Current Status, Distribution, and Future Directions of Natural Products against Colorectal Cancer in Indonesia: A Systematic Review

In 2020, an estimated 19.3 million new cancer cases and nearly 10 million cancer deaths have occurred worldwide, with colorectal cancer ranking as the third most frequently diagnosed (10.0%). Several attempts have been conducted against cancer, including surgery, radiation, monoclonal antibodies, and chemotherapy. Many people choose natural products as alternatives against cancer. These products will not only help in human life preservation but also work as a source of up-to-date information, leading people away from incorrect information. We discuss the current status, distribution, and future implications of protecting populations with natural products as an alternative against colorectal cancer in Indonesia. Thirty-eight studies were included in this review for data extraction. The distribution of natural products in Indonesia that have potential activity against colorectal cancer cells was predominated by terpenoids, followed by phytosterols, phenolics, alkaloids, and polyisoprenoids. The type of cell line utilized in the cytotoxic activity analysis of natural products was the WiDr cell line, followed by HT-29 cells and HCT-116 cells. This review showed that MTT in vitro assay is a general method used to analyze the cytotoxic activity of a natural product against colorectal cancer cells, followed by other in vitro and in vivo methods. The systematic review provided predictions for several secondary metabolites to be utilized as an alternative treatment against colorectal cancer in Indonesia. It also might be a candidate for a future co-chemotherapy agent in safety, quality, and standardization. In addition, computational methods are being developed to predict the drug-likeness of compounds, thus, drug discovery is already on the road towards electronic research and development.


Introduction
Cancer is a deadly disease that has become a burden to everyone. There were 9.8 million cancer deaths reported in 2018. Colorectal cancer (CRC) is the third most common type of cancer globally, with 1.80 million cases, and it ranks second with the highest mortality in the world, i.e., 862,000 deaths [1]. The increase in CRC in developing countries is possibly due to an increase in the aging population, modern living habits, dietary habits, and an increase in risk factors for CRC, which include genetic diseases, smoking, alcohol, and lack of exercise. The percentage of CRC deaths in Indonesia in 2014 was 10% of the • What evidence literature and data exist on the application of natural products from Indonesian plants in colorectal cancer?
The secondary review questions are: • What are the available trends and study distribution of colorectal cancer in Indonesia? • What are the used approaches and indicators for colorectal cancer derived from natural products in Indonesia? • What are the challenges and gaps in the science and application of natural products in colorectal cancer in Indonesia?

The Scope of the Review
The systematic review is focused on studies that have reported the utilization of natural products in colorectal cancer in Indonesia. The data was gathered from published literature obtained through preselected bibliographic databases such as Scopus and PubMed, as well as search engines such as Google Scholar. To determine the scope of the review, this study used a globally applied standard approach for systematic review, by first defining the Population, Intervention, Comparator, and Outcome (PICO) of the review [15]. The detailed description of PICO used for this systematic review is as follows: • Population: Colorectal cancer in Indonesia. • Intervention: Any secondary metabolites and natural products used for colorectal cancer from Indonesian plants. These common approaches include in vitro and in vivo.

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Comparator: Any indicators used to monitor the success of anti-colorectal cancer activity by the use of natural products or secondary metabolites. • Outcome: Inhibited colorectal cancer or no proliferation activity of colorectal cancer.

Methods
Most of the methodological steps in this systematic review follow previous systematic reviews on the health topic or pharmacy practice [16,17]. In addition, the reporting guidelines for systematic reviewers in the medical and health sciences are used in this systematic review report [18].

Literature Search
Before conducting a literature search in the bibliography database, we composed a search string by identifying the relevant keywords following the defined PICO. However, we only considered keywords from Population and Intervention categories to avoid the narrowed search string and reduce the number of papers. Literature searches in Scopus and PubMed were conducted by using the English search string, while we used both an English and Bahasa Indonesia search string for Google Scholar (see Tables 1 and 2). To avoid the inclusion of further irrelevant studies in Google Scholar results, we only selected the first 50 publications, following the most relevant order.

Literature Screening
The study's relevance was determined by using the inclusion criteria presented in Table 3. The studies must meet the following relevant criteria: population, intervention, comparator, and outcome of interest. After duplicates were removed from the search results, all studies went through a two-stage screening process at the combined title and abstract, and full-text levels three reviewers. All screening stages used predefined questions-formulated following PICO-to select which publications satisfy the scope of the review.

Reporting and Presentation
The reporting and presentation of the results of this systematic review followed a standardized reporting approach for the systematic review of health and pharmacy studies [16][17][18]. A database containing systematic review screening results is complementarily presented in addition to the report.

Results
The PRISMA flow diagram of the publication screening is presented in Figure 1. Initially, 465 publications were identified through the systematic literature search string described in Table 1. Following the relevance of the initial papers to the defined research questions for this study, the literature screening, which consisted of title, abstract, and full-text screenings, resulted in 38 final papers for further data extraction. Overall, the screening processes only included 10% of the initially identified papers in Scopus, PubMed, and Google Scholar publication databases. The literature search results from major scholar databases such as Scopus as well as Google Scholar suggested the approximate publications that were recorded out of publications with duplicates ( Figure 1). The cut-off date of the literature search was 13 February 2021.
As presented in Table 4, the research publication on colorectal cancer based on natural products derived from Indonesian plants has increased since 2008. The trend of publications seems to have increased significantly, starting in 2016, and it was the highest in 2018. Afterward, the positive trend continued until 2020, with the most publications in the form of journal articles. Table 5 exhibits that the distribution of natural products having the potential activity against colorectal cancer cells in Indonesia is dominated by terpenoids, followed by phytosterols, phenolics, alkaloids, and polyisoprenoids. The type of cell line that is most often utilized in the cytotoxic activity analysis of natural products against colorectal cancer cells is the WiDr cell line (followed by HT-29 cells and HCT-116 cells). WiDr cells are human colorectal cancer cells isolated from the colon of a 78-year-old woman, a derivative of other colorectal cancer cells, namely HT-29 cells [19]. This data led to the MTT in vitro assay as a general method that was used to analyze the cytotoxic activity of a natural product against colorectal cancer cells, followed by other in vitro and in vivo methods. Commonly known anticancer methods include the MTT assay method, apoptosis, enzymatic inhibition by the in vitro method, and the use of mice and rats in the in vivo method. Azoxymethane, a metabolite of dimethyhydrazine (DMH), has been used extensively by many investigators to induce colon tumors and to study the effects of nutritional factors and chemopreventive agents in colon carcinogenesis. Azoxymethane (AOM) induces colon cancer in experi-mental animals; in a mechanism that is mediated by glutathione (GSH) depletion and impairing the total antioxidant capacity in colonic cells of rats [20,21]. In anticancer studies, researchers prefer the in vitro method, as can be seen from the larger research ratio than the in vivo method from results screened in the Scopus, Pubmed, and Google Scholar databases. A more complete explanation of the distribution of the types of methods, objects used, and natural products analyzed from 38 articles is summarized in Table 6.    ---Control of cell proliferation in ACFs and/or normal-appearing crypts of rats exposed to AOM [23].

Rats
Ethanol extract of Phaleria macrocarpa fruits (mostly flavonoids contains) [24] --- The crude ethanolic extract of P. macrocarpa had high antioxidant activity and it modulated the oxidative stress as proved by the up-regulation of glutathione-s-transferase and superoxide dismutase [24]. ---Lissoclibadin 1 suppressed tumor growth in nude mice. Lissoclibadin 1 induced cell death via apoptosis due to the mitochondrial cytochrome dependent activation (intrinsic pathway) of the caspase-9 and caspase-3 cascade pathway [26].
The compounds had antioxidant activity leading to a cytoprotective effect. The mechanisms of this chemoprevention included up-regulation of Bax and proliferation-promoting proteins (PCNA) [24].
Lissoclibadins (polysulfur aromatic alkaloids) from Ascidian lissoclinum [26] 5 µM 4.0 µM Significant / strong Lissoclibadin 1 exerted the most potent cytotoxic effects and mainly promoted apoptosis through an intrinsic pathway with the activation of a caspase-dependent pathway in HCT-15 cells [26].   The ethanol extract of P. crocatum had an activity to induce apoptosis and suppress COX-2 protein expression in WiDr cells [37].
Peptides from Platycephalus fuscus [50] 0.005 mg protein/mL 91.04% (inhibition) Not classified The further cell-based study is essential to observe the mechanistic pathways and structure or function relationship of peptides in stimulating apoptosis [50].
Cycloart-24-ene-26-ol-3-one from Aglaia exima leaves [49] 0.39-200 µM 2.4 µM (HT-29); 5.6 µM (CaCo-2) Significant / strong It is bound to tumor necrosis factor-receptor 1 (TNF-R1) leading to the initiation of caspase-8 and, through the activation of Bid, in the activation of caspase-9. This activity causes a reduction in mitochondrial membrane potential (MMP) and the release of cytochrome-C. The activation of caspase-8 and -9 both acts to commit the cancer cells to apoptosis through downstream caspase-3/7 activation, PARP cleavage and the lack of NFkB translocation into the nucleus [49]. 16.82 µg/mL (Eucheuma Sp.) [46] Significant / strong The presence of cucurbitacin type triterpenoid could be a marker compound for Begonia plant species. It exhibited potent cytotoxic activity against HCT-116 via apoptosis induction with a significant percentage of early and late apoptosis [29].

Discussion and Future Directions
Cancer is the second leading cause of death in the world after cardiovascular disease. Meanwhile, colorectal cancer ranks in the top three in the number of causes of death and is ranked as the second most common cancer type in men and third in women [58,59]. A comprehensive therapy development to treat colorectal cancer is needed to reduce patient mortality. This therapy development is expected to be capable of overcoming the resistance of conventional chemotherapy agents that already exist today. Resistance of WiDr cells to 5-fluorouracil (5-FU)-an antimetabolite chemotherapeutic agent-is mediated by an increased expression of thymidylate synthetase enzyme which is the main inhibitory target of 5-FU [60,61]. WiDr is also one of the cells that have low sensitivity to treatment with 5-FU. WiDr cells are widely applicable in Indonesia due to proper carcinogenesis and tumorigenicity studies and anti-tumor testing on potency disclosure of bioactive compounds from natural products for further development. Besides, WiDr cells are identical toHCT-15 and HT-29 cell lines because they are derived from the same patient and most likely from the same tumor [61]. Overall, WiDr cells are suitable to be used as models in screening the novel compound as a co-chemotherapeutic agent with 5-FU. Combination therapy (co-chemotherapy) of 5-FU with a chemo-preventive agent is an alternative to overcome resistance, increase efficacy, and reduce adverse effects. These facts that lead the research and development of natural products become important for the future directions of medicinal plants in colorectal cancer therapy.
Plants are one of the largest sources of natural products that are used to discover and develop novel chemotherapeutic agents. In particular, several discovered novel compounds from plants have unique mechanisms of action, greater efficacy, or lower adverse effects compared to the conventional chemotherapy drugs currently in use. Bioactive compounds from medicinal plants in Indonesia have also been studied to exhibit anticancer activities in the colorectal, namely terpenoids, phytosterols, alkaloids, phenolics, flavonoids, and polyisoprenoids (the basic structure for one of several derivatives from those bioactive compounds is presented in Figure 2).
As previously described in the research on the anti-colorectal cancer mechanism of polyisoprenoid compounds, polyisoprenoid was able to inhibit the colorectal cancer cells proliferation through up-regulation mechanisms, i.e., increased p53 gene expression (generating the cancer cell cycle blockade), induced apoptosis, and through down-regulation mechanisms, i.e., inhibition of PI3K gene expression, AKT1 gene, mTOR gene, and inhibition of EGF receptor expression [39,53]. The increase of p53 gene expression played a key role in response to cellular stress, for example, exposure to carcinogens, and also inhibited the proliferation of abnormal cells that have initiated carcinogens to prevent the development of neoplasms. The p53 gene also regulated apoptosis, inhibited angiogenesis, and regulated DNA repairment. According to the previous investigation, the increased level of the polyisoprenoid dose was able to provide a significantly increased percentage of p53 expression [48]. In addition, polyisoprenoid also has the potential to inhibit colorectal cancer cell metastasis through blockade of COX-2 expression [40,52]. It seems that colorectal cancer in Indonesia is still far beyond common understanding and more gene expression studies are needed to reveal the heterogeneous mechanisms of its process.
In the current paper, we conducted a systematic review that provided predictions for several secondary metabolites sourced from natural products to be utilized as an alternative treatment against colorectal cancer in Indonesia. They are also prospective candidates for future co-chemotherapy agents in safety, quality, standardization, and efficaciousness. This finding emphasized the potential of several natural products as anticancer agents against colorectal cancer cells.
The rich biodiversity of Indonesian plants derived from tropical rainforests provides a diversity of structural compounds that can be used as anti-colorectal cancer agents. At the same time, this review challenged Indonesian researchers of colorectal cancers to support drug-independence programs as these national products can be a part of the competitive multinational market. The utilization of Indonesia's natural resources, which are rich in plant biodiversity, seems to be less than optimal compared to the number of traditional medicines that have been proven to have benefits and are scientifically proven. Indonesia has more than 30,000 species of plants and animals that have the potential as medicines and 9,600 species of plants and animals that are known to have medicinal properties. This potential can be developed to meet the growing demand for traditional medicines and health supplements based on natural ingredients [62,63].
Traditional medicine in Indonesia is popular with the name Jamu. Jamu is usually used as a plant that has medicinal properties and has served various Indonesian generations for centuries. The Indonesian government categorizes traditional medicinal preparations into three categories, namely Jamu, standardized herbal medicine, and fitofarmaka (phytomedicine). Herbal medicine is categorized as a traditional medicine that has been used empirically but has not yet been scientifically tested; standardized herbal medicine are those plants with medicinal properties that have been proven by preclinical testing; fitofarmaka is a medicinal plant that has been tested to the clinical stage. Until 2019, 23 natural products have been registered and have been proven in clinical trials. Therefore, they are categorized as fitofarmaka in Indonesia [63,64]. Herbal medicine has become a part of the culture in Indonesia. This has been done for generations. Its use in the long term has proven its safety and benefits empirically in the community. Despite having been used for centuries, herbal medicine had no place in the formal healthcare system in Indonesia before 1987. Since 1988, the Indonesian government, through the Ministry of Health, has been regulating and encouraging efforts to use scientific Jamu as an alternative and additional medicine. The Indonesian government has also established a body under the name Directorate of Traditional, Alternative, and Complementary of Health Care (BINA YANKES TRADKOM) [65][66][67]. However, in practice, the use of traditional medicine informal medicine is not a priority and is even avoided sometimes. This is due to the absence of guidelines, legal basis for use, and support within the Health Insurance Agency in Indonesia [68]. Another reason for this may be because the dose of the natural product to date to induce cytotoxicity is less than established chemotherapy. Therefore, there is a need to look for a highly toxic natural compound that may be conjugated to cancer antigen-specific antibodies. Designing a clinical trial to test the efficacy of adding herbal products in combination with chemotherapy is also a challenge. Many herbal compounds demonstrate antioxidant activity which contradicts the mechanism of chemotherapy, which is generally a pro-oxidant agent.
Leading the discovery is the main component of today's early pharmaceutical research. The exploration of novel drugs for colorectal cancer therapy that has a fast progression can be a drug repurposing strategy to bypass preclinical steps that usually require laborious and resource-intensive work. In addition, it is also considered that the development of existing agents in the future can be more easily utilized by the community. For those purposes, the exploration of Indonesia's frequently used natural resources biodiversity is the best choice. In the past, drug development is based on trial and error, so it was costly and time-consuming. Today, molecular modeling, with the aid of computer hardware and software (computational method), has reduced the risk and the process of discovery is more effective in cost and time [69].
Molecular docking is the computational simulation of a ligand binding to a receptor, which helps to predict binding molecule to the protein target to predict the affinity and activity. The aim of target discovery is the identification and validation of suitable drug targets for therapeutic intervention. Structure-based drug design (SBDD) is responsible for the toxicity and activity of a compound in the body. The SBDD utilizes information from the target protein structure to find the active site of the protein that binds to the drug compound. This is a challenge for a natural product such as Jamu which is generally not a pure compound. There is a need to purify and synthesize specific compounds that are originally derived from exotic plants to conserve the environment. Based on the prediction of the active site, compounds can be designed with the expectations to bind to the target protein and have biological activity. These techniques support those novel drug developments which have specific activities by the mechanism of drug-receptor interaction. Computer-aided drug design (CAAD) helps to identify small molecules by orienting and scoring them in the active binding site of a protein [70].
In silico approaches contribute significantly to early pharmaceutical research and are especially important in target discovery and its lead. The need for timely adaptation and application of in silico approaches in pharmaceutical research has been recognized to design and discover novel active substances better than previous compounds and is expected to improve further the overall efficiency of drug discovery [71].

Conclusions
In summary, this review revealed bioactive compounds from natural products of Indonesian plants that have been researched and have potential as anticancer agents that are most commonly experienced in men and women including colorectal cancer. The general method used for the analysis of the cytotoxic activity of colorectal cancer cells is the in vitro method using the MTT assay, the most widely used cell line is WiDr. The most studied bioactive compounds that have any activity against colorectal cancer are terpenoids, phytosterols, phenolics, alkaloids, and polyisoprenoids, but other natural products may have the potential to be developed from this study.