Molecular screening of Chinese medicinal plants for progestogenic and anti-progestogenic activity HM MANIR AHMED1 , JAN-YING YEH2 , YI-CHIA TANG3 , WINSTON TENG-KUEI CHENG1,4 and BOR-RUNG OU1,4,* 1
Department of Animal Science and Biotechnology, 3Department of Food Science, and Tunghai Green Energy Development and Management Institute (TGEI), Tunghai University, No. 1727, Sec. 4, Taiwan Boulevard, Xitun District, Taichung City 40704, Taiwan 2 Department of Biotechnology, Asia University, 500, Lioufeng Rd., Wufeng, Taichung 41354, Taiwan 4
*Corresponding author (Fax, +886-4-23590385; Email,
[email protected])
Estrogen and progestins have adverse effects, and many of these adverse effects are caused by progestins. Due to this, many women choose to use botanical alternatives for hormone replacement therapy, which does not trigger steroidogenic properties. Therefore, it is necessary to screen these herbs for progestogenic and anti-progestogenic properties. Extract of 13 Chinese medicinal plants were analysed for progestogenic and anti-progestogenic activities by using progesterone response element-driven luciferase reporter gene bioassay. MTT assay was carried out to investigate the cytotoxic effect of herb extract on PAE cells. Among the 13 herbs, Dipsacus asperoides extract exhibited progestogenic activity, and 10 species – Cortex eucommiae, Folium artemisiae argyi, Glycyrrhiza uralensis, Angelica sinensis, Atractylodes macrocephala koidz, Scutellaria baicalensis, Cuscuta chinensis, Euscaphis japonica, Ailanthus altissima, and Dioscorea opposita – were recognized to have anti-progestogenic like activities. Extract of Dipsacus asperoides demonstrated dose-dependent progestogenic activity, and the progestogenic activity of 100 μg/mL extracts was equivalent to 31.45 ng/mL progesterone activity. Herbs extracts that exhibited anti-progestogenic-like activity also inhibited the 314.46 ng/mL progesterone activity in a dose-response manner. None of the herb extracts shown significant toxic effect on PAE cells at 40–100 μg/mL compared to control. This discovery will aid selection of suitable herbs for hormone replacement therapy. [Ahmed HMM, Yeh J-Y, Tang Y-C, Cheng WT-K and Ou B-R 2014 Molecular screening of Chinese medicinal plants for progestogenic and antiprogestogenic activity. J. Biosci. 39 453–461] DOI 10.1007/s12038-014-9434-z
1.
Introduction
Progesterone is essential for mammalian reproduction. Progesterone and estrogen act on the central nervous system, ovary and uterus, and are important for ovulation, fertilization, implantation of embryo, maintenance of pregnancy, mammary gland development and lactation (Lydon et al. 1995 ; Graham and Clarke 1997). Natural progesterone is often unavailable, is poorly active orally and has short half-life (Stanczyk 2003). The poor
Keywords.
pharmacological profile of natural progesterone leads to develop synthetic compounds, progestins that are biologically similar to progesterone, comparatively resistant to enzymatic degradation, have long half-life and are orally active (Stanczyk 2003). Synthetic progestins have wide range of uses, especially for the negative feedback of progesterone on the hypothalamic hyperphysical axis: progestins are used in oral contraceptives in women (Cogliano et al. 2005) and estrus synchronizing drug (SáFilho et al. 2010) as well as for growth promoter
Anti-progesterone; bioassay; medicinal plants; progesterone
http://www.ias.ac.in/jbiosci
Published online: 1 May 2014
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(Kreikemeier and Mader 2004) in the animal industry. Progesterone is also used for the treatment of endometrial hyperplasia (Hammond and Johnson 2004), endometrial cancer (Yang et al. 2011), premenstrual headaches, depression (Dickerson et al. 2003), body fluid retention (Stachenfeld 2008), mystodynia (Winkler et al. 2001), endometriosis (Banno et al. 2012), luteal phage defects and luteal support to assist in reproduction cycles for infertile patients (Kaul et al. 1995). Main sources of progesterone are ovaries that secrete functional levels of progesterone in a periodic manner up to a certain age. At menopause, estrogen and progesterone suddenly drop. During this period, some symptoms such as hot flashes, decreased vaginal lubricant, thin vaginal mucosa, insomnia, osteoporosis, urinary problems, vaginal irritation and painful intercourse are prompted by the decline of estrogen and progesterone levels (Palace et al. 1999; Cheung et al. 2004; Tong 2013). For preventing menopause symptoms, estrogen and progestins are commonly used in hormone replacement therapy (Cogliano et al. 2005). Use of synthetic progesterone along with estrogen generates some adverse effects (Turgeon et al. 2004; Jaakkola et al. 2010). The risks of coronary heart diseases, breast cancer and pulmonary embolism are higher when progestins and estrogen are used together in hormone replacement therapy (Rossouw et al. 2002; Rohan et al. 2008; Shufelt and Merz 2009). These adverse effects are due to progestagen (Freeman et al. 1993). Mifepristone (RU486) is pure anti-progesterone in McPhail test, but it also exhibits progesterone receptor agonist activity (Klein-Hitpass et al. 1991). Mifepristone is extensively used in gynecology, specifically for termination of early pregnancy, menstrual regulation, fetal death management and emergency contraception (Spitz 2003). RU486 is also recommended for treatment of Cushing diseases, glaucoma, viral diseases, hormonedeepened tumour, uterine myom, and endometriosis (Spitz and Bardin 1993; Hazra and Pore 2001; Spitz 2003). RU486 has some unwanted side effects, such as heavy bleeding in early pregnancy termination cases, nausea, vomiting, abdominal pain, and fatigue and skin rashes (Lamberts et al. 1992; Hazra and Pore 2001). Under these circumstances, women are not motivated to continuously use of synthetic progesterone (Hillman et al. 2004) and anti-progesterone. Instead, many women are using botanical extracts as alternatives to synthetic steroid hormone replacement therapy (Beck et al. 2005). Most of the Chinese medicinal plants have been screened for phytoestrogen, but research and literature on the screening of herbs for progesterone and antiprogesterone are few. The objective of this study was
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to screen the extracts of Chinese herbs for progestogenic and anti-progestogenic activities. 2.
Materials and methods 2.1
Materials
Progesterone (P), mifepristone (RU486), 3-(4,5-dimethyl-2yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Alrich (St. Louis, MO, USA). Lipofectamine TM 2000, Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), trypsin/ ethylenediaminetetraacetic acid (EDTA), penicillin/streptomycin, and sodium pyruvate were bought from Invitrogen (Carlsbad, NY, USA). Progesterone responsive reporter plasmid pGL3-2PRE-TATA was previously developed in our laboratory as described previously by Ahmed et al. (2014). The pCH110 control vector, luciferase enzyme assay system, β-galactosidase enzyme assay system, and restriction enzymes were purchased from Promega (Madison, WI, USA). Bradford protein assay kit was bought from Bio-Rad (Hercules, CA, USA). T47D human mammary adenocarcinoma cell line was obtained from American Type Culture Collection (ATCC) (Manassas, Virginia, USA) and porcine aortic endothelial cell (PAEC) was previously developed in our lab as described by Yeh et al. (2002). 2.2
Herbal extraction
All plant materials (table 1) were obtained from Sun-Ten Pharmaceutical Co. Ltd. (Taichung, Taiwan, ROC). The voucher specimens were deposited in the Pharmaceutical Industry Technology and Development Center, Taiwan. Ten gram plant materials were grinded and soaked with 100% ethanol for 72 h at RT. The extracts were filtered and kept in a hood for rapid evaporation of ethanol. Dried extracts were diluted to 40 mg/mL in 100% ethanol as stock and kept in −80°C. Stock solutions were mixed with 10% charcoal stripped FBS fortified phenol red free DMEM or MI99 medium at desired concentrations (10, 20, 40 and 100 μg/mL) by keeping final concentration of ethanol 0.1% of medium. 2.3
Sample preparation
Progesterone, anti-progesterone (RU486) were diluted at concentration 314.46 and 429.60 μg/mL in 100% ethanol as a stock and kept in −20°C. Stock solutions were mixed with 10% charcoal stripped FBS fortified phenol red free DMEM or MI99 medium at desired concentrations by keeping final concentration of ethanol 0.1% of medium. To use as vehicle control, 10% charcoal stripped FBS supplemented
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Table 1. List of Chinese medicinal plants used for screening of progestogenic and anti-progestogenic activity Species (plant family)
Plant part
Extraction yield (%) (dry weight)
Dipsacus asperoides C.Y. Cheng et T.M.Ai. (Dipsacaceae ) Cortex eucommiae Oliv. (Eucommiaceae) Folium artemisiae argyi Levl. et Vant. (Asteraceae) Pueraria mirifica Airy Shaw et. Suvatab. (Fabaceae) Cuscuta chinensis L. (Convolvulaceae) Glycyrrhiza uralensis Fisch. (Leguminosae) Euscaphis japonica Thunb. (Staphyleaceae) Ailanthus altissima (Mill.) Swingle (Simaroubaceae) Dioscorea opposita Thunb. (Dioscoreaceae) Angelica sinensis (Oliv) Diels (Apiaceae) Atractylodes macrocephala koidz DC. (Asteraceae) Perilla frutescens (L.) Britton. (Lamiaceae) Scutellaria baicalensis Georgi. (Lamiaceae)
Root Bark Leaf Root Seed Root Stem Stem Root Root Stem Stem Root
3.6 4.4 9.6 5.0 2.0 4.2 7.6 8.4 7.0 9.0 8.2 9.0 5.2
phenol red free DMEM or MI99 medium was fortified with 0.1% ethanol.
2.4
Cell culture
T47D, the progesterone receptor-positive human mammary adenocarcinoma cell line, was routinely cultured in DMEM supplemented with 10% FBS, 1% P/S and 1% 100 mM sodium pyruvate. Porcine aortic endothelial cell (PAEC) was maintained as described previously by Yeh et al. (2002). All cells were cultured in a humidified incubator of 5% CO2 and 37ºC.
2.5
Cell transfection and luciferase assay
2.6
Cytotoxicity study
The cytotoxicity of herbal extracts was investigated in PEC cells by MTT assay. The cells were plated in 96-well plates at 1×104 cells/well. After 24 h, the cells were treated with ethanolic herbal extract, and treatment of 0.1% ethanol was the negative control. After 24 h of treatments, 10 μL of MTT reagent (final concentration 0.5 mg/mL) was added to each well and incubated for 4 h to form purple formazan crystals. After 4 h, 100 μL solubilized solution (10% SDS in 0.01 M HCl) was added into each well and incubated overnight to dissolve the purple formazan crystals. Spectrophotometric absorbance of the formazan products was measured with 595 nm wavelength. The cytotoxicity of effects was expressed as the percentage of cell survival as follows: Cell viabilityð%Þ ¼ ðAbsorbance of the treated group= Absorbance of the vehicle control groupÞ 100:
Two days before transfection, T47D cells were plated at 2.5×105 cells per well into six-well plates with DMEM containing 2% charcoal-stripped FBS and incubated for 48 h. Cells were co-transfected with 2 μg of pGL32PRE-TATA and 1 μg of pCH110 control vector using Lipofectamine TM 2000, according to the manufacturer’s protocol. After 6 hr of transfection, cells were treated with different concentrations of progesterone, antiprogesterone or herbal extracts. Twenty-four hr later, the cells were lysed and luciferase and β-galactosidase activities were determined using luciferase and βgalactosidase assay kits, respectively, according to the manufacturer’s instructions. Protein contents were determined by Bradford protein assay kit. Luciferase activity was normalized by β-galactosidase activity and protein content.
2.7
Data analysis
Progestogenic or anti-progestogenic activities were determined by normalizing the luciferase activities for different treatments (progesterone, anti-progesterone, herbal extracts) with vehicle control. Thus, the absolute effect of components or dried-extract was observed. All data were summarized from at least three independent experiments with triplicates. Data were analysed by using Statgraphics software (STATGRAPHICS Centurion XVI, Warrenton, VA, USA). Each value is presented as the mean ± SEM. Mean values were compared by analysis of variance (ANOVA) with Fisher’s LSD tests for comparing group. A Student’s t-test was used to evaluate the differences between the groups. A significant level of 0.05 was adopted.
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Figure 1. Progestogenic activity of Chinese herb extracts. T47D cells were treated with 40 μg/mL ethanolic herbal extracts of herb. Progesterone 31.45 ng/mL was used as positive control and 0.1% alcohol used as control. Progestogenic activities (for absolute amount components or dried extract) were determined by normalizing the luciferase activities for different treatments (progesterone and herbal extracts) with vehicle control (ethanol). Data are represented as the mean-fold induction compared to vehicle control ± SEM. Vehicle control (C), Progesterone (P), Dipsacus asperoides (DA), Cortex eucommiae (CE), Folium artemisiae (FA), Pueraria mirifica (PM), Cuscuta chinensis (CC), Glycyrrihiza uralensis (GU), Euscuaphilis japonica (EJ), Ailanthus altissima (AA), Dioscorea opposit (DO), Angelica sinensis (AS), Atractylodes macrocephala koidz (AM), Parilla frutescens (PF), Scutellaria baicalensis (SB). Differences (P
