Information on the studies of Pulsatilla chinensis (Bge.) Regel (PC) is collected from scientific journals and reports via library and electronic data search (PubMed, Baidu Academic, Google Scholar, Science Direct, ACS, Web of Science, and CNKI). Meanwhile, it is also obtained from published works of folk records, ethnopharmacological literature, Ph.D. and Master’s Dissertation. The specific search results are as follows.

The keywords of the second part are “chemical composition of PC”. A total of 32 pieces of literature were retrieved. The keywords of the third part are “PC anti-tumor”, “PSA anti-tumor”, “AB4 anti-tumor”, and “PSA anti-tumor”. A total of 79 pieces of literature were retrieved. After the screening, 62 kinds of literature met the requirements. The keywords of the fourth part are “anti-tumor of PC” and “clinical application of PC”. A total of 7 literature were retrieved. 3 literature were screened to meet the requirements. The keywords of the fifth part are “PC pharmacokinetics”. A total of 11 pieces of literature were retrieved. The keywords of the sixth part are “network pharmacology of PC”, “modern technology of PC”, and “molecular docking technology of PC”. A total of 3 literature were retrieved.

Traditional Chinese medicine (TCM) has been an integral part of healthcare in China for thousands of years. In modern medicine, Chinese herbs also have been used to treat a variety of diseases. For example, the anti-cancer effect of Pulsatilla chinensis (Bge.) Regel (PC) had been found in previous research studies. PC is a common TCM, which belongs to the plant genus Pulsatilla of the Ranunculaceae family. Because PC likes to be born in fields, wetlands, riverbanks, and damp grasses, it is widely distributed in China, such as Sichuan, Hubei, Jiangsu, Jilin, Heilongjiang, etc. In addition, PC has also been found in North Korea and the Russian Far East.

The first description of PC was recorded in the Shijing as early as the Western Zhou Dynasty. In addition, PC was first documented as a medicinal material in Shen Nong’s Herbal Classic, recording that it tasted bitter and cold. It is mainly used for treating malaria fever, dispelling dampness and heat, cooling blood, relieving pain, and treating malignant sores. Among them, concretions and gatherings were referred to as a type of tumor in modern medicine.

In recent years, a large number of literatures have reported that the pentacyclic triterpenoid saponins in PC (PC saponins) have significant anti-tumor activities in vitro and in vivo, such as inhibition of cell proliferation, effects of signal transduction, apoptosis and tumor invasion, etc (1). Modern pharmacological studies have shown that a large number of researchers have conducted extensive research on PC saponins, for their cancer chemopreventive potential against various cancers, for instance, gastric cancer, lung cancer, breast cancer, cervical cancer, hepatocellular carcinoma, pancreatic cancer (2), leukemia, colon cancer, multiple myeloma, etc (3). In particular, pulsatilla saponin A (PSA) (4), Anemoside B4 (AB4) (5), raddeanoside R13 (R13) (6), and Pulsatilla saponins D (PSD) (7) proved to have strong anti-tumor effects too. At the same time, it has also been widely studied.

As people pay more and more attention to the anti-cancer effects of PC, a lot of researchers had been conducted on its pharmacological effects and clinical applications. However, the literature lacked a systematic and comprehensive review of the anti-tumor effects of PC. Considering the complexity of TCM, more scientific research was needed to determine its chemical composition, biological activity, and potential molecular mechanism. In this review article, the mechanisms and biological effects of PC were reviewed from seven aspects: cell proliferation, apoptosis, cell cycle, energy metabolism, drug resistance, autophagy, and angiogenesis. And through the evaluation of its curative effect and therapeutic use, its clinical application value was discussed. This article also discussed the pharmacokinetic and bioinformatic studies of PC and proposes future research directions, to make this review article a useful reference resource for researchers.

In recent years, more and more scientists have paid attention to the bioactive molecules and anti-tumor activity of PC. Early literature reported that PC contained Anemonin, Okinalin, and Okinalein, etc (8). Subsequently, triterpene acids, lignans, carotinosides (9), non-O-linked glycoprotein components PCG-A (10), and a toxic protein AME (11) were gradually isolated from PC. According to the literature, the anti-tumor components of PC were mainly pentacyclic triterpenoid saponins. Chen Wenkan (12, 13), Yoshihirol (14, 15), etc. respectively extracted and separated various lupinane triterpenoid saponins and oleanane triterpenoid saponins from PC. And their aglycones were mainly divided into three types: oleanolic acid sapogenin, ivy sapogenin, and 2,3-hydroxy betulinic acid sapogenin (16). The names of these constituents were listed in Table 1 , and their chemical structures were shown in Figure 1 . In addition, the structure-activity relationship indicated that oleanane-type saponins had better cytotoxic activity than lupinane-type saponins. This was inseparable from the free carboxyl group on the aglycon C-28. Similarly, the length and bonding degree of the ethanol chain on the aglycon C-3 also had an important influence on the cytotoxic activity (30).

Some studies showed that Pulsatilla saponin D (PSD) and Pulsatilla saponin A (PSA) had the strongest anti-tumor activity among PC saponins, and their IC₅₀ for NCI-H460 cells were 5.2, and 7.9 μg/mL, respectively (31). Among these biologically active molecules, the release of the carboxyl group at position C-28 of PSD was essential for the anti-tumor activity of saponins. At the same time, the type of aglycon, the number of sugar groups, and the sugar chain sequence were rha(1→2)[glc(1→4)ara saponin, which had strong anti-tumor activity (32). Notably, to better apply the active compounds of PC saponins in clinics, PSA and PSD derivatives with C ring or C-28 or C-3 modifications were synthesized (32). In addition to PSD and PSA, raddeanoside R13 (R13) had clear anti-tumor activity and their IC₅₀ for NCI-H460 cells was 4.6 μg/mL.

Among the PC, anemoside B4 (AB4) had the highest content in the herb, and it was used as a quality control marker for the PC, quantified as over 4.6% (33). At present, AB4 is the most studied active compound in PC. And some researchers reviewed the pharmacological effects of AB4 (34). However, the anti-tumor effect of AB4 had not been elucidated in detail. Particularly, studies have confirmed that AB4 can reduce the toxicity of cisplatin. After 10 days of intraperitoneal injection at a dose of 3 mg/kg, the inhibitory rate of AB4 (98.4%) on tumor cells was greater than that of cisplatin (95.1%) (35). This will provide a direction for the future anti-tumor research of AB4.

This article provides a comprehensive review of the literature on the anticancer activity of PC. The anti-tumor mechanism of PC is mainly to inhibit cell proliferation, induce apoptosis, inhibit cell cycle, regulate cell energy metabolism, reverse drug resistance, induce autophagy and inhibit angiogenesis. The specific molecular pathways are shown in Table 2 and Figure 2 .

At present, after treatment with PC compound, adverse reactions such as neutropenia, vomiting and diarrhea were improved in 66 patients (97). It is reported that this may be related to the inhibition of zDHHC9 expression and the restricted function of the Ras gene (98). In addition, studies have shown that the TCM compound mainly based on PC is also used to treat complications caused by radiotherapy in 22 patients with cervical cancer (99). In short, the TCM compound mainly based on PC has achieved good clinical effects in the adjuvant treatment of colorectal cancer patients.

Pharmacokinetic studies on PC are vital to clarify the principle of compatibility. The mechanisms contribute to deciphering a reasonable dose and lower clinical adverse reactions. Studies have shown that the main part of intestinal absorption of PC saponin is the duodenum (100). Simultaneously, the efficacy of PC saponin is concentration-dependent (101).

Bioinformatics is a common method to study the multi-target characteristics of TCM. It was reported that 11 active compounds in PC were found to overlap with colorectal cancer target genes through network pharmacology analysis. Among them, 21 core genes play an anticancer role through 11 major pathways including the p53 signal transduction pathway and the central carbon metabolism pathway in cancer. Furthermore, molecular docking results showed that the saponins of PC had a good binding effect on the target genes of colorectal cancer. Among them, PSA can bind to colorectal cancer proteins HSP90, KIT, and SIRT1 (113).

In addition, some researchers explored the bioinformatics of Baitouweng Decoction, a TCM compound based on PC. It is reported that 5 cases of colon cancer patients were purified by blood samples before and after treatment with Baitouweng Decoction. Agilent chip and computer software were used to screen 159 differentially expressed genes, including 38 up-regulated genes and 121 down-regulated genes. It is mainly manifested in Jak-STAT, MAPK, and other tumor-related signaling pathways (114). Another study predicted by network pharmacology that Baitouweng decoction may affect tumor cell proliferation, migration, apoptosis, and angiogenesis by regulating signaling pathways such as TNF, PI3K-Akt, HIF1, and vascular endothelial growth factor (115). These results provide a reference for discussing the anti-tumor mechanism of PC.

In this review, we summarized the reports on the anticancer effect and molecular mechanisms of active compounds and extracts of PC in both laboratory and clinic. In recent years, the anticancer effect of PC extracts against liver, lung, breast, gastric, and colon cancer, etc has been widely investigated. The majority of the scientific literature reported that PC exerted anticancer effects through suppressing cell proliferation, inducing apoptosis, inhibiting migration, inhibiting invasion, inducing autophagy, and restraining angiogenesis. To determine the molecular mechanism of the anticancer effect of PC, detailed studies have been carried out. The reported molecular mechanisms include Notch, PI3K/AKT/mTOR, AKT/mTOR, and MEK/ERK signaling pathways. PC can down-regulate the expression levels of IL-6R, TNF-α, NF-kB, Bcl-2, and Caspase-3, meanwhile, up-regulate the expression of autophagy-related molecules Beclin1 and Atg5. In addition, PC can reduce the expression of HIF-1α and VEGF and effectively inhibit the phosphorylation of PI3K downstream factors (such as Akt, mTOR, and p70S6K). Finally, it induces tumor cell apoptosis by inhibiting angiogenesis. Interestingly, PC regulates energy metabolism of tumor cells by reducing glucose consumption, lactic acid production and adenosine triphosphate content. At the same time, PC can also down-regulate key glycolytic proteins GLUT1, HK2, MCT4, MCT1, c-Myc and HIF-1a. It can be seen from the literature that the curative effect of TCM and the molecular mechanisms involved are very complicated. Although the biological role of the anticancer activity of PC has been reported in detail, the mechanisms of anti-tumor effects of PC saponins remain to be further identified. More explorations remain to be performed, such as the effects of PC saponins on cancer metastasis or immunity. It may be hoped that further studies will be conducted on PC saponins to identify more effective anti-tumor components.

PC extract has been widely applied in China as an alternative medicine against diseases. This review provides the latest progress in the anti-tumor activity and mechanism of PC. At the same time, the clinical application, pharmacokinetics and bioinformatics of PC are also described. Among them, the compounds PSA, PSD, AB4, and R13 have high anti-tumor activity. Some studies have confirmed that the anti-tumor effect of R13 is comparable to that of cisplatin (53). Meanwhile, the effect of R13 on apoptosis was stronger than that of PSD. However, as far as the existing literature is concerned, there are few significant antitumor studies of R13. Therefore, it is imminent to study the molecular mechanism of the antitumor effect of active compounds in PC. In addition, it is an important research topic to find the optimal process conditions for the further separation and purification of PC saponins monomer.

Moreover, studies based on UPLC-QTOF-MS serum metabolomics studies have confirmed that long-term oral PC total saponins can cause chronic liver damage, and its safety needs further attention (116). To be sure, the development of highly efficient and low toxic anti-cancer drugs is one of the most urgent problems in the medical field. PC compounds have the characteristics of natural, low toxicity, and high efficiency, which allows them a promising antitumor drug.

HL wrote the full article and provided ideas. Meanwhile, according to the requirements of editors and reviewers, HL revised the whole process. LW provided the idea for the article. XZ modified the language of the article. WX and XZ modified Figure 2 . HS and XF conducted the article for guidance. All authors contributed to the article and approved the submitted version.

Study on the material basis and mechanism of effect of national medicine Thymus quinquecostatus on ischemic cardio-cerebrovascular diseases; National Natural Science Foundation of China(81760769); Study on anti-reperfusion injury metabolism of polyphenols extracted from Chinese medicinal herb Thymus quinquecostatus; Key research and development plan of autonomous region(2020BFG03007); Special Talents Initiation Project of Ningxia Medical University(XT2013003).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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