Dysmenorrhea, the painful menstruation of uterine origin, is the most common complaint in adolescent girls and women of reproductive age during their gynecological visits [1]. Primary dysmenorrhea (PD) is characterized by a spasmodic, cramping sensation at the lower abdomen without any identifiable pelvic pathology [2, 3]. Typically, painful sensation usually occurs after a few months when ovulatory cycles are attained and are accompanied by systemic symptoms such as fatigue, nausea, vomiting, diarrhea, and sleep disturbance [3]. The prevalence rate varies based on different assessment methods. Generally, PD has been estimated to affect 50% to 95% of females around the world [4, 5]. As the leading cause of absenteeism in school, PD is associated with loss of productivity at work; menstrual cramp also increases the risk of developing depressive symptoms and chronic pain disorders [6, 7]. Given the high prevalence and negative consequences, PD has drastically affected the social relationships of young females by interfering with their daily activities and has become a public health concern [8].

The pathomechanism of PD remains complex and has not been fully elucidated. The over-production of uterine prostaglandins (PG) appears to be the main cause of the hypercontractility of smooth muscles in vessels and uterine myometrium [9]. Based on the PG-dominated etiology, non-steroid anti-inflammatory drugs have been prescribed as the first-line treatment, followed by combined oral contraceptives [2]. However, both pharmacological agents are not suitable for long-term administration and have adverse effects on the digestive tract and central nervous system [10]. Additionally, conventional use of hormone contraceptives is associated with an increased risk of venous thromboembolism [11]. These concerns show an urgent need for alternative approaches to managing menstrual pain.

There’s mounting evidence supporting the application of complementary and alternative medicine in the pain management field as it contains a wide range of medical practices that have been greatly developed and evolved in recent years. A systematic review found the pulsed electromagnetic fields had a significant inhibitory effect on the pain scores in patients with osteoarthritis when compared with the sham group (standardized mean differences [SMD] = 0.71, 95% confidence interval [CI]: 0.08–1.34, p = 0.03) [12]. Moreover, the association between acupuncture analgesia and the endogenous opioid system has been corroborated by many studies [13]. Results from a randomized trial also showed rubbing oil could provide additional benefits to people suffering from low back pain [14]. In China, it has been reported that over half of the patients with painful periods use traditional Chinese medicine (TCM) to treat menstruation-associated discomforts and TCM shows significant efficacy [15]. Based on the theory of conventional Chinese medicine, PD is the clinical manifestation of Qi stagnation and blood stasis syndrome [16]. TCM acts on pain perception in a holistic way with multiple components and targets, in contrast to western painkillers with a single targeted pathway [17]. The Dingkun pill (DKP), one of the well-known TCM, was developed during the Qing dynasty (A.D. 1730) and has been used as a treatment strategy for various gynecological diseases over the past three centuries in East Asia [18]. DKP, comprising thirty herbs and animal-orient ingredients, has been used in the clinical setting for its Qi-nourishing and blood-activating features [19]. In the Chinese literature, there is strong evidence that DKP has an analgesic effect on dysmenorrhea [18, 20]. However, most of them are empirical reports and do not strictly comply with the standard of the CONSORT statement, which makes it difficult to support the conclusion that DKP is therapeutic in dysmenorrhea. Here, we hypothesized that DKP could alleviate PD compared with placebo and aimed to investigate the clinical efficacy, safety, and mechanism of action of DKP in PD using high-level evidence.

It was the first randomized, double-blinded, placebo-controlled study that assessed the clinical effectiveness and safety of DKP in the treatment of PD. Comprehensive indicators including pain scores, pain biochemical mediators, ovarian hormonal profiles, and uterine artery flow indices were evaluated. We found that DKP produced significantly more relief of menstrual pain compared with the placebo. The levels of pain mediators in the DKP group were significantly improved in comparison with those in the placebo group, which further validated the subjective pain score with objective parameters. No abnormal results were found in safety indexes after the intervention. Therefore, our study suggests that DKP has an analgesic effect on dysmenorrheal pain with little systemic toxicity.

DKP has exhibited diverse pharmacological activities by targeting multiple pathways through its multiple ingredients [21, 22]. Previously, 234 chemical compounds were isolated and identified from the 30 crude ingredients of DKP; most of the ingredients were triterpenoid saponins, flavonoids, and alkaloids [21]. Notably, in vitro experiments suggested the triterpenoid saponins extracted from Ilex oubescens roots were able to inhibit nitric oxide (NO) and prostaglandin E${}_2$ (PGE${}_2$) production [23]. Flavonoids were also found with anti-inflammatory effects by inhibiting the release of PGs [24]. Serological examination in rats further confirmed the 18 prototype constituents in 6 primary ingredients namely Ginseng Radix et Rhizoma Rubra, Rhizoma Ligustici Chuanxiong, Rhizoma Corydalis, Radix Glycyrrhizae, Angelicae Sinensis, and Radix Scutellariae. Those original ingredients, identified in other TCMs, have anti-inflammatory, and immunomodulatory properties and promote blood circulation and estrogen-receptor activation for pain management [18].

Although DKP has a wide clinical application in polycystic ovarian syndrome, female subfertility, and menstrual disorders, studies investigating the efficacy of DKP in PD are extremely scarce [25, 26]. There is only one randomized controlled study that investigated the role of DKP in dysmenorrhea [20]. Ma et al. [20] found that DKP remarkably relieved the pain severity in women experiencing dysmenorrhea; a significantly higher proportion of participants in the DKP group achieved over 50% reduction in VAS scores compared with the control group. In addition, the DKP group also showed a more significant decrease in the serum levels of PGF2$\alpha$ and endothelin which are the contributing factors of myometrial hyperactivity than the control group. However, their study used another TCM (Fuke Zaizao capsule) as positive drug control, rather than an inert one, and no allocation concealment was mentioned. Given that pain sensation can be influenced by psychological factors [27], such limitations compromised the scientific merit of their findings. Our study corroborates the benefit of DKP in treating PD by direct comparison with a placebo group. The pain-relieving effect of DKP mainly existed in women with moderate and severe pain while it was not effective in patients with mild symptoms. However, even in mild cases, the gaps in pain reduction between groups also increased with time, suggesting there might be a potential benefit of DKP if the treatment duration is extended.

Based on previous findings, many components in DKP have been found involved in the symptomatic regression for pain management. For example, the biologically active constituents such as baicalein, ethyl acetate fraction, and glabridin in Radix Scutellariae, Angelicae Sinensis, and Radix glycyrrhizae have been found to reduce the COX-2 mRNA or protein expression, and to inhibit the release of PG [28, 29, 30]. In vitro experiments indicated both Radix Scutellariae and Radix Glycyrrhizae had a spasmolytic effect on the smooth muscle of rat uterine tissue [31, 32]. Furthermore, as the primary extract of Rhizoma Corydalis, tetrahydropalmatine displays antinociceptive action on different types of pain syndromes by inhibiting the protein expression of inducible nitric oxide synthase (iNOS) which is responsible for the inflammatory cascade [33, 34]. Future research is needed to unravel the intricate molecular mechanism behind the antinociceptive effect of DKP in PD.

Though the pathogenesis of PD has not been fully elucidated to date, previous studies have shown that this complicated process is modulated by many biological factors [35]. In general, PGF2$\alpha$, vasopressin, and oxytocin are associated with increased contractility of smooth muscle in the uterus and vessels [2]. Numerous studies have shown that PD is correlated with an elevated concentration of PGF2$\alpha$ in both serum and endometrium, and the pain severity seemed to be positively related to its level [36]. Oxytocin and vasopressin are mainly secreted from the posterior lobe of the pituitary, supraoptic, and paraventricular nuclei of the hypothalamus. Both hormones are potent stimulants of uterine contraction, leading to a restriction on uterine blood supply [37]. Similar to previous findings, the present study showed the serum concentrations of PGF2$\alpha$, vasopressin, and oxytocin were significantly lower in the DKP group compared to the control group, suggesting that DKP exerts the analgesic function through an integral regulation of vasoconstrictive substances [20].

Conversely, beta-endorphin, a neurotransmitter primarily produced in the anterior lobe of the pituitary gland, has morphine-like effects on opioid receptors throughout the body by inhibiting the irritation of peripheral somatosensory fibers [38]. It’s been believed that anxiety disorders induced by the painful event are related to decreased peripheral beta-endorphin [39]. Fedele et al. [27] discovered women suffering from PD had positive feedback to placebo and postulated that a surge in endogenous beta-endorphin might result in short-term attenuation. Intriguingly, we indeed detected a significant elevation of 3.05 $\pm$ 0.43 pg/mL in serum beta-endorphin from baseline in the sham group. It’s speculated that the placebo treatment may induce positive expectations which attenuate the mental stress during painful episodes and thereby restore the beta-endorphin levels. Moreover, the growth in beta-endorphin concentrations was significantly higher in the DKP group as compared with the placebo control. This finding indicates that beta-endorphin could probably involve the regulation of the pain matrix in PD as a pain reliever during DKP treatment. Briefly, the underlying mechanism of DKP in pain suppression is mediated through a variety of biochemical factors, and generally, those substances are predominantly regulated at the molecular level. Therefore, in-depth exploration is needed to investigate which signaling and metabolic pathways are involved in the pain-relieving process during DKP treatment.

Doppler sonographic studies suggest dysmenorrheal women have higher uterine impedance than eumenorrheic women throughout the whole menstrual cycle [40]. In the present study, patients in the DKP group showed favorable changes in hemodynamic parameters, inhibition of vasoconstrictors, and improvement in uterine blood flow (spasmolytic effect of DKP). Similar to our findings, a previous study observed that DKP treatment decreased PI and RI of the uterine artery [20, 41]. However, it remains unclear whether this vasodilative function is a direct effect of DKP or secondary sequalae indirectly mediated by the diminishing levels of vasoconstrictors.

Up to date, our study is the first and largest, randomized, double-blinded, placebo-controlled trial investigating the analgesic effect of DKP on PD based on a clinical setting. Notably, in this research, not only the subjective pain rating score but also the objective indicators, biochemical substances involved in pain pathogenesis and uterine artery flow indices reflecting vascular contractility, were evaluated thoroughly, which provided a more comprehensive understanding of the bioactive effect of DKP. Additionally, we included patients with different severity ranging from mild to severe pain and performed subgroup analysis. This approach ensured less selection bias and offered more pragmatic information of reference in daily practice.

Despite those advantages mentioned above, the results of our study should be interpreted with caution considering the following limitations. First, we took the blood sample for the measurement of biochemical parameters associated with pain perception, which might to some extent cause potential bias since the serum concentration of these biofactors could be regulated by systemic metabolic status. Further clinical trials investigating samples from menstruation blood or endometrial biopsy should be encouraged because they might provide more specific details on the local effect of DKP on the uterus. Second, the subjects who participated in our study are all Chinese Han women, which make it hard to extrapolate these therapeutic effects to all women of different ethnicity. Third, although the PD diagnosis in our study has been clinically verified via personal history, pelvic examination, and ultrasonographic screening, some pathological conditions that may cause secondary dysmenorrhea, like peritoneal endometriosis, can barely be identified at the very early stage. Therefore, the possibility of recruiting a dysmenorrheic woman with organic pathological conditions cannot be completely ruled out. Fourth, no pilot study has been done to conducted in this study. Last but not least, there are still major gaps in knowledge about the mechanism of the antinociceptive effect that DKP had on PD. In the current study, we did not explore the intervention of active components in DKP on the targets of different biological functions involved in this painful disorder. Additional bench research on the DKP targets of pain genesis is expected to elucidate its potential pain-killing mechanism. Hopefully, based on that, further trials might be proposed to explore whether PD patients who are refractory to first-line treatment could benefit from DKP treatment.

In conclusion, a pronounced reduction of pain intensity was observed in patients with PD administrating DKP, compared with the placebo, especially for patients with moderate to severe symptoms. With the elevation in beta-endorphin and inhibition of PGF2$\alpha$, oxytocin, and vasopressin, its therapeutic effect seems to be associated with a synergistic combination of vasoconstrictors and neurological stress hormone. Investigation into the molecular mechanism of DKP behind pain attenuation may provide further evidence for alternative therapy in PD management.

PD, Primary dysmenorrhea; DKP, Dingkun pill; VAS, visual analog scale; PG, prostaglandins; PGF2$\alpha$, prostaglandin F2 alpha; PGE${}_2$, prostaglandin E${}_2$; FSH, follicular stimulating hormone; LH, luteinizing hormone; TCM, traditional Chinese medicine; ELISA, enzyme-linked immunosorbent assay; RI, resistance index; PI, pulsatility index; S/D, peak-systolic and end-diastolic; SEM, standard error of the mean; NO, nitric oxide; iNOS, inducible nitric oxide synthase; Lif, leukemia inhibitory factor.

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

AS contributed to the conceptualization and design of the study, the data collection and interpretation. SZ and XD wrote and editing the manuscript. SZ and XM contributed to the protocol planning, data collection and analysis. SZ, XD, JG, YD and YW contributed to patient recruitment and data collection. All authors contributed to editorial changes in the manuscript. All authors read and approved the final version of the paper.

All patients recruited were fully informed, and the informed consent was signed by each participant. he protocol of this trial was evaluated and approved by the Ethics Committee of Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Science (No.ZS-1913).

We are grateful to all the women who participated in our study, and appreciate the arduous work performed by the clinicians and nurses in cooperating hospitals, namely Beijing Obstetrics and Gynecology Hospital, First People’s Hospital of Hangzhou affiliated to Zhejiang University, the Second Affiliated Hospital of Nanchang University, the Hubei Maternal and Child Health Hospital, the First Affiliated Hospital of Chongqing Medical University, Fujian Maternal and Child Health Hospital, and Liuzhou Maternal and Child Health Hospital.

This study received funding from the Capital’s Funds for Health Improvement and Research (CFH: 2020-2-40113), the Natural Science Foundation of China (No. 82074143), and the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (NO. 2020-PT320-003).

The authors declare no conflict of interest.