Danggui-Shaoyao-San, a traditional Chinese prescription, suppresses [PGF.sub.2.[alpha]] production in endometrial epithelial cells by inhibiting COX-2 expression and activity.

Abstract

Danggui-Shaoyao-San, a famous traditional Chinese prescription, has been widely used in China for treating various gynecological inflammatory diseases including dysmenorrhea, but it is still poorly understood how it works on those inflammatory disorders. Prostaglandin [F.sub.2.[alpha]] (PG[F.sub.2.[alpha]]), one important mediators of inflammation, plays crucial roles in the pathological mechanism responsible for dysmenorrhea. Here, we demonstrate that Danggui-Shaoyao-San significantly, suppresses oxytocin-evoked [PGF.sub.2[alpha]] production of rat endometrial epithelial cells in a concentration-dependent manner. Furthermore, Danggui-Shaoyao-San-mediated down-regulation of cyclooxygenase-2 message RNA transcription, protein expression and enzyme activity in endometrial epithelial cells may be involved in the inhibitory effect on [PGF.sub.2[alpha]] production. Our study provides a possible mechanism for the bioactivity of Danggui-Shaoyao-San for treating dysmenorrhea and other gynecological disorders. [c] 2008 Elsevier GmbH. All rights reserved.

Keywords: Danggui-Shaoyao-San; Dysmenorrhoea; Prostaglandin [F.sub.2[alpha]]; Cyclooxygenase-2; Oxytocin 

Introduction

Dysmenorrhea is a common gynecologic disorder in women of reproductive age. The prevalence of dysmenorrhea is highest in adolescent women (French, 2005). There are two types of dysmenorrhea: Primary dysmenorrhea with menstrual pain but without pelvic pathology occurring during ovulatory menstrual cycles and secondary dysmenorrhea with menstrual pain associated with pelvic pathology. Primary dysmenorrhea is by far the most common gynecologic problem in menstruating women; reported prevalence rates are as high as 90% (Coco, 1999; French, 2005). It is generally thought that prostaglandins have a crucial role in primary dysmenorrhea, especially prostaglandin [F.sub.2[alpha]] ([PGF.sub.2[alpha]]). [[PGF.sub.2[alpha]] levels are elevated in women with primary dysmenorrhea compared with asymptomatic controls and is directly related to the pain (Ghodgaonkar et al., 1979; Rosenwaks et al., 1981). Rapidly synthesized prostaglandins exert a direct effect on the myometrium, causing the uterine musculature to contract resulting in constriction of small endometrial blood vessels, tissue ischemia, endometrial disintegration, bleeding and pain. This may be the underlying cause of dysmenorrhea (Coco, 1999). There are two types of cells in the endometrial: stromal cells and epithelial cells. A study showed that epithelial cells were the main location of [PGF.sub.2[alpha]] [production. [PGF.sub.2[alpha]] production was greater than prostaglandin [E.sub.2] production in epithelial cells (Fang et al., 1998). The cyclooxygenase (COX) protein is a rate-limiting enzyme involved in the biosynthesis of prostaglandins using arachidonic acid as its principal substrate (DeWitt, 1991; Smith, 1992). Two genes (COX-1 and COX-2) encode this enzyme. The constitutive isoform, COX-1, is expressed in all tissues (Needleman et al., 1986) and most, if not all, nucleated cells. On the other hand, the inducible form, COX-2, is present only after induction by a variety of factors such as chorionic gonadotropin, cytokines, and tumor promoters (Asselin et al., 1997). High COX-2 expression leading to increased prostaglandin formation during menstruation is probably the mechanism responsible for dysmenorrhea and excessive bleeding and explains the therapeutic efficacy of selective COX-2 inhibitors and oral contraceptives in ameliorating these conditions (Coll Capdevila, 1997; Sales and Jabbour, 2003). COX-2 expression in the endometrium is detected mainly in the cytoplasm of epithelial cells in the glandular and surface epithelium, and is absent in the stroma (Maia et al., 2005). In nonpregnant rat uterus, COX-2 is more abundantly expressed in epithelial cells than in stromal cells (Fang et al., 1998). The pulsatile release of oxytocin by the neurohypophysis stimulates the production of uterine [PGF.sub.2[alpha]] in ruminants. Oxytocin could induce a dose-dependent increase of both [PGF.sub.2[alpha]] production and COX-2 gene expression in epithelial but not stromal cells (Asselin et al., 1997).

Danggui-Shaoyao-San (DSS) is one representative of the famous prescriptions derived from "Jingui Yaolue" ("Synopsis of Golden Chamber"), a medical classic written by Zhongjing Zhang in the Eastern Han Dynasty. This prescription comprises six drugs: Angelica sinensis Dials, Paeonia lactiflora Pallas, Ligusticum chuanxiong Hort, Hoelen, Rhizoma Alismatis and Rhizoma Atractylodis Lanceae. In clinical use, DSS has been proven to be effective in treating gynecological disorders such as dysmenorrhea, hypermenorrhea, infertility, etc. (Guo et al., 2003; Li and Yuan, 2005). However, as seen with most other traditional Chinese prescriptions, DSS is also lacking scientific evidence about the exact mechanism through which it exerts its effects. A clinical study on dysmenorrhea patients indicated that DSS obviously reduced the level of [PGF.sub.2[alpha]] in blood and menstrual fluid during menstraion period (Xie and Wang, 1990). Recent studies have demonstrated that DSS has the antagonistic action on both [PGF.sub.2[alpha]] and acetylcholine induced uterine contraction (Hsu et al., 2006). However, the detailed mechanism of this prescription for treatment of the dysmenorrhea is still unknown. In this study, therefore, we examined the mechanisms of DSS in regulating [PGF.sub.2[alpha]], one of the important inflammatory mediators responsible for dysmenorrhea, with regard to COX-2 expression and activity.

Materials and methods

Materials and reagents

Tissue culture plates were purchased from Corning Inc. (Corning, NY). Tissue culture medium DMEM/F12 was purchased from GIBCO (Grand Island, NY). Fetal bovine serum (FBS) was purchased from Sijiqing Biological Engineering Materials Co., Ltd. Specific COX-2 inhibitor (NS-398) [PGF.sub.2[alpha]] enzyme immunoassay and COX Inhibitor Screening Assay (catalog no. 760111) were all purchased from Cayman Chemical Co. (Ann Arbor, MI). Dexamethasone, Oxytocin, Collage-nase type II, Dispase, Pancreatin, dithiothreitol (DTT), Triton X-100 and HEPES were all purchased from Sigma (St. Louis, MO). Cytokeratin 13 immunocyto-chemistry kit was purchased from BM0784 Boster Bioengineering Company (Wuhan, China). Anti-COX-2 antibody and anti-[alpha]-tubulin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). M-MLV Reverse Transcriptase was purchased from Promega (Madison, WI). SYBR Green I was purchased from Molecular Probes (Eugene, OR).

Preparation of the extract

Crude herbs, Radix Angelicae, Rhizoma Ligustici, Radix Paeoniae, Rhizoma Atractylodes, Poria cocos, Rhizoma Alismatis, were purchased from Minxian (Gansu), Pengzhou (Sichuan), Haige'er (Neimeng), Songyang (Zhejiang) and Pengzhou (Sichuan), respectively. All the crude drugs were identified by one of the authors, Dr. Jin-ao Duan (Nanjing University of Chinese Medicine, Nanjing, China). All the voucher specimens (No. NJUTCM-20060811 20060816) were deposited in the herbarium of Nanjing University of Chinese Medicine. The 1.0 kg mixed crude herbs at the weight ratio of 3:8:16:4:4:8 were crushed into small pieces. The mixture was refluxed with 101 of 50% ethanol for 2 h. The filtrates were collected and the residues were then refluxed twice in 101 of 50% ethanol for 1.5 h. Two batches of filtrates were combined. The solvent was removed at 70 [degrees] C under vacuum and the residue was then dried at room temperature in an oven under vacuum to give original extract powder. The dried powder of DSS extract was stored at 4 [degrees] C and made up to a final concentration of 29.75 mg/ml in methanol and filtered through a 0.45[mu]m membrane before being used for the HPLC/DAD analysis.

HPLC-DAD analysis

Waters-2695 Alliance HPLC instrument (Waters Corporation, Milford, MA) equipped with an on-line degasser, an auto-sampler and a 2996 photodiode array detector (DAD) was used. UV detection was achieved in the scale of 210-360 nm. A Waters Sun Fire[TM] C18 column (4.6mm x 250mm, 5pm, Serial No. 186002560 Waters Corporation, MA) was used. The mobile phase consisted of (A) C[H.sub.3]CN and (B) (C[H.sub.3]COOH:[H.sub.2]O = 0.5:100) using a linear gradient of 90-80% of B in 35 min, 80-56% of B in 35-60 min, 56-20% of B in 60-85 min. The solvent flow rate was 0.8 ml/min with UV absorbance detection 280 nm. The column temperature was set at 30 [degrees] C. Fig. 1 shows the HPLC fingerprint of Danggui-Shaoyao-San. All assigned peaks were identified by coinjection test with authentic samples and compared with UV spectral data.

[FIGURE 1 OMITTED]

Animals

Non-pregnant female Sprague-Dawley rats (190-210g) were obtained from Jiangsu Laboratory Animal center. The animals had free access to food and water and were exposed to a 12 h light: 12 h darkness cycle. Rats were pretreated with estradiol benzoate (s.c., 5mg/kg) 24 h prior to the study. Animal welfare and experimental procedures were carried out strictly in accordance with the guide for the care and use of laboratory animals (The ministry of science and technology of China, 2006) and the related ethical regulations of our university. All efforts were made to minimize animal's suffering and to reduce the number of animals used.

Epithelial cells isolation and culture

The endometrial epithelial cells were separated by the procedure described by (McCormack and Glasser, 1980). Cells were plated at 5 x [l0.sup.5] cells (in 0.5ml DMEM/F12 containing 10% FBS) in matri-gel-coated 24-well plates (For [PGF.sub.2[alpha]] measurements) or 6-well plates (for protein and RNA extraction). At the time of plating, the viability of cell was greater than 95%. The cell types of epithelial cells were identified by Cytokeratin 13 immunocytochemistry experiments. Immunocy-tochemical analysis indicated that the purity of these cell preparations was greater than 95%.

After the cells reached confluency, the medium was replaced with fresh serum-free DMEM/F12 containing increasing doses of DSS, NS-398 ([10.sup.-6] M in 24-well plates for [PGF.sub.2[alpha]] measurements) or dexamethasone ([10.sup.-6] M in 6-well plates for protein and RNA extraction) in triplicates; Oxytocin ([10.sup.-7]M) was added to each wells except basal group. Cells were then incubated at 37 [degrees]C in an atmosphere of 5% [CO.sub.2]:95% air for 24h. For all experiments, at the end of the incubation period, the supernatants were collected for PG measurements and stored at -20[degrees]C until further processing. Cells were recovered and processed to obtain protein and RNA.

Enzyme immunoassays of prostaglandin [F.sub.2[alpha]]

For [PGF.sub.2[alpha]] measurements, an enzyme immunoassay technique was used. Prostaglandin [F.sub.2[alpha]] Kit (catalog no. 516011), which used acetylcholinesterase-linked PG tracers, was quantified according to the manufacturer's instructions. A standard curve was developed simultaneously with standards of [PGF.sub.2[alpha]], and determination of [PGF.sub.2[alpha]] concentrations relative to those standards was calculated. Samples were assayed in triplicates.

Measurement of COX-2 inhibition activity

COX-2 activity was quantified using COX-2 inhibitors assays according to the manufacturer's instructions. The Colorimetric COX Inhibitor Screening Assay was used in this study. In this assay, the COX-2 activity was evaluated by monitoring the appearance of oxidized N, N, N', N'-telramethyl-p-phenylenediamine (TMPD) at 590 nm.

Reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time PCR 

Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer. Single-stranded cDNA was synthesized from 2 [mu]g of total RNA by reverse transcription using 0.5 [mu]g primer of oligo [(dT).sub.18]. Then the amplification was performed using the following primers (Invitrogen, Shanghai, China): GAPDH (NM_017008) 5'-TATCTGGACGCCTGGTTAC and 3'-CGTTCAAGTTGCCGTGTC, COX-2 (AF233596) 5'-TGGGATGACGAGCGACTG and 3'- ATAGTCTTGGCGTAACGG. The PCR cycle conditions were: 94 [degrees] C for 30 s, 58 [degrees] C for 30 s, and 72 [degrees] C for 30 s for 28 cycles. After the amplification, PCR products were separated by electrophoresis on 1.5% agarose gels and visualized by ethidium bromide dying. For quantitative real-time PCR analysis, message levels were quantified using the ABI 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Amplification was carried out for 35 cycles of the same PCR condition mentioned above and product was detected using SYBR Green I dye (Molecular Probes Inc., Oregon). Samples were run in triplicate, and their relative expression was determined by normalizing expression of each target to GAPDH and then comparing this normalized value to the normalized expression in a reference sample to calculate a fold-change value.

Western blot

Thirty [mu]g of protein was loaded on 10% SDS-PAGE. Proteins were then transferred to a polyvinylidene difluoride membrane. The membrane was incubated in blocking solution (5% dry milk in PBS/0.05% Tween 20) for 1 h at room temperature and subsequently exposed to antibodies overnight at 4[degrees]C. Then the membrane was incubated with an HRP-coupled secondary antibody for 2 h at room temperature. Detection was performed using a LumiGLO chemiluminescent substract system (KPL, Guildford, UK).

Statistical analysis

Results are presented as mean [+ or -] standard deviation (s.d.). Statistically evaluated by Student's t-test when only two value sets were compared, and one-way ANOVA followed by Dunnett's test when the data involved three or more groups. p < 0.05 was considered to be significant.

Results and discussion

The present study shows that DSS obviously inhibited [PGF.sub.2[alpha]] production in epithelial cells in vitro. As shown in Fig. 2, when endometrial epithelial cells were stimulated by oxytocin ([10.sup.-7]M), production of [PGF.sub.2[alpha]] was increased by 10-fold. Combined with oxytocin, NS-398 ([10.sup.-6]M) inhibited the stimulation of [PGF.sub.2[alpha]] production (P < 0.01). Addition of DSS resulted in a significant inhibition of oxytocin-induced [PGF.sub.2[alpha]] production. During endometrial sloughing, the disintegrating endometrial cells release [PGF.sub.2[alpha]] as menstruation begins. [PGF.sub.2[alpha]] stimulates myometrial contractions, ischemia and sensitization of nerve endings (Coco, 1999). Our result provides support for the use of DSS in the treatment of menstrual pain.

[FIGURE 2 OMITTED]

COX is the rate-limiting enzyme involved in the biosynthesis of prostaglandins. It has been demonstrated that COX-2, not COX-1, is involved in the mechanism by which OT regulates [PGF.sub.2[alpha]] production in the endometrium (Asselin et al., 1997). The results of the present study, therefore, would suggest that the inhibition of COX-2 maybe play a key role in the inhibition of [PGF.sub.2[alpha]] production. Some evidence could be obtained as to the effect of OT regulates [PGF.sub.2[alpha]] production was blocked by NS-398, a specific inhibitor of COX-2. To explore further the mechanisms of DSS in regulating prostaglandins production, the activity and expression of COX-2 were examined since COX-2 play such a key role in the inhibition of [PGF.sub.2[alpha]] production. As shown in Fig.3, DSS dose-dependently inhibited COX-2 activity. The [EC.sub.50] (95% Confidence Limits) was 25.62 (21.82-29.88) [mu]g/ml.

[FIGURE 3 OMITTED]

To document the mechanism by which the modulation of [PGF.sub.2[alpha]] production described above is effected, steady-state levels of the mRNA for COX-2 were determined. RT-PCR results demonstrated that [10.sup.-7]M oxytocin induced a significant increase of COX-2 mRNA in endometrial epithelial cells. Compared with the oxytocin group, mRNA expression of COX-2 was significantly inhibited with the addition of DSS (10, 100[mu]g/ml) and [10.sup.-6]M of dexamethasone (Fig.4). To further confirm this result, the quantitative real-time PCR analysis was performed. As a result, COX-2 mRNA expression was remarkably decreased when endometrial epithelial cells were treated with 10 and 100 [mu]g/ml of DSS. The reductions of COX-2 mRNA expression in the DSS-treated group were significantly different compared with those of the oxytocin group (P < 0.01, Fig. 5). To determinate the effect of DSS on COX-2 protein expression, western blot method was used. As shown in Fig. 6, oxytocin obviously induced COX-2 protein expression in epithelial cells. DSS (10, 100[micro]g/ml) and dexamethasone markedly reduced the expression of COX-2 protein in epithelial cells induced by oxytocin.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

[FIGURE 6 OMITTED]

These results suggested that DSS inhibited COX-2 obviously by restraining COX-2 activity and diminishing COX-2 expression, not only at the transcription but also at the translation level. COX-2 inhibitors can diminish menstrual bleeding and treat dysmenorrhea (Morrison et al., 1999). Oral contraceptives are highly efficacious for treating menstrual pain and their use is associated with a reduction of over 80% in the levels of prostaglandin metabolites in menstrual blood. This inhibitory effect on prostaglandin production is due to the decrease in COX-2 expression in the endometrium (Maia et al., 2005). These findings indicate that the inhibition of COX-2 might contribute to the treatment of dysmenorrhea, and might be one important mechanism of DSS. This result is in agreement with the inhibitory effect of [PGF.sub.2[alpha]] production in endometrial epithelial cells. Dexamethasone, used in this study as a standard agent, also inhibits COX-2 gene and protein expression. Similar results have also been observed in IL-1-induced COX-2 mRNA expression (Ristimaki et al., 1996).

DSS consists of 6 crude drugs. At present, our knowledge of active components in DSS still remains insufficient. We identify 8 components in DSS. Vanillic acid, caffeic acid, ferulic acid, senkyunolide I and ligustilide come from the crude drugs of Radix Angelica and Rhizoma Ligusticum. Albiflorin, paeoniflorin and tetragalloylglucose come from Radix Paeonia. Phenolic acids such as vanillic acid, caffeic acid, ferulic acid, reportedly have anti-platelet aggregation effects as well as anti-oxidant effects. Caffeic acid is also a selective inhibitor for leukotriene biosynthesis (Koshihara et al., 1984). Ferulic acid showed an inhibitory effect on uterine movement (Ozaki and Ma, 1990). As selectively combining components with red blood cell, ferulic acid and senkyunolide I were reported to alleviate ConA induced erythrocytic deformability damage (Hong et al., 2003; Dong et al., 2005). Ligustilide possesses a nonspecific antispasmodic function, as well as antinociceptive and anti-inflammatory activities (Du et al., 2007). Paeoniflorin suppresses rat adjuvants arthritis at least partly by reducing COX-2 expression in synovium (Zheng et al., 2007). This evidence maybe supports our previous findings. Taken together, we suggested that multiple bioactive components were associated with the effect of DSS.

As a famous traditional Chinese prescription, DSS is widely applied in many gynecological diseases, from adolescent to menopause. DSS can stimulate ovarian steroidgenesis and the ovulatory process by including the secretion of CINC/gro with IL-l beta and TNF-alpha in vitro (Irahara et al., 2000). DSS also has neuroprotective effects on aged mice (Kou et al., 2005). Dysmenorrhea is the basic indication of DSS. It has been demonstrated that DSS has the antagonistic action on both [PGF.sub.2[alpha]] and acetylcholine induced uterine contraction (Hsu et al., 2006). Our study demonstrated that DSS reduced the generation of [PGF.sub.2[alpha]], and gives strong evidence to explain the mechanism by which DSS treats dysmenorrhea and other problems in pregnancy such as threatened abortion or for the prevention and treatment of premature labor.

For the selective COX-2 inhibitors, there are clear contraindications and warnings in Europe, since they increase the risk of thrombotic cardiovascular events. This kind of side effects has never been reported in the clinical use of DSS This may be an interesting issue for further study.

In conclusion, the present results demonstrate that DSS significantly reduces the generation of PG[F sub 2x] in endometrial epithelial cells induced by oxytocin in vitro The mechanisms may involve the inhibition of COX-2 m RNA transcription, protein expression and enzyme activity in endometrial epithelial cells. The present study provides a novel mechanistic explanation for the anti-inflammatory activity of DSS for treating dysmenorrhea and other gynecological disorders.

Acknowledgements

We thank Dr. Yang Sun for experimental assistance. We also thank Profs. Quan Zhu, Shaoping Li, Anwei Ding and Min Hong for their helpful assistance. This work was supported by the 2006 Great Basic Science Research Project of Jiangsu College and University (no 06KJA 3622) and the 2006 "Qinglan Project" Scientific and technological innovation team training program of Jiangsu College and University.

References.

Asselin, E., Drolet, P., Fortier, M.A., 1997. Cellular mechanisms involved during oxytocin-induced prostaglandin F2al-pha production in endometrial epithelial cells in vitro; role of cyclooxygenas-2 Endocrinology 138, 4798-4805.

Coco, A.S., 1999, Primary dysmenorrhea. Am Fam Physican 60,489-496.

Coll Capdevila, C., 1997. Dysfunctional uterine bleeding and dysmenorrhea. Eur. J Contracept Reprod Health Care 2 229-237.

DeWitt, D.L., 1991, Prostaglandin endoperoxide synthase: regulation of enzyme expression. Biochim. Biophys. Acta 1083, 121-134.

Dong Z.B., Li, S.P., Hong, M., Zhu, Q., 2005. Hypothesis of potential active components in Angelica sinensis by using biomembrane extraction and high performance liquid chromatography J. Pharm. Biomed Anal 38, 664-669.

Du, J. Yu., Ke, Y., Wang C., Zhu L., Qian, Z.M., 2007. Ligustilide attenuates pain behavior induced by acetic acid or formalin. J. Ethnopharmacol. 112, 211-214.

Fang, L., Chatterjee, S., Dong, Y.L., Gangula, P.R., Yallamplli C., 1998. Immunohistochemical localization of constitutive and inducible cyclo-oxygenases in rat uterus during the oestrous cycle and pregnancy. Histochem. J.30, 383-391.

French, L., 2005 Dysmenorrhea. Am Fam. Pysician 71, 285-291.

Ghodgaonkar, R.B., Dubin, N.H., Blake, D.A., King, T.M., 1979, 13, 14-Dihydro-15-keto-prostaglandin F2ealpha concentrations in human plasma and amniotic fluid. Am J Obstet Gynecol 134, 265-269.

Guo, X., Wang, Y., Zhang, Y., Li, P., 2003. Epidemiologic study of 132 primary dysmenorrhea and the therapeutic effect of Danggui-Shaoyao-San. J. Math. Med 16, 409.

Hong, M., Dong, Z. B., Zhu, Q., 2003 Effects of ferulic acid, Senkyunolide H and Senkynolide I on erythrocytes. Lishizhen Med. Mater. Med. Res 28, 738-739.

Hsu, C.S., Yang, J.K., Yang, L.L., 2006 Effect of "Dang-Qui-Shao-Yao-San" a Chinese medicinal prescription for dysmenorrhea on uterus contractility in vitro Phytomedicine 13, 94-100.

Irahara, M., Yasui, T., Tezuka, M., Ushigoe, K., Yamano S., Kamada., M., Aono, T., 2000. Evidence that Toki-shakuyaju-san and its ingredients enhance the secretion of a cytokine-induced neutrophil chemoattractant (CINC/gro) in the ovulatory process. Methods Find. Exp Clin. Pharmacol. 22, 725-730.

Koshihara, Y., Neichi, T., Murota, S., La, A., Fujimoto Y., Tatsuno, T., 1984. Caffeic acid is a selective inhibitor for leukotriene biosynthesis. Biochim. Biophys. Acta 792, 92-97.

Kou, J., Zhu, D., Yan, Y., 2005 Neuroprotective effects of the aqueous extract of the Chinese medicine Danggui-Shaoyao-san on aged mice. J. Ethnopharmacol. 97, 313-318.

Li, J., Yan, L.X., 2005. Review of Danggui-Shaoyao-San clinic research. Chin Arch. Tradit Chin. Med (chin) 23, 971-973.

Maia. Jr., Hr., Maltez., A., Studard, E., Zausner, B., Athayde, C., Coutinho, E., 2005 Effect of the menstrual cycle and oral contraceptives on cyclooxygenase-2 expression in the endometrium. Gynecol. Endocrionl 21, 57-61.

McCormack, S.A., Glasser, S.R., 1980. Differential response of individual uterine cell types from immmature rats treated with estradiol. Endocrinology 106, 1634-1649.

Morrison, B.W., Daniels, S.E., Kotey, P., Cantu, N., Seidenberg, B., 1999. Rofecoxib, a specific cyclooxygenase-2 inhibitor, in primary dysmenorrhea: a randomized controlled trial. Obstet. Gynecol. 94, 504-508.

Needleman, P., Turk, J., Jakschik, B.A., Morrison, A.R., Lefkowith, J.B., 1986. Arachidonic acid metabolism. Annu. Rev. Biochem. 55, 69-102

Ozaki, Y., Ma, J.P., 1990. Inhibitory effects of tetramethyl-pyrazine and ferulic acid on spontaneous movement of rat uterus in situ. Chem. Pharm. Bull. (Tokyo) 38, 1620-1623.

Ristimaki, A., Narko, K., Hla, T., 1996. Down-regulation of cytokine-induced cyclo-oxygenase-2 transcript isoforms by dexamethasone: evidence for post-transcriptional regulation. Biochem. J. 318 (Pt. 1), 325-331.

Rosenwaks, Z., Jones, G.S., Henzl, M.R., Dubin, N.H., Ghodgaonkar, R.B., Hoffman, S., 1981. Naproxen sodium, aspirin, and placebo in primary dysmenorrhea. Reduction of pain and blood levels of prostagladin F2-alpha metabolite. Am. J. Obstet. Gynecol. 140, 592-598.

Sales, K.J., Jabbour, H.N., 2003. Cyclooxygenase enzymes and prostaglandins in pathology of the endometrium. Reproduction 126, 559-567.

Smith, W.L., 1992. Prostanoid biosynthesis and mechanisms of action. Am. J. Physiol. 263, F181-F191.

Xie, C.G., Wang, X.H., 1990. Effect of Danggui-Shaoyao-San on hemorheology and PGFs[alpha] level of dysmenorrhea patients. Zhongguo Zhong Xi Yi Jie He Za Zhi (chin) 10, 410-412.

Zheng, Y.Q., Wei, W., Zhu, L., Liu., J.X., 2007. Effects and mechanisms of Paeoniflorin, a bioactive glucoside from paeony root, on adjuvant arthritis in rats. Inflamm. Res. 56, 182-188.

Y.Q. Hua (a), (b), (c), S.L. Su (b), J.A. Duan (a), (b), *, Q.J. Wang (d), **, Y. Lu (b), L. Chen (c)

(a) School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210038, China

(b) Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China

(c) National Standard Laboratory of Pharmacology for Chinese Materia Medica, Nanjing University of Chinese Medicine. Nanjing 210029, China

(d) Department of Physiology, China Pharmaceutical University, Nanjing 210009, China

* Corresponding author at: Jiangsu Key Laboratory for TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing, 210046, China. Tel./fax: + 86258511116.

** Corresponding author. Tel./fax: + 862583271341.

E-mail addresses: [e-mail] (J.A. Duan), [e-mail] (Q.J. Wang).

0944-7113/$-see front matter [c] 2008 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed.2008.06.010