title: Traditional Chinese medicine Guizhi Fuling capsule used for therapy of dysmenorrhea via attenuating uterus contraction.
abstract: BACKGROUND
  Guizhi Fuling formula, a well-known Chinese herbal formula recorded in the Eastern Han Dynasty, is composed of Cinnamomum cassia (L.) J.Presl (Cassia bark), Poria cocos (Schw.) Wolf (Poria), Paeonia suffruticosa andrews (Moutan Cortex), Paeonia lactiflora Pall (Herbaceous peony), and Amygdalus persica L.(Persicae Semen). It has clinical efficacy of activating blood circulation to dissipate blood stasis and is commonly used for the treatment of primary dysmenorrhea. However, its therapeutic mechanism has not been clearly elucidated. The aim of this study is to reveal molecular mechanisms of action using in vivo and in vitro experimental models.
  
  
  METHODS
  The ICR mouse uterine contraction was induced by oxytocin exposure following estradiol benzoate pretreatment. Mice were given GZFLC (0.54, 1.08g/kg) by gavage. The levels of NO, PGF2α and Ca(2+) in uterine tissue were determined according to instructions. Cyclooxygenase-2 (COX-2) and oxytocin receptor (OTR) proteins in uterine tissue were assessed by Western Blot. Mouse isolated uterus strips were mounted in tissue organ baths containing Locke's solution. The contractile responses were recorded with Power Lab recording system. The effect of GZFLC on spontaneous uterine contraction, and uterine contraction induced by oxytocin, PGF2α was observed. Myometrial cells were exposed to oxytocin (5U/L) to induce calcium release, and the effect of GZFLC and its components (PL, PGG, CA) on intracellular Ca(2+) was analyzed with fluorometry imaging.
  
  
  RESULTS
  In vivo study demonstrated that GZFLC significantly reduced oxytocin-induced writhing responses with a maximal inhibition of 55%. It also decreased the levels of NO, PGF2α and Ca(2+) in oxytocin-induced mice uterine tissue. Moreover, Western blot analysis showed that COX-2 and OTR expressions in uterine tissue of dysmenorrhea mice were significantly reduced. GZFLC inhibited spontaneous uterus contractions in a dose-dependent manner, and the IC50 value was 0.99mg/ml. The IC50 values of GZFLC on PGF2α, oxytocin-induced contractions were 1.45mg/ml, 3.53mg/ml, respectively. Further in vitro studies indicated that GZFLC and its components (PL, PGG, CA) could restrain intracellular calcium levels in favour of uteri relaxation.
  
  
  CONCLUSIONS
  Both in vivo and in vitro results indicated that GZFLC possessed a significant spasmolytic effect on uterine tetanic contraction. The present study provides in vivo and in vitro experimental evidence to support the use of GZFLC for the clinical treatment of primary dysmenorrheal (PD).
authors.affiliation: Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing, Jiangsu 211198, PR China; State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China. Electronic address: sunlan2012@126.com.
authors.forename: Lan
authors.initials: L
authors.lastname: Sun
authors.affiliation: State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China.
authors.forename: Lina
authors.initials: L
authors.lastname: Liu
authors.affiliation: State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China.
authors.forename: Shaobo
authors.initials: S
authors.lastname: Zong
authors.affiliation: State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China.
authors.forename: Zhengzhong
authors.initials: Z
authors.lastname: Wang
authors.affiliation: State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhou
authors.affiliation: State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China.
authors.forename: Zhiliang
authors.initials: Z
authors.lastname: Xu
authors.affiliation: State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China.
authors.forename: Gang
authors.initials: G
authors.lastname: Ding
authors.affiliation: State Key Laboratory of New-tech For Chinese Medicine Pharmaceutic Process, Jiangsu Kanion Pharmaceutical Co., Ltd., Lianyungang, Jiangsu 222001, PR China. Electronic address: xw_kanion@163.com.
authors.forename: Wei
authors.initials: W
authors.lastname: Xiao
authors.affiliation: Jiangsu Provincial Key Laboratory for TCM Evaluation and Translational Research, China Pharmaceutical University, Nanjing, Jiangsu 211198, PR China. Electronic address: junpingkou@cpu.edu.cn.
authors.forename: Junping
authors.initials: J
authors.lastname: Kou
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015237:Dinoprost / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D004412:Dysmenorrhea / Q000188:drug therapy* / Q000378:metabolism / Q000503:physiopathology; D004958:Estradiol / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D066298:In Vitro Techniques; D008813:Mice, Inbred ICR; D009569:Nitric Oxide / Q000378:metabolism; D010120:Oxytocics / Q000494:pharmacology; D010121:Oxytocin / Q000494:pharmacology; D010276:Parasympatholytics / Q000494:pharmacology*; D018045:Receptors, Oxytocin / Q000378:metabolism; D015149:Tocolytic Agents / Q000494:pharmacology*; D014590:Uterine Contraction / Q000187:drug effects*; D014599:Uterus / Q000187:drug effects* / Q000378:metabolism / Q000503:physiopathology
chemical_list: D004365:Drugs, Chinese Herbal; C576074:OXTR protein, mouse; D010120:Oxytocics; D010276:Parasympatholytics; D018045:Receptors, Oxytocin; D015149:Tocolytic Agents; C525079:guizhi-fuling; C074283:estradiol 3-benzoate; D009569:Nitric Oxide; D004958:Estradiol; D010121:Oxytocin; D015237:Dinoprost; C077623:Ptgs2 protein, mouse; D051546:Cyclooxygenase 2; D002118:Calcium
keywords: 1,2,3,4,6-Penta-O-galloyl-beta-d-glucopyranose (Pubchem CID: 65238); Albiflorin (Pubchem CID: 162355); Benzoic acid (Pubchem CID: 243); Benzoylpaeoniflorin (Pubchem CID: 46882878); Ca(2+); Cinnamic acid (Pubchem CID: 444539); Cinnamic aldehyde (Pubchem CID: 637511); Ethyl gallate (Pubchem CID: 13250); Gallic acid (Pubchem CID: 370); Guizhi Fuling capsule; Paeoniflorin (Pubchem CID: 442534); Paeonol (Pubchem CID: 11092); Primary dysmenorrhea; Uterine contraction; Uterine smooth muscle cells