Screening And Pharmacological Research Of The Active Ingredients Of Dang-gui-shao-yao-san For Dysmenorrhea

Dysmenorrhea is defined as cramping pain in the lower abdomen occurring before or during menstruation, accompanied with headache, nausea or vomiting. It is very common in adolescent, unmarried or married women without child, and often severely affects women's health and work efficiency. Nowadays non-steroidal anti-inflammatory drugs (NSAIDs), selective COX-2 inhibitors and oral contraceptives are the main drugs for dysmenorrhea. But these drugs are not suitable for regular use as they often have many side effects such as liver, kidney or cardiovascular toxicity. Chinese traditional medicine such as Shaofu Zhuyu Decoction, Wenjing Decoction and Dang-gui-shao-yao-san (DSS) are feasible alternative with great priority.DSS was recorded in Jinguiyaolue, a book written by a famous doctor Zhang Zhong-jing in Han dynasty. Composing of Paeonia lactiflora Pallas, Angelica sinensis Dials, Ligusticum chuanxiong Hort, Poria cocos Wolf, Atractylodes macrocephala Koidzumi, Alisma arientale Juzepczuk, the priscription was effective for dysmenorrhea. However its active ingredients remain unclear. Our previous studies showed that DSS had analgesic effect in writhing test induced by acetic acid and could inhibit spontaneous contraction of isolated rat uterine strips. According to these results, we extracted four ingredients, DSS-A-W-W, DSS-W, DSS-A-E and DSS-A-N-30 from DSS, and screened out active ingredients using acetic-acid-induced writhing test and isolated uterine contraction test. Then menorrhalgia model in vitro and in vivo were used to evaluate their activity, and their possible mechanism for treating dysmenorrhea was also discussed in this study.1. Screening out the active ingredients of DSS 1.1 Effect of extracted ingredients of DSS on acetic acid-induced writhing testAll the test drugs, DSS-A-W-W, DSS-W, DSS-A-E and DSS-A-N-30, coulddecrease the number of writhing significantly as well as the positive control Asrpin and DSS. The data suggested that the four extracted ingredients of DSS might have analgesic effect.1.2 Effect of extracted ingredients of DSS on spontaneous contraction of isolated rat uterine strips in vitroDysmenorrhea was closely related to the function status of uterus. Thus the effect of the four extracted ingredients of DSS on spontaneous contraction of non-gravid isolated rat uterine strips in estrus stage was observed. Spontaneous contraction was inbibited by DSS-A-E (16.4, 32.8, 49.2, 65.7 mg/L) and DSS-A-N-30 (283.0 mg/L) in single administration as well as DSS (7140.0 mg/L). And DSS-W (0-643.0 mg/L) tended to slightly decrease the spontaneous contraction, while no obvious effect of DSS-A-W-W (0-2957.0 mg/L) was observed when they were administrated cumulatively. These data implied that the extracted ingredients DSS-A-E and DSS-A-N-30 could directly inhibit the spontaneous contraction of isolated rat uterine strips. Basingon above-all-mentioned results, DSS-A-E and DSS-A-N-30 were screened out as the active ingredients of DSS for the further studies.2. Analgesic effect of DSS-A-E and DSS-A-N-30The analgesic effect of DSS-A-E and DSS-A-N-30 on acetic-acid-induced writhing test and menorrhalgia model induced by oxytocin was evaluated in this part of research.2.1 Acetic acid-induced writhingDSS-A-E (115.0, 230.0, 460.0 mg/kg) and DSS-A-N-30 (19.8, 39.6, 79.2 mg/kg), as Asprin (100.0 mg/kg) and DSS (9000.0 mg/kg), decreased the number of acetic acid-induced abdominal constrictions in mice significantly in contrast to control group, suggesting DSS-A-E and DSS-A-N-30 might have analgesic effect.2.2 Oxytocin induced-menorrhalgia modelThe effect of DSS-A-E and DSS-A-N-30 on menorrhalgia model induced by oxytocin was observed. Results showed that DSS-A-E (230.0, 460.0 mg/kg) and DSS-A-N-30 (19.8 mg/kg) decreased the number of writhing significantly. These results imply that DSS-A-E and DSS-A-N-30 might have analgesic effect on menorrhalgia.3. Effect of DSS-A-E and DSS-A-N-30 on uterine contraction of isolated rat uterine stripsThe effect of DSS-A-E and DSS-A-N-30 on spontaneous contraction and uterine agnonists (oxytocin, PGF_2α, ACh)-induced contraction of non-gravid isolated rat uterine strips was observed. Then two models, K⁺ deplorization-induced contraction and oxytocin-induced contraction in calcium-free solution, were established to investigate the effect of drugs on the intracellular realease and extracellular influx of calcium ions.3.1 Effect of DSS-A-E and DSS-A-N-30 on spontaneous contraction of isolated rat uterine stripsThe vehicle had no obvious effect on spontaneous contraction of isolated rat uterine strips. DSS at 7140.0 mg/L inhibited the spontaneous contraction after 10-min treatment and maintained at least to 30 min. DSS-A-E (16.4, 32.8, 49.2, 65.7 mg/L) have inhibition effect concentration-dependently, reached the maximal inhibition (60%) at 20 min and mainted to 30 min. The inhibition effect of DSS-A-N-30 (141.5, 283.0,566.0 mg/L) was similar to DSS-A-E, except that the maximal inhibition (40%) reached at 10 min then weakened a little later. These results implicated that DSS-A-E and DSS-A-N-30 can directly inhibit spontaneous contraction of isolated rat uterine strips.3.2 Effect of DSS-A-E and DSS-A-N-30 on the contraction induced by uterine agonists3.2.1 Effect of DSS-A-E and DSS-A-N-30 on oxytocin-induced contractionSpontaneous contraction of normal isolated rat uterine strips became intense after the treatment of oxytocin (10 IU/L), as contracting area under curve (AUC), frequency and tension increased immediately, which could be reduced by DSS at 7140.0 mg/L. DSS-A-E (16.4, 32.8, 49.2, 65.7 mg/L) significantly decreased the increasing of AUC, frequency and tension concentration-dependently. DSS-A-N-30 (141.5, 283.0, 566.0 mg/L) decreased the increase of AUC and frequency siginificantly and concentration-dependently, but only high concentration (566.0 mg/L) could decrease the increasing tension.These data showed that DSS-A-E and DSS-A-N-30 could inhibit the intense contraction induced by oxytocin.3.2.2 Effect of DSS-A-E and DSS-A-N-30 on PGF_2α-induced contractionPGF_2α (1 mg/L) could enhance the spontaneous contraction of isolated rat uterine strips, which could be countered by 7140.0 mg/L-DSS. DSS-A-E inhibited the increase of AUC from 49.2 to 65.7 mg/L and inhibited the increase of tension from 16.4 to 65.7 mg/L in a concentration-dependent manner, but had no obvious effect on the increase of frequency. DSS-A-N-30 (141.5-566.0 mg/L) inhibited the increase of AUC. Concentrations from 283.0 to 566.0 mg/L could inhibite the frequency at the same time, but only high concentration (566.0 mg/L) could inhibit the increase of tension significantly. These results indicated that DSS-A-E and DSS-A-N-30 can inhibit the intense contraction induced by PGF_2α.3.2.3 Effect of DSS-A-E and DSS-A-N-30 on Ach-induced contractionIntense contraction of isolated rat uterine strips was induced by ACh (10μmol/L), which could be weakened significantly by 7140.0 mg/L-DSS. DSS-A-E of 32.8 to 65.7 mg/L inhibited the increase of AUC and 49.2 to 65.7 mg/L could also inhibit the frequency and tension concentration-dependently. Effect of DSS-A-N-30 was similar to that of DSS-A-E, except that only high concentration (566.0 mg/L) could inhibit the increase of tension significantly. These data implicated that DSS-A-E and DSS-A-N-30 can inhibit the intense contraction induced by ACh.3.3 Effect of DSS-A-E and DSS-A-N-30 on the contraction induced by depolarizationCompared with control group, DSS of 7140.0 mg/L relaxed the contraction induced by K⁺ depolarization significantly, and maximal relaxation reached 50% 30 minutes after treatment. Maximal inhibition was induced by DSS-A-E in concentration from 32.8 to 65.7 mg/L. No significant effect of DSS-A-N-30 (141.5-566.0 mg/L) was observed. These data strongly suggest that DSS-A-E, but not DSS-A-N-30, can inhibit influx of Ca²⁺ from extracelluar fluid.3.4 Effect of DSS-A-E and DSS-A-N-30 on oxytocin-induced contraction in Ca²⁺-free solutionSustained contraction induced by oxytocin (10 IU/L) in calcium-free solution could maintaine stable in 15 min. DSS (7140.0 mg/L) significantly decreased tension from 5 min to 15 min. Sustained contractile was decreased significantly by DSS-A-E of 65.7 mg/L at 5 min and 49.2 to 65.7-mg/L at 15 min. For DSS-A-N-30, sustained inhibition was observed by concentration of 141.5, 283.0 and 566.0 mg/L at 5 min and 566.0 mg/L at 15 min. These data implicate that DSS-A-E and DSS-A-N-30 can inhibit cytosolic Ca²⁺ realeased from sarcoplasmic reticulum to relax uterine smooth muscle.The data showed that spontaneous contraction and agonists-induced intense contraction of isolated uterine strips were inhibited by DSS-A-E and DSS-A-N-30, indicating both of the two active ingredients can inhibit uterine conctration, which is one most important cause of dysmenorrhea. DSS-A-E, but not DSS-A-N-30, could inhibite the uterine contraction induced by K⁺-depolarization, while both DSS-A-E and DSS-A-N-30 could inhibit the sustained contraction induced by oxytocin in calcium-free solution, indicating that DSS-A-E might reduce uterine smooth muscle contration by arresting both the intracellular realease and extracellular influx of calcium ions, while DSS-A-N-30 mainly by holding the intracellular release of calcium ions.4. Modulation of DSS-A-E and DSS-A-N-30 on the productive endocrine systems in miceProductive-endocrine disequilibrium such as high levels of serum E² in luteal phase or premenstrual phase, overproduction of PGF_2α or vasopressin is the important factors to cause dysmenorrhea. In this study, normal ICR mice were orally administered with DSS, DSS-A-E and DSS-A-N-30 for 12 days, and the effect of these drugs on the regulation of endocrine systems was observed. 4.1 Effect of DSS-A-E and DSS-A-N-30 on the oestrus cycle and the weight of uterus and ovary in normal ICR miceResults showed that DSS-A-E (115.0, 230.0, 460.0 mg/kg), DSS-A-N-30 (19.8, 39.6, 79.2 mg/kg) and DSS (9000.0 mg/kg) had no obvious effect on the oestrus cycle, the weight of uterus and ovary in normal mice.4.2 Effect of DSS-A-E and DSS-A-N-30 on the concentration of serum E₂ in nomal miceThe results showed that DSS-A-E (115.0, 230.0, 460.0 mg/kg) and DSS-A-N-30 (19.8, 39.6, 79.2 mg/kg) reduced serum E₂ dose-dependently, especially that of mice in estrus stage. These results implied that DSS-A-E and DSS-A-N-30 can reduce serum E₂ of normal mice in estrus stage.4.3 Effect of DSS-A-E and DSS-A-N-30 on the expression of pituitary LH in nomal ICR miceProtein expression of LH in pituitary was slightly increased by DSS at 9000.0 mg/kg, while DSS-A-E (115.0, 230.0 mg/kg) and DSS-A-N-30 (19.8, 39.6 mg/kg) significantly decreased LH expression in pituitary, implying DSS-A-E and DSS-A-N-30 can regulate the expression of LH in pituitary.4.4 Effect of DSS-A-E and DSS-A-N-30 on uterine PGF_2α concentration in menorrhalgia model miceUterine PGF_2α concentration in menorrhalgia model mice was decreased significantly by DSS-A-N-30 (79.2 mg/kg). It is suggested that decreasing the uterine PGF_2α concentration is one of the mechanism for DSS-A-N-30 in treating dysmenorrhea.5. Conclusion5.1 DSS-A-E and DSS-A-N-30 had analgesic effect on acetic-acid-induced writhing test and oxytocin-induced writhing test in mice, suggesting that DSS-A-E and DSS-A-N-30 might probably have analgesic effect on dysmenorrhea. 5.2 DSS-A-E and DSS-A-N-30 could inhibit the spontaneous uterine contraction and intense uterine contraction induced by oxytocin, PGF_2α and ACh, indicating they might have antispasmodic effect in normal or pathological uterine smooth muscle.5.3 DSS-A-E inhibited the contraction induced by K⁺-depolarization, but DSS-A-N-30 failed. Both DSS-A-E and DSS-A-N-30 inhibited the contraction induced by oxytocin in calcium-free solution. It is implicated that DSS-A-E can decrease cytosolic Ca²⁺ concentration by inhibiting Ca²⁺ realeased from sarcoplasmic reticulum and Ca²⁺ entered from the extracelluar fluid, but DSS-A-N-30 mainly inhibited cytosolic Ca²⁺ realeased from sarcoplasmic reticulum.5.4 DSS-A-E and DSS-A-N-30 decreased serum E₂ of normal mice in estrus stage and LH level in pituitary but had no obvious effect on the normal oestrus cycle and the weight of uterus and ovary, implicating that DSS-A-E and DSS-A-N-30 can balance the ovarian hormones.5.5 Uterine PGF_2α concentration was reduced by DSS-A-N-30. It is implied that decreasing uterine PGF_2α concentration might be one of the menchanism for DSS-A-N-30 to treat dysmenorrhea.5.6 DSS-A-E and DSS-A-N-30 are the active ingredients of DSS in treating dysmenorrhea, and analgesia, inhibiting uterine contraction and balancing the productive endocrine system might be the possible mechanism.