Medicinal Plants in the Prevention and Treatment of Colon Cancer

The standard treatment for cancer is generally based on using cytotoxic drugs, radiotherapy, chemotherapy, and surgery. However, the use of traditional treatments has received attention in recent years. The aim of the present work was to provide an overview of medicinal plants effective on colon cancer with special emphasis on bioactive components and underlying mechanisms of action. Various literature databases, including Web of Science, PubMed, and Scopus, were used and English language articles were considered. Based on literature search, 172 experimental studies and 71 clinical cases on 190 plants were included. The results indicate that grape, soybean, green tea, garlic, olive, and pomegranate are the most effective plants against colon cancer. In these studies, fruits, seeds, leaves, and plant roots were used for in vitro and in vivo models. Various anticolon cancer mechanisms of these medicinal plants include induction of superoxide dismutase, reduction of DNA oxidation, induction of apoptosis by inducing a cell cycle arrest in S phase, reducing the expression of PI3K, P-Akt protein, and MMP as well; reduction of antiapoptotic Bcl-2 and Bcl-xL proteins, and decrease of proliferating cell nuclear antigen (PCNA), cyclin A, cyclin D1, cyclin B1 and cyclin E. Plant compounds also increase both the expression of the cell cycle inhibitors p53, p21, and p27, and the BAD, Bax, caspase 3, caspase 7, caspase 8, and caspase 9 proteins levels. In fact, purification of herbal compounds and demonstration of their efficacy in appropriate in vivo models, as well as clinical studies, may lead to alternative and effective ways of controlling and treating colon cancer.


Introduction
An uncontrolled growth of the body's cells can lead to cancer. Cancer of the large intestine (colon) is one of the main cause of death due to cancer. While the numbers for colon cancer are somewhat equal in women (47,820) and men (47,700), it will be diagnosed in (16,190) men (23,720) more than women. Multiple factors are involved in the development of colorectal cancer, such as lack of physical activity [1], excessive alcohol consumption [2], old age [3], family history [4], high-fat diets with no fiber and red meat, diabetes [5], and inflammatory bowel diseases, including ulcerative colitis and Crohn's disease [6].
Prevention of colorectal cancer usually depends on screening methods to diagnose adenomatous polyps which are precursor lesions to colon cancer [7]. The standard treatment for cancer is generally based on using cytotoxic drugs, radiotherapy, chemotherapy, and surgery [8]. Apart from these treatments, antiangiogenic agents are also used for the treatment and control of cancer progression [9].
Colon cancer has several stages: 0, I, II, III, and IV. Treatment for stages 0 to III typically involves surgery, while for stage IV and the recurrent colon cancer both surgery and chemotherapy are the options [10]. Depending on the cancer stage and the patient characteristics, several chemotherapeutic drugs and diets have been recommended for the management of colorectal cancer. Drugs such as 5-fluorouracil , at the base of the neoadjuvant therapies folfox and folfiri, are used together with bevacizumab, panitumumab, or cetuximab [7].
Chemotherapy works on active cells (live cells), such as cancerous ones, which grow and divide more rapidly than other cells. But some healthy cells are active too, including blood, gastrointestinal tract, and hair follicle ones. Side effects of chemotherapy occur when healthy cells are damaged. Among these side effects, fatigue, headache, muscle pain, stomach pain, diarrhea and vomiting, sore throat, blood abnormalities, constipation, damage to the nervous system, memory problems, loss of appetite, and hair loss can be mentioned [11].
Throughout the world, early diagnosis and treatment of cancer usually increase the individual's chances of survival. But in developing countries, access to effective and modern diagnostic methods and facilities is usually limited for most people, especially in rural areas [12]. Accordingly, the World Health Organization (WHO) has estimated that about 80% of the world population use traditional treatments [13]. One of these treatments is phytotherapy, also known as phytomedicine, namely, the use of plants or a mixture of plant extracts for the treatment of diseases. The use of medicinal plants can restore the body's ability to protect, regulate, and heal itself, promoting a physical, mental, and emotional well-being [14][15][16]. Various studies have shown the therapeutic effects of plants on fertility and infertility [17], hormonal disorders, hyperlipidemia [18], liver diseases [19], anemia [20], renal diseases [21], and neurological and psychiatric diseases [22]. Therefore, due to all the positive effects showed by medicinal plants, their potential use in cancer prevention and therapy has been widely suggested [23][24][25].
Since the current treatments usually have side effects, plants and their extracts can be useful in the treatment of colon cancer with fewer side effects. The aims of this review are to present and analyse the evidence of medicinal plants effective on colon cancer, to investigate and identify the most important compounds present in these plant extracts, and to decipher underlying molecular mechanisms of action.

Literature Search Methodology
This is a narrative review of all research (English full text or abstract) studies conducted on effective medicinal plants in the treatment or prevention of colon cancer throughout the world. Keywords, including colon cancer, extract, herbs, plant extracts, and plants, were searched separately or combined in various literature databases, such as Web of Science, PubMed, and Scopus. Only English language articles published until July 2018 were considered.
In the current narrative review, studies (published papers) were accepted on the basis of inclusion and exclusion criteria. The inclusion criterion was English language studies, which demonstrated an effective use of whole plants or herbal ingredients, as well as studies which included standard laboratory tests. In vivo and in vitro studies that were published as original articles or short communications were also included. The exclusion criteria included irrelevancy of the studies to the subject matter, not sufficient data in the study, studies on mushrooms or algae, and the lack of access to the full text. Reviews, case reports/case series, and letters to editors were also excluded but used to find appropriate primary literature.
The abstracts of the studies were reviewed independently by two reviewers (authors of this study) on the basis of the inclusion and exclusion criteria. In case of any inconsistency, both authors reviewed the results together and solved the discrepancy. Data extracted from various articles were included in the study and entered into a check list after the quality was confirmed. This check list included some information: authors' name, year of publication, experimental model, type of extract and its concentration or dose, main components, and mechanisms of action (if reported).

Medicinal Plants and Colon
Cancer. Overall, 1,150 articles were collected in the first step and unrelated articles were excluded later on according to title and abstract evaluation. Moreover, articles that did not have complete data along with congress and conference proceedings were excluded. Accordingly, a total of 1,012 articles were excluded in this step. Finally, 190 articles fulfilled the criteria and were included in this review. These papers were published within 2000-2017. A total of 190 plants were 2 Oxidative Medicine and Cellular Longevity included in this study. Based on literature search, 172 experimental studies and 71 clinical cases were included.
Overall, results indicate that grape, soybean, green tea, garlic, olive, and pomegranate are the most effective plants against colon cancer. In these studies, fruits, seeds, leaves, and plant roots were used for in vitro and in vivo studies.
3.1.1. In Vitro Studies. Out of 172 studies, 75 were carried out on HT-29, 60 on HCT116, and 24 on Caco-2 cells (Table 1). On HT-29 cells, both Allium sativum root extracts and Camellia sinensis leaf extracts induced cell apoptosis by two different mechanisms, respectively. In fact, the former showed inhibition of the PI3K/Akt pathway, upregulation of PTEN, and downregulation of Akt and p-Akt expression, while the latter was involved in attenuation of COX-2 expression and modulation of NFκB, AP-1, CREB, and/or NF-IL-6. Moreover, an antiproliferative activity has also been detected in Olea europaea fruit extracts, which increased caspase 3-like activity and were involved in the production of superoxide anions in mitochondria. An antiproliferative activity, by means of a blockage in the G2/M phase, has also been reported in Caco-2 cells by Vitis vinifera fruit extracts. Concerning HCT116 cells, several plants, such as American ginseng and Hibiscus cannabinus, induced cell cycle arrest in different checkpoints.

Studies in Animal Models.
The most used animal model is the murine one (Tables 2(a) and 2(b)). In particular, studies were carried out above all on HT-29 and HCT116 cells. The effects of the different medicinal plants and their extracts are essentially the same detected in in vitro studies. In particular, plant extracts were able to induce apoptosis and inhibit proliferation and tumor angiogenesis by regulating p53 levels and checkpoint proteins with consequent cell cycle arrest and antiproliferative and antiapoptotic effects on cancerous cells.
The main mechanisms of action of medicinal plants are summarized in Figure 1.
In in vitro studies, it has been found that grapes, which contain substantial amounts of flavonoids and procyanidins, play a role in reducing the proliferation of cancer cells by increasing dihydroceramides and p53 and p21 (cell cycle gate keeper) protein levels. Additionally, grape extracts triggered antioxidant response by activating the transcriptional factor nuclear factor erythroid 2-related factor 2 (Nrf2) [27].
Grape seeds contain polyphenolic and procyanidin compounds, and their reducing effects on the activity of myeloperoxidase have been shown in in vitro and in vivo studies. It has been suggested that grape seeds could inhibit the growth of colon cancer cells by altering the cell cycle, which would lead eventually to exert the caspase-dependent apoptosis [180].
Another plant that attracted researchers' attention was soybean, which contain saponins. After 72 h of exposure of colon cancer cells to the soy extract, it was found that this extract inhibited the activity and expression of protein kinase C and cyclooxygenase-2 (COX-2) [34]. The density of the cancer cells being exposed to the soy extract significantly decreased. Soybeans can also reduce the number of cancer cells and increase their mortality, which may be due to increased levels of Rab6 protein [216].
Green tea leaves have also attracted the researchers' attention in these studies. Green tea leaves, with high levels of catechins, increased apoptosis in colon cancer cells and reduced the expression of the vascular endothelial growth factor (VEGF) and its promoter activity in in vitro and in vivo studies. The extract increased apoptosis (programmed cell death) by 1.9 times in tumor cells and 3 times in endothelial cells compared to the control group [182]. In another in vitro study, the results showed that green tea leaves can be effective in the inhibition of matrix metalloproteinase 9 (MMP-9) and in inhibiting the secretion of VEGF [183].
Garlic was another effective plant in this study. Its roots have allicin and organosulfur compounds. In an in vitro study, they inhibited cancer cell growth and induced apoptosis through the inhibition of the phosphoinositide 3-kinase/Akt pathway. They can also increase the expression of phosphatase and tensin homolog (PTEN) and reduce the expression of Akt and p-Akt [32]. Garlic roots contain S-allylcysteine and S-allylmercaptocysteine, which are known to exhibit anticancer properties. The results of a clinical trial on 51 patients, whose illness was diagnosed as colon cancer through colonoscopy, and who ranged in age from 40 to 79 years, suggest that the garlic extract has an inhibitory effect on the size and number of cancer cells. Possible mechanisms suggested for the anticancer effects of the garlic extract are both the increase of detoxifying enzyme soluble adenylyl cyclase (SAC) and an increased activity of glutathione S-transferase (GST). The results suggest that the garlic extract stimulates mouse spleen cells, causes the secretion of cytokines, such as interleukin-2 (IL2), tumor necrosis factor-α (TNF-α), and interferon-γ, and increases the activity of natural killer (NK) cells and phagocytic peritoneal macrophages [200].
The results of in vitro studies on olive fruit showed that it can increase peroxide anions in the mitochondria of HT-29 cancer cells due to the presence of 73.25% of maslinic acid and 25.75% of oleanolic acid. It also increases caspase 3-like activity up to 6 times and induces programmed cell death through the internal pathway [217]. Furthermore, the olive extract induces the production of reactive oxygen species (ROS) and causes a quick release of cytochrome c from mitochondria to cytosol.
The pomegranate fruit contains numerous phytochemicals, such as punicalagins, ellagitannins, ellagic acid, and other flavonoids, including quercetin, kaempferol, and luteolin glycosides. The results of an in vitro study indicate the anticancer activity of this extract through reduction of phosphorylation of the p65 subunit and subsequent inhibition of nuclear factor-κB (NFκB). It also inhibits the activity of TNF receptor induced by Akt, which is needed for the activity of NFκB. The fruit juice can considerably inhibit the expression of TNF-α-inducing proteins (Tipα) in the COX-2 pathway in cancer cells [43]. The effective and important compounds in pomegranate identified in these 104 studies are flavonoids, polyphenol compounds, such as caffeic acid, catechins, saponins, polysaccharides, triterpenoids, 3 Oxidative Medicine and Cellular Longevity   6 Oxidative Medicine and Cellular Longevity   9 Oxidative Medicine and Cellular Longevity 10 Oxidative Medicine and Cellular Longevity  [87] 11 Oxidative Medicine and Cellular Longevity 12 Oxidative Medicine and Cellular Longevity 13 Oxidative Medicine and Cellular Longevity 14 Oxidative Medicine and Cellular Longevity 15 Oxidative Medicine and Cellular Longevity 16 Oxidative Medicine and Cellular Longevity Luffa echinata 17 Oxidative Medicine and Cellular Longevity 18 Oxidative Medicine and Cellular Longevity 19 Oxidative Medicine and Cellular Longevity Red grape   22 Oxidative Medicine and Cellular Longevity  24 Oxidative Medicine and Cellular Longevity   (ii) Modulated activation of mitogen and protein kinases, p38, and c-Jun NH2-terminal kinase [168] 26 Oxidative Medicine and Cellular Longevity Lycium barbarum L.   Green coffee

28
Oxidative Medicine and Cellular Longevity Inhibition of edema formation correlated to attenuation of COX-2 expression and promoter analysis revealed [36] 29 Oxidative Medicine and Cellular Longevity  [59] 30 Oxidative Medicine and Cellular Longevity        [198] 37 Oxidative Medicine and Cellular Longevity   (iv) Regulation of proteins [169] 40 Oxidative Medicine and Cellular Longevity 41 Oxidative Medicine and Cellular Longevity alkaloids, glycosides, and phenols, such as quercetin and luteolin, and kaempferol and luteolin glycosides.
In a systematic review of the plants being studied, some mechanisms were mainly common, including the induction of apoptosis by means of an increase of expression and levels of caspase 2, caspase 3, caspase 7, caspase 8, and caspase 9 in cancer cells, increasing the expression of the proapoptotic protein Bax and decreasing the expression of the antiapoptotic proteins.
Many herbal extracts block specific phase of the cell cycle. For instance, the extract prepared from the leaves of Annona muricata inhibits the proliferation of colon cancer cells and induces apoptosis by arresting cells in the G1 phase [53].
They can also prevent the progress of the G1/S phase in cancer cells [74]. In general, the herbal extracts reported here have been able to stop cancer cells at various stages, such as G2/M, G1/S, S phase, G0/G1, and G1 phase, and could prevent their proliferation and growth.
Other important anticancer mechanisms are the increase of both p53 protein levels and transcription of its gene. Even the increase of p21 expression is not without effect [137]. In an in vitro study on the Garcinia mangostana roots, the results were indicative of the inhibitory effect of the extract of this plant on p50 and P65 activation [93]. Moreover, reduction of cyclin D1 levels and increase of p21 levels are among these mechanisms [137], as well as inhibition of NFκB  Figure 1: Cell damage and cancer trigger p53 activation. The p53 protein activates the apoptotic protein Bax. Bax inhibits the antiapoptotic protein Bcl-2. During apoptosis, cytochrome c is released from mitochondria. To activate the Apaf-1 protein, the interaction between these proteins and cytochrome C is necessary. Pro-caspase 9 attaches to Apaf-1 and activates caspase 9. Caspase 9 activates caspases 3 and 7 and apoptosis occurs. 42 Oxidative Medicine and Cellular Longevity and reduction of the transcription of its genes, which contribute to reduce the number of cancerous cells [127]. Other important anticancer mechanisms are the inhibition of COX-2, as well as the reduction of the protein levels in this pathway [34]. In addition to this, in some cases, the inhibition of MMP-9 can be mentioned as the significant mechanism of some herbal extracts to kill cancer cells [183].

Conclusion and Perspectives
The findings of this review indicate that medicinal plants containing various phytochemicals, such as flavonoids, polyphenol compounds, such as caffeic acid, catechins, saponins, polysaccharides, triterpenoids, alkaloids, glycosides, and phenols, such as quercetin and luteolin, and kaempferol and luteolin glycosides, can inhibit tumor cell proliferation and also intduce apoptosis. Plants and their main compounds affect transcription and cell cycle via different mechanisms. Among these pathways, we can point to induction of superoxide dismutase to eliminate free radicals, reduction of DNA oxidation, induction of apoptosis by inducing a cell cycle arrest in S phase, reduction of PI3K, P-Akt protein, and MMP expression, reduction of antiapoptotic Bcl-2, Bcl-xL proteins, and decrease of proliferating cell nuclear antigen (PCNA), cyclin A, cyclin D1, cyclin B1, and cyclin E. Plant compounds also increase the expression of both cell cycle inhibitors, such as p53, p21, and p27, and BAD, Bax, caspase 3, caspase 7, caspase 8, and caspase 9 proteins levels. In general, this study showed that medicinal plants are potentially able to inhibit growth and proliferation of colon cancer cells. But the clinical usage of these results requires more studies on these compounds in in vivo models. Despite many studies' in vivo models, rarely clinical trials were observed among the studies. In fact, purification of herbal compounds and demonstration of their efficacy in appropriate in vivo models, as well as clinical studies, may lead to alternative and effective ways of controlling and treating colon cancer.

Conflicts of Interest
There is no conflict of interest regarding the publication of this paper.

Authors' Contributions
Dr. Paola Aiello and Maedeh Sharghi contributed equally to this work. Shabnam Malekpour Mansourkhani and Azam Pourabbasi Ardekan contributed equally to this work.