Effect of a dysmenorrhea Chinese medicinal prescription on uterus contractilityin vitro

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PHYTOTHERAPY RESEARCH Phytother. Res. 17, 778–783 (2003) Published online in Wiley InterScience (www.interscience.wiley.com). C.-S. HSU ET AL.DOI: 10.1002/ptr.1235

Effect of a Dysmenorrhea Chinese Medicinal Prescription on Uterus Contractility in vitro Chun-Sen Hsu1, J-Kay Yang 2 and Ling-Ling Yang2,3 1

Department of Obstetrics and Gynecology, Taipei Wan-Fang Hospital-affiliated Taipei Medical University, Taipei, Taiwan Graduate Institute of Pharmaceutic Science, Taipei Medical University, Taipei, Taiwan 3 Graduate Institute of Biotechnology, Life Science College, National Chiayi University, Chiayi, Taiwan 2

Dysmenorrhea is a common gynecologic complaint. After their first menstrual period, 30%–60% of American women suffer from some level of discomfort. It is estimated that 6 billion work hours are lost in this manner every year in the United States which equals an economic loss of nearly US$200 million. Dysmenorrhea is not only a problem for women but also one which affects quality of life and even reduces productivity in general. Dysmenorrhea is directly related to elevated levels of PGF2α (prostaglandins F2α) and is treated using nonsteroid anti-inflammatory drugs in Western medicine. Though efficacy of the latter is rapid, there are many side effects to the liver, kidney, and digestive system. The anti-inflammatory effect is temporary, and such drugs are unable to provide a long-term cure. Because of this, Chinese medicinal therapy is being considered as a feasible alternative medicine. In this study, Wen-Jing Tang (one of the dysmenorrhea Chinese medicinal prescriptions) was selected. A 50% alcoholic solution was used to extract active ingredients and create a freeze-dried product. At first, WenJing Tang was used to suppress spontaneous contractions and prostaglandins F2α -induced contractions of rat uterine smooth muscle in vitro. Then, an assessment was performed to determine the mechanism of the prescription. Acetylcholine, ergonovine, propranolol, oxytocin, and KCl were used to analyze the physiological mechanisms of WJT. The results show that antagonism of both PGF2α and ACh are the major mechanisms for treating dysmenorrhea by Wen-Jing Tang. Furthermore, the antagonistic effect of KCldepolarization contractions may be an auxiliary mechanism of the curative effect. Copyright © 2003 John Wiley & Sons, Ltd. Keywords: PGF2α, ACh; Ergonovine; Propranolol; Oxytocin; KCl, Wen-Jing Tang.

INTRODUCTION Dysmenorrhea is a common gynecologic disorder that affects approximately 30%–60% of menstruating women in America. An estimate of 7%–15% of these women suffer from serious pain which leads to the inability to function professionally for 1–3 days each month. About 6 billion work hours are lost in this manner every year in the US which equals an economic loss of nearly US$200 million (Dawood, 1990; Harlow et al., 1996). It can be seen that menstruation is not only a problem for women but also one which affects the quality of life and even reduces national productivity. Dysmenorrhea manifests in patients as facial paleness during or around menstruation, and regular spasmodic pain often accompanied by headaches, nausea, vomiting, fatigue, cold sweats, lower back pain, and so forth (Andrea, 1996).

* Correspondence to: Prof. L.-L. Yang, Office of Life Science, College of National Chiayi University, 300 University Road, Chiayi, Taiwan, Republic of China. Tel: 886-5-2717930. Fax: 886-5-2717931. E-mail: [email protected] Copyright © 2003 John Wiley & Sons, Ltd. Copyright © 2003 John Wiley & Sons, Ltd.

Dysmenorrhea has been classified into primary and secondary forms. Primary dysmenorrhea is a disorder of young women, occurring more frequently during the teens and early twenties when menstrual pain is not associated with recognizable pelvic pathology. This kind of dysmenorrhea generally begins with the onset of ovulatory cycles, typically 6–12 months after menarche (Andrea, 1996; Montero et al., 1996). Secondary dysmonorrhea is defined as pain that occurs during menstruation and that is presumably due to an anatomic or pelvic abnormality. Common causes include endometriosis, uterine leiomyomas, the installment of intrauterine contraceptive devices (IUDs), etc. (Maxson et al., 1993). Typical menstrual contractions can be reproduced with long-term infusion of prostaglandin F2 alpha (PGF2α) (Roth-Brandel et al., 1970). PGF2α levels are elevated in women with primary dysmenorrhea compared with asymptomatic controls (Ghodgaomkar et al., 1979; Rosenwaks et al., 1981). Prostaglandin synthesis is initiated by lysosomal enzymes that are released in the late luteal phase of the menstrual cycle. Rapidly synthesized prostaglandins exert a direct myometrial effect, causing the uterine musculature to contract resulting in constriction of small endometrial blood vessels, tissue ischemia, endometrial disintegration, January(2003) 2001 Phytother.Received Res. 17,11 778–783 Accepted 26 March 2002

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bleeding, and pain. This may be the underlying cause of dysmenorrhea (Maxson et al., 1993). Dysmenorrhea is usually treated with nonsteroid anti-inflammatory drugs (NSAIDs) in clinical medicine. Although their efficacy is rapid, there are many side effects that affect the liver (Tolman, 1998), kidney (Radford et al., 1996), and digestive system (GarciaRodriguez et al., 1994a), and can even cause acute liver injury (Garcia-Rodriguez et al., 1994a), acute renal failure (Perez-Gutthann et al., 1996), and inflammatory bowel disease (Evans et al., 1997). Intolerance to NSAIDs is also troublesome. A contraceptive has been use in the management of dysmenorrhea, but it has side effects of nausea and water retention. Because of these shortcomings, Chinese medicinal therapy is considered as a feasible alternative (Brody and Schwartz, 1993a). A project was proposed to investigate the mechanism of dysmenorrhea Chinese medicinal prescriptions (DCMPs). We chose one named Wen-Jing Tang (WJT) which is composed of Pinellia ternata Breitenbach, Panax ginseng CA Meyer, Asini gelatinum, Cinnamomum cassia Blume, Glycyrrhiza uralensis Fischer, Ophiopogon japonicus, Paeonia lactiflora Pallas, Paeonia suffruticosa, Evodia rutaecarpa Bentham, Angelica sinensis Diels, Ligusticum chuanxiong Hort, and Zingiber officinale Rodcoe. A 50% alcohol solution was used to extract active ingredients and create a freeze-dried product. This was applied to inhibit spontaneous rat uterine smooth muscle contractions, and prostaglandins F2α-induced contractions alternatively in vitro. Then we used acetylcholine, oxytocin, and others agents to analyze the physiological mechanisms of the prescription.

METERIALS AND METHODS Drugs. The following drugs were used: prostaglandin F2α (Shoya Pharmacy, Japan, Prostamon inject), acetylcholine chloride (Sigma, USA, A-6625, lot 5H0784, approx. 99%), ergonovine maleate (Sigma, E-6500, lot 54H1077), propranolol hydrochloride (Sigma, P-8688, lot 88H4092), and oxytocin (N.V. Organon Oss, Holland, Piton-S Ad inject). All drugs were prepared daily in distilled and deionized water. Extract preparation. A 50% alcohol solution was used to extract active ingredients by decoction from WenJing Tang. The extract rate was 35.73%. The extract was used to create a freeze-dried product. This was applied to rat uterine smooth muscle in vitro for the investigation.

force displacement transducers (Kent Scientific, USA) using MP100 workstation software (Biopac Systems, USA) on a PC.

EXPERIMENTAL PROCEDURES Spontaneous uterine contractions A uterine horn was incubated in Locke’s solution and equilibrated for 45 min. Wen-Jing Tang was added until the amplitude was stable. Agonist-induced contraction of the uterus A uterine horn was incubated in Locke’s solution and equilibrated for 45 min. Contractions were induced by submaximal concentrations of PGF2α (1 ug/ml) or ACh (10−5 M) or oxytocin (0.01 U/ml) or ergonovine (7.5 × 10−4 M), and propranonol (1 uM). WJT was added to the incubation solution and allowed to react for 15 min before a more-concentrated agent was added. Concentration-response curves of the prescription (0.125–4 mg/ ml) were plotted against the phasic response to PGF2α, ACh, oxytocin, ergonovine, and propranonol. Oxytocin-induced Ca+ +-free contractions A uterine horn was equilibrated for 1 h in a RingerLocke solution. The solution was then replaced by a Ca++-free solution containing 3 mM EDTA, and incubation continued for 50 min. Subsequently, the solution was replaced by Ca++-free solution containing 1 mM EDTA, and the uterus was incubated for an additional 20 to 30 min. A sustained contractile response to oxytocin (0.01 U/ml) was obtained, and cumulative amounts of WJT were added. K+-depolarized uterus The organ was immersed in Jalon-Ringer solution and equilibrated for 20 min. This solution was replaced by a depolarizing solution (KCl, 56.3 mM) that caused rapid contraction, followed by slight relaxation and a prolonged contraction plateau. When the plateau was reached, cumulative concentrations of WJT were administered and concentration-related relaxations were observed. Solutions

Animals used and method of preparation. We used female Wistar rats weighing 250–350 g which had been estrogenized (with 5 mg/kg s.c. of estradiol benzoate 24 h before the experiments). The animals were sacrified by decapitation; uterine horns were excised and cut into two halves (1 cm). The preparations were placed in isolated organ baths, incubated in a physical solution at 37 ± 1 °C, and bubbled with carbogen (95% O2, 5% CO2). The preload was 1 g, and the equilibration period was not less than 45 min (Zafra-Polo et al., 1993; Gordan et al., 1997). Contractions were recorded by Copyright © 2003 John Wiley & Sons, Ltd.

The following solutions were used: Locke solution (mM): NaCl 154, KCl 5.63, NaHCO3 1.79, CaCl2·2H2O 2.55, glucose 5.55; Jalon-Ringer solution (mM): NaCl 154, KCl 5.63, NaHCO3 5.95, CaCl2·2H2O 0.648, glucose 2.77; Depolarizing solution (mM): NaCl 103.3, KCl 56.3, NaHCO3 5.95, CaCl2·2H2O 0.648, glucose 2.77; Ringer-Locke solution (mM): NaCl 154, KCl 5.63, NaHCO3 5.95, CaCl2 2.16, MgCl2 2.1, glucose 5.55; and Ca2+-free solution (mM): NaCl 154, KCl 5.63, NaHCO3 5.95, MgCl2 2.1, glucose 5.55, EDTA 3 or 1. Phytother. Res. 17, 778–783 (2003)

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Statistical analysis The results are expressed as the mean ± SEM of several preparations (n) from different animals. Agonists were added until the steady, largest amplitudes were maximal contractions. In accordance with the reduction of contraction of the dose-response curve the concentration of WJT giving 50% of maximal contraction (IC50 value) was estimated. At the end of the experiments, the relaxation response was quantified by percent papaverine-induced maximum relaxation. Relaxation was expressed as a percentage of the maximum tension obtained by adding the agonist. Statistical significance of differences between groups was assessed using Student’s t-test for unpaired data. P values less than 0.05 were considered significantly different.

tion has inhibitory effects on uterine contractions induced by ACh, and a linear dose-response relationship was eventually maintained between them. The IC50 of the prescription at 10−5 M of ACh was 1.85 ± 0.53 mg/ml. The effect of WJT on ergonovine (7.5 × 10−4 M)induced uterine contractions is shown in Figure 3. The prescription has inhibitory effects on uterine contractions induced by ergonovine, and a linear dose-response relationship was shown. The IC50 of the prescription at 7.5 × 10−4 M of ergonovine was 0.71 ± 0.04 mg/ml. The effects of WJT on propranolol (β -adrenergic receptor antagonist)-induced uterine contractions is shown in Figure 4. The prescription had inhibitory effects on uterine contractions induced by propranolol, and it also showed a linear dose-response relationship. However, at a concentration of 0.125 mg/ml, Wen-Jing Tang was more effective inducing uterine contractions.

RESULTS Effects of WJT on spontaneous uterine contractions were evaluated in this study. We found that the amplitude of uterine contractions slightly decreased with WJT, but the decrease did not significantly differ compared with the control group. PGF2α is the major substance inducing uterine contractions in dysmenorrhea. The effects of WJT (1 mg/ml) on in vitro uterine contractions induced by PGF2α (1 µg/ml) were observed in our study. Percentages of the inhibitive effect exceeded 50% in this group (35.06% ± 4.81% of maximal contractions). As the concentration of WJT increased, we observed the effects against PGF2α-induced uterine smooth muscle contractions. Results are shown in Figure 1. WJT has inhibitory effects on uterine contractions induced by PGF2α, and a linear dose-response relationship was eventually maintained between them. The IC50 of WJT at 1 ug/ml of PGF2α was 0.45 ± 0.05 mg/ml. The effect of WJT against ACh (10−5 M)-induced uterine contractions is shown in Figure 2. The prescrip-

Figure 1. Effects of WJT on phasic contractions of rat uterus induced by PGF2α (1 µg/ml). Vertical bars represent the SEM (n = 4). Copyright © 2003 John Wiley & Sons, Ltd.

Figure 2. Effects of WJT on phasic contractions of rat uterus induced by ACh (10−5 M). Vertical bars represent the SEM (n = 4).

Figure 3. Effects of WJT on phasic contractions of rat uterus induced by ergonovine (7.5 × 10−4 M). Vertical bars represent the SEM (n = 4). Phytother. Res. 17, 778–783 (2003)

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Figure 4. Effects of WJT on phasic contractions of rat uterus induced by propranolol (1 µM). Vertical bars represent the SEM (n = 4).

Figure 5. Effects of WJT on phasic contractions of rat uterus induced by oxytocin (0.01 U/ml). Vertical bars represent the SEM (n = 4).

The IC50 of the prescription at 1 uM of propranolol was 1.49 ± 0.16 mg/ml. The effect of WJT on 0.01 U/ml oxytocin-induced uterine contractions is shown in Figure 5. It had inhibitory effects on oxytocin-induced uterine contractions, and it also showed a linear dose-response relationship. The IC50 of the prescription at 0.01 U/ml of oxytocin was 0.92 ± 0.12 mg/ml. The effect of WJT on 0.01 U/ml oxytocin-induced calcium release from smooth muscle in a solution without calcium on uterine contractions is shown in Figure 6. It showed a linear dose-response relationship. The IC50 of Wen-Jing Tang was 1.33 ± 0.26 mg/ml. The effect of WJT on a high concentration of potassium (KCl 56.3 mM)-induced uterine contractions is shown in Figure 7. WJT had inhibitory effects on uterine contractions induced by high potassium, and it also showed a linear dose-response relationship. The IC50 of WJT at 56.3 mM of KCl was 0.36 ± 0.05 mg/ml. Copyright © 2003 John Wiley & Sons, Ltd.

Figure 6. Effects of WJT on contractions of rat uterus induced by oxytocin (0.01 U/ml) on Ca++ – free solution. Vertical bars represent the SEM (n = 4).

Figure 7. Effect of WJT on tonic contractions of rat uterus induced by KCl (56.3 mM). Vertical bars represent the SEM (n = 4).

DISCUSSION Evaluation of the DCMP model Macht and Davis (1934) demonstrated that an extract of menstrual fluids, called ‘menstrual toxin’, had a potential effect on the contraction of the rat vas deferens. Pickles proposed that the extractable substance from the menstruating uterus directly stimulated myometrial contractions and could be responsible for primary dysmenorrhea (Pickles et al., 1965). Typical menstrual pain was later reproduced with long-term infusion of PGF2α (Roth-Brandel et al., 1970). Ghodgaonkar et al. (1979) and Rosenwaks et al. (1981) found that PGF2α levels are elevated in women with primary dysmenorrhea. Therefore, we know that prostaglandins are implicated in causing dysmenorrhea. Phytother. Res. 17, 778–783 (2003)

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There are several studies which evaluated the effect of DCMPs. In 1991, Calixto et al. investigated the effect of Leonotis nepetaefolia (Labiatae) on rat uterus in vitro; Perez-Vallina et al. (1995) investigated the effect of NSAIDs on rat uterus contractions induced by PGF2α or KCl in vitro. Our research shows that Wen-Jing Tang inhibits PGF2α in vitro-induced uterus contractions in vitro but has no effect on spontaneous contractions. Mechanisms of action When the intracellular Ca++ ion concentration in uterus smooth muscle exceeds 10−6 M (Brody and Schwartz, 1993), uterine contractions will be induced. The intracellular Ca++ ion concentration is regulated by two different Ca++ channels: receptor- and voltage-operated channels (Bolton, 1979; Hurwitz, 1986; Triggle et al., 1989; Pilar D’Ocon et al., 1991). This study focused on PGF2α, ACh, ergonovine (αadrenergic agonist), and propranolol (β -adrenergic antagonist). In this investigation, controlling the receptor-operated channels (ROCs) allowed the intracellular calcium concentration to increase to a level at which uterus contraction took place. The results from our study suggest that Wen-Jing Tang has a doseresponse relationship antagonistic to the induction of uterine contractions. Oxytocin is regulated by ROCs to induce uterus contractions. After oxytocin binds with the receptor, the chemical structure of the G-protein on the uterus cell membrane of the smooth muscle changes and activates phospholipase C; then phospholipase hydrolyzes PIP2 (phosphatidyl 4,5-bisphosphate) to produce IP3 (inositol 1,4,5-trisphosphate) and DAG (1,2-diacylglycerol) [Carsten and Jordan, 1987; Rodger, 1992; Horowitz et al., 1996]. DAG activates protein kinase C which opens ROCs to allow the influx of extracellular Ca++ ions and induce contractions. IP3 is released into the cytoplasm. The other mechanism is by IP3 receptors on the SR (sarcoplasmic reticulum) resulting in the release of Ca++ ions of the SR to induce contractions. This research focused on oxytocin as a tool to investigate the pathway of the Ca++ flow. The results indicate that WJT has a dose-response relationship antagonistic to the effects of oxytocin. Oxytocin in Ca++-free EDTAcontaining physical solutions accelerates the release of Ca++ ions in the SR resulting in the induction of uterine contractions. Experimental results show that Wen-Jing Tang is a prescription that demonstrates a significant linear dose-response relationship in its inhibitory effects

on the induction of uterine contractions. This is shown not only in the suppression of the influx of Ca++ ions, but also in the suppression of SR Ca++ ion release. With a high concentration of K+ ions, VOCs allow the cell to depolarize, and the channels open up. Then, extracellular Ca++ ions migrate into the cell resulting in the induction of uterine contractions (Ballejo et al., 1986; Triggle et al., 1989). A high concentration of KCl (56.3 mM) was used to induce contractions of uterine smooth muscle in an isolated rat uterus. Then WJT was added to gradually regulate contractions. Results from this study suggest that WJT effectively inhibits uterine contractions, and shows a dose-response relationship. In conclusion, Wen-Jing Tang works through two pathways involving both ROCs and VOCs. Treatment implications In clinical reports, Wen-Jing Tang may be used to treat dysmenorrhea. It can also be applied for a threatened abortion or in the prevention and treatment of premature labor. The experiment indicates that Wen-Jing Tang has multiple effects on uterine smooth muscles. These include PGF2α and ACh antagonistic functions which are similar to the mechanisms of indomethacin and piperidolate. Wen-Jing Tang also has β -adrenergic agonist characteristics similar to those of ritodrine. The depolarization antagonist functions to stabilize cell membrane polarity similar to how magnesium sulfate decrease uterine muscle polarity. Gap junctions increase in late pregnancy as the uterus in more sensitive to oxytocin (Brody and Schwartz, 1993b). Hence the antagonist effect of WJT plays an important role in this mechanism. Thereby, Wen-Jing Tang has an effect on preterm labor. Antagonism of PGF2α and ACh of the ROC pathway in uterine smooth muscle is considered a mechanism of action in treating dysmenorrhea. The antagonist effect of WJT on VOCs stabilizes the membrane potential of uterine smooth muscle cells, and subsequently decreases uterine contractions by decreasing the membrane action potential. This effect is considered an alternative function in the treatment of dysmenorrhea. The WJT prescription has antagonistic effects on reactions in uterine smooth muscle caused by PGF2α and ACh. It also stabilizes the electrophysiology of cell membranes of uterus smooth muscle. Moreover, WenJing Tang may be used to treat preterm labor and postpartum uterine contraction pain.

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