The Anticancer Effect of Phytochemicals and Potential of Breynia cernua : An overview

Cancer treatment still has challenges from its expense, side effect, and survival rate. One of the actions to improve this is searching for new anticancer agents. Medicinal plants are a candidate source since they have traditionally been used to treat illness. Phytochemicals of medicinal plants play a significant role in exhibiting anticancer effects. Literature studies of the phytochemicals of existing medicinal plants can be a clue to finding out the potential other plants whose studies are still limited, such as Breynia cernua, a plant with anticancer effects used traditionally. This study will provide information on the phytochemicals effect of medicinal plants or other compounds against cancer and their anticancer mechanisms. The agents are collected based on their compound's group, and each group's anticancer mechanism is resumed. The results showed that phytochemicals (flavonoids, alkaloids, saponins, quinone, tannins, and terpenoids) affect cancer cell through variant mechanism; induction of apoptosis, inhibition of cell growth, inhibition of cell migration, and induction of autophagic pathway. Most of the studies used methanol extracts, and most showed very strong toxicity to cancer cells. For further study, we suggest using isolated compounds from methanol, ethanol, or N-hexane extracts of Breynia cernua to get better anticancer activity, especially compounds belonging to the flavonoid or quinone group.

Cancer has accounted for 9.5 million deaths worldwide and ranks 2 nd in the world's leading cause of death. 1,2 Some of the challenges in cancer treatment such as high cost, unfavorable side effects (e.g. fatigue, soreness, nausea, vomit, tumor lysis syndrome, cardiac arrest, disturb life quality, anxiety or depressive symptoms), and low survival rates in poor countries up to 60%. [3][4][5][6][7][8][9][10] Seeing this, the research to develop cancer treatment still need to be done. One of the action is to find new candidates as adjuvant or even new drug candidates. According to WHO, 80% of world's population uses traditional medicine to treat disease. 5 One of the source of traditional medicine is a plant that can be utilized directly or by extracting it. 7 National Cancer Institute (NCI) noted that 3,000 of 35,000 species were identified to have anticancer activity. 11 The plants' phytochemicals exhibit cancer cell proliferation inhibitory activity, cell cycle arrest, or induce apoptotic cell death. 12 The vinca alkaloid is one example of plant-derived anticancer agent in clinical use. 11 Besides alkaloids, several group compounds reported having anticancer potential, including flavonoids, saponins, quinone, tannins, and terpenoids. [12][13][14][15][16] One of the medicinal plants that contain those phytochemicals is Breynia cernua which also has a history of traditional use as a medicine for cough, swelling, wound, and soreness. [17][18][19] There is also a clinical study showing this plant's cytotoxicity against MCF-7 cancer cell line. 17 Furthermore, this plant also grows well in an extreme areas such as coal reclamation, so it has the potential to be cultivated easily as a source of medicinal raw materials. 20 The study aims to obtain information about the anticancer effect of the mentioned phytochemicals and to observe the anticancer potential of Breynia cernua from its latest studies to have a better development of this plant in the future.

Material and Methods
The search strategy uses several keywords combined with boolean operators in online databases, as stated in table 1. We included original articles published before 2016 about flavonoids, alkaloids, saponins, quinone, tannins, and terpenoids against cancer, whether in English or Indonesian. The same applies to the Breynia cernua's species, except the year of publication is not limited because the study of Breynia cernua is scarce. (table 2).

results and discussion
From the previous methodology, fortyseven articles have been obtained, as described in figure 1. Thirteen original articles used samples classified as flavonoids, fifteen as alkaloids, twelve as saponins, two as quinone, three as tannins, and two as terpenoids.
Most studies use cancer cell lines (in vitro), and a few use animal models. Most articles use the compound (isolated compound, synthesized compound, or purchased compound) as a sample, and only a few use fraction or extraction. There are twenty-one types of cancer cell lines used to evaluate the toxicity of these agents. In addition, some of them investigated the anticancer mechanism. (table 3)

Phytochemical and It's anticancer effect
Plants produce non-nutritive chemical compounds called phytochemicals or secondary ("flavonoids" OR "alkaloids" OR "saponins" OR "quinone" OR "terpenoids" Google Scholar OR "tannins") AND ("anticancer" OR "antitumor" OR "antineoplastic") Sciencedirect Medline Springer Links Cochrane ("flavonoid" OR "alkaloid" OR "saponin" OR "kuinon" OR "terpenoid" Google Scholar Indonesia OR "tannin") AND ("antikanker" OR "antitumor" OR "antineoplastik") Portal Garuda ("breynia cernua" OR "ironstone range" OR "coffee bush") Google Scholar AND ("anticancer" OR "antitumor" OR "antineoplastic") Sciencedirect Medline Springer Links Cochrane ("breynia cernua" OR "katuk hutan" OR "sugi-sugi") Google Scholar Indonesia AND ("antikanker" OR "antitumor" OR "antineoplastik") Portal Garuda metabolites. 68 Until now, more than a thousand plant species, including Breynia cernua, have been identified with anticancer potential. 17,69 Phytochemicals had anti-tumor, such as induced apoptosis, inhibiting cell growth, or inhibiting cell migration. 70 Three types of cell death are involved in this study; apoptosis, necrosis, or autophagy. 71 Necrosis is morphologically characterized by cell swelling, organelle dysfunction, and cell lysis. 71 Autophagy is a process that begins with the formation of autophagosomes. 71 Apoptosis is characterized by cell shrinkage, nuclear condensation, nuclear fragmentation, dynamic membrane blebbing, and loss of adhesion to neighbors or extracellular matrix through intrinsic or extrinsic pathway. 71 There are various descriptions regarding the anticancer mechanism of samples in this study, most of which lead to the intrinsic pathway of apoptosis.

Flavonoids
Flavonoids are a class of plant secondary metabolites that have a polyphenolic structure. 72 This phytochemical is found in many fruits and vegetables and has broad biological activities. 72 In this study, two samples used crude extracts, one used fraction, and the rest used pure compounds obtained by isolation, synthesis, or purchase. Gunawan Indrayanto et al. showed that most of the samples were considered (IC50 value is below 200 ìM or 100 ìg/mL), and there was only one sample above that value, morin-7-sulphate sodium. 24,73 The compound with the strongest cytotoxic value is oncamex with IC50 value 0.4150 ìM, which is considered to have very strong activity. 30 Oncamex  . 30 The mechanism of oncamex involved several pathways; involvement of caspase protein, production of superoxide, and altered gene expression that related to cell cycle and apoptosis. 30 Interestingly, strong cytotoxicity was also demonstrated by crude extract of Dodonaea viscosa. 33 This study indicated that the anticancer activity of flavonoids is related to inhibiting cell invasion and cell growth or inducing apoptosis. The generation of ROS seems to start the process of apoptosis, which then triggers mitochondrial membrane leakage and activates the caspase-dependent intrinsic pathway of apoptosis. 22,25,26,28,30,32 Some studies also proposed that mitochondrial membrane leakage is caused by disruption of Bax/Bcl balance, which is triggered by disabling the binding of P53 protein with E6 or EGFR protein. 29,34,41,42 Another study showed the involvement of caspase-8, indicating that an extrinsic apoptosis pathway may also happen. 66 Inhibition of some proteins (RXRa transcription and STAT3) also leads to apoptosis, although the detailed mechanism needs more investigation. 22,31 STAT3 has been shown to regulate several genes involved in the cell cycle; therefore, its inhibition may lead to the induction of cell cycle arrest and will inhibit the growth of cancer cells. 22 Inhibition of cell growth may also be indirectly caused by factors such as inhibition of TMEM16A, Phosphorylation of ERK protein caused by ROS generation, and inhibition of inflammation. 21,24 Suppression of vimentin and MMP-9 also appears to correlate to cell invasion inhibition. 24 Briefly, most of the compounds used in this study indicate that flavonoids play a role against cancer cells, either in crude extracts form (such as crude extract of Dodonaea viscosa) or pure compounds (such as oncamex, hesperetin, or galangin). 21,27,30,33 Twelve of thirteen articles proposed an anticancer mechanism; induced apoptosis, inhibited cell growth or migration, as depicted in figure 2. alkaloids Alkaloids are secondary metabolites that usually consist of basic nitrogen atoms. 74 In   these articles on alkaloids, most studies evaluated pure compounds obtained by isolation, synthesis, or purchased, the rest using crude extract. Gunawan Indrayanto et al. showed that most of the agents had good activity against cancer cells, except two compounds, vincamine and crebanine. 35,44,73 The strongest cytotoxic activity was demonstrated by Loonamycins A (obtained from bacteria, Nocardiopsis flavescens) with IC50 value of 31.4 nM. 37,73 The second strongest is the Khasuanine A (obtained from plant, Melodinus khasianus) with IC50 value of 0.74 ± 0.03 ìM. 34,73 Eight of fifteen articles proposed an anticancer mechanism; leads to induction of apoptosis, inhibition of cell growth, and induction of autophagic pathway, as depicted in figure  3. Khasuanine A appears to induce an intrinsic apoptotic pathway involving several proteins such as Bcl-2, caspase 3, and P53. 34,37 These studies showed that alkaloids have good cytotoxicity towards cancer cells in the form of crude extracts such as in ethanol extract of Solanum blumei Nees ex Blume and pure compounds khasuanine A of Melodinus khasianus. Figure 3 shows alkaloids against cancer cells by inhibiting cell growth, inducing autophagic pathways, and inducing apoptosis. Apoptosis pathway in a caspase-dependent manner seems related to stress oxidative affected by ROS generation. In addition, the redox stress may cause depolarization of mitochondrial membrane potential, resulting in mitochondrial membrane leakage. Then the release of cytochrome-c from mitochondria will initiate intrinsic apoptosis by activating caspase 7 and caspase 3. [34][35][36]38,41,42 ROS also appears to mediate the autophagic pathway. However, the detailed mechanism needs to be investigated. 38 It also appears that the decrease of Bcl-2, caused by increasing P53, also contributes to mitochondrial membrane leakage, activating caspase-3, resulting in the intrinsic pathway of apoptosis. 34,41 HMGB1 reported promotes cancer cell growth; therefore, inhibition in this will lead to inhibition of cell growth. 39 saponins Saponins are a groups of natural plant products which have a form of glycosides of triterpenes and steroids. 75 These studies evaluated the cytotoxic effect of pure compounds, crude extracts, fraction, or crude saponins. Most of them were active except fraction Dia-80 from Astragalus glycyphyllos L, saponin-containing fractions 1 -6 from Astragalus glycyphyllos, and crude extract of organic culture Spirulina. 52,53,60,73 The most potent compound is the Parquispiroside from leaves of Cestrum parqui with IC50 value of 3.3 ± 0.63 ìM. 55,73 The methanol extract of Zanthoxylum armatum also showed potent cytotoxicity with IC50 value of 0.87 ± 0.4 ìg/mL. 51,73 Interestingly, the methanol extract which was further processed into   6. Mechanism chart of terpenoids crude saponins was found to have lower activity against cancer cells (IC50 13.92 ± 3.6 g/ml). 51 These findings indicate that the saponin group's interaction with other compounds makes their work more effective against cancer. 76 Eight of twelve articles in this study on saponins mentioned the anticancer mechanism of action. Overall, the anticancer agents of the saponins group involves an intrinsic pathway in induction of apoptosis and other routes, such as inhibiting cell growth. (Figure 4). Anticancer evaluation of one of the most potent agents, Paris saponin 1, showed changes in PI3K, pAKT, Bcl-2, Bax, caspase-3, and capsase-9. 49 Therefore, Xinhai Zhu et al. proposed in their article that Paris saponins could target the PI3K/AKT pathway to activate the apoptotic pathway. 49 Figure 4 shows that saponins have two effects on cancer cells; inhibit cell growth and induce apoptosis. ROS generation is reported to trigger mitochondrial membrane leakage. 54 Further studies demonstrating mitochondrial membrane depolarization, the release of cytochrome c, and activation of caspase 9 and 3, can be done to prove the apoptotic pathway in a caspasedependent manner. Saponin seems involved in decreasing PI3K/Akt in inducing apoptosis, although the mechanism has not been explained in detail. 49 Nonetheless, inactivation of the PI3K/ Akt signaling pathway has been reported to be involved in apoptosis via the generation of ROS. 77 The contribution of PI3K/AKT in apoptosis is also associated with activating pro-apoptotic protein Bax. 78 In addition, saponins were reported to be involved in cell growth inhibition which seems to be due to anti-inflammatory and immune modulating activities. 50

Quinone
Quinone derivatives from natural products display various compounds that have been used as cytotoxic agents for anticancer therapy, such as daunorubicin, doxorubicin, geldanamycin, and mitomycin C. [79][80][81][82] Quinones plays an important role in the aerobic metabolism of most cell because of its capacity on their one-or two-reduction to generate Reactive Oxygen Species (ROS), or its arilation capacity through quinone-thiol formation (Michael adduct formation). 80,[83][84][85][86] Based on these activities, many studies have used compounds from the quinone group to understand the role of proteins involved in fighting cancer. 74,83,84,[87][88][89][90] In addition, the researchers are looking for new anti-cancer candidates from the quinone group. In this study there are two articles that investigate the anticancer effects of quinones derived from medicinal plants. They investigated samples from the root of the plant species Maytenus ilicifolia; then they isolated Maylenin and 22-b-hydrodymaytenin compounds. 61 Both compounds were considered to have very strong cytotoxicity. 61,73 The other study did a direct synthesis to get fourteen derivatives; compounds 9h, 9k, 9i, and 9b exhibited very good anticancer activities. 62 Further analysis showed that maylenin induced apoptosis with the involvement of caspase 3 and caspase 7 (figure 5). 61 All of these results reinforce the potential of quinone as a source of new drug candidates. Figure 5 shows that the quinone group of medicinal plants appears to induce apoptosis in a caspase-dependent manner, involving activation of caspase 7 and caspase 3.61 Further studies using the pan-caspase inhibitor ZVAD-FMK (N-benzyloxy carbonyl-Val-Ala-Asp-fluoromethyl ketone) can be carried out for confirmation this pathway. 91,92 Another important thing to do is to observe the morphological changes of apoptosis in cells treated with candidate anticancer compounds, such us the round-up of cells, apoptotic bodies formation, cell shrinkage, or chromatin condensation. 93 tannins Tannins are a group of natural phenolic biomolecules that protect plants against fungi and insects. 94 In this study, methanol extract of three plant species, Rubus niveus, Rubus Fairholmianus, and Rubus ellipticus showed very strong cytotoxicity. 63,73 Another section with various solvents (such as ether, petroleum, methanol, hexane, and water) of Azadirachta indica A. Juss and Melia azedarach Linn. 64 Both species exhibit lower anticancer activity with IC50 value more than 100 ìg/mL. 64,73 All studies did not mention the mechanism of action against cancer cells. Further purification is needed to get the active compound of methanol extract of the Rubus genus.

Terpenoids
Terpenoids are a vast group of natural compounds which form a major constituent of essential oil from plants and it can be classified according to the number of their isoprene unit. 95 In this study, two samples used crude extract, one used fractions, and one used compound. In the first study, they isolated BC I compounds which is considered to have a moderate cytotoxicty 65,73 The second study investigate samples from the plant species Stachys pilifera; then, they proceed its extract and fraction. 66 Both of them were considered to have moderate cytotoxicity 66,73 The third study investigate samples from the fruit of the plant species Luffa echinata Roxb. 67 They used LC50 as a cytotoxicity parameter and it is stated that the extracts showed a remarkable anticancer activity. 67 Two of three articles proposed an anticancer mechanismwhich involve arrest of cell cycle phase, involvement of NO concentration and also involvement of caspase proteins, as stated in the figure 6. 65,67 All of these results reinforce the potential of terpenoids to be an anticancer agents. Figure 6 demonstrates that terpenoids inhibit cell growth and inducing apoptosis. Intrinsic and extrinsic apoptosis pathways have been reported to be affected by terpenoids, with the involvement of caspase 9 and caspase 8. 66 There was also a decrease in the concentration of nitric oxide (NO), which caused a decrease in NF-kB p65 levels and could lead to the induction of apoptosis. 66 In this pathway, terpenoids may activate ROS production because increased ROS levels seem to suppress NO synthase (NOS), reducing NO. 96 NF-êB is commonly known to mediate cell proliferation and survival, which is also associated with apoptosis. In vitro studies identified that NF-êB inhibition is induces apoptosis in leukemic stem cells and intrinsic apoptotic pathways in leukemia cell lines. 97,98 Nonetheless, other studies have also reported necrosis induction by terpenoids.
Terpenoids are also reported to inactivate cell growth by inhibiting the cell cycle. 65

The Potential of Medicinal Plant Breynia cernua
It was recorded that from 35,000 plant species studied by the National Cancer Institute (NCI), 3,000 species have anticancer activity. 11 The compounds contained in this plant include flavonoids, alkaloids, saponins, quinones, tannins, terpenoids, and many more. [12][13][14][15][16] These compounds were also detected in the Breynia cernua. [17][18][19] Nasrul Wathan et al. revealed that Breynia cernua contains saponins, flavonoids, and quinones. 18 Another study using Thin Layer Chromatography (TLC) and column chromatography indicated that this plant showed the presence of alkaloids, saponins, terpenoids, flavonoids, and tannins. 17 Breynia cernua, also known as Katuk Hutan or Sugi-sugi, is a small shrub or tree with a height of 3 -5 m. 18,99,100 It has thin green leaves with prominent veins and red to purple berry-like fruits. 18,100 This plant is found in tropical areas such as Indonesia (Java, Papua, or Kalimantan province), Philippines, East Malesia, Northern Australia and Solomons. 18,100 It can be cultivated around the house but primarily grows under primary forest and also commonly found on the hills in secondary forest. 100 This plant can grow in an area with reduced soil fertility, an ex-mining or reclamation land. 20, 101 Our preliminary study showed that Breynia cernua is one of the dominant medicinal plants growing in the coal reclamation area at Tanah Bumbu-South Kalimantan, Indonesia. 20 According to the WHO catalog, this plant is traditional used to treat diseases. 100 People used its leaves directly or with various methods (e.g., crush, bake, heat, or decoct the leaves) to relieve illnesses such as cough, soreness, ulcer, and fever. 100 People from Jayapura and Timika also used this plant as an alternative breast and cervical cancer treatment. 99 Two studies in this review showed that Breynia cernua has cytotoxic activity, as stated in table 4. In the first study, seven species, including Breynia cernua, were obtained from some public forest in West Papua Province, Manokwari; Merauke: Sentani; Serui and Jayapura City (Indonesia). 99 Their cytotoxicity was evaluated using in vitro Brine Shrimp Lethality Test (BSLT). The ethanol extract of Breynia cernua showed toxicity with a Lethal Concentration (LC50) value of 255.76 ppm. 99 Second study obtained fresh leaves of Breynia cernua from Jayapura City, Papua (Indonesia). 17 The study showed that the extract or fraction of Breynia cernua showed cytotoxic activity against MCF-7 tumor cell line, with the highest activity recorded in the n-hexane fraction. 17 Furthermore, the extract or fraction of B. cernua has cytotoxicity to cancer cells. Although research is still limited, phytochemical studies of medicinal plants provide clues that the phytochemicals contained in B. cernua have anticancer potential. Therefore, further studies need to be carried out, for example, purification of the n-hexane fraction to obtain the active compound. In addition, this plant has additional values , such as it can grow easily in infertile soil and also its history of medical use. The flavonoid or quinone group of Breynia cernua may play a role in causing cytotoxic effects on cancer cell lines. The most common mechanism that appear in this study were intrinsic pathway of apoptosis which involved caspase and other pro-apoptotic protein. This pathway might be the one that will appear in the further study about this plant towards cancer cell. There are also various pathway in inhibiting cancer cell proliferation, which one them might also be the mechanism of action of anticancer agents inside Breynia cernua.
Nanoparticles have been extensively studied to increase anticancer activity by strengthening the bioavailability. 43,102 Anna Grebinyk et al. used berberine (Ber) with a carbon nanomaterial, C60 fullerenes (C60), and formed C60-Ber nano complexes. 43 This complex amplified its toxic effect in a low concentration range and potentiated its effect in vivo. 43 Ruma Baksi et al. also developed quercetin-containing chitosan. Nanoparticles (QCT-CS NPs) with enhanced encapsulation efficiency and sustained release property. 102 This suggests that QCT-CS NPs have increased efficacy over free quercetin in reducing the tumor size of mice containing lung and breast tumor xenografts. 102 Gold and silver are also widely used as nanoparticle study materials to increase the cytotoxic activity of natural products.

conclusion
In this present study, phytochemicals has various anticancer effect towards cancer cell. The flavonoid group is involved in the induction of apoptosis through caspase activity, inhibition of cell growth, and inhibition of cell migration. The alkaloid group induces apoptosis in the intrinsic pathway, cell growth inhibition, and autophagic induction. The saponins group can target the PI3K/AKT pathway to activate intrinsic apoptotic. Anticancer properties of quinones lead to the generation of Reactive Oxygen Species (ROS) associated with apoptosis in a dependent or independent caspase manner. Few studies have discussed the potential of the tannin group as an anticancer candidate. Mechanism against cancer cells of terpenoid group involves cell cycle arrest, NO concentration, and caspase proteins. Seven of the twelve extracts used in this study were methanolic extracts, and most of them have very strong cytotoxicity. Meanwhile, no studies have been conducted on the methanol extract of Breynia cernua, so using this extract seems promising. In addition, nine of twenty-three samples with low IC50 (strong activity) were isolated or purchased compounds. Therefore, we recommend isolating compounds from methanol, ethanol, or N-hexane extracts of Breynia cernua to get better anticancer activity, especially compounds belonging to the flavonoid or quinone group. Furthermore, using nanoparticles such as carbon, chitosan, silver, or gold may increase the bioavailability of anticancer agents. In summary, medicinal plants with limited studies, such as Breynia cernua, deserve to be explored for their anticancer potential based on their history of traditional use and phytochemical content. Therefore, the use of methanol extract needs to be investigated, with the target of its phytochemical content being flavonoids or quinones.