Unknown active constituents and unclear mechanism-of-actions have sparked criticism when Chinese medicine (CM) formula is getting more popular today [1, 2]. Thus, investigating the pharmacology is important to the modernization of CM formula. However, the pharmacological study of a CM formula is much more complicated than that of a single compound. With a single compound study, researchers need only determine which biological target(s) it acts on, and which disease pathway(s) it alters (Fig. 1a). With a formula study, there is much more to be done and many more factors to be considered. Firstly, the CM formula is comprised of several herbs, each of which contains hundreds, possibly thousands, of compounds, many of which could be unique to that herb. Secondly, not all the compounds from herb are involved in the pharmacological activity—some of them are removed during preparation, while some of them are just passed by. Thirdly, most compounds from herbs are weak modulators of biological targets, thus the effect of an individual compound is hard to determine. Fourthly, the herbal compounds may have multiple pharmacological actions, some of which are not directly correlated with symptom improvement; to identify the targets and pathways that are truly involved is not easy. Lastly, the complex interactions (synergistic or antagonistic) between herb compounds are hard to determine.

Multiple approaches have been utilized and developed for investigating the pharmacology of CM formula, including network pharmacology, pharmaco-genomics, -proteomics, and -metabolomics. These approaches have been successfully applied in studying the pharmacology of the Liu-Wei-Di-Huang pill, Qing-Luo-Yin, and other CM formulas [3–11]. In particular, there are reviews discussing the theory, methodology and applications of CM network pharmacology [12–19]. We used several of these methodologies to investigate the pharmacology of a CM formula MaZiRenWan (MZRW, also known as Hemp Seed Pill) [20, 21]. Based on the nature, we observe that most of these approaches fall into one of two categories in terms of how they tackle the problem: bottom-up, or top-down (Fig. 2).

In the context of medicinal herb research, by bottom-up, we mean starting with the many smaller units, i.e. isolated constituents, and determining their larger role in a disease pathway (Fig. 2a). By top-down, we mean starting with the disease pathway, and determining which constituents are involved in regulating it (Fig. 2b). These two contrasting approaches are equally effective—and are seen in other contexts such as nanotechnology, neuroscience, psychology, public health, ecology, management, and organization [22]. For example, in cognitive process, bottom-up cognition is focusing on details primarily, then the whole landscape. While a top-down approach is used by the person who focus on the big picture first and from that figure out details to support it [23]. In this article, we will compare these two distinct approaches in the investigation of pharmacology of one CM formula, MZRW for functional constipation (FC).

MZRW is an herbal formula for constipation from traditional chinese medicine (TCM). About 2000 years ago, MZRW was firstly recorded in Discussion of Cold-Induced Disorders (Shang Han Lun) [24, 25]. It is comprised of six herbs, namely Fructus cannabis (Huo Ma Ren), Radix et rhizoma rhei (Da Huang), Semen Armeniacae Amarum (Ku Xing Ren), Radix paeoniae Albo (Bai Shao), Cortex magnolia officinalis (Hou Pu), and Fructus aurantii immaturus (Zhi Shi) [26]. In TCM theory, MZRW can drain heat, unblock the bowel, promote the movement of Qi, and moisten the intestines [26].

We chose MZRW because a systematic review of the published literature showed that MZRW is the most frequently used TCM formula for constipation [27] yet there is little if any strict clinical evidence of its efficacy. To that end, we demonstrated that MZRW is significantly better than placebo in improvement of bowel movement during drug treatment, while such effect is more sustainable than placebo during 8 weeks follow-up, in the randomized, placebo-controlled clinical study with 120 FC patients [26]. Recently, we have finished a larger clinical study including 291 FC patients to compare the efficacy of MZRW with that of Senna (commonly used laxative in Hong Kong) and placebo [28]. The results showed that, both MZRW and Senna are better than placebo during the treatment period; while the efficacy of MZRW is more sustainable than that of Senna and placebo in the follow-up period. We also identified ten major compounds from MZRW in rat plasma by UPLC–MS/MS [29] to facilitate the pharmacokinetic study of MZRW in healthy volunteers [30].

On top of this solid clinical evidence and pharmacokinetic data, we set out to elucidate the pharmacology of MZRW for FC. We tried different methodologies to determine (1) which active compound(s) are in MZRW, and how they act (2) on which biological target(s), (3) through which signaling pathway(s) to alter the bowel movement, as slow bowel movement is the major symptom of FC patients. Doing this work eventually we realized that every methodology has its own advantages and disadvantages, but they can be compared in an efficient way: bottom-up versus top-down (Table 1). In the following sections we will first describe these two different approaches; then describe their application in the analysis of MZRW; and conclude with the take-home lessons for doing similar research on other CM formulas.