimetric method of the protein assay according to the Bradford method [27]. Extracted proteins were denatured by boiling for 5 min in SDS buffer (0.125 M Tris-base, 4% SDS, 0.001% bromophenol blue, 12% sucrose, and 0.15 M dithiothreitol) [28]. The proteins (20 g) in the samples were separated on

12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 75 V for 15 min and then at 150 V for 40 min using a running buffer. The proteins were electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (NEN Life Science

Products, Inc., Boston, MA, USA) using a Trans-Blot SD semi-dry transfer cell

(170-3940, Bio-Rad, Hercules, CA, USA) at 64 mA (for 8 mm ×10 mm membrane) for 45 min in a blotting solution.

10 Real Time RT-PCR analysis

The same source of total RNA used to define gene expression profiles was used in real time RT-PCR experiments, following all instructions outlined in the

LightCycler-RNA amplification kit SYBR Green I manual (Roche Molecular

Biochemicals, Mannheim, Germany). cDNA synthesis was carried out in LightCycler

(Roche) in a capillary as follows: 20 µl mix reaction containing 500 ng of DNase I treated total RNA, 4 µl of LightCycler-RT-PCR reaction mix SYBR Green I (final concentration 1X), 5 mM MgCl2, 0.4 µl LightCycler-RT-PCR enzyme mix, and 5.0 pmol forward and reverse primers for both genes. The expected PCR products would be 246 bp for the StAR cDNA; 536 bp for the rat P450scc, and 194 bp for the rat

PRL19. For reverse transcription, the reaction was incubated at 55°C for 30 min and at 95°C for 30 sec.

Effects of adlay extracts on progesterone and estradiol release in vivo

Diestrous or estrous rats were catheterized via the right jugular vein [29;30].

Twenty hours later, they were injected with saline (1ml/kg), AHM-EA-G (2 mg/ml/kg), hCG (5 IU/ml/kg), or hCG plus AHM-EA-G via the jugular catheter.

Blood samples (0.5 ml each) were collected at 0, 15, 30, 120, 180, 240 and 360 min after the challenge. Plasma was separated by centrifugation of blood samples at

10,000xg for 1 min. The concentration of progesterone and estradiol in plasma was measured by RIA.

11

Effects of adlay extracts on in vitro progesterone and estradiol release by rat GCs

For study about adlay extracts on secretion of progesterone in rat GCs. The granulosa cells were aliquoted in 24-well plates at approximately 1×105 cells per well and incubated at 37 ℃ with 5% CO2 -95% air for 2 days, and then incubated with 1 ml BSA-MED-199 medium containing adlay extracts (0, 0.1, 1, 10 and 100 μg/ml) in the presence of vehicle (0.1% DMSO) or hCG (0.5 IU/ml) or forskolin (10-5 M) or

8-Br-cAMP (10-4 M) for 120 min. The media were collected and the concentrations of progesterone were measured by RIA.

For studying the accumulation of cAMP in response to adlay extracts, the rat granulosa cells were incubated IBMX (phosphodiesterase inhibitor, 1 mM) and adlay extracts presence or absence of hCG (0.5 IU/ml) for 1 h. At end of the incubation, the cells were homogenized in 0.5 ml of 65% ice-cold ethanol by polytron (PT-3000,

Kinematica AG, Luzern, Switzerland), and then centrifuged at 2,000 × g for 10 min.

The supernatants were lysophilized in a vacuum concentrator (Speed Vac, Savant,

Holbrook, NY, USA), then reconstituted with assay buffer (0.05 M sodium acetate buffer with 0.01% azide, pH 6.2) before measuring the concentration of cAMP by

EIA.

For studying the influence of adlay extracts on the early step of enzyme activity of steroidogenesis, the granulosa cells were aliquoted in 24-well plates at approximately

12 5 1×10 cells per well and incubated at 37 ℃ with 5% CO2 -95% air for 2 days, and then incubated with 1 ml BSA-MED-199 medium containing adlay extracts (0 and

100 μg/ml) in the presence of vehicle (0.1% DMSO) or precursors of steroidogenesis such as 25-OH-cholesterol (10-5 M) or Pregnenolone (10-5 M) or 3β-HSD inhibitor

Trilostane (10-5 M) for 120 min, and progesterone or pregnenolone were measured by

RIA.

For study about adlay extracts on secretion of estradiol in rat GCs. The androgen substrate (androstenedione 20 μg/ml) is added to the BSA-MED-199 medium containing adlay hull extracts (0, 1, 10, and 100 μg/ml) in the presence of vehicle

(0.1% DMSO) for 120 min. The media were collected and the concentrations of estradiol were measured by RIA. All adlay extracts were dissolved in DMSO.

Hormone and cAMP assays

The concentration of progesterone in the medium was determined by RIA as described elsewhere [31;32]. With anti-progesterone serum No. W5, the sensitivity of progesterone RIA was 5 pg per assay tube. Intra- and interassay coefficients of variation (CV) were 4.8% (n=5) and 9.5% (n=4), respectively.

The concentration of pregnenolone in the medium was determined by RIA as described elsewhere [33;34] With anti-pregnenolone serum, the sensitivity of pregnenolone RIA was 16 pg per assay tube. Intra- and interassay coefficients of

13 variation (CV) were 2.5% (n=4) and 3.9% (n=5), respectively.

The concentration of estradiol in the medium was determined by RIA as described elsewhere [35]. With anti-estradiol serum No. W1, the sensitivity of estradiol RIA was

1 pg per assay tube. Intra- and interassay coefficients of variation (CV) were 6.0%

(n=5) and 5.9% (n=5), respectively.

The intracellular levels of cAMP were measured in rat granulosa cells using commercial EIA kit.

Statistical analysis

Date values are given as the mean ± SEM for a minimum of three values. In some cases, the treatment means were tested for homogeneity by two-way ANOVA, and the difference between specific means was tested for significance by Duncan’s multiple-range test [36]. In other cases, Student’s t test was used. A difference between two means was considered statistically significant when P<0.05.

14 Results

Effect of adlay hull extract on the concentration of plasma progesterone and estradiol

The effects of AHM-EA-G injection on plasma progesterone levels are shown in (Fig.

2A). The levels of plasma progesterone were not altered by saline injection. The plasma concentration of progesterone decreased gradually from 30-240 min after an intravenous injection of AHM-EA-G (2 mg/ml/kg). A single I.V. injection of 5 IU/ml hCG stimulated the concentration of plasma testosterone by 1.6 folds at 120 min. The concentration of plasma progesterone returned to basal three hours following hCG challenge. Two hour after administration of AHM-EA-G plus hCG markedly decreased the secretion of progesterone at 120 and 180 min as compared with basal level. (Fig. 2A).

The effects of AHM-EA-G injection on plasma estradiol levels are shown in (Fig. 2B).

The levels of plasma estradiol were not altered by saline injection. The plasma concentration of estradiol decreased gradually from 30-180 min after an intravenous injection of AHM-EA-G (2 mg/ml/kg). A single I.V. injection of 5 IU/ml hCG stimulated the concentration of plasma testosterone by 2.1 folds at 30 min. The concentration of plasma estradiol returned to basal one hour following hCG challenge.

Two hour after administration of AHM-EA-G plus hCG markedly decreased the secretion of progesterone at 30-180 min as compared with basal level (Fig. 2B).

15

Effect of adlay extracts on progesterone release by rat granulosa cells

To determine whether AHM affect progesterone biosythesis in granulosa cells,

Figure 3A shows that hCG at 0.5 IU/ml increased progesterone release by 2.5-fold

in rat GCs. Administration of different doses of AHM (0.1-100 μg/ml) with or

without hCG for 2 h incubation indicating that AHM at 100 μg/ml elicited an

inhibition of progesterone release by granulosa cells (P<0.01). Both 8-Br-cAMP and

forskolin stimulated progesterone release significantly in GCs (Fig. 3A, P<0.01).

Furthermore, AHM (100 μg/ml) inhibited not only basal but also 8-Br-cAMP- and

forskolin-stimulated progesterone release in rat GCs (P<0.01). Neither ATM nor

PAM had inhibitory effect on progesterone released in rat GCs (data not shown).

Effect of subfractions or compounds from AHM on progesterone release by rat GCs

To confirm the inhibitory effect of major active fraction in adlay of hulls on progesterone production, AHM was further partitioned into four subfractions including AHM-Wa, AHM-Bu, AHM-EA and AHM-Hex. (saw the flow chart in Fig.

1A). Administration of different doses of AHM-EA (0.1-100 μg/ml) with or without hCG for 2 h incubation indicating that AHM at 0.1, 1, 10 and 100 μg/ml elicited an inhibition of progesterone release by granulosa cells (P<0.01). Furthermore, AHM-EA

(100 μg/ml) inhibited not only basal but also 8-Br-cAMP and forskolin-stimulated

(P<0.01) progesterone release in GCs (Fig. 3B). However, AHM-Wa, AHM-Bu or

16 AHM-Hex did not alter progesterone production in vitro (data not shown).

Subsequently, we fractionated the AHM-EA portion into thirteen fractions (A to M,

Fig. 1A) with normal-phase silica gel column chromatography and determined their activity. Fractions E to G all had inhibitory effects on progesterone secretion. Fraction

G was found to be the most potent fraction among them. Administration of different doses AHM-EA-G (0.1-100 μg/ml) with or without hCG for 2 h incubation indicating

AHM at 0.1-100 μg/ml elicited an inhibition of progesterone release by granulosa cells (P<0.05 or P<0.01). Furthermore, AHM-EA-G (100 μg/ml) inhibited not only basal but also 8-Br-cAMP and forskolin-stimulated (P<0.01) progesterone release in

GCs (Fig. 3C).

Effect of AHM-EA-G on accumulation of cAMP in rat GCs

For studying the accumulation of cAMP in response to AHM-EA-G in rat granulosa cells, incubation of rat granulosa cells with IBMX (1 mM) in the presence or absence of hCG (0.5 IU/ml) or forskolin (10-5 M) for 1 h increased cellular cAMP production.

The AHM-EA-G (100 μg/ml) inhibited not only basal but also hCG-stimulated, forskolin-stimulated (P<0.05 or P<0.01) cellular cAMP production by rat granulosa cells (Fig. 4).

Effect of AHM-EA on biosynthesis pathway of progesterone by rat GCs

17 To investigate the effects of AHM-EA on P450scc activity, both 25-OH-cholesterol

(10-5 M) and pregnenolone (10-5 M) were used to challenge the rat GCs. The

25-OH-cholesterol and pregnenolone significantly increased progesterone release by

GCs (P<0.01). The AHM-EA and AHM-EA-G (10 and 100 μg/ml) inhibited basal,

25-OH-cholesterol- or pregnenolone-induced release of progesterone by granulose cells (P<0.01) (Fig. 5). This result indicates that ABM-Bu might have a direct inhibitory effect on P450scc and/or 3β-HSD activity. To further confirm whether

ABM-Bu affects P450scc and 3β-HSD activities in granulosa cells, trilostane

(3β-HSD inhibitor, 10-5 M) was employed. incubated with or without

25-OH-cholesterol (10-5 M) to inhibit turnover of pregnenolone to progesterone. The

25-OH-cholesterol with or without trilostane significantly increase pregnenolone release by GCs (P<0.05 or P<0.01). However, the AHM-EA and AHM-EA-G (10 and

100 μg/ml) inhibited basal, 25-OH-cholesterol- or pregnenolone-induced release of progesterone by granulose cells (P<0.01) (Fig. 6).

Effect of AHM-EA-G on the expression of cytochrome P450scc and StAR mRNA by rat GCs

The mRNA expressions of P450scc and StAR protein in rat granulosa cells administrated with AHM-EA-G were investigated. L19 was used as an internal control and was not affected by ABM-Bu and ABM-Bu-G. Administration of

AHM-EA-G inhibited basal or forskolin-stimulated mRNA expression of P450scc and

18 StAR protein in rat granulosa cells (Fig. 7).

Effect of AHM-EA-G on the expression of PKA, cytochrome P450scc and StAR protein by rat GCs

To determine the effect of ABM-Bu on expressions of PKA, P450scc and StAR protein in rat granulosa cells, the western blotting was used to determine which steroidogenic enzymes or proteins were altered at protein level. In Figure 8, β-actin signal was used as an internal control. Bands of PKA, P450scc and StAR were detected in rat granulosa cells. The results showed that both PKA, P450scc and StAR levels were decreased by 2 h treatment with 100 μg/ml of AHM-EA-G.

Effect of adlay extracts on estradiol release by rat GCs

In Figure 9, administration of AHM (10-100 μg/ml) elicited an inhibition on estradiol release by rat GCs (0.16 ± 0.03 to 0.07 ± 0.03 ng/1×105 cells/2 h, n=6). The maximal inhibition caused by 100 μg/ml AHM in rat granulosa cells was 73% (0.07 ±

0.03 ng/1×105 cells /2 h versus basal release 0.27 ± 0.03 ng/1×105 cells /2h, n=6,

P<0.01). However, AHM-Wa, AHM-Bu or AHM-Hex did not alter estradiol production in vitro (data not shown). These results showed that AHM-EA portion significant activity on estradiol secretion in rat GCs. Subsequently, we fractionated the

AHM-EA portion into thirteen fractions (A to M, Fig. 1A) with normal-phase silica gel column chromatography and determined their activity. Administration of

19 AHM-EA elicited an inhibition of estradiol release by rat GCs (0.24 ± 0.02 to 0.03 ±

0.02 ng/1×105 cells/2 h, n=6) (Fig. 9). Fractions E to I all had inhibitory effects on estradiol secretion. Fraction F and G were found to be the most active fraction among these. Administration of AHM-EA-F and AHM-EA-G elicited an inhibition of estradiol release by rat GCs (Fig. 9). Neither ATM nor PAM had any inhibitory effect on estradiol released in rat GCs (data not shown).

Effect of adlay extracts on estradiol release by rat GCs

To determine the effect of AHM-EA-F, AHM-EA-G and narinigenin on aromatase activity in rat granulosa cells, the aromatase activity assay was used to determine aromatase activity. In Figure 10, AHM-EA-F, AHM-EA-G and narinigenin inhibited aromatase activity in the presence or absence of forskolin–stimulated by rat granulosa cells.

20 Discussion

Adlay has long been consumed as both an herbal medicine and a food supplement in China and Japan. However, the relationship between its action and endocrine function has not yet been reported. In the present results, we found that administration of AHM and its subfractions in rats significantly 1) inhibited the spontaneous and hCG-stimulated secretion of progesterone and estradiol in vivo and in vitro, 2) decreased secretion of progesterone and estradiol in rat granulosa cells partly via a mechanism involving decrease of accumulation of cAMP or aromatase activity, and 3) decreased the activities of P450scc and 3β-HSD. 4) decreased the PKA, P450scc and

StAR protein expressions and P450scc and StAR mRNA expressions. To our knowledge, this is the first report demonstrating the effects of adlay hull extracts on steroid hormone secretion in vivo and in vitro, thus partially explaining the modulatory effects of adlay extracts on female reproductive functions. Apparently, different components of adlay seed extracts may have different chemicals to regulate endocrine functions.

It has been well established that hCG increases cyclic AMP generation and then stimulates progesterone secretion in vivo [37] and in vitro [38] and increases granulosa cellular cAMP content. We found that AHM inhibited hCG-stimulated production of progesterone by rat granulosa cells. The decrease in progesterone was not attributed to the cytotoxicity of adlay extracts. The administration of AHM,

AHM-EA and subfraction (100 μg/ml) did not cause a releasing of lactate

21 dehydrogenase (LDH) from rat granulosa cells (data not shown). We also assess that cell viability with recovery study. We determine if removing the substance and refeeding the cells plus stimulus results in reversal of the inhibition. From our results, we found that AHM, AHM-EA and subfraction (100 μg/ml) treated granulosa cells can restore progesterone and estradiol production once the extracts were removed

(data not shown). To examine the mediation of cyclic AMP pathway in the effect of

AHM on rat granulosa cells, both an adenylyl cyclase activator, forskolin, and a membrane permeable cyclic AMP analog 8-Br-cAMP, were employed. Since the administration of AHM decreased 8-Br-cAMP- and forskolin-induced production of progesterone, which indicates that the inhibition may be located at not only the post-adenylyl cyclase but also the post-cAMP pathway of progesterone biosynthesis in rat granulosa cells. However, in present study, we measured cAMP directly in rat granulosa cells. We found that AHM-EA-G decreased not only hCG- but also forskolin-induced cellular cAMP production. These results suggested that one of actions of AHM-EA was beyond the membrane receptor to inhibit the formation of cAMP in rat granulosa cells.

In rat granulosa cells, progesterone biosynthesis is via conversion of pregnenolone under the catalyzation of microsomal enzyme 3β-HSD after transformation of cholesterol to pregnenolone by P450scc (the rate limiting enzyme) [39]. In the present study, the administration of either 25-OH-cholesterol or pregnenolone stimulated progesterone secretion in rat granulosa cells. In the granulosa cell cluture, a significant effect of 25-OH-cholesterol (10-5 M) or pregnenolone (10-5 M) on

22 progesterone release has been observed [40;41]. However, administration of AHM-EA inhibited progesterone production caused by 25-OH-cholesterol or pregnenolone.

These data suggested that the function of P450scc and/or 3β-HSD might be affected by AHM-EA or AHM-EA-G. To further examine if the function of either P450scc or

3β-HSD is altered by AHM-EA or AHM-EA-G, we administrated 25- OH-cholesterol with or without AHM-EA or AHM-EA-G. After inhibiting the function of 3β-HSD by trilostane, the pregnenolone accumulation in rat GCs was measured and used as an index of the activity of P450scc. In our results showed that both AHM-EA and

AHM-EA-G inhibited pregnenolone accumulation in rat GCs. These results suggest that AHM-EA or AHM-EA-G inhibited progesterone secretion in rat GCs, at least in part, by reduction of P450scc and 3β-HSD activity in progesterone steroidogenesis.

The AHM is a crude extract containing much characterization of chemical components to regulate endocrine functions. To confirm which characterization of chemical decreases progesterone secretion by rat granulosa cells, we partitioned AHM into four subfractions. The present results indicated that AHM-EA could decrease both basal and hCG-stimulated progesterone secretion by rat granulosa cells.

AHM-EA and AHM-EA-G inhibited the forskolin- and 8-Br-cAMP-induced production of progesterone in rat granulosa cells. AHM-EA-G also inhibited the forskolin- and hCG-induced production of cAMP in rat granulosa cells, which implied that AHM, AHM-EA or AHM-EA-G possess some specific chemicals to decrease adenylyl cyclase activity and cAMP function in rat granulosa cells.

23 The biosynthesis of estradiol is from androstenedione to testosterone and then followed by the conversion of testosterone to estradiol via aromatase catalyzation [42].

We found that the AHM, AHM-EA and subfractions reduced progesterone and estradiol production. The cause of reduction of estradiol may be a decreased aromatase activity or substrate supplement during for estrogen synthesis. In our results demonstrated that AHM, AHM-EA and subfractions reduced aromatase activity than could inhibited estradiol synthesis. Therefore, AHM, AHM-EA and their subfractions might decrease aromatase activity or progesterone firstly and then affect estradiol synthesis.

Progesterone and estradiol are essential for blastocyst implantation and maintenance of pregnancy in several species. Inhibition of the biosynthesis of both progesterone and estradiol or a blockade of receptor binding will affect endometrial development and function [43-45]. Some reports have suggested that adlay seeds should be avoided to use during pregnancy, although no scientific evidence was given

[21]. It has been reported that AHM possesses some low molecular weight and moderately polar substances that have anti-oxidation effects [46]. Moreover, it has been shown that there are at least six classes of chemical constituents of

AHM-, lignan, , , polysaccharide and

[47-49]. Flavonoid have been shown to possess an inhibitory effect on steroidogenic enzymes in human adrenocortical H295 cells [50]. We suggested that the flavonoid phytochemicals in AHM and AHM-EA may play an important role to inhibit progesterone or estradiol secretion in rat granulosa cells. The phenolic

24 compounds like p-coumaric acid, and ferulic acid have also been isolated and quantified in AHM [51;52]. The p-coumaric acid has been found to have antifertility effects on early gestation of mice [53]. The p-coumaric acid has been shown to be a reproductive inhibitor in male rat [54]. In the present study, AHM,

AHM-EA and subfractions (E, F, G, H, I) could inhibit progesterone or estradiol secretion in granulosa cells. We have further observed the quantification of components in AHM-EA-F and A HM-EA-G. The AHM-EA-F contained quercetin, and gallic acid. The AHM-EA-G contained naringenin, quercetin, vanillin, , gallic acid, syringic acid and p-coumaric acid. In the present results, we demonstrated that naringenin one of flavonoid was to inhibit progesterone secretion by rat granulosa cells. Taken together, in the present study we found that AHM contained different components to inhibit progesterone and estradiol production in rat granulosa cells. These results provided some evidence to explain the reason why adlay should not be used during pregnancy.

In summary, the present data demonstrated that the partitioned fractions of adlay extracts possess both hypogonadal in vitro and in vivo. The reduction of the production of progesterone and estradiol is due in part to a decrease of adenylyl cyclase activity, cAMP functions, P450scc and 3β-HSD activity or protein and mRNA expressions in rat granulosa cells.

25 Acknowledgements

The technical assistance provided by Dr. Shiow-Chwen Tasi is appreciated. This study was supported by a grant [ No. DOH92-TD-1002, No. DOH93-TD-1014 and

No. DOH93-TD-F-113-053-(2) ] from the Department of Health, Executive Yuan,

Taiwan, Republic of China.

26 Figure legends

Figure 1. Scheme for extraction, partitioning and fractionation of anti-progesterone and anti-estradiol fractions from adlay hulls (A). Flow chart of quantification of components from AHM-EA-F and AHM-EA-G (B). Fraction (Fr.) E to H showed the most potent activity on inhibition of progesterone secretion. Fraction E to I showed the most potent activity on inhibition of estradiol secretion.

Figure 2. Effects of AHM-EA-G on the basal and hCG-stimulated level of plasma progesterone (A) and estradiol (B) in female rats. Rats were given a single intravenous injection of vehicle, AHM-EA-G, hCG, or hCG plus AHM-EA-G via right jugular catheter and bled at different time intervals after injection. The concentration of progesterone and estradiol was measured by RIA. *P<0.01; **P < 0.01 compared with vehicle or hCG group. + P < 0.05; ++ P < 0.01 compared with time = 0 min. Each value represents mean ± SEM.

Figure 3 Effects of different doses of AHM (A) ,AHM-EA (B), and AHM-EA-G (C) on the release of progesterone in the presence or absence of 8-Br-cAMP-, hCG- and forskolin–stimulated progesterone release in rat granulosa cells. *P<0.01; **P<0.01 versus AHM = 0 μg/ml. +P<0.05; ++ P<0.01 compared with vehicle-treated group

(containing 0.1% DMSO in medium). Each column represents mean ± SEM.

Figure 4. Effects of AHM-EA-G on the basal, hCG-stimulated and forskolin-stimulated cellular cAMP production by rat granulosa cells. Cells were treated with hCG or forskolin and with or without AHM-EA-G for 1 h incubation.

Basal, hCG-stimulated and forskolin-stimulated *P<0.05 and ** P<0.01 compared with ABM-Bu at 0 μg/ml. ++ P<0.01 compared with vehicle-treated group. Each 27 column represents mean ± SEM.

Figure 5. Effects of different doses of AHM-EA on the release of progesterone in the presence or absence of 25-OH-cholesterol– and pregnenolone-stimulated progesterone release in rat granulosa cells. * P<0.05; **P<0.01 versus AHM-EA = 0

μg/ml. +P<0.05; ++ P<0.01 compared with vehicle-treated group. Each column represents mean ± SEM.

Figure 6. Effects of AHM-EA and AHM-EA-G on progesterone release in rat granulosa cells treated with or without trilostane(10-6 M), 25-OH-Cholesterol (10-5 M) and trilostane plus 25-OH-Cholesterol. * P<0.05; **P<0.01 versus AHM-EA or

AHM-EA-G = 0 μg/ml. +P<0.05; ++ P<0.01 compared with vehicle-treated group.

Each column represents mean ± SEM.

Figure 7. Effect of AHM-EA-G on StAR and P450scc mRNA levels in rat granulosa cells. Cells were treated for 30 min with AHM-EA-G (100 μg/ml) in the presence or absence of forskolin (10-5 M).

Figure 8. Effect of AHM-EA-G on P450scc, PKA and StAR protein levels in rat granulosa cells. Cells were treated for 2 h with or without AHM-EA, and then protein levels of P450scc and StAR were measured by western blotting. This experiment was repeated two times with similar results.

Figure 9. Effects of different doses of AHM, AHM-EA and AHM-EA-F on the release of estradiol in rat granulosa cells. * P<0.05; ** P<0.01 versus AHM,

AHM-EA or AHM-EA-F = 0 μg/ml. Each column represents mean ± SEM.

28 Figure 10. Effects of different compounds on the aromatase activity in rat granulosa cells. * P<0.05; ** P<0.01 versus compound= 0 μg/ml. Each column represents mean ± SEM.

29 Reference

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34 A Adlay Hull 7.3kg

MeOH Extract (AHM) 74.5g partitioned (Hexane/H2O)

Hexane Fr. H2O Fr. (AHM-Hex) 19.05g partitioned (Ethyl acetate/H2O)

EA Fr. H2O Fr. (AHM-EA) 11.1g partitioned (Butanol/H2O)

Butanol Fr. 9.98g H2O Fr. 20.35g (AHM-Bu) (AHM-Wa)

column chromatography

ABCDEFGHIKLM J ＊ ＊ ＊ ＊ # # # # #

＊ :Inhibits progesterone secretion in rat granulosa cells # :Inhibitsestradiol secretion in rat granulosa cells

B AHM-EA-F AHM-EA-G

HPLC HPLC

quercetin quercetin naringenin naringenin gallic acid gallic acid syringaldehyde vanillin syringic acid p-coumaric acid

Fig 1 (Hsia, et al.)

35

+

30 + A

20

∗ ∗ 10 + + + Plasma Plasma Progesterone (ng/ml) ∗ ∗∗ Saline (n=5)

+ ∗∗

+ AHM-EA-G (2 mg/ml/kg, n=5) + 0 B hCG (5 IU/ml/kg, n=5)

+ hCG + AHM-EA-G (2 mg/ml/kg, n=5) 3

2

Plasma Estradiol (ng/ml) ∗ 1 ∗ + ∗ ∗ ∗ ∗ ∗ ∗ 0 + 0 60 120 180 240 0 60 120 180 240 Time after injection (min)

Fig 2A and 2B (Hsia, et al.)

36

Vehicle 8-Br-cAMP (10-4 M) hCG (0.5 IU/ml) Forskolin (10-5 M)

4 A (n=6) 4 B (n=6) 4 C (n=6) 2 2 2 ∗

∗ ∗∗ ∗∗ 0 0 0

4 4 + 4 + + + + + + + + + + + + 3 3 ∗ ∗∗ 3 ∗ ∗∗ 2 ∗∗ 2 2 cells/2 h) ∗∗ 5 1 1 1 0 0 0 8 8 8 ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ 6 6 ∗∗ 6 ∗∗ ++ ++ ∗∗ + ∗ ∗ ++ ∗

4 ∗∗ 4 4 ∗∗ ∗∗ 2 2 2

0 0 0 10 10 10 ++ ++ ++ ++ ++ 8 8 8 ++ ++ ++ ++ ++ ++ ++ 6 6 6 ∗∗ ++ + ∗∗ + Progesterone Release (ng/1x10 4 4 4 ∗∗

2 2 ∗∗ 2

0 0 0

0 0.1 1 10 100 0 0.1 1 10 100 0 0.1 1 10 100 AHM (μg/ml) AHM-EA (μg/ml) AHM-EA-G (μg/ml)

Fig 3A, 3B and 3C (Hsia, et al.)

37 2 Vehicle (n=6)

1 ∗∗

cells/ h) 0 5 40 hCG (0.5 IU/ml, n=6) Forskolin (10-5 M, n=6) 20 cells/ h)

18 ++ 5 30

16

14 ++ 20 ++

cAMP Accumulation cAMP (pmole/1x10 0 12 ∗∗ 0 100 AHM-EA-G (μg/ml) 10 ++ 8 ∗∗

6 cAMP Accumulation (pmole/1x10

4

2

0 0 1 10 100 AHM-EA-G (μg/ml)

Fig 4 (Hsia, et al.)

38

Fig 5 (Hsia, et al.)

39

Fig 6 (Hsia, et al.)

40

Vehicle (n=3) Forskolin (10-5 M, n=3) 4

3 + 2

P450/L19 ∗ 1 ∗

0

5 ++

4

3

StAR/L19 2

1 ∗ ∗∗ 0 0100 AHM-EA-G (μg/ml)

Fig 7 (Hsia, et al.)

41

Fig 8 (Hsia, et al.)

42

0.4 AHM (n=6) 0.3

0.2 ∗ ∗∗

0.1 ∗∗

0.0 AHM-EA (n=6) 0.3 cells/2 h) 0.2 ∗∗ 5

0.1 ∗∗

0.0 AHM-EA-F (n=6) 0.3 ∗∗ 0.2 ∗∗

0.1 ∗∗ Estradiol Release (ng/1x10 0.0 AHM-EA-G (n=6) 0.3

0.2

0.1 ∗∗

0.0 0 1 10 100 Methanolic Extract of Adlay Hull (μg/ml)

Fig 9 (Hsia, et al.)

43

Fig 10 (Hsia, et al.)

44 Reference List

[1] Li S.C. Pen-t's sao kang mu (Systematic Pharmacopoeia). In. China: 1596.

[2] Tanimura A. Studies on the anti-tumor component in the seeds of Coix lachryma-jobi L. var. ma-yuen (Roman.) Stapf. The structure of coixenolide. Chem Pharm Bull (Tokyo) 1961; 9: 47-53.

[3] Otsuka H, Hirai Y, Nagao T, Yamasaki K. Anti-inflammatory activity of benzoxazinoids from roots of Coix lachryma-jobi var. ma-yuen. J Nat Prod 1988; 51: 74-79.

[4] Takahashi M, Konno C, Hikino H. Isolation and hypoglycemic activity of coixans A, B and C, glycans of Coix lachryma-jobi var. ma-yuen seeds. Planta Med 1986; 64-65.

[5] Park Y, Suzuki H, Lee YS, Hayakawa S, Wada S. Effect of coix on plasma, liver, and fecal lipid components in the rat fed on lard- or soybean oil-cholesterol diet. Biochem Med Metab Biol 1988; 39: 11-17.

[6] Hidaka Y, Kaneda T, Amino N, Miyai K. Chinese medicine, Coix seeds increase peripheral cytotoxic T and NK cells. Biotherapy 1992; 5: 201-203.

[7] Check JB, K'Ombut FO. The effect on fibrinolytic system of blood plasma of Wister rats after feeding them with Coix mixed diet. East Afr Med J 1995; 72: 51-55.

[8] Otsuka H, Hirai Y, Nagao T, Yamasaki K. Anti-inflammatory activity of benzoxazinoids from roots of Coix lachryma-jobi var. ma-yuen. J Nat Prod 1988; 51: 74-79.

[9] Kondo Y, Nakajima K, Nozoe S, Suzuki S. Isolation of ovulatory-active substances from crops of Job's tears (Coix lacryma-jobi L. var. ma-yuen STAPF.). Chem Pharm Bull (Tokyo) 1988; 36: 3147-3152.

[10] Kuo CC, Chiang W, Liu GP, Chien YL, Chang JY, Lee CK, Lo JM, Huang SL, Shih MC, Kuo YH. 2,2'-Diphenyl-1-picrylhydrazyl radical-scavenging active components from adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) hulls. J Agric Food Chem 2002; 50: 5850-5855.

[11] Kuo CC, Shih MC, Kuo YH, Chiang W. Antagonism of free-radical-induced damage of adlay seed and its antiproliferative effect in human histolytic

45 lymphoma U937 monocytic cells. J Agric Food Chem 2001; 49: 1564-1570.

[ 12] Chiang W, Cheng C, Chiang M, Chung KT. Effects of dehulled adlay on the culture count of some microbiota and their metabolism in the gastrointestinal tract of rats. J Agric Food Chem 2000; 48: 829-832.

[13] Hsu HY, Lin BF, Lin JY, Kuo CC, Chiang W. Suppression of allergic reactions by dehulled adlay in association with the balance of TH1/TH2 cell responses. J Agric Food Chem 2003; 51: 3763-3769.

[14] Hung WC, Chang HC. Methanolic extract of adlay seed suppresses COX-2 expression of human lung cancer cells via inhibition of gene transcription. J Agric Food Chem 2003; 51: 7333-7337.

[15] Lin H, Tsai SC, Chen JJ, Chiao YC, Wang SW, Wang GJ, Chen CF, Wang PS. Effects of evodiamine on the secretion of testosterone in rat testicular interstitial cells. Metabolism 1999; 48: 1532-1535.

[16] Tsai SC, Chiao YC, Lu CC, Wang PS. Stimulation of the secretion of luteinizing hormone by ginsenoside-Rb1 in male rats. Chin J Physiol 2003; 46: 1-7.

[17] Chen JJ, Wang PS, Chien EJ, Wang SW. Direct inhibitory effect of digitalis on progesterone release from rat granulosa cells. Br J Pharmacol 2001; 132: 1761-1768.

[18] Wang SW, Pu HF, Kan SF, Tseng CI, Lo MJ, Wang PS. Inhibitory effects of digoxin and digitoxin on corticosterone production in rat zona fasciculata-reticularis cells. Br J Pharmacol 2004; 142: 1123-1130.

[19] Wang SW, Lin H, Tsai SC, Hwu CM, Chiao YC, Lu CC, Chen JJ, Wang GJ, Chou CJ, Lin LC, Chen CF, Wang PS. Effects of methanol extract of Chansu on hypothalamic-pituitary-testis function in rats. Metabolism 1998; 47: 1211-1216.

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