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Adjuvant effects of Chinese medicinal tonics on gastric, liver, and colorectal cancers—OMICs-based contributions to understanding their mechanism of action

Gastric, liver, and colorectal cancers belong to gastrointestinal (GI) cancers, one of the most threatening diseases in the world. The tonics class in Chinese medicines plays a critical role in antigastrointestinal cancer as adjuvants. However, it is a challenge to study the effects and underlying mechanisms of tonics due to their multiple components and multiple targets; OMICs were introduced to facilitate the investigation of the complex mixture of tonics. In this review, the online databases PubMed, ProQuest, Web of Knowledge, China National Knowledge Infrastructure (CNKI), Chongqing VIP, and Wanfang were retrieved from 1 January 2011 to 31 May 2022, in an aim to summarize and discuss the research progress of the effects and, especially, the underlying mechanisms of tonics for antigastrointestinal cancers via OMICs. The results showed that through the combination of OMICs and other technologies, tonics have been used for gastrointestinal cancer by targeting cancer hallmarks, enhancing body resistance to carcinogenesis, enhancing therapeutic effects, and/or decreasing side effects. In conclusion, tonics may play a promising role in gastric, liver, and colorectal cancers as adjuvants and can be well investigated via the combination of OMICs and other technologies, which deserves further study.


Introduction
Gastrointestinal (GI) cancers are one of the most threatening diseases in the world. There were approximately 5,142,192 new cases and 3,628,920 deaths from GI cancers in the world in 2020 (Sung et al., 2021). Based on disease sites, GI cancers are divided into two families: upper digestive tract cancers (including esophageal, stomach, pancreatic, liver, gallbladder, and lymphoma involving the mucosa-associated lymphoid tissue, gastrointestinal stromal, and biliary tree) and lower cancers (including colorectal, anal, and gastrointestinal carcinoid). The order of the mortality rate from high to low for cancer sites was liver (8.3%), stomach (7.7%), colon (5.8%), esophagus (5.5%), pancreas (4.7%), rectum (3.4%), gallbladder (0.9%), and anus (0.2%) (Sung et al., 2021). The total new deaths of GI cancers (36.7%) exceeded those of lung cancer (18.0%) and GI cancers ranked as the leading cause of death (Sung et al., 2021). The conventional treatments of GI cancers include surgical resection, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. However, some patients cannot tolerate surgical resection. Chemotherapy and radiotherapy induce toxicity and side effects. Targeted therapy and immunotherapy are often expensive, especially for patients in developing countries. Thus, natural medicines have drawn attention due to their lower toxicity and effectiveness as adjuvant strategies and have been widely used in clinical practice to adjust patients' constitution and reduce toxicity and side effects after surgery, chemotherapy, and radiotherapy.
Tonics of Chinese medicines (CMs) refer to medicines that can supplement Qi, blood, Yin, and Yang of the human body, relieve deficiency and weakness syndromes, enhance visceral function, and improve the body's ability to resist disease. Their pharmacological actions include enhancing immunofunction, regulating metabolism of substances, improving the endocrine system, anti-aging, and anticancer (Chen, 2017). Tonics are divided into four categories, Qi tonics (e.g., Panax ginseng C.A.Mey and Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao), blood tonics (e.g., Angelica sinensis (Oliv.) Diels and Polygonum multiflorum Thunb.), Yin tonics (e.g., Lycium barbarum L. and Ophiopogon japonicus (L. f) Ker-Gawl.), and Yang tonics (e.g., Epimedium brevicomu Maxim.) (Chen, 2017). Some Chinese formulas are also believed to be tonics such as Liu Wei Di Huang pills (Xiong et al., 2022). There are 72 tonics out of 371 CMs for anti-GI cancers, which ranked the second class of the most frequently used CMs following the clearing heat and detoxifying class   (Figure 1).
However, it was difficult for the research community to explore the effects, and particularly the underlying mechanisms of tonics due to their multiple components Yang et al., 2019), complex pharmacokinetic (PK) processes Wang et al., 2021b), and multiple targets until OMICs were introduced Wang and Lu, 2019;Li et al., 2020;Liu et al., 2021a;Li et al., 2022b). OMICs are novel technologies that have been dramatically developed in the last 2 decades. Small size samples and large-scale and high-throughput screening make OMICs possible to apply to various disciplines in biology including complex pharmacological mechanisms (Li et al., 2022a). OMICs include genomics, proteomics, metabolomics (or metabonomics), metagenomics, transcriptomics, epigenomics, glycomics, and lipomics. In this review, we retrieved online databases, aiming to summarize and discuss the OMICs-based research progress of adjuvant effects, especially underlying mechanisms, and provide insights into the complex multiple components and targets of tonics on GI cancers.

Data retrieval and collection
The keywords "OMICs or genomics/proteomics/ metabolomics/metabonomics/metagenomics/transcriptomics/ epigenomics/glycomics/lipomics" and "tonics" and "gastrointestinal cancer" were used to retrieve studies of tonics for GI cancers from the online databases of PubMed, ProQuest, Web of Knowledge, China National Knowledge Infrastructure (CNKI), Chongqing VIP, and Wanfang from 1 January 2011 to 31 May 2022. Duplicates were discarded. The effects, overall efficacy, and underlying mechanisms via OMICs technologies in these studies were summarized and analyzed. All plant names were checked with the World Flora Online (www. worldfloraonline.org) or MPNS (http://mpns.kew.org).

Inclusion criteria
The inclusion criteria were as follows (Ding et al., 2019): • The contents of the literature involve the in vitro, in vivo, and clinical effects of tonics on GI cancers • The references included pure compounds, single herbal fractions, and formulas of tonics • The methodologies were designed using OMICs

Exclusion criteria
The exclusion criteria were as follows (Ding et al., 2019): • The literature was associated with neither tonics nor OMICs • The pure compounds were not naturally from tonics but were chemical derivatives • The species of tonics were not clearly presented, or the plant names were not checked in the "World Flora Online" (www.worldfloraonline.org) or MPNS (http://mpns. kew.org) • The fractions and/or formulas of tonics were described with neither the extraction methodology nor quality control • The components of the formulas were not given • The concentration/dose of tonics was not given • The clinical studies were not randomized and controlled • The in vivo and clinical studies did not claim any ethical approvals, and the clinical studies were conducted without the declaiming of patients' agreement or signing informed consent 3 OMICS for adjuvant effects and mechanisms of tonics on gastric, liver, and colorectal cancers

Gastric cancer
The effects and mechanisms of tonics on gastric cancer via OMICs are listed in Table 1.

Transcriptomics
Angelicae Sinensis Radix (roots of Angelica sinensis (Oliv.) Diels, Danggui) is a blood-tonifying CM . It was reported that Angelicae Sinensis Radix can treat patients with gastric cancer. The transcriptomic results showed that n-butylidenephthalide, the active compound in Angelicae Sinensis Radix, induced REDD1 (regulated in development and DNA damage responses 1) and consequently inhibited its downstream factor mammalian target of rapamycin (mTOR) in gastric cancer (Liao et al., 2018).
Another role of genomics is in the shortage of chemotherapy-drug resistance. How to reverse drug resistance via OMICs has drawn attention from the medical community. Paeoniae Radix Alba is one of the blood tonics used for nourishing the blood and regulating menstruation, astringing Yin and checking sweeting, emolliating the liver, relieving pain, and depressing the liver Yang. Although Paeoniae Radix Alba reversed the drug resistance of GC Frontiers in Pharmacology frontiersin.org 03 cells, the mechanism was unknown until paeonol (the active compound in Paeoniae Radix Alba) was reported using genomics-data showed that paeonol inhibited the malignancy of apatinib-resistant GC cells through the LINC00665/miR-665/MAPK1 axis (Li et al., 2022b).

Metabolomics
Dendrobium officinale Kimura et Migo (Tiepishihu) is one of the sources of Dendrobii Caulis (Shihu). To investigate the substantial basis of Dendrobii Caulis on gastric cancer, blood metabolites were analyzed to screen the active compounds using UPLC-Q-TOF-MS. The metabolomics results showed that among five candidate metabolites, phingosine-1-phosphate (S1P) inhibited GC angiogenesis by inhibiting VEGF, SPHK1, and S1PR1 mRNA in rats (Zhao et al., 2017). Another urine metabolomics study showed that Dendrobii Caulis aqueous extracts inhibited the progression of gastric precancerous lesions. The mechanism is related to porphyrin metabolism, tryptophan metabolism, folic acid and pterin biosyntheses, and galactose and arachidonic acid metabolisms .
Yiwei decoction is a tonic formula that includes Astragalus membranaceus ( showed that Yiwei decoction intervened in gastric precancerous lesions by regulating 13 metabolites involved in the biosynthesis of unsaturated fatty acids, biosynthesis of valine, leucine, and isoleucine, sphingolipid metabolism, arachidonic acid metabolism, and steroid hormone synthesis (Dong et al., 2020). The Yiqi Fusheng recipe includes Atractylodis Macrocephalae Rhizoma, Astragali Radix, Myristicae Semen (seeds of Myristica fragrans Houtt., Roudoukou), Codonopsis is reported to enhance body resistance to GC. The serum metabolomics results showed that this effect was associated with an increase in the serum levels of αlinolenic acid, linoleic acid (LA), and arachidonic acid (AA) (Xu et al., 2021). LA can be metabolized to AA. LA/ AA plays an important role in enhancing body resistance, i.e., the inflammatory response and immune function (e.g., natural killer cell activity). Although certain arguments show the relationship between LA/AA and breast cancer (Gago-Dominguez et al., 2003;Murff et al., 2011), dietary intake of LA/AA was reported to decrease the risk of colorectal cancer (Kuriki et al., 2006) and liver cancer (Bao et al., 2017). This indicates that the Jianpi Yangzheng Xiaozheng recipe may enhance immunofunction through LA/AA metabolism.

Colorectal cancer
OMICs for the adjuvant effects and mechanisms of tonics on colorectal cancer (CRC) are listed in Table 2.

Transcriptomics
There is an interesting concept, namely, the Chinese herb pair (Yao Dui). In this case, two herbs are commonly included at an appropriate ratio in some formulas for enhancing effects and/ or decreasing toxicity. Gegen Qinlian decoction (GQD) is an ancient formula from the Han dynasty. Since it consists of four herbs, Puerariae Lobatae Radix (roots of Pueraria lobata (Willd.) Ohwi, Gegen), Scutellariae Radix (roots of Scutellaria baicalensis Georgi, Huang Qin), Coptidis Rhizoma (rhizomes of Coptis chinensis Franch., Coptis deltoidea C. Y. Cheng et Hsiao and Coptis teeta Wall., Huanglian), and Glycyrrhizae Radix et Rhizoma (roots and rhizomes of Glycyrrhiza uralensis Fisch., Glycyrrhiza inflata Bat., or Glycyrrhiza glabra L., Gancao), the dominant herbs have not yet been clarified. The transcriptomics data by Li et al. (2021b) showed that two active compounds, puerarin (PUE) and glycyrrhetinic acid (GLY) without other active compound pairs, influenced the Wnt signaling pathway by upregulating GSK3B and downregulating CTNNB1 synergistically in colon SW480 cells. As PUE and GLY are the main components of Puerariae Lobatae Radix and Glycyrrhizae Radix et Rhizoma, respectively, the results confirmed the pharmacological role of the herb pair, Puerariae Lobatae Radix and Glycyrrhizae Radix et Rhizoma, in GQD.

Genomics
Accumulating evidence suggests that aberrant DNA methylation and gene silencing of tumor suppressors are pervasive in GI cancers (Cao et al., 2020). ATP4a is an important tumor suppressor gene, encoding H + , K + -ATPase, and there is an inverse correlation between methylation and expression in ATP4a. Genomics evidence showed that isoliquritigenin (ILTG), an active compound in Glycyrrhiza glabra L., exhibited a demethylating activity on HT-29 colon cancer by increasing ATP4a (Zorko et al., 2010).

Metabolomics
Astragalus membranaceus and Curcuma wenyujin (AC) are classic Chinese herb pairs for colon cancer metastasis. Metabolomics data indicate that AC inhibits liver and spleen metastases of colon cancer by disturbing energetic dysfunction including valine, leucine, and isoleucine biosyntheses, aminoacyl-tRNA biosynthesis, caffeine metabolism pathway, and retinol metabolism pathways .

Multi-OMICs
Jujubae Fructus (Dazao) is the fruit of Zizyphus jujuba Mill. for tonifying Qi in CMs. The transcriptomic and metabolomic profiles showed that its polysaccharide consumption prevented mouse CRC and decreased colon mortality, reduced proinflammatory cytokines, increased the concentration of total short-chain fatty acids (SCFAs) and gut microbiota Bifidobacterium, Bacteroides, and Lactobacillus, and decreased gut microbiota Firmicutes/Bacteroidetes in mouse feces, indicating that Jujubae Fructus polysaccharides prevented inflammation-mediated carcinogenesis by restoring the balance of the gut microbiota in CRC (Ji et al., 2019;Ji et al., 2020).
Cyclophosphamide (CTX) is a widely used chemotherapy drug. However, it may result in complicated adverse effects including vomiting, diarrhea, and abdominal pain, related to the disruption of the mucosal barrier, bacterial translocation, and changes in microbial composition. Polysaccharides and ginsenosides are the two classes of active compounds in American ginseng. Through metabolomic and transcriptomic analyses, these polysaccharides and ginsenosides were found to exert synergistic effects to ameliorate CTX-induced intestinal immune disorders and gut barrier dysfunctions .

Liver cancer
OMICs for the adjuvant effects and mechanisms of tonics on liver cancer are listed in Table 3.

Proteomics
Concerning complications, patients with liver cancer may suffer from depression, which is also an inducing factor in hepatocellular carcinogenesis. The Jiawei Xiaoyao pulvis is a formula and was reported as an adjuvant to relieve liver carcinogenesis-induced depression. The proteomic results showed that Jiawei Xiaoyao pulvis reversed depression-like behaviors by regulating GSTM1, PDK1, and HSP90AB1 (Wen et al., 2022) (Table 3).

Transcriptomics
Astragalus protein is the active compound from the tonics Astragalus membranaceus (Fisch.). Transcriptomic data in combination with qRT-PCR and WB showed that Astragalus protein induced programmed necrosis of liver cancer HepG2 cells via the p53 signaling pathway (Wang et al., 2020b). Transcriptomics and network pharmacology studies have shown that daidzein (an active compound in Astragali Radix) induces ferroptosis by downregulating MT1G in liver cancer .

Metabolomics
According to Chinese medicine philosophy, one of the typical syndromes of liver cancer is spleen deficiency, with which patients may suffer from cancer pain, ascites, fatigue, etc. . Sijunzi decoction (Ginseng Radix et Rhizoma, Atractylodis Macrocephalae Rhizoma, Poria, and Glycyrrhizae Radix et Rhizoma) is a tonifying formula for treating spleen deficiency. However, because of the complex mixture of multiple components and multiple targets, how to profile spleen deficiency and how to interpret Chinese herbs treating such a syndrome pharmacologically are challenges. Based on blood plasma metabolomics using UPLC-HDMS, Wang et al. found that Sijunzi decoction treated spleen deficiency with metabolic dysfunctions in liver cancer via regulating the metabolisms of amino acids, arachidonic acid, fatty acids, and glutathione (Wang et al., 2020a).

Multi-OMICs
Using metabolomics for fecal metabolites and transcriptomics for gut microbiota, Panax Ginseng was found to regulate bile acid biosynthesis, unsaturated fatty acid biosynthesis, tryptophan metabolism, arachidonic acid metabolism, pyrimidine metabolism, and vitamin B6 metabolism. Furthermore, 25 species of bacteria with significant differences in effective parts in liver cancer and 23 species of bacteria with significant differences in synergistic action of ginsenosides and polysaccharides indicated that Qi deficiency liver cancer was associated with bile acid biosynthesis, unsaturated fatty acid biosynthesis, tryptophan metabolism, arachidonic acid metabolism, pyrimidine metabolism, vitamin B6 metabolism, and certain gut microbiota (Hou et al., 2022).

Discussion
Due to the heterogeneity of cancer, different molecular targets achieve different effects, and even cancers of one organ require different treatment strategies. This leads to the challenges for anti-GI cancers, especially in terms of understanding their underlying mechanisms. Thus, Hanahan introduced a new concept, hallmark, to distinguish the different mechanisms and potential targets for anticancer (Hanahan and Weinberg, Frontiers in Pharmacology frontiersin.org 09 2011). Tonics are effectively complementary and alternative medicines to conventional treatments with less toxicity. OMICs are novel technologies with a small sample size, and large-scale and high-throughput screening. These properties make it possible to apply OMICs for exploring the pharmacological mechanisms of tonics, especially targeting cancer hallmarks. In this study, the data showed that the targets of tonics included sustaining proliferative signaling, resisting cell death, activating invasion and metastasis, inducing angiogenesis, deregulating cellular energetics, inflammation-mediated carcinogenesis, and genomic instability and mutation (Table 1, Table 2, and Table 3; Figure 2).
However, there are still some challenges to limit the applications of OMICs. To further explore the role of OMICs in tonics on GI cancer, we focused on the active compounds, mechanisms, and compatibility of tonics, combined with basic experiments and novel technologies, and emphasized minimal injury methodologies.
First, tonics in clinical use are herbs, especially in formulas. This means that tonics for anti-GI cancer have multiple components and targets (signaling pathways) and complex mixtures. Thus, OMICs for tonics in anti-GI cancer research should focus on the active compounds, mechanisms, and compatibility of tonics to determine the relationship between the active compounds of tonics and their effects and underlying mechanisms, e.g., using comprehensive two-dimensional liquid chromatography (2DLC), Qiao et al. found 311 compounds from the extract of Glycyrrhiza uralensis Fisch. within 40 min, of which the method was superior to high-performance liquid chromatography (HPLC). Then, chemicalomics and metabolomics are matched, where metabolomics facilitates the exploration of the active compounds and analysis of the responding signaling pathways via metabolites, while transcriptomics easily unveils the differentially expressed genes via RNA sequencing (Gong et al., 2012). Another strategy for exploring the compound-herb-disease relationship is polypharmacokinetics (Poly-PK), a novel technology for OMICs comparison. For example, Xie et al. identified 84,292 and 532 compounds in extracts of Huang Qin decoction (including Scutellaria baicalensis Georgi and Glycyrrhiza uralensis Fisch.) and serum metabolites before and after oral administration, respectively. Among these compounds, 485 were changed after oral administration, of which 56 were from the extract of Huang Qin decoction, 292 were metabolites in the PK process, and 166 were metabolites from endogenous components. This methodology may profile a complex network between tonics, drug metabolites, and body metabolism function and provide insights into the holistic effect of a complex of tonics formulas (Xie et al., 2018).
Second, there are often inconsistencies between different results of OMICs, at least partially caused by the small size of the sample and systematic deviation. It is necessary to integrate basic experimental methods such as real-time PCR and WB, to confirm OMICs data and obtain more accurate results. Additionally, only genomics, transcriptomics, epigenomics, metabolomics, and proteomics among OMICs were used in this study, while glycomics and lipomics were not applied. Moreover, with the development of modern technologies, novel methods can be introduced in the research of tonics on GI cancer. For example, hepatocellular carcinoma (HCC) is characterized by high heterogeneity and metastatic potential and leads to poor prognosis. Thus, single-cell transcriptomic and proteomic data may identify mutations in small populations of cells and distinguish metastatic potential cells from HCC. A number of studies show that Chinese medicines can be used for HCC metastasis (Guo et al., 2022), and most of the mechanisms have been unknown, single-cell multi-OMICs may be a useful tool to unveil the metastatic mechanisms (Peng et al., 2018;Sun et al., 2021c;Wang et al., 2022) of tonics including Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao, Panax quinquefolium L., Atractylodes macrocephala Koidz., Glycyrrhiza uralensis Fisch., and Polygonum multiflorum Thunb , Yang et al., 2022. These potential findings may offer more individual and precise strategies for patients with GI cancer (Lu et al., 2021).
Third, decreased quality of life may worsen for patients suffering from GI cancers, so it is essential to choose simple, non-invasive, or minimally invasive ways for subjects to reduce injuries. The methods include metabolomic or genomic analysis of a small sample of blood, metabolomic analysis of urine, feces, and saliva, or transcriptomics combined with X-ray, computed tomography (CT), magnetic resonance imaging (MRI) scanning, or small animal in vivo imaging technology. OMICs in combination with network pharmacology and bioinformatics are another effective way to obtain ideal results .
Of note, GI cancers include two families based on disease sites, upper digestive tract cancers (including esophageal, stomach, pancreatic, liver, gallbladder, and lymphoma involving the mucosa-associated lymphoid tissue, gastrointestinal stromal, and biliary tree) and lower cancers (including colorectal, anal, and gastrointestinal carcinoid); however, to the best of our knowledge, only gastric cancer, liver cancer, and colorectal cancer have been reported using tonics via OMICs. The literature of tonics on the other kinds of GI cancers may have not been reported using OMICs, or, unfortunately, it may be filtered via inclusive and/or exclusive criteria because of low quality of the studies.
Another limitation of this review is that the primary therapies in most studies were not introduced and discussed although most CMs, including tonics, are add-on therapies. In this review, Ginseng Radix et Rhizoma treated colon cancer patients to reduce gastrointestinal symptoms after laparoscopic colectomy (Hanada et al., 2021). The Quxie capsules enhanced body resistance to colorectal cancer after chemotherapy, radiotherapy, targeted therapy, and immunotherapy (Sun et al., 2021a). Polysaccharides and ginsenosides in American Ginseng had effects on CTX-induced intestinal immune disorders and gut barrier dysfunctions   (Table 2). However, for most in vitro and in vivo studies in this review, in which tonics were used for exploring mechanisms, few primary therapies are Frontiers in Pharmacology frontiersin.org discussed. This indicates that high quality studies and more evidence are necessary for tonics as adjuvants. Furthermore, for a high quality of pharmacological study, detailed information (positive and negative controls, minimal active concentration, the model used, concentration or dose, duration, extract process, in vitro/in vivo/ clinical study, etc.) are essential. In this study, 12 out of 34 studies (Tables 1, Table 2, and Table 3) were in vitro, although they were screened via inclusion and exclusion criteria. This may be the result from that the mechanism studies did not involve in in vivo and clinical data. However, high-quality studies should be guaranteed.

Conclusion
With the data from OMICs, tonics were found to be adjuvants for gastric, liver, and colorectal cancers with mechanisms including for targeting cancer hallmarks (sustaining proliferative signaling pathways, resistance to cell death, activation of invasion and metastasis, inducing angiogenesis, deregulating cellular energetics, inflammation-mediated carcinogenesis, genomic instability, and mutation), enhancing body resistance to carcinogenesis, and enhancing therapeutic effects and/or decreasing side effects via drug interactions. However, more investigations and evidence are necessary for tonics being used as adjuvants.
Author contributions XW, YF, and ML designed the study; ZZ, JJ, and HL collected and double-checked the data and wrote the draft; and RS, DW, K-WZ, HN, X-GW, ML, WL, YF, and XW revised the manuscript. All authors reviewed and approved the submission of this manuscript.

FIGURE 2
Tonics targeting hallmarks as adjuvants in gastric, liver, and colorectal cancers via OMICs.

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Funding
This study was financially supported by the National Natural Science Foundation of China (81874356 and 82274155), the Open Project of Hubei Key Laboratory of Wudang Local Chinese Medicine Research from Hubei University of Medicine (WDCM2018002; WDCM201917; and WDCM201918), the Chinese Medicine Project of Health Commission of Hubei Province (ZY2021Z010 and WJ2021M055), and the Advantages Discipline Group (Medicine) Project in Higher Education of Hubei Province (2022XKQY3). The funders did not play any role in the design of this study or in the collection, analysis, and interpretation of data and writing of the manuscript, which are completely the responsibilities of the authors.