In vivo and in vitro metabolism study of traditional Chinese medicine formula Dingkun Dan in rats by using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Dingkun Dan (DKD), a reputable traditional Chinese medicine formula, has been used to treat gynecological diseases and showed significant clinical effects since ancient times. However, the application and development of DKD are seriously hampered by the unclear active substances. Structural characterization of compounds absorbed in vivo and their corresponding metabolites is significant for clarifying the pharmacodynamic material basis. In this study, an integrated strategy using ultra-performance liquid chromatography, coupled with quadrupole time-of-flight mass spectrometry and UNIFI™ software, was used to identify prototypes and metabolites after oral administration of DKD in rats. As a result, a total of 261 compounds, including 140 prototypes and 121 metabolites, were tentatively characterized in rat plasma, urine, and feces. The metabolic pathways of prototypes have been studied to clarify their possible transformation process in vivo. Moreover, an in vitro metabolism study was applied for verifying the metabolites under simulating the metabolic environment in vivo. This first systematic metabolic study of DKD is important for elucidating the metabolites and metabolic pathways and could provide a scientific basis for explaining the integrative mechanism in further pharmacology study.

[Influence of Dingkun Dan Combined with Estradiol Valerateon Wnt/ß-catenin Signaling Pathway in Rats with Thin Endometrium with Kidney-Yang Deficiency].

OBJECTIVE: To explore the influence for combination of Dingkun Dan with estradiol valerateon in treating rats with thin endometrium with Kidney-Yang deficiency based on Wnt/ß-catenin signaling pathway. METHODS: The estrous period 40 rats were randomly divided into the normal control group, the model group, the Dingkun Dan group, the estradiol valerateon group and the combination group, with 8 rats in each group. In addition to the normal control group, the rat model of thin endometrium with Kidney-Yang deficiency was established in other groups. The control group used free diet, the model group was given distilled water, the estradiol valerateon group was treated with progynova by gavage at 0.3 mg/(kg·d) , Dingkun Dan group was treated with Dingkun Dan by gavage at 2.26 g/(kg·d), and the combined group was given Dingkun Dan at 2.26 g/(kg·d) on the basis of progynova at 0.3 mg/(kg·d). After 3 estrous cycles, the rats were killed and harvested. HE staining was used to observe histopathologic changes in endometrium. The expression of VEGF in rats endometrium were detected by immunohistochemistry. The expression of ß-catenin, E-cadherin and MMP-9 protein in rat endometrium was detected by Western blot. RESULTS: Compared with the control group, the uterine cavity was narrowed or enlarged, the endometrium glands and blood vessels were sparse, and the endometrium was thinner significantly in the model group ( P<0.01); the levels of VEGF was decreased significantly ( P<0.01), while ß-catenin, E-cadherin and MMP-9 were increased significantly ( P<0.01). Compared with the model group, more endometrial glands, rich intimal vessels, the endometrium were thickened significantly in the 3 treatment groups ( P<0.01 or P<0.05); the levels of VEGF was increased differently. The protein levels of ß-catenin and E-cadherin were significantly decreased in each treatment group ( P<0.01), and MMP-9 were significantly decreased in the Dingkun Dan group and in the combination group ( P<0.01). Compared with the estradiol valerateon group, the level of ß-catenin in Dingkun Dan group was higher, and MMP-9 was lower ( P<0.05 or P<0.01). The levels of ß-catenin and MMP-9 in the combination group were significantly decreased ( P<0.01). Compared with the combination group, the levels of ß-catenin was increased significantly, while decreased signicantly in Dingkun Dan group ( P<0.01 or P<0.05). CONCLUSION: Dingkun Dan combined with estradiol valerateon can increase the thicken of the endometrium by up-regulation of VEGF, while down-regulate of ß-catenin, E-cadherin and MMP-9 in rats with Shen-Yang deficiency and thin endometrium.

[Effects of Dingkun Dan combined with Western medicine on endometrial ß-catenin, VEGF and MMP-9 mRNA expression in rats with multiple lesions].

The aim of this paper was to investigate the effect of Dingkun Dan on endometrial receptivity in rats with multiple lesions. Forty SD female rats with regular sexual cycle were randomly divided into 5 groups, control group, model group, progynova group, Dingkun Dan group and combination group. The thin endometrium model of kidney-yang deficiency was established in all the other rats except normal control group. The rats in normal control group were free to drink and eat; the rats in the model group were administered with distilled water; the rats in the progynova group were treated with progynova; rats in Dingkun Dan group were treated with Dingkun Dan, and the rats in combination group were treated with Dingkun Dan and progynova. After 15 days, serum levels of OPN, VEGF and MMP-9 were measured by ELISA. HE staining, immunohistochemistry and RT-PCR were used to analyze endome-trial morphology, endometrial thickness and the treatment mechanism of Dingkun Dan. As compared with the control group, the serum levels of OPN, VEGF and MMP-9 in the model group were significantly increased(P<0.01). As compared with the model group, the serum levels of OPN and MMP-9 were decreased in Dingkun Dan group(P<0.05, P<0.01). As compared with the control group, endometrial stromal cells were fewer, the endometrium glands and blood vessels were sparse, and the endometrium was thinner significantly in the model group(P<0.01). As compared with the model group, there were more endometrial glands, rich intimal vessels, and dense stromal cells in various treatment groups, and the endometrium were thickened significantly in the treatment groups(P<0.01). As compared with the control group, the expression area of CK19 in the model group was decreased significantly(P<0.01). As compared with the model group, the expression area of CK19 in each treatment group was increased significantly(P<0.05). As compared with the control group, endometrial ß-catenin and MMP-9 mRNA expression levels were increased significantly in the model group(P<0.05), while VEGF mRNA expression was decreased(P<0.05). As compared with the model group, MMP-9 mRNA expression was decreased significantly in the progynova group and the combination group(P<0.05). Dingkun Dan combined with progynova can improve endometrial receptivity by up-regulating expression of ß-catenin, VEGF mRNA and down-regulating the expression of MMP-9 mRNA in the injury rats.

Chemical profliling of Dingkun Dan by ultra High performance liquid chromatography Q exactive orbitrap high resolution mass spectrometry.

Dingkun Dan (DKD) has been widely used for a variety of gynecological disease. However, the systematic analysis of the chemical constituents of DKD has not been well established because of the complexity of the formula and confidentiality. In this paper, liquid chromatography Q Exactive high resolution accurate mass spectrometry (UHPLC-QE-HRMS) with automated MetaboLynx analysis was established to characterize the chemical constituents of DKD. The analysis was performed on a Water Acquity UPLC® HSS T3 using a gradient elution system. Full scan ranged 100-1500 m/z in positive and negative ion mode combined with MS/MS fragmentation for top 5 ions was proposed for aiding the structural identification of the components. All of the peaks were tentatively characterized by not only comparing the retention time and MS data with those from reported literature and database, but also summarizing the fragmentation pathways and promoting to other ingredients identification. Additionally, the network pharmacology study had been used to analysis the identified ingredients and DKD's clinical diseases. In this work, a total of 121 components and isomers were characterized, including amino acids, phenolic acids, lactones, terpenoids, alkaloids, saponins, flavonoids, and other compounds. Network pharmacology analysis showed that identified compounds, such as ginsenosides and notoginsenosides, crocin I, echinacoside, rutin and verbascoside, could be responsible for the pharmacological activity of DKD by regulating the hormone with related metabolism pathways, estrogen signaling pathways and serotonergic synapse pathways. It could indicate that UHPLC-MS showed obvious superiority used to find the potential bioactive compounds of complicated TCM formula without the process of extraction and isolation.

Effects of Dingkun Dan combined with Western medicine on endometrial β-catenin, VEGF and MMP-9 mRNA expression in rats with multiple lesions / 中国中药杂志

The aim of this paper was to investigate the effect of Dingkun Dan on endometrial receptivity in rats with multiple lesions. Forty SD female rats with regular sexual cycle were randomly divided into 5 groups, control group, model group, progynova group, Dingkun Dan group and combination group. The thin endometrium model of kidney-yang deficiency was established in all the other rats except normal control group. The rats in normal control group were free to drink and eat; the rats in the model group were administered with distilled water; the rats in the progynova group were treated with progynova; rats in Dingkun Dan group were treated with Dingkun Dan, and the rats in combination group were treated with Dingkun Dan and progynova. After 15 days, serum levels of OPN, VEGF and MMP-9 were measured by ELISA. HE staining, immunohistochemistry and RT-PCR were used to analyze endome-trial morphology, endometrial thickness and the treatment mechanism of Dingkun Dan. As compared with the control group, the serum levels of OPN, VEGF and MMP-9 in the model group were significantly increased(P<0.01). As compared with the model group, the serum levels of OPN and MMP-9 were decreased in Dingkun Dan group(P<0.05, P<0.01). As compared with the control group, endometrial stromal cells were fewer, the endometrium glands and blood vessels were sparse, and the endometrium was thinner significantly in the model group(P<0.01). As compared with the model group, there were more endometrial glands, rich intimal vessels, and dense stromal cells in various treatment groups, and the endometrium were thickened significantly in the treatment groups(P<0.01). As compared with the control group, the expression area of CK19 in the model group was decreased significantly(P<0.01). As compared with the model group, the expression area of CK19 in each treatment group was increased significantly(P<0.05). As compared with the control group, endometrial β-catenin and MMP-9 mRNA expression levels were increased significantly in the model group(P<0.05), while VEGF mRNA expression was decreased(P<0.05). As compared with the model group, MMP-9 mRNA expression was decreased significantly in the progynova group and the combination group(P<0.05). Dingkun Dan combined with progynova can improve endometrial receptivity by up-regulating expression of β-catenin, VEGF mRNA and down-regulating the expression of MMP-9 mRNA in the injury rats.

[Effect of clomiphene citrate and Dingkun Dan on ovulation induction and clinical pregnancy of polycystic ovary syndrome].

The evaluation is based on clomiphene citrate (CC)+gonadotropin (Gn), clinical study on CC and Dingkun Dan's treatment on ovulation induction and clinical pregnancy effect of PCOS, and to provide ideas and methods for traditional Chinese medicine assisted reproductive treatment. This study selected 60 PCOS infertility patients treated with ovulation induction in reproductive medicine clinic, Jiangsu Province Hospital of traditional Chinese medicine during 2015-10-01-2017-04-23. They were randomly divided into two groups： Group A (CC+Gn+HCG) and Group B (CC+Gn+Dingkun Dan). These results were observed and compared including cycle ovulation rate, cycle cancellation rate, cycle pregnancy rate, cumulative pregnancy rate, endometrial thickness, duration of Gn, total amount of Gn, the occurring rate of luteinized unruptured follicle syndrome and ovarian hyperstimulation syndrome. Group A had lower cycle ovulation rate, cycle pregnancy rate, cumulative pregnancy rate and endometrial thickness, compared with Group B, the difference was statistically significant(P＜0.05). However, Group A had higher cycle cancellation rate, duration of Gn and total amount of Gn, compared with Group B, the difference was statistically significant(P＜0.05). In this study, no case of LUFS or OHSS was found in all patients. CC and Dingkun Dan had the effect of promoting ovulation on PCOS infertility patients, and CC+Gn+Dingkun Dan could elevate clinical pregnancy rate.

[Impact of prostaglandin F2α and endothelin, pulsation index and resistance index of uterine artery blood flow on dysmenorrhea patients of cold stagnation and blood stasis syndrome with Dingkun Dan].

This research apply Dingkun Dan to treat patients with dysmenorrhea of cold stagnation and blood stasis syndrome. This study observed its effectiveness and safety of the treatment of the disease and its influence on the serum prostaglandin F2α, endothefin, pulsatility index and resistant index of uterine artery blood, to explore the possible mechanism of effect of Dingkun Dan in the treatment of dysmenorrhea and provide scientific basis for clinical application. The 75 patients with dysmenorrhea of cold stagnation and blood stasis who met the inclusion criteria, were divided into treatment group (n=37) and control group (n=38) by using random number remainder grouping method. In the treatment group patients were treated with Dingkun Dan, the other group were given Fuke Zaizao Jiaonang. Two groups have same time to take the medicine, three days prior to the menstruation for ten days. Medication for three menstrual cycles was seen as a course of treatment. To observe and compare the two groups of patients before and after treatment VAS score, syndrome integral, serum levels of prostaglandin F2α and endothelin, pulsation index and resistance index of uterine artery blood flow and related safety index changes. Finally makes statistical analysis. It has been identified that, Treatment group and control group can reduce pain symptom of dysmenorrhea patients and improve the syndromes scores, compare with control group, effect of the treatment group is more significant(P<0.01). VAS pain curative effect: the treatment group and control group total effective rate respectively were, 97.22%, 69.44%, markedly effective rate were 83.33%, 30.56%, comparison between two groups, treatment group is better than that of control group(P＜0.01). Syndromes curative effect: the treatment group and control group total effective rate respectively were 97.22%, 94.44%, markedly effective rate was 66.67%, 2.78%, respectively. The comparison between two groups, the total effective rate has no obvious difference, but markedly effective rate of treatment group is better than that of control group(P＜0.01). The treatment group can significantly reduce the patients' serum level of prostaglandin F2α(P<0.01), but no obvious difference was found in the control group before and after treatment. Both groups can significantly reduce the serum level of endothelin(P<0.01), comparison between two groups, the treatment group is more significant(P<0.01).Both treatment group and control group were significantly lower left and right pulsation index and resistance index of uterus artery blood flow(P<0.01). Between groups to compare the effect, the treatment group is more significant(P<0.01). Both treatment group and control group in the security check before and after treatment found no significant anomalies. Dingkun Dan in treating dysmenorrhea with cold stagnation and blood stasis syndrome seems to have remarkable clinical curative effect and safety, which may be achieved by significantly reducing the serum level of prostaglandin F2αand endothefin of the patients, and reducing the pulsation index and resistance index of uterine artery blood flow of the patients, to improve uterine artery condition of blood, and correcting local tissue ischemia to relieve pain.

Effect of clomiphene citrate and Dingkun Dan on ovulation induction and clinical pregnancy of polycystic ovary syndrome / 中国中药杂志

The evaluation is based on clomiphene citrate (CC)+gonadotropin (Gn), clinical study on CC and Dingkun Dan's treatment on ovulation induction and clinical pregnancy effect of PCOS, and to provide ideas and methods for traditional Chinese medicine assisted reproductive treatment. This study selected 60 PCOS infertility patients treated with ovulation induction in reproductive medicine clinic, Jiangsu Province Hospital of traditional Chinese medicine during 2015-10-01-2017-04-23. They were randomly divided into two groups： Group A (CC+Gn+HCG) and Group B (CC+Gn+Dingkun Dan). These results were observed and compared including cycle ovulation rate, cycle cancellation rate, cycle pregnancy rate, cumulative pregnancy rate, endometrial thickness, duration of Gn, total amount of Gn, the occurring rate of luteinized unruptured follicle syndrome and ovarian hyperstimulation syndrome. Group A had lower cycle ovulation rate, cycle pregnancy rate, cumulative pregnancy rate and endometrial thickness, compared with Group B, the difference was statistically significant(P＜0.05). However, Group A had higher cycle cancellation rate, duration of Gn and total amount of Gn, compared with Group B, the difference was statistically significant(P＜0.05). In this study, no case of LUFS or OHSS was found in all patients. CC and Dingkun Dan had the effect of promoting ovulation on PCOS infertility patients, and CC+Gn+Dingkun Dan could elevate clinical pregnancy rate.

Impact of prostaglandin F2α and endothelin, pulsation index and resistance index of uterine artery blood flow on dysmenorrhea patients of cold stagnation and blood stasis syndrome with Dingkun Dan / 中国中药杂志

This research apply Dingkun Dan to treat patients with dysmenorrhea of cold stagnation and blood stasis syndrome. This study observed its effectiveness and safety of the treatment of the disease and its influence on the serum prostaglandin F2α, endothefin, pulsatility index and resistant index of uterine artery blood, to explore the possible mechanism of effect of Dingkun Dan in the treatment of dysmenorrhea and provide scientific basis for clinical application. The 75 patients with dysmenorrhea of cold stagnation and blood stasis who met the inclusion criteria, were divided into treatment group (n=37) and control group (n=38) by using random number remainder grouping method. In the treatment group patients were treated with Dingkun Dan, the other group were given Fuke Zaizao Jiaonang. Two groups have same time to take the medicine, three days prior to the menstruation for ten days. Medication for three menstrual cycles was seen as a course of treatment. To observe and compare the two groups of patients before and after treatment VAS score, syndrome integral, serum levels of prostaglandin F2α and endothelin, pulsation index and resistance index of uterine artery blood flow and related safety index changes. Finally makes statistical analysis. It has been identified that, Treatment group and control group can reduce pain symptom of dysmenorrhea patients and improve the syndromes scores, compare with control group, effect of the treatment group is more significant(P<0.01). VAS pain curative effect: the treatment group and control group total effective rate respectively were, 97.22%, 69.44%, markedly effective rate were 83.33%, 30.56%, comparison between two groups, treatment group is better than that of control group(P＜0.01). Syndromes curative effect: the treatment group and control group total effective rate respectively were 97.22%, 94.44%, markedly effective rate was 66.67%, 2.78%, respectively. The comparison between two groups, the total effective rate has no obvious difference, but markedly effective rate of treatment group is better than that of control group(P＜0.01). The treatment group can significantly reduce the patients' serum level of prostaglandin F2α(P<0.01), but no obvious difference was found in the control group before and after treatment. Both groups can significantly reduce the serum level of endothelin(P<0.01), comparison between two groups, the treatment group is more significant(P<0.01).Both treatment group and control group were significantly lower left and right pulsation index and resistance index of uterus artery blood flow(P<0.01). Between groups to compare the effect, the treatment group is more significant(P<0.01). Both treatment group and control group in the security check before and after treatment found no significant anomalies. Dingkun Dan in treating dysmenorrhea with cold stagnation and blood stasis syndrome seems to have remarkable clinical curative effect and safety, which may be achieved by significantly reducing the serum level of prostaglandin F2αand endothefin of the patients, and reducing the pulsation index and resistance index of uterine artery blood flow of the patients, to improve uterine artery condition of blood, and correcting local tissue ischemia to relieve pain.