Clinical antitumor application and pharmacological mechanisms of Dahuang Zhechong Pill.

Introduction

Cancer, a top cause of death in humans, represents an essential barrier to life expectancy increase, whose incidence and mortality rates are quickly rising worldwide ( Bray et al., 2021 , Siegel et al., 2022 ). According to GLOBOCAN 2020, an estimated 19.3 million incident cancer cases and almost 10.0 million deaths associated with malignant diseases were reported globally in 2020. The cancer burden is projected to reach 28.4 million cases in 2040, marking a 47% increase over the current figure ( Sung et al., 2021 ). Despite a series of breakthroughs in cancer treatment in the past few years, cancer remains the leading cause of mortality. Routine and new modalities utilized for cancer treatment, including surgery, radiotherapy, chemotherapy, targeted therapy, and immunotherapy, have secondary effects on decreasing the quality of life ( Arvind et al., 2021 , Mun, Babiker, Weinberg, Kirson, & Von Hoff, 2018 ). Additionally, the toxic effects of current treatment options significantly hamper tolerability and compliance ( Rodriguez-Frias & Lee, 2007 ). Hence, more potential drugs with low toxicity for the improvement of clinical outcomes of cancer patients are urgently needed.

Traditional Chinese medicine (TCM) is gradually being accepted as a potent treatment alternative for cancer ( Xiang, Guo, Zhu, Chen, & Huang, 2019 ). Chinese medicine preparations show obvious advantages as available, efficient and relatively safe products. Abundant evidence confirms that TCM is capable of alleviating patients' clinical symptoms, reducing adverse reactions from chemotherapy and improving immunity ( Pan et al., 2020 , Zhang et al., 2021 ).

Dahuang Zhechong Pill (DHZCP) represents a great classical Chinese medical preparation derived from the Synopsis of the Golden Chamber by Zhongjing Zhang (150–219 CE, Fig. 1 ), which includes twelve traditional Chinese medicines, including Eupolyphaga Steleophaga (Tubiechong in Chinese), Rhei Radix et Rhizoma (Dahuang in Chinese), Scutellariae Radix (Huangqin in Chinese), Glycyrrhizae Radix et Rhizoma (Gancao in Chinese), Persicae Semen (Taoren in Chinese), Armeniacae Semen Amarum (Kuxingren in Chinese) , Paeoniae Radix Alba (Baishao in Chinese), Rehmanniae Radix (Dihuang in Chinese), Toxicodendri Resina (Ganqi in Chinese), Tabanus bivittatus Matsumura (Mengchong in Chinese), Hirudo (Shuizhi in Chinese), and Holotrichia diomphalia Bates (Qicao in Chinese) ( Zhang, Liu, Wen, & Liu, 2009 ). The original prescription mainly treats the symptoms of consumptive disease and stagnation of blood stasis. Moreover, the characteristics of DHZCP, such as removing blood stasis for promoting tissue regeneration, moderating middle-Jiao and tonifying deficiency, and eliminating evils to support healthy energy are particularly suitable for healing the symptoms of stagnant blood, weakened qi and blood, deficiency of healthy atmosphere presented by various diseases such as cancer, thus reducing pain, prolonging life expectancy, and improving immune function. As such, modern clinics often use DHZCP to treat tumors, chronic viral hepatitis, cirrhosis, chronic hepatitis, liver fibrosis, chronic renal failure, angina pectoris, ventricular premature contractions and traumatic hematomas, among others. In recent years, multiple pharmacological properties and clinical applications for DHZCP have been reported, especially in the treatment of tumors, with the advantages of prescription in tumor suppression and adjuvant therapy ( Ren & Jiang, 2019 ). The antitumor effects of DHZCP mostly involve suppressed cell proliferation, induced cell apoptosis, regulated immune function, inhibited angiogenesis, decreased metastasis and reversal of cell resistance. DHZCP is primarily adopted to treat malignant tumors such as liver, gastric, esophageal and lung cancers ( Ni et al., 2019 ). Fig. 1 Origin and evolution of Dahuang Zhechong Pill. (A) Portrait of Zhongjing Zhang, a famous Chinese medical practitioner (150–219 CE); (B) Ancient version of Synopsis of the Golden Chamber , a classical literature of Chinese medicine written by Zhongjing Zhang; (C) Packaging and appearance of Dahuang Zhechong Pill.

Material basis of DHZCP

Chemical constituents of DHZCP

In recent years, with the continuous maturation and development of high-performance liquid chromatography (HPLC) and UPLC-MS/MS technologies, the chemical components in the whole formula of DHZCP have been fully characterized and accurately identified. According to all available literature reports ( Guo et al., 2019 , Huang et al., 2019 , Li et al., 2017 , Sun & Qiu, 2020 , Wu et al., 2016 , Wu et al., 2019 , Wu et al., 2022 , Zhang & Wang, 2019 , Zhang, 2019 ), a total of 140 chemical constituents are contained in DHZCP, of which 13 components ( 1 – 13 ) are derived from Rhei Radix et Rhizoma , mostly anthraquinone derivatives ( Fig. 2 ); A total of 12 compositions ( 14 – 25 ) are related to Persicae Semen or Armeniacae Semen Amarum , consisting of flavonoids, organic acids, etc.; 11 ingredients ( 26 – 36 ) are attributed to Scutellariae Radix , all of which are flavonoids ( Fig. 3 ); 15 components ( 37 – 51 ) are affiliated with Rehmanniae Radix , with the majority of iridoid glycosides and phenylethanoid glycosides ( Fig. 4 ); 13 compounds ( 52 – 64 ) are originated from Paeoniae Radix Alba , comprising terpenoids and phenolic acids ( Fig. 5 ); 14 components ( 65 – 78 ) are generated from Glycyrrhizae Radix et Rhizoma , with most being pentacyclic triterpenes, flavonoids and steroids ( Fig. 6 ); A total of 51 compounds ( 79 – 129 ) are from animal medicines in the DHZCP which contain alkaloids, organic acids and vitamins ( Fig. 7 ); The remaining 11 candidates ( 130 – 140 ) could not be precisely attributed to medicinal herbs, most of them are organic acids ( Fig. 8 ). The common names, sources, and molecular formulas of all the chemical substances in DHZCP were summarized in Table 1 . Fig. 2 Chemical structures of components 1–13 from Rhei Radix et Rhizoma . Fig. 3 Chemical structures of components 14–25 from Persicae Semen or Armeniacae Semen Amarum and components 26–36 from Scutellariae Radix. Fig. 4 Chemical structures of components 37–51 from Rehmanniae Radix. Fig. 5 Chemical structures of components 52–64 from Paeoniae Radix Alba. Fig. 6 Chemical structures of components 65–78 from Glycyrrhizae Radix et Rhizoma. Fig. 7 Chemical structures of components 79–129 from animal medicines. Fig. 8 Chemical structures of other components 130–140. Table 1 Summarized information of chemical components in DHZCP. No. Source attribution Compounds Molecular formula Whether detected in blood No. Source attribution Compounds Molecular formula Whether detected in blood 1 Rhei Radix et Rhizoma Emodin C 15 H 10 O 5 Y 71 Glycyrrhetinic acid C 30 H 46 O 4 N 2 Chrysophanol C 15 H 10 O 4 Y 72 Isoliquiritin C 21 H 22 O 9 Y 3 Rhein C 15 H 8 O 6 Y 73 Glycyrrhizic acid ammonium salt C 42 H 65 NO 16 N 4 Physcion C 16 H 12 O 5 Y 74 Stigmasterol C 29 H 48 O N 5 Aloe emodin C 15 H 10 O 5 Y 75 Rhapontisterone C 27 H 44 O 8 Y 6 Polydatin C 20 H 22 O 8 Y 76 Betulinic acid C 30 H 48 O 3 N 7 Lindleyin C 23 H 26 O 11 Y 77 Oleanic acid C 30 H 48 O 3 N 8 Isolindleyin C 23 H 26 O 11 Y 78 Hederagenin C 30 H 48 O 4 N 9 Emodin-8-glucoside C 21 H 20 O 10 N 79 Animal medicines Cinnamic acid C 9 H 8 O 2 Y 10 Gallic acid C 7 H 6 O 5 N 80 Myristic acid C 14 H 28 O 2 Y 11 Rhapontin C 21 H 24 O 9 Y 81 Vitamin A C 20 H 30 O Y 12 Physion 8- O - β - D -glucoside C 22 H 22 O 10 N 82 Vitamin E C 29 H 50 O 2 Y 13 Sennoside A C 42 H 38 O 20 N 83 Vitamin K C 31 H 46 O 2 Y 14 Persicae Semen & Armeniacae Semen Amarum Amygdalin C 20 H 27 NO 11 Y 84 Vitamin C C 6 H 8 O 6 N 15 Naringenin C 15 H 12 O 5 Y 85 DL -tyrosine C 9 H 11 NO 3 Y 16 Kaempferol C 15 H 10 O 6 Y 86 DL -Lysine C 6 H 14 N 2 O 2 Y 17 Campesterol C 28 H 48 O Y 87 Uracil C 4 H 4 N 2 O 2 Y 18 Oleic acid C 18 H 34 O 2 Y 88 Uridine C 9 H 12 N 2 O 6 Y 19 Linoleic acid C 18 H 32 O 2 Y 89 Hypoxanthine C 5 H 4 N 4 O Y 20 Heptanoic Acid C 7 H 14 O 2 Y 90 Inosine C 10 H 12 N 4 O 5 Y 21 Palmitoleic acid C 16 H 30 O 2 Y 91 Xanthine C 5 H 4 N 4 O 2 Y 22 Palmitic acid C 16 H 32 O 2 Y 92 DL -Phenylalanine C 9 H 11 NO 2 Y 23 Stearic acid C 18 H 36 O 2 Y 93 Adenosine C 10 H 13 N 5 O 4 Y 24 L -tryptophane C 11 H 12 N 2 O 2 Y 94 Proline C 5 H 9 NO 2 Y 25 Prunin C 21 H 22 O 10 Y 95 Valine C 5 H 11 NO 2 Y 26 Scutellariae Radix Wogonin C 16 H 12 O 5 Y 96 L -isoleucine C 6 H 13 NO 2 Y 27 Baicalein C 15 H 10 O 5 Y 97 Methyl 12-methylmyristate C 16 H 32 O 2 Y 28 Baicalin C 21 H 18 O 11 Y 98 14-Methylpentadecanoic Acid Methyl Ester C 17 H 34 O 2 Y 29 Wogonoside C 22 H 20 O 11 Y 99 Methyl stearate C 19 H 38 O 2 Y 30 Skullcapflavone Ⅰ C 17 H 14 O 6 Y 100 3-Methoxy-4-methylbenzoic acid C 9 H 10 O 3 Y 31 Chrysin C 15 H 10 O 4 Y 101 Cytosine C 4 H 5 N 3 O Y 32 Norwogonin C 15 H 10 O 5 Y 102 Thymine C 5 H 6 N 2 O 2 Y 33 Rivularin C 18 H 16 O 7 Y 103 11-Octadecenoic acid C 18 H 34 O 2 Y 34 Apigenin C 15 H 10 O 5 N 104 8, 11, 14-Eicosatrienoic acid C 20 H 34 O 2 Y 35 Tenaxin I C 18 H 16 O 7 Y 105 Leucine C 6 H 13 NO 2 Y 36 Cosmetin C 21 H 20 O 10 Y 106 Vitamin B1 C 12 H 17 ClN 4 OS N 37 Rehmanniae Radix Rehmannioside A C 21 H 32 O 15 Y 107 Riboflavin C 17 H 20 N 4 O 6 Y 38 Rehmapicroside C 16 H 26 O 8 Y 108 Elaidic acid C 18 H 34 O 2 Y 39 Acteoside C 29 H 36 O 15 Y 109 1,2-Cyclohexanediol C 6 H 12 O 2 Y 40 Forsythoside A C 29 H 36 O 15 Y 110 Pyridine-3-carboxamide C 6 H 6 N 2 O Y 41 Salidroside C 14 H 20 O 7 Y 111 Oxindole C 8 H 7 NO Y 42 Pentadecanoic acid C 15 H 30 O 2 Y 112 Methyl oleate C 19 H 36 O 2 Y 43 L -Pyroglutamicacid C 5 H 7 NO 3 Y 113 2-Piperidone C 5 H 9 NO Y 44 Sterol C 17 H 28 O N 114 4-Methoxyphenylacetic acid C 9 H 10 O 3 Y 45 Isomartynoside C 31 H 40 O 15 N 115 Vanillic acid C 8 H 8 O 4 Y 46 Aucubin C 15 H 22 O 9 N 116 Phloretic Acid C 9 H 10 O 3 Y 47 Melittoside C 21 H 32 O 15 N 117 Allantoin C 4 H 6 N 4 O 3 N 48 Ajugol C 15 H 24 O 9 N 118 L -Glutamic acid C 5 H 9 NO 4 Y 49 Catalpol C 15 H 22 O 10 N 119 Methyl palmitate C 17 H 34 O 2 Y 50 Echinacoside C 35 H 46 O 20 N 120 Methyl palmitelaidate C 17 H 32 O 2 Y 51 Mussaenosidic acid C 16 H 24 O 10 Y 121 4-Hydroxycinnamic acid C 9 H 8 O 3 Y 52 Paeoniae Radix Alba Paeoniflorin C 23 H 28 O 11 Y 122 Gentisic acid C 7 H 6 O 4 N 53 Albiflorin C 23 H 28 O 11 Y 123 Syringic acid C 9 H 10 O 5 N 54 Pyrogallol C 6 H 6 O 3 Y 124 Salicylic acid C 7 H 6 O 3 N 55 Di-(2-ethylhexyl) phthalate C 24 H 38 O 4 Y 125 4-Hydroxybenzoic acid C 7 H 6 O 3 N 56 Lactiflorin C 23 H 26 O 10 Y 126 Nicotinic acid C 6 H 5 NO 2 N 57 Palbinone C 22 H 30 O 4 Y 127 L -Arginine C 6 H 14 N 4 O 2 N 58 Benzoic Acid C 7 H 6 O 2 Y 128 4-Hydroxyphenylacetic acid C 8 H 8 O 3 N 59 Oxypaeoniflora C 23 H 28 O 12 Y 129 L -Alanine C 3 H 7 NO 2 N 60 Paeonol C 9 H 10 O 3 N 130 Unknown Citric acid C 6 H 8 O 7 Y 61 Benzoylpaeoniflorin C 30 H 32 O 12 N 131 β - D -fructopyranose C 6 H 12 O 6 Y 62 1,2,3,4,6- O -Pentagalloylglucose C 41 H 32 O 26 N 132 Succinic acid C 4 H 6 O 4 Y 63 Ethyl gallate C 9 H 10 O 5 N 133 Nervonic acid C 24 H 46 O 2 Y 64 Catechin C 15 H 14 O 6 N 134 Hexadecenoic acid C 16 H 30 O 2 Y 65 Glycyrrhizae Radix et Rhizoma Liquiritin C 21 H 22 O 9 Y 135 6, 8-Diprenylorobol C 25 H 26 O 6 N 66 Glycyrrhizic acid C 42 H 62 O 16 Y 136 Diosmetin C 16 H 12 O 6 N 67 Formononetin C 16 H 12 O 4 Y 137 Methyl heptadecanoate C 18 H 36 O 2 N 68 Liquiritigenin C 15 H 12 O 4 Y 138 Salicin C 13 H 18 O 7 N 69 Isoliquiritigenin C 15 H 12 O 4 Y 139 Norharmane C 11 H 8 N 2 N 70 7-Hydroxycoumarin C 9 H 6 O 3 Y 140 Salicyl alcohol C 7 H 8 O 2 N Note: Y, detected in the blood; N, not detected in the blood.

Quality markers of DHZCP

By reviewing the quality control requirements for each medicinal material in DHZCP from the Chinese Pharmacopoeia , the primary quality markers of DHZCP could be initially inferred to be aloe emodin, rhein, emodin, chrysophanol, physcion, baicalin, liquiritin, glycyrrhizic acid, paeoniflorin, amygdalin, catalpol, and rehmannioside D. In addition, Fu et al. (2021) showed that quality control markers included rhein, emodin, aloe emodin, and allantoin in the principal drug rhubarb, allantoin in Eupolyphaga Steleophaga , hypoxanthine, p -methoxyphenylacetic acid, cinnamic acid, naringenin in the minister medicines Hirudo , T. bivittatus , H. diomphalia and Persicae Semen .

Metabolism of DHZCP in vivo

According to published accessible investigations ( Chen, Du, Wang, Wu, & Yin, 2022 , Ni et al., 2020 ), 100 components of DHZCP were recognized by the UPLC-HRMS technique in serum or plasma following oral administration to animals, detailed information of which were displayed in Table 1 . Moreover, Wu et al. (2020) dissected the pharmacokinetic profiles of 19 active ingredients ( 1 , 2 , 3 , 4 , 5 , 15 , 26 , 28 , 29 , 32 , 39 , 40 , 41 , 52 , 66 , 67 , 80 , 90 , and 114 ) in DHZCP using a highly sensitive UHPLC-QqQ-MS/MS technique and evaluated their characteristic absorption behaviors in vivo , suggesting that DHZCP exerted antitumor effects as a result of the combined and enhanced intervention of multiple effective constituents.

Clinical applications of DHZCP in treatment of cancer

DHZCP is clinically suitable for treating many malignancies such as leukemia, uterine fibroids, and liver, gastric, esophageal, lung, pancreatic and cervical cancers, substantially increasing the patient’s quality of life and slowing the development of tumors.

Liver cancer

According to the random and double-blind principle, 60 liver cancer patients were divided into two groups (gemcitabine and oxaliplatin (GEMOX), combination GEMOX with DHZCP). The combination’s effective rate was 66.67%, while that of GEMOX was 43.33%. The results displayed that DHZCP has good effects on improving the patients' symptoms and quality of life, enhancing the functional status, and reducing bone marrow suppression and gastrointestinal reactions caused by chemotherapy ( Zhou, 2014 ). A 52 patients with advanced primary liver cancer were randomized into the DHZCP + chemotherapy and chemotherapy groups. The results showed that cases treated with DHZCP (3 g per dose, twice a day for 56 d) were significantly improved compared with the second group in terms of short-term curative effect and 0.5 to 2-year survival rate after two treatment cycles. At the same time, the toxic and side effects of the DHZCP were lower than another ( Wu & Shen, 2011 ). The symptoms of 15 patients with primary liver cancer were relieved after DHZCP (3 g once, three times a day, for a total of 1.5 months therapy) treatment, with both AFP and AKP decreasing after therapy. The tumor shrinkage rate of patients was (14.72 ± 11.26)% ( Zhao, Tao, & Chen, 2006 ). Finally, DHZCP (3 g at a time, 3 times a day, treated for 56 d) has a certain ameliorating function on liver fibrosis caused by interventional management in patients with liver cancer ( Xie, Zhao, & Liu, 2014 ).

Lung cancer

Totally 60 lung cancer patients with blood stasis treated based on TCM were randomly divided into the TP regimen chemotherapy and DHZCP combination groups. It was found that DHZCP has a definite antitumor effect on improving the clinical symptoms of patients and their living quality, reducing the side effects of chemotherapy, adjusting cellular immune function, preventing tumor high viscosity state and adjusting vascular endothelial factor, which provides a reliable clinical basis for the treatment of lung cancer with DHZCP (6 g per day, twice daily) ( Qi, Li, & Li, 2011 ). DHZCP treatment for primary lung cancer was performed in 62 cases, with an effective rate of 54.8%, an overall effective rate of 61.3%, and a survival rate of 32.3% for over one year. Among cases, lung squamous cell carcinoma had the best response, followed by lung adenocarcinoma ( Shen et al., 1982 ). Yang applied flavor-added DHZCP to treat lung cancer and achieved satisfactory results ( Yang, 2013 ).

Gastric cancer

Liu et al. employed DHZCP (30 g per day, but divided into two doses) to treat 17 gastric cancer cases. It was found that immune function, clinical efficacy and the life-quality were remarkably improved via clinical assessments ( Liu & Chen, 2001 ). In addition, Wen et al. treated a gastric cancer patient with DHZCP with or without prescriptions, and the condition of patients was significantly improved. The survival time and living quality of the patient were also enhanced ( Wen, Liao, & Yang, 2004 ).

Pancreatic cancer

Totally 74 pancreatic cancer cases were treated with DHZCP, and clinical syndromes and lesion sizes were ameliorated, while the incidence of adverse reactions was dramatically reduced. Based on the favorable efficacy of DHZCP, it is worthy of clinical promotion ( Sun, 2014 ). Additionally, DHZCP combined with chemotherapy could markedly alleviate the symptoms of clinical pancreatic cancer cases, increase the quality of life, help stabilize and gain weight, increase the body's immune function, improve blood hypercoagulability, and diminish blood and digestive system toxicity ( Xu, 2010 ).

Other tumors

A total of 120 patients with uterine fibroids were treated with DHZCP in combination with TCM. Interestingly, 72 cases were cured clinically, accounting for 60% ( Li, 2011 ). Zhao used DHZCP (taking 6 g everyday, twice a day) combined with Zhongjiefeng Tablets to treat 38 patients with ovarian cysts. Of these, 24 cases (63.16%) were cured and 12 cases (31.58%) with effective performance. Follow-up data showed that all patients were healed without recurrence, and there was no apparent increase in cyst volume on those with effective remission ( Zhao, 2008 ). In 28 patients with multiple myeloma, DHZCP could strengthen the function of the reticuloendothelial system, improve hyper-viscosity and hypercoagulability, enhance immunity, reduce antigens, promote macrophages to engulf immune complexes, increase renal blood flow to reduce kidney damage, augment the hematopoietic function of the bone marrow, and exert a specific positive effect on hematopoietic stem cells ( Zheng, 2009 ). Liu et al. administered DHZCP in leukemia patients based on hydroxyurea treatment, observed clinical symptoms and detected peripheral blood leukocytes and lymphocytes, lymphocytes in the bone marrow, platelets, hemoglobin and other indicators. The results suggested that functional mechanisms of DHZCP might involve the inhibition of platelet aggregation, anti-thrombosis and immune reinforcement ( Liu & Sun, 2012 ).

Antitumor mechanism of DHZCP

Inhibition of tumor cell growth

The capability of persistent and unrestricted proliferation represents an important hallmark of cancer. Abnormal cellular proliferation is an essential feature of the cancer phenotype ( Bading & Shields, 2008 ). Targeting cells with high proliferation rate is critical in multiple aggressive malignancies ( Cardano, Tribioli, & Prosperi, 2020 ). MTT and clonogenic assays are generally performed to assess the proliferative ability of cancer cells ( McCauley, Zivanovic, & Skropeta, 2013 ). It was demonstrated that DHZCP l dose-dependently inhibits the proliferation of K562 cells by the MTT assay. As the concentration rose, the suppression rate of clonogenicity also increased gradually ( Qin et al., 2015 ). Examining the inhibitory effect of DHZCP on colorectal cancer LOVO cells, it was found that treatment with DHZCP at 160 mg/mL resulted in an increased inhibition rate to 55.3%, which could efficiently restrain the growth of colorectal cancer cells ( Qu, 2016 ).

Cell cycle arrest

Cell division normally occurs through the cell cycle, which encompasses multiple highly controlled processes orchestrated molecularly by certain cyclins that are associated with cyclin-dependent kinases (CDKs) ( Ingham & Schwartz, 2017 ). Aberrant cell cycle distribution is considered a major mechanism underpinning carcinogenesis, indicating cell cycle markers represent invaluable molecular targets in cancer treatment ( Liu, Peng, & Wei, 2022 ). The PI3K/Akt pathway plays an important role in signal transduction, activating downstream mTOR and nuclear factor kappa-B (NF- κ B), inactivating caspases and inducing the transcription of cyclin and the production of transcriptional regulators ( Chang et al., 2003 ). DHZCP can block K562 cells at G0/G1 and induce apoptosis to suppress cancer cells, which may be achieved via PI3K/AKT pathway inhibition ( Qin et al., 2015 ). It was found that breast cancer MCF7 cells treated with DHZCP are blocked in the S phase for cancer cell suppression as detected by flow cytometry ( Qu, 2016 ). Preliminary clinical and experimental studies have confirmed that DHZCP may be a promising candidate for the treatment of BCR/ABL-negative myeloproliferative neoplasms, inducing cell cycle arrest at G0/G1, concentration-dependently ( Qin, 2015 ).

Induction of apoptosis

Apoptosis, a sort of programmed cell death, takes place in an arranged manner to efficiently remove injured cells, e.g., after DNA damage ( Pistritto, Trisciuoglio, Ceci, Garufi, & D'Orazi, 2016 ). Deregulated apoptosis leads to tumorigenesis and tumor progression as well as treatment resistance ( Carneiro & El-Deiry, 2020 ). Survivin, a member of the IAP family, represents a protein that prevents cells from self-destruction; It is expressed in most tumors but not or lowly expressed in normal tissues, with association with poor prognosis in many human cancers ( Martínez‐García, Manero‐Rupérez, Quesada, Korrodi‐Gregório, & Soto‐Cerrato, 2019 ). Ai et al. selected tumor-bearing mice as research objects to demonstrate the anti-tumor and apoptosis-inducing effects of DHZCP ( Ai & Tian, 2012 ). It was found that DHZCP invoked tumor apoptosis by decreasing the expression of survivin in tumor cells. Wang et al. found that treatment with 82 g/kg DHZCP results in a tumor inhibition rate reaching 50% in a S180 sarcoma animal model. After administration, apoptotic cells in tumor tissues were obviously elevated. It was revealed that DHZCP can inhibit tumor growth and induce apoptosis ( Wang, Ma, Tian, & Ai, 2011 ). Tian et al. observed that tumor volume in mice treated with DHZCP was significantly reduced; Microvilli on the cell surface were decreased or even disappeared and apoptotic bodies were discovered. These data indicated that DHZCP could induce tumor cell apoptosis ( Tian, Wang, & Ai, 2011 ).

Induction of autophagy

Autophagy, an important process in which the cellular content is degraded by lysosomes, controls the basal turnover of cells and provides the energy and macromolecules required by cells ( Levy, Towers, & Thorburn, 2017 ). Autophagy maintains the functions of various organelles, inhibits the toxic effects of waste products, and supplies substrates to maintain metabolic pathways during starvation. To some extent, autophagy could suppress carcinogenesis ( White, 2015 ). DHZCP significantly reduces inflammation and mortality in colitis-associated cancer, which may be related to highly increased LC3B I/II and autophagy protein 5 (ATG5) levels and reduced SQSTM1 expression to promote intestinal autophagy in colorectal cancer ( Dai et al., 2021 ).

Metastasis inhibition

Metastatic malignancies are very hard to cure, accounting for most deaths caused by cancers. Metastasis to distant sites is a very dynamic and asynchronous process involving both tumor and host factors. Effective treatment targets blocking metastatic events suppress major pathways indispensable for many steps of metastasis ( Stoletov, Beatty, & Lewis, 2020 ). As the predominant cause of cancer-associated deaths, metastasis serves as a great challenge in current efforts against malignant diseases ( Wan, Pantel, & Kang, 2013 ). With treatment by high-dose DHZCP, the spontaneous lung metastasis inhibition rate reached 57.44%, which could greatly lower the metastasis rate of Lewis lung cancer in C57BL mice. This was related to vascular endothelial growth factor (VEGF) downregulation and CD44 activity suppression, thereby inhibiting tumor angiogenesis and weakening invasion in tumor cells ( Shu, Pan, & Li, 2009 ). The number of lung and lymphatic metastases in H22 liver cancer model mice was remarkably decreased after DHZCP treatment, especially the large-DHZCP dose group. In comparison with carmofur-treated animals, DHZCP administration showed no distinct difference in terms of efficacy but had less toxicity and side effects. The study indicated that DHZCP constrained lung and lymph node metastases of H22 liver tumors ( Lv & Zhai, 2010 ). Finally, DHZCP inhibited liver metastasis of colorectal cancer (CRC) by curbing C-C motif chemokine 2 (CCL2) mediated M2-skewed paradigm and improved the pro-fibrotic microenvironment ( Chen et al., 2019 ).

Anti-angiogenic effect

Excessive abnormal angiogenesis, a hallmark of solid tumors, plays a vital role in tumor progression. This is due to an imbalance between pro- and anti-angiogenic events with hypoxia-induced VEGF overexpression contributing majorly. VEGF-associated pathway has rapidly emerged as a key anti-angiogenic treatment target in cancer therapy ( Jászai & Schmidt, 2019 ). Anti-angiogenic treatment therefore represents a promising strategy for multiple solid tumors ( Itatani, Kawada, Yamamoto, & Sakai, 2018 ). It was shown that DHZCP significantly inhibited tumors and prolonged survival in mice harboring Lewis lung cancer cells. The expression of COX-2 was decreased by immunohistochemistry, RT-PCR and Western blotting. The mechanism of DHZCP’s antitumor effect may be connected with COX-2 repression, thereby preventing tumor angiogenesis and weakening the invasion of tumor cells ( Shu, Li, Wang, & Zhu, 2011 ). Li et al. detected the expression of VEGF, COX-2 and CD34, vascular endothelial cell markers, to assess tumor angiogenesis by immunohistochemical staining ( Li & Shu, 2015 ). The mechanism of DHZCP’s suppressive effects on tumors might be associated with VEGF and COX-2 downregulation and the reduction of tumor angiogenesis. Evidence suggests that DHZCP markedly prolongs survival in mice with experimental liver cancer based on H22 cells, effectively inhibits cancer cell growth and improves the life-quality. The chick embryo chorioallantoic membrane test revealed that DHZCP may exert anti-liver tumor effects by controlling angiogenesis ( Song, 2011 ). Furthermore, Chen et al. combined the UPLC-Q-TOF-MS/MS technique with the partial least squares method to investigate the spectral effect relationship between the fingerprint of DHZCP and pharmacodynamics indexes such as HIF-1α, VEGF, Ang2, and Tie2, discovering that various components of DHZCP could facilitate vascular normalization ( Chen et al., 2022 ).

Enhancement of immunity

Cancer immunotherapy manipulates the immune system to induce antitumor effects ( Papaioannou, Beniata, Vitsos, Tsitsilonis, & Samara, 2016 ). In the past decade, immunotherapy has been considered a robust treatment option for cancer. The immune system identifies and destroys newly formed tumor cells through cancer immunosurveillance, a critical defense mechanism against cancer ( Vesely, Kershaw, Schreiber, & Smyth, 2011 ). After treatment with DHZCP and hepatic arterial chemoembolization (TACE) in primary liver cancer cases, CD4 + , CD4 + /CD8 + and CD3 + cells and other immune indicators were elevated. Meanwhile, AFP, CA199 and other tumor indicators, as well as ALT, TBiL, VEGF, transforming growth factor-β1 (TGF-β1) and matrix metalloproteinases-2 (MMP-2) levels were decreased. These findings indicated that DHZCP combined with TACE surgery could help ameliorate immune function, protect hepatic function, reduce the risk of metastasis, and decrease the occurrence of adverse events induced by chemotherapy ( Dai, Jin, & Zhang, 2021 ). In a recent study, it was proved that DHZCP could promote the differentiation of Th1 cells and the secretion of IFN-γ through modulating the levels of IL-2 and IL-12, reduce the infiltration of local Treg cells, activate CD8 + T cells, attenuate the tumor immune escape, and thus inhibit the growth of hepatocellular carcinoma, hence, DHZCP actually possesses the capability to enhance the body's anti-tumor immune function ( Chen et al., 2022 ).

Wu et al. found that DHZCP could reduce the symptoms of H22 liver cancer mice, improve the quality of life and inhibit tumor growth. The thymus in which T cells differentiate and the spleen in which all kinds of immune cells reside have essential functions in the synthesis of immune active substances ( Wu, Qi, & Tao, 2008 ). In addition, DHZCP increased thymus and spleen weights in mice bearing liver cancer (H22 cells), suggesting that DHZCP reduces tumor-induced damage and improves the host's immune function. Studies have found that DHZCP prolongs the survival of H22 tumor-harboring mice, increases the proliferation of T lymphocytes and reverses Th1 shift to Th2 as examined by MTT and flow cytometry, revealing that DHZCP can improve the survival status by regulating cellular immune function ( Zhai, 2007 ). Regulatory T cells (Tregs) in the tumor tissue are key mediators of tumor immunosuppression, which is related to disease progression and poor prognosis in cancer patients. DHZCP lessens the accumulation of Tregs in the tumor tissue, decreases CCL17 expression, CCR4 expression rate on the surface of Treg cells, and NF-κB p65 levels in colon cancer cells to suppress colon cancer development in CT26 mice ( Ruan, Ren, Xu, Lai, & Su, 2021 ). Zhang confirmed that DHZCP exerts better effects on the general condition, body weight and organ index changes of Lewis lung cancer tumor-bearing mice compared with cisplatin, which may occur by increasing IL-2 amounts and reducing Foxp3 expression and CD4 + CD25 + Foxp3 + Treg accounts in spleen CD4 + T cells ( Zhang, 2015 ).

Reversal of drug resistance

Drug resistance constitutes a major hurdle to cancer treatment. Several therapies often fail to produce a durable tumor response clinically and ultimately lead to treatment resistance and tumor relapse ( Bukowski et al., 2020 , Chatterjee & Bivona, 2019 ). Current solutions aim to reverse drug resistance and increase the efficacy of chemotherapy while decreasing toxicity for prolonging survival in cancer cases ( Huang, Cole, Cai, & Cai, 2016 ). DHZCP and low- or middle-dose DOX had synergistic antihepatoma effects by downregulating essential enzymes involved in oxidative phosphorylation and reversing the elevation of intracellular ATP amounts induced by DOX, suggesting DHZCP has anti-hepatocarcinoma properties and reverses drug resistance by suppressing energy metabolism ( Wu et al., 2017 ). Wu et al. showed that DHZCP could significantly inhibit metabolic enzymes in the mitochondria of doxorubicin-resistant SMMC-7721 cancer cells and reduce ATP levels in the tumor tissue. In addition, DHZCP dramatically regulated the expression of apoptosis- and angiogenesis-associated proteins, reversing drug resistance in liver cancer ( Wu et al., 2018 ).

From the above literature deliberations, it is clear that DHZCP exhibits different therapeutic mechanisms for a variety of cancers, and the pathways through which it exerts its curative effects also appear to vary. The similarities and differences in the mechanisms of DHZCP on different cancers were dissected as shown in Table 2 . The mechanisms of action and the regulatory pathways involved in the treatment of various cancers and tumors with DHZCP were visually displayed in Fig. 9 . Table 2 Therapeutic mechanisms and pathways of action of DHZCP on different cancers. Cancer types Anticancer effect Pathways Liver cancer Anti-metastasis, anti-angiogenesis, immunity and reversal of drug resistance HIF-1α, VEGF, Ang2, Tie2, TGF-β1, MMP-2, CCL17 and CCR4 Lewis lung cancer Anti-metastasis, anti-angiogenesis CD44, VEGF and COX-2 Breast cancer Cell cycle arrest − Colorectal cancer Antiproliferation, autophagy LC3B I/II, ATG5 and SQSTM1 Chronic myelocytic leukemia Antiproliferative effect, cell cycle arrest, apoptosis PI3K/AKT pathway Sarcoma Apoptosis Survivin Blood cancer Cell cycle arrest − Fig. 9 Antitumor mechanisms of Dahuang Zhechong Pill.

Conclusion and perspectives

In this review, we firstly outline the chemical substances composition, quality control criteria, quality markers, in vivo absorption behaviors and pharmacokinetic profiles of DHZCP, which can not only thoroughly clarify the therapeutic material basis of DHZCP, elucidate its biotransformation and dynamic processes in vivo , but also further promote the optimization of its quality control system, which is beneficial for clinical safety monitoring. Furthermore, we have extensively reviewed the available clinical data on DHZCP for the treatment of cancer and tumors, showing that DHZCP has unique advantages in dealing with malignant tumors such as leukemia, uterine fibroids, liver cancer, gastric cancer, esophageal cancer, lung cancer, pancreatic cancer and cervical cancer, and can significantly improve patients’ life-quality, slow down the worsening progression of the disease, effectively prolong longevity, with few side effects and excellent tolerability, which are difficult to achieve with traditional chemotherapy methods and conventional drug administration. Finally, we also retraced in detail the basic research reports on the utilization of DHZCP in treating cancer cells and model animals, categorized and summarized its mechanisms of action and implicated pathways, and concluded that the mechanisms of DHZCP in curing cancer mainly include inhibition of cancer cells proliferation, induction of cell cycle arrest, enhancement of apoptosis, triggering of autophagy, suppression of metastasis, anti-angiogenic effect, strengthening of immunity and reversal of drug resistance, indicating that DHZCP exerts its therapeutic effects through a multi-pathway, multi-target and holistic integrated approach, which is in line with the whole-site discernment thinking of TCM.

Admittedly, it is obvious that DHZCP, as a promising classical formula of Chinese medicine excavated from the treasury of TCM to supplement cancer treatment, has been clinically proven to be beneficial initially, but there is still more space for deeper exploration of its potential medical possibilities. (1) Since the practice of DHZCP is based on important underlying theories of TCM, adequate theoretical support is necessary for both daily use and clinical management. Hence, there is an urgent task to collate the ancient literature that has emerged over the past few thousand years, to clarify the conceptual relevance and pertinence of DHZCP's overall prescription effects and cancer categories, so as to formulate a complete and abundant theoretical system of TCM therapy to guide clinical application; (2) At present, there is still a shortage of research reports on the chemical components of DHZCP, which leads to the ambiguity of its material basis of action. The modern extraction, separation and spectral identification techniques should be fully exploited to accurately profile and characterize the substance compositions of DHZCP, which can also lay a solid foundation for further quality standard establishment and pharmacodynamic mechanism mining; (3) Nowadays, modern pharmacological research is in full swing, but there are few studies on the in-depth mechanisms of DHZCP targeting cancer therapy. Metabolomics, gene networks, proteomics and other advanced macroscopic means consistent with TCM ideology can be adopted to thoroughly investigate the mechanisms of DHZCP, enabling us to deeply understand its therapeutic potential and grasp its key curative pathways and action targets. We believe that based on continuous research and exploration, DHZCP, as a representative of anti-cancer Chinese patent medicine, will definitely keep shining brightly and erecting an influential milestone in the history of cancer treatment.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Acknowledgements

This work was financially supported by National Science and Technology Major Projects (No. 2018ZX09201-011, 2018ZX09301-011-003), the National High Technology Research and Development Program of China (No. 2019YFC1711400).