A novel mechanism: Erxian Decoction, a Chinese medicine formula, for relieving menopausal syndrome

Journal of Ethnopharmacology 123 (2009) 27–33

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A novel mechanism: Erxian Decoction, a Chinese medicine formula, for relieving menopausal syndrome S.C.W. Sze a , Y. Tong a,∗ , Y.B. Zhang a , Z.J. Zhang a , A.S.L. Lau b , H.K. Wong a , K.W. Tsang a , T.B. Ng c a b c

School of Chinese Medicine, LKS Faculty of Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong, China Molecular Chinese Medicine Laboratory, LKS Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China

a r t i c l e

i n f o

Article history: Received 21 November 2008 Received in revised form 22 January 2009 Accepted 14 February 2009 Available online 4 March 2009 Keywords: Antioxidant Chinese medicine Endocrine function Erxian Decoction Menopause

a b s t r a c t Ethnopharmacological relevance: Many clinical and experimental reports demonstrated that Erxian Decoction (EXD) was effective in relieving menopausal syndrome. Aim of the study: The mechanisms of action of EXD were explored on the endocrine and antioxidant regimen. Materials and methods: Menopause causes a decline in both endocrine function and activities of antioxidant enzymes. In this study, 12-month-old female Sprague–Dawley-rats (SD-rats) with a low serum estradiol level were employed. Their endocrine functions after treatment with EXD were assessed by the determination of their serum estradiol level and ovarian mRNA levels of aromatase, which is a key enzyme for biosynthesis of estradiol. Meanwhile, superoxide dismutase-1 (SOD), catalase (CAT) and glutathione peroxidase (GPx-1) in the liver were also determined to assess the effect of EXD on the antioxidant regimen. Results: Results revealed a significant elevation in serum estradiol level and the mRNA level of ovarian aromatase and liver CAT in the EXD-treated menopausal rat model. Conclusions: The results obtained from mRNA and estradiol level of the present investigation revealed that the EXD relieves the menopausal syndrome involved an increase of endocrine and antioxidant function through, at least, the activation of aromatase and CAT detoxifying pathways. © 2009 Elsevier Ireland Ltd. All rights reserved.

1. Introduction There were more than 477 million menopausal women in the world in 1998, and the number expected to reach 1.1 billion by 2025 (Uhl, 2008). Menopause is a process of normal aging, during which the level of estradiol secreted by the ovaries gradually declines, and menopausal women therefore have irregular menses and much higher incidences of physiological and mental changes, which severely disrupt their quality of life (Burger et al., 2007). The solution to overcome menopausal symptoms seems simple—hormone replacement therapy. Although the therapy has been widely prac-

Abbreviations: AAS, atomic absorption spectrophotometer; CAT, catalase, cDNA; Ct , threshold cycle; EXD, Erxian Decoction; FSH, follicle-stimulating hormone; GPx-1, peroxidase; HPLC, high performance liquid chromatography; HPLC-DAD, high-performance liquid chromatography with photodiode array detection; mRNA, messenger ribonucleic acid; RT-PCR, reverse-transcribed-polymerase chain reaction; RNA, ribonucleic acid; PCR, polymerase chain reaction; SD-rats, Sprague–Dawley-rats; SOD, superoxide dismutase-1; SDS, sodium dodecyl sulfate; UV, ultra violet. ∗ Corresponding author. Tel.: +852 25890436; fax: +852 28725476. E-mail address: [email protected] (Y. Tong). 0378-8741/$ – see front matter © 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2009.02.034

tised for the past 50 years, it increases the risk of breast cancer and cardiovascular disease etc. (Ross et al., 2000; Vera and Rada, 2002). Therefore, it is desirable and urgent to explore the use of alternative medicine, especially Chinese medicine (Newton et al., 2002; Low Dog, 2005; Zhang et al., 2005). Erxian Decoction (EXD), a popular Chinese medicine formula, consists of six Chinese medicinal herbs, Curculigo orchioides (Hypoxidaceae, rhizome), Epimedium brevicornum (Berberidaceae, whole herb), Morinda officinalis How (Rubiaceae, root), Angelica sinensis (Umbelliferae, root), Phellodendron chinense (Rutaceae, bark) and Anemarrhena asphodeloides (Anthericaceae, rhizome) (Ban, 2005). It has been clinically used in treatment of menopausal syndrome for more than 50 years. Nian et al. (2006) and Qin et al. (2008) report that EXD and its chemical constituents possess antiosteoporotic activity in ovariectomized rats and it can increase the estradiol level in serum. Many clinical reports demonstrated that EXD was effective in relieving menopausal syndrome via increasing the circulatory estradiol level (Li et al., 2007). Our recent systematic review also indicated that the efficacy of EXD was better than other forms of non-hormone replacement therapy, while there was no significant difference between the efficacy of EXD and hormone replacement therapy groups (Chen et al., 2008).

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However, the mechanism of action of EXD on stimulation of estradiol secretion for relieving menopause has not been reported so far. Estradiol is bio-synthesized in ovarian granulosa cells. Ovarian aromatase Cyp19 is a critical enzyme for estradiol bio-synthesis (Simpson et al., 1994). In menopause, although the circulating level of FSH remains high for several years, it does not induce an increase of circulating estradiol level. Interestingly, previous studies showed that the estradiol level increased after administration of EXD (Fang et al., 1992; Nian et al., 2006; Chen et al., 2008). Along with aging and menopause, the activities of steriodogenic enzyme, aromatase, and antioxidant enzymes are also lowered (Okatani et al., 1993). We therefore hypothesize that EXD is effective in relieving menopausal syndrome through increasing the activation of aromatase and antioxidant enzymes. In this study, we propose to: (1) evaluate the steriodogenic activity of EXD by measuring the estradiol level in serum and mRNA level of aromatase in ovary and (2) evaluate the antioxidant activity of EXD by measuring the mRNA level of antioxidant enzymes, including superoxide dismutase-1 (SOD), catalase (CAT) and glutathione peroxidase (GPx-1), in liver. Besides, the levels of main elements with reproductive toxicity, including arsenic (Chattopadhyay et al., 1999), cadmium (Nagata et al., 2005) and lead (WHO, 1995), were assessed using atomic absorption spectrophotometer (AAS) to ensure the safety of EXD extracts. A simple, rapid and valid chromatographic fingerprint with marker standards was developed using high-performance liquid chromatography with photodiode array detection (HPLC-DAD) for assessing quality consistency among the EXD extracts.

are well-known compounds in EXD (Ban, 2005), were employed and compared to the fingerprint of EXD. Reproducibility and linearity were estimated by performing repetitive injections. The external standard method using a series of mixed standard solutions with concentrations ranging from 2.25 to 60 ␮g/ml was examined. A reversed-phase column (XBridge® C18 , 5 ␮m, 250 mm × 4.6 mm i.d., Thermo, USA) was used and the mobile phase consisted of acetonitrile (A) and 0.05% sodium dodecyl sulfate (SDS) in 0.1% acetic acid (B) using gradient program of 95–70% (B) in 0–30 min, 70–70% (B) in 30–35 min, 70–50% (B) in 35–50 min, 50–45% (B) in 50–65 min. The flow rate was 1.0 ml/min. DAD detector was set at 345 nm for obtaining chromatograms with maximum number of peaks. UV spectra were acquired from 200 to 400 nm. Chromatogram and peak integration were analyzed by the software of Millennium32 Chromatography Manager Version 3.2. 2.3. Animals Twelve-month-old female Sprague–Dawley (SD)-rats with a low serum estradiol level were employed as a menopausal animal model in this study. Female SD-rats, aged 8 months, were purchased from the Animal Laboratory Unit, the University of Hong Kong. The animals were housed in an air-conditioned room at an ambient temperature of 24 ◦ C and 50–65% relative humidity with automatic 12 h light:dark illumination cycles. They were acclimated for 4 months and their serum estradiol level monitored before the experiment. The experiment was approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of Li Ka Shing Faculty of Medicine, the University of Hong Kong.

2. Materials and methods 2.4. Drug administration, sera and organs collections 2.1. Herbal materials The six plant materials, Curculigo orchioides, Epimedium brevicornum, Morinda officinalis How, Angelica sinensis, Phellodendron chinense and Anemarrhena asphodeloides (composition ratio: 12:12:10:10:9:9) (voucher numbers. CME140706-03, CME150906-11, CME050406-02, CME090706-12, CME100606-09 and CME180806-01) were collected from various sources and their identity was confirmed by Professor Zhengtao Wang, Department of Pharmacognosy, China Pharmaceutical University and voucher specimens were kept in the China Pharmaceutical University.

Rats were randomly divided into four groups of 10 animals each. Chinese medicine EXD extract (0.76 and 1.52 g/kg) and Western medicine Premarin capsule (31.25 mg/kg) (each capsule containing 0.3 mg of estrogen) were administered orally everyday for 6 weeks. The control group received orally an equal volume of water instead of EXD. At the end of the experiment, the rats were anesthetized by an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg) dissolved in 0.9% saline. Their sera, ovaries and livers were collected and stored at −80 ◦ C for further analysis. 2.5. Detection of serum estradiol level

2.2. Preparation of EXD and quality analysis One kilogram of the six medicinal materials in the mixture was extracted separately by decocting it with distilled water, 10:1 (v/w) at 100 ◦ C for 1 h. The extraction was repeated twice. The filtrates were lyophilized in a freeze drier (Labconco, Freezone) and kept at 4 ◦ C for quality control and molecular studies. The yield of dried extract from the starting crude materials was 20%. For detection of toxic elements in the EXD extract, all standards, including arsenic, cadmium and lead, were diluted to working concentrations ranging from 3.125 to 200 ppb in milli-Q water. EXD powder (1.5 g) was burnt to ashes by flame for 30 min. This was followed by addition of 4 ml 70% nitric acid for dissolving the metal constituents in the samples for 30 min, dilution to 100 ml, and filtration for AAS furnace analysis (Chi et al., 1993). For evaluation of the quality consistency among EXD extracts, three batches of EXD extracts were weighed and extracted with 10 ml of methanol in a water bath at 60 ◦ C for 15 min, followed by ultrasonication for 30 min. After centrifugation, the supernatant was filtered by a 0.45 ␮m Millex® Syringe filter unit, and then injected in a volume of 10 ␮l in HPLC. Five standard chemicals of jatorrhizine, berberine, palmatine, ferulic acid and icariine, which

The estradiol level in serum samples was determined using the electro-chemiluminescence immunoassay (Elecsys 1010; Roche Diagnostics), following the manufacturer’s instruction. 2.6. RNA isolation Total RNA was isolated from the ovary and liver using the TRIZOL® Reagent according to the manufacturer’s instructions (Invitrogen Life Technologies). The purity and concentration of RNA were determined by measuring the relative absorbance at 260/280 nm and the absorbance at 260 nm, respectively. 2.7. Quantitative real-time RT-PCR One microgram of total RNA was reverse-transcribed to cDNA using random hexamers (Promega) and reverse transcriptase II (Invitrogen Life Technologies) following the manufacturer’s instructions. Real-time PCR was performed for quantification of expression of aromatase, SOD, CAT and GPx-1 mRNAs using the Platinum® Quantitative PCR SuperMix-UDG (Invitrogen Life Technologies) in a final reaction volume of 25 ␮l in 0.25 X SYBR green

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Table 1 Sequences of the PCR primers of target genes. Gene

Sequence

Size of PCR products 



Aromatase Cyp19

Forward primer Reverse primer

5 -GCCTGTCGTGGACTTGGTCAT-3 5 -GGGTAAATTCATTGGGCTTGG-3

143-bp

SOD

Forward primer Reverse primer

5 -TGGGTTCCATGTCCATCAATA-3 5 -TTCCAGCATTTCCAGTCTTTGT-3

296-bp

CAT

Forward primer Reverse primer

5 -GTCACTCAGGTGCGGACATTC-3 5 -TCTTAGGCTTCTGGGAGTTGT-3

202-bp

GPx-1

Forward primer Reverse primer

5 -AGGAGAATGGCAAGAATGAAGA-3 5 -AGGAAGGTAAAGAGCGGGTGA-3

136-bp

␤-Actin

Forward primer Reverse primer

5 -CCTCTATGCCAACACAGTGC-3 5 -ATACTCCTGCTTGCTGATCC-3

211-bp

Table 2 Analysis of main toxic elements analysis in EXD extracts using AAS. Mean values ± S.D. and relative standard deviation (R.S.D., %), n = 3. Heavy metals in EXD

EXD (ppm or ␮g/g) (mean ± S.D.) (%R.S.D.)

Calibration correlation coeff. (%)

Arsenic Cadmium Lead

0.0552 ± 0.0063a (11.4130%) 0.0400 ± 0.0087a (21.7500%) −0.2020 ± 0.0406a (20.0990%)

99.68 99.91 99.95

Safety standard set by Hong Kong Department of Health (ppm) <2.0 <0.3 <5.0

a The individual toxic element content in EXD extracts is below the safety standard set by the Department of Health of the Government of Hong Kong Special Administrative Region.

(Molecular Probes) according to the manufacturer’s protocol. The sequences of the PCR primers are shown in Table 1. A series of 10-fold dilution of all PCR products were prepared to generate a standard curve (plot of Ct values/crossing points of different standard dilutions against the log of the amount of standard). The data were expressed as the number of threshold cycle (Ct ). The following thermocycler program was used for real-time PCR: preincubation at 94 ◦ C for 15 min, followed by 40 cycles of incubation at 94 ◦ C for 20 s, 57 ◦ C for 20 s, and 72 ◦ C for 20 s. Following the amplification process, a melting curve analysis was performed by raising the temperature from 72 to 95 ◦ C gradually, 1 ◦ C per 5 s to ensure that the specific products were also amplified. The PCR products were also verified by ethidium bromide-stained agarose gel electrophoresis. For each real-time PCR sample was performed for target gene and housekeeping gene (␤-actin). The relative quantification of the target gene was determined by calculating the

ratio between the concentration of the target gene and that of the housekeeping gene. For each real-time PCR analysis, the individual experiments were performed in triplicate. 2.8. Calculation of copy number and statistical analysis The software installed in the PCR instrument was used to calculate the standard curve for each run based on the Ct for each standard. Based on these values, a linear regression line was plotted and the resulting equation was used to calculate the log starting quantity and copy number for the unknown samples. Data are reported as the mean ± standard deviation. Comparisons for statistical significance were made with one-way ANOVA. A level of P < 0.05 was considered to be statistically significant. Calculations were made with the commercially available software SPSS.

Fig. 1. Overlaid HPLC chromatographic fingerprints of three batches of EXD extracts monitored at a 345 nm. Five standard chemicals of ferulic acid, icariine, jatorrhizine, palmatine and berberine, and their respective retention times were shown.

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Table 3 The contents of five standard chemicals in EXD extracts. EXD batch no.

Berberine (␮g/mg)

Ferulic acid (␮g/mg)

Icariine (␮g/mg)

Jatorrhizine (␮g/mg)

Palmatine (␮g/mg)

EXD 1 EXD 2 EXD 3

1.429 1.505 1.534

0.557 0.507 0.519

1.671 1.688 1.746

0.077 0.079 0.082

0.892 0.940 0.960

Mean

1.489

0.528

1.702

0.079

0.931

S.D. R.S.D.

0.054 3.641

0.026 4.947

0.039 2.311

0.003 3.172

0.035 3.755

3. Results:

Table 4 Effect of EXD on serum estradiol level (n = 10, mean ± S.D.).

3.1. The quality control

Groups

Estradiol level (pg/ml)

Young rat group (3-month-old SD-rat) Control group (12-month-old SD-rat) EXD-treated group (0.76 g/kg) EXD-treated group (1.52 g/kg)

225.6 58.9 93.2 170.2

The contents of main toxic elements, including arsenic, cadmium and lead, were lower in EXD formulations compared with the standard in the Department of Health of the Government of Hong Kong Special Administrative Region. The results are summarized in Table 2. For exploring most of detectable peaks in the HPLC chromatogram, the spectra of all eluted peaks found in the chromatogram of EXD were investigated with photodiode array detection. The chromatograms were generated under the detection wavelength of 345 nm. A chromatographic fingerprint showing the elution peaks of five standard compounds and other common peaks are shown in Fig. 1. Three batches of EXD preparations were examined by HPLC using the optimum running conditions. The results of their contents are shown in Table 3. Inter-assay relative standard deviation (R.S.D.) values were less than 5%.

## * **

± ± ± ±

73.6 21.2## 33.5* 54.6**

P < 0.01 compared with young rat group. P < 0.05. P < 0.01 compared with old rat group.

3.2. Effect of estradiol level in sera after EXD treatment Results of serum estradiol level in rats of different groups are shown in Table 4. A significant reduction in serum estradiol level in 12-month-old female SD-rat when compared with 3-month-old female SD-rat (## P < 0.01) was found. EXD (1.52 and 0.76 g/kg) treatment improved the sera estradiol level compared with the control group (*P < 0.05, **P < 0.01).

Fig. 2. Effects of EXD (EXD-L: 0.76 g/kg; EXD-H: 1.52 g/kg) and Premarin capsule (P: 0.03125 g/kg) on mRNA level of (A) ovarian aromatase, (B) liver SOD, (C) liver GPx-1 and (D) liver CAT of 12-month-old female SD-rats (n = 10). Data denoted are means ± S.D., n = 10, *P < 0.05 and **P < 0.01 compared with control by one-way ANOVA.

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3.3. Effects on gene expression levels of aromatase, SOD, CAT and GPx-1 The mRNA level of ovarian aromatase was increased 10-fold and 5-fold in EXD-treated (1.76 g/kg) group and Premarin-treated group, respectively, compared with the control group (*P < 0.01). EXD treatment up-regulated the mRNA level of ovarian aromatase significantly in a dose-dependent manner (Fig. 2A). Meanwhile, the mRNA level of liver CAT was only significantly increased in EXD-treated (1.52 g/kg) group compared with the control group (*P < 0.05) (Fig. 2D). However, the mRNA level of SOD (Fig. 2B) and GPx-1 (Fig. 2C) in all treatment groups was not significantly different compared with the control group.

4. Discussion In this study, an atomic absorption spectrophotometer and a reversed-phase HPLC with a photodiode array detection assay were used to evaluate the main toxic elements and the quality consistency among different batches of EXD extracts respectively. The contents of the main reproductive toxic elements in Chinese herbs, including arsenic, cadmium and lead, have been assessed in this study. Usually, higher contents of these toxic elements in Chinese herbs may be attributed to the uptake of these elements from polluted soil due to industrial and antropogenic activities (Wang et al., 1996). Exposure to arsenic has been associated with reduction in serum estradiol and ovarian steroidogenesis (Chattopadhyay et al., 1999). Exposure to cadmium is associated with increased testosterone levels that have been associated with the risk of breast cancer in postmenopausal women (Nagata et al., 2005). Exposure to lead can adversely affect the reproductive system (WHO, 1995). Our results demonstrated that the levels of these three main reproductive toxic elements in the EXD extract were lower compared with the permitted level in the Department of Health of the Government of Hong Kong Special Administrative Region. This evaluation ensures the safety of our EXD preparation. Also, a novel, simple, accurate, reliable and reproducible method to evaluate the quality consistency of EXD among different batches was developed by using the five separated compounds jatorrhizine, berberine, palmatine, ferulic acid and icariine as markers in a single chromatographic run at the detection wavelength of 345 nm (Fig. 1). Ferulic acid, icariine and berberine possess estrogenic activity. Ferulic acid treatment slightly increases the serum levels of estrogen and increases the bone mineral density in ovariectomized female rats of the Sprague–Dawley strain (Sassa et al., 2003). Besides, it has been shown to be effective in treating hot flashes in menopausal women. (Philip, 2003). Icariine has been reported to possess an estrogenic effect (Ye and Lou, 2005). Berberine chloride has antiosteoporotic effects on ovariectomized rats by increasing the production of estradiol (Li et al., 2003; Qin et al., 2008). In addition, these five marker compounds possess antioxidant activities (Kim et al., 2000; Liu et al., 2004; Lucia et al., 2004; Barone et al., 2009). This chromatographic fingerprint with five marker compounds is used as a reference standard, indicating the purity, identity and quality consistency among the EXD extracts. Our result showed that the relative standard deviations of the amount of the five standards are less than 5% in our three batches of EXD (Table 3). This result indicated the quality consistency among different batches of EXD extracts as well as excluded the influence of any unknown variability or instability found in the composition of the active constituents in the molecular investigation of the EXD extract. EXD has been used clinically for half a century with proven clinical efficacy in human subject (Chen et al., 2008). Previous publications have also shown that the serum level of estradiol was

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significantly increased after EXD treatment in both menopausal woman and animal model (Zhang, 1965; Dong and Fang, 2003; Zhang, 2004; Dong et al., 2006; Nian et al., 2006). Our results also confirmed the previous findings and revealed that EXD (1.52 and 0.76 g/kg) treatment improved the serum estradiol level compared with that in the control group (Table 4). However, its underlying mechanism has not yet been elucidated. In the present study, the mechanism of action of EXD responsible for estradiol biosynthesis in the ovary has been elucidated by using 12-month-old female spontaneously aging SD-rats with low serum estradiol level (Table 4), which stop cycling in a manner comparable to menopausal women (Fortepiani et al., 2003). This animal model has been used to mimic reproductive failure in menopausal women due to the gradual decline of neuroendocrine status, including ovarian steroidogenesis as the physiological condition in rats is similar to human (Danilovich et al., 2002; Wu et al., 2005; Danilovich and Sairam, 2006). Reproductive aging has been studied in numerous vertebrate species. Although there are wide variations in reproductive strategies and hormone cycle components, many of the fundamental changes that occur during aging are similar. The level of estradiol in serum and function of ovarian steroidogensis decline in menopause (Kamiyama et al., 1992). Ovarian aromatase is the key steroidogenic enzyme responsible for estradiol biosynthesis. Its activity in menopausal women showed a sharp decline and a significant negative correlation with age (Uno, 1987; Purohit and Reed, 2002; Zimon et al., 2006). Interestingly, the mRNA level of aromatase and the estradiol level in serum were increased by 10-fold and 2.8-fold in EXD-treated (1.52 g/kg/day; EXD/rat) group, respectively, compared with the control group (Fig. 2A and Table 4). This finding allows us to elucidate that the molecular action of EXD on estrogen bio-synthesis entails, at least, the ovarian aromatase pathway. Results obtained from this study would shed new light on establish the mechanismdriven anti-menopausal screening system to test more effective drugs and active ingredients of EXD in the future. EXD treatment increases the ovarian steroidogensis machinery for promoting estradiol biosynthesis. It does not supply estradiol directly, thus the level of biosynthesis of estradiol in ovary can be regulated physiologically by a feedback mechanism that maintains a proper balance of hormones, and prevents excessive estradiol secretion. Higher levels of estradiol increase the risk of breast cancer. Hormone replacement therapy only supplies exogenous estradiol to menopausal women; its amount in menopausal women could not be regulated physiologically, therefore the major side effect is breast cancer after long-term treatment (DiamantiKandarakis, 2004). Moreover, the previous study supports that the mechanisms of normal aging in menopausal women are linked not only to estradiol loss but also to high levels of oxidative stress (Okatani et al., 1993; Droge, 2002, 2003). In addition, during aging, the activities of antioxidant enzymes will be reduced. Oxidation of non-saturated lipids and the increase of lipid peroxidation that destroy DNA (Ji et al., 1988). Most reactive oxygen is produced as the superoxide anion, which rapidly dismutates to hydrogen peroxide and oxygen by SOD (Ray and Husain, 2002). Hydrogen peroxide is detoxified by CAT to water and oxygen. GPx-1 also promotes the removal of hydrogen peroxide (Droge and Schipper, 2007). Antioxidant enzymes can detoxify these free radicals. In the present study, the most efficient enzymatic antioxidants, including SOD, CAT and GPx-1 in the major organ, liver, of the experimental animals were also detected to ascertain the effect of EXD on the antioxidant regimen. Our results demonstrated that EXD treatment significantly increased the mRNA of CAT in EXD-treated (1.52 g/kg/day; EXD/rat) group compared with the control group (*P < 0.05) (Fig. 2D). The mRNA levels of SOD and GPx-1 in all treatment groups were not significantly different compared with the

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control group (Fig. 2B and C). The effect of EXD on CAT was one of the mechanisms responsible for its antioxidant property. A high level of CAT expression has been shown to be protective against oxidant damage (Ji et al., 1988) and is closely associated with anti-aging, which can protect cells from hydrogen peroxide. Also, a previous study suggested that overexpression of catalase resulted in lifespan extension, a slower rate of mortality acceleration, and a delayed loss in physical performance (Orr and Sohal, 1994). Another previous publication revealed that free radicals may have an important role in the decrease of estrogen production through the effects on the aromatase activity with menopause and age (Okatani et al., 1993). In this study, treatment of menopausal SD-rats with EXD increased CAT mRNA level, thus preventing the animal from the damaging effect of hydrogen peroxide and possibly preventing the decrease of aromatase activity in menopause due to free radical. This study may provide useful information in gaining insights into the molecular mechanisms of action of EXD used for treating menopausal syndrome and aid in the establishment of a simple and rapid molecular mechanism-driven pharmacological discovery platform for screening more effective anti-perimenopausal drugs and active constituents derived from EXD, which will be conducted in our future studies. Moreover, the enzymatic activities revealed the functional status of enzyme, the enzymatic activities of aromatase, SOD, CAT and GPx-1 will thus be investigated in our next study. 5. Conclusion In this study, we developed the comprehensive quality control methods to examine the main reproductive toxic elements, including arsenic, cadmium and lead, and to evaluate the quality consistency of EXD among different batches of EXD extract. These ensure the safety and quality of EXD extract. Furthermore, we explored the novel mechanism of EXD for relieving menopausal syndrome. EXD treatment up-regulated the mRNA levels of aromatase and CAT, and displayed estrogenic activity. These findings also confirmed that EXD was clinically effective in relieving menopausal syndrome via increasing the circulatory estradiol level. Further studies on the isolation of estrogenic fractions and constituents in EXD are in progress. Acknowledgements This research was supported in part by a grant from Small Project Funding (no. 200607176131), Seed Funding Programme for Applied Research (no. 200802160025), the University of Hong Kong and Liu Hao Tsing Foundation Ltd. We also thank Ms. Cindy Lee for expert technical assistance. References Ban, Z.A., S, X., 2005. Pharmacopoeia of the People’s Republic of China. Beijing Chemical Industry Press, pp. 66, 229, 289, 255, 214, 148. Barone, E., Calabrese, V., Mancuso, C., 2009. Ferulic acid and its therapeutic potential as a hormetin for age-related diseases. Biogerontology 10, 97–108. Burger, H., Woods, N.F., Dennerstein, L., Alexander, J.L., Kotz, K., Richardson, G., 2007. Nomenclature and endocrinology of menopause and perimenopause. Expert Review of Neurotherapeutics 7, S35–43. Chattopadhyay, S., Ghosh, S., Chaki, S., Debnath, J., Ghosh, D., 1999. Effect of sodium arsenite on plasma levels of gonadotrophins and ovarian steroidogenesis in mature albino rats: duration-dependent response. Journal of Toxicological Sciences 24, 425–431. Chen, H.Y., Cho, W.C.S., Sze, S.C.W., Tong, Y., 2008. Treatment of menopausal symptoms with Er-xian decoction: a systematic review. Amercian Journal of Chinese Medicine 36, 233–244. Chi, Y.W.C.S., Yang, M.H., Hwang, R.C., Chu, F M.L., 1993. Heavy metals in traditional Chinese medicine: Ba-pao-neu-hwang-san. Acta Paediatrica Sinica 34, 181–190.

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