Insight into Potential Anticancer Activity of Algal Flavonoids: Current Status and Challenges

Flavonoids are some of the most precious phytochemicals, believed to be found largely in terrestrial plants. With the advancement of phytochemical research and marine bioprospecting, flavonoids have also been reported by the research of microalgae and macroalgae. High growth rate with minimal nutritional and growth requirement, saving arable land and rich metabolic profile make microalgae an excellent repertoire of novel anticancer compounds, such as flavonoids. In addition, marine algae, especially seaweeds contain different types of flavonoids which are assumed to have unique chemical structures and bioactivities than their terrestrial counterparts. Flavonoids are not only good antioxidants but also have the abilities to kill cancer cells by inducing apoptosis and autophagy. However, the study of the anticancer properties of flavonoids is largely limited to terrestrial plants. This review offers an insight into the distribution of different classes of flavonoids in eukaryotic microalgae, cyanobacteria and seaweeds with their possible anticancer activities. In addition, extraction and purification methods of these flavonoids have been highlighted. Finally, prospects and challenges to use algal flavonoids as anticancer agents have been discussed.


Introduction
Cancer is ranked as the second-highest cause of death and accounts for about 10 million death in 2020 [1]. Based on the severe negative effects, chemo-resistance, high expenditure and scarcity of novel anticancer compounds, anticancer drug discovery is now more inclined to the investigation of natural sources. About eighty percent of all approved chemotherapeutics and fifty percent of all drugs are derived from natural origin. Natural drugs are accepted as a safer option than synthetic drugs because of their presence in the human diet and broad availability. Furthermore, natural drugs have reduced side effects and the potential to target various tumorigenesis-related signaling pathways. Considering all of these advantages, natural products research is burgeoning to search for novel anticancer compounds not only from terrestrial plants and microorganisms but also from marine organisms [2]. Marine organisms are offering a huge array of pharmaceutically important natural products that can be used to treat different kinds of human diseases, especially cancer. The chemical structures of marine-derived bioactive compounds are unique and highly diversified. Marine organisms show novel molecular scaffolds than terrestrial products [3]. Currently, a total of 14 marine-derived drugs are available on the market and 9 of them are used for cancer therapy. Another 19 compounds from the marine organisms are in different phases of cancer clinical trials. The anticancer compounds derived from marine compounds mainly come from mollusk/cyanobacterium, sponge, tunicate, bacterium, fungus and sea animals. Mollusk/cyanobacteria account

Flavonoids
Flavonoids are a large group of polyphenolic metabolites which is widely dispersed throughout the plant species. Among many of the plant pigments, flavonoids are classed as edible. They are derived from derivatives of 2-phenyl-benzo-γ-pyrone [41]. Flavonoids have a common flavan structure that is a 15-carbon phenylpropanoid chain (C6-C3-C6), which is assembled into two aromatic rings and linked with another O 2 containing pyrene ring. This basic structure is changed through oxidation and also modified in substituents to the pyrene ring that leads to the formation of other flavonoid classes [42]. Two aromatic rings are condensed to form a chalcone. Cyclization of chalcone forms flavanone which is the primary compound for the synthesis of other classes of flavonoids. Flavonoids are categorized into six different types. Flavanols (catechin, epicatechin, epigallocatechin), Flavanones (naringenin and hesperetin), Flavonols (quercetin, kaempferol or myricetin), Isoflavones (genistein and daidzein), Flavones (apigenin and luteolin) and Anthocyanidins are the groups (Figure 2) found in plant and algae [41].

Algal Flavonoids
Flavonoids are widespread in algae and the concentration of flavonoids can be further increased through different manipulation or enhancement techniques. For example, increased salt concentration has a significant effect on the accumulation of phenolics and flavonoids. The growth of cyanobacterial species Plectonema boryanum, Anabaena doliolum and Oscillatoria acuta were decreased when treated with high NaCl concentration but the accumulation of rutin was increased [13]. Metal stress can also help in the accumulation of flavonoids in microalgae. Dunaliella tertiolect has been reported to have increased catechin and epicatechin with an augmented amount of copper and these flavonoids are thought to be acted as a protector of microalgae cells from metal toxicity. The extracts containing these flavonoids also showed antioxidant activity [19]. With the increased concentration of nitrate and growth factor L-phenylalanine, Spirulina maxima showed accumulation of a higher amount of flavonoids, such as quercetin and kaempferol. The presence of these flavonoids along with other phenolics gave antioxidative protection against lipid peroxidation of hepatic microsomes in rats which were induced by carbon tetrachloride, an oxidizing agent. The results were comparable to the commercial antioxidant BHT and BHA [43]. Yadavalli et al. also reported the accumulation of flavonoids, like quercetin and catechin, in Chlorella vulgaris under nitrate-stressed-condition with supplementation with L-phenylalanine. Depletion of nitrate in the media upregulates the synthesis of flavonoids and the rate has been accelerated after the addition of L-phenylalanine due to its direct link in the flavonoid synthesis pathway [44]. In Leptolyngbya sp., catechin, apigenin, naringenin, luteolin and luteolin-7-glucoside were found to be the most common flavonoids. Among them, naringenin was the highest, 4.1 ± 0.01 mg/g [27].
Singh et al. investigated twenty terrestrial cyanobacteria species and detected three main flavonoids namely, quercetin, kaempferol and rutin. Rutin was found at highest level in Microcheate tenera (29.4 ± 0.7 µg/g) and the lowest amount in Chroococcus sp.
Seaweeds, especially red and brown algae, are often considered a superfood. Different seaweed species are consumed as food and supplements worldwide. Likewise, eukaryotic microalgae Chlorella spp. and cyanobacteria Spirulina sp. are also now quite popular in the market of food supplements. These edible macro-and microalgae are rich in polyphenols, where one-third is phenolic acids and the rest two-thirds are flavonoids. A study showed that flavonoid, particularly epicatechin, was the most frequently found phenolic compound in commercial algal food products, which was detected in each red and brown seaweeds, as well as in Chlorella pyrenoidosa and Spirulina platensis. Other than this flavonoid, catechin gallate, epicatechin gallate, epigallocatechin, epigallocatechin gallate and pyrocatechol was measured in those algae. However, the most interesting thing is that the antioxidant capacity of these water-soluble compounds (ACW) of brown algae, Eisenia bicyclis, was found more than bilberry, strawberry and kiwi [32]. A very comprehensive study was done by Yoshie et al., where twenty-seven Japanese seaweeds from red, green, brown types were evaluated for their flavonoids contents. They found mainly catechin, epicatechin, epigallocatechin and epigallocatechin gallate in different seaweed species. From their study, catechin was found in most of the red and brown species but detected in the highest in Acetabularia ryukyuensis, which is a green alga [45]. In another study, they found rutin, quercetin, myricetin and two other unique flavonoids, hesperidin and morin. Morin was found in each species. Similarly, hesperidin was also detected in most of the seaweeds, even more than rutin, quercetin and myricetin. The highest amount of hesperidin was detected in A. ryukyuensis, Gracilaria texorii and Gracilaria asiatica [25].
Rutin kills cancer cells through an increased level of ROS that causes oxidative stress in the cells. It can inhibit PI3K/Akt and Ras/Raf/MAPK signaling pathways that ultimately cause cell cycle arrest and, finally, apoptosis. Upregulation of Bax and downregulation of MMP-2 and Bcl-2 by rutin can also lead to apoptosis through caspase-3 activation. In the animal model, rutin can effectively reduce doxorubicin-induced neurotoxicity and nephrotoxicity [46].
Quercetin also have strong anticancer activity. Quercetin has been reported to arrest the cell cycle by releasing p53 that augments p21, GADD45 and Bax expression, as well as impedes CDK2, cyclin A and B activity. In addition, it hindered the migration of cancer cells through upregulation of the expression of E-cadherin and downregulation of N-cadherin, Vimentin, MMP-2, -7 and Snail-dependent Akt activation pathway. On top of that quercetin can induce autophagic cell death by modulating LC3I. Quercetin induced apoptotic and autophagic cell death in the mouse model as well [48]. Furthermore, quercetin potentiated the efficacy of sorafenib in lower doses in thyroid cancer therapy by increasing the expression of E-cadherin and decreasing the expression of N-cadherin [49].
Kaempferol can induce apoptosis in ovarian cancer cells through increased expression of apoptotic proteins, caspase-3, -8, -9, Bax, or by stimulating death receptors/FADD/ Caspase-8 pathway. In these cancer cells, it stopped the progression of the cell cycle in G2/M phase by inducing the Chk2/Cdc25C/Cdc2 and Chk2/p21/Cdc2 pathways [50,51]. In pancreatic cancer cells, it induced apoptosis by activating tissue transglutaminase (TGM2) mediated Akt/mTOR signaling pathway that increased the ROS production in the cells [52].
Morin exerted anticancer activity against chronic myeloid leukemia via downregulation of the PI3K/AKT signaling pathway and also miR-188-5p which led to apoptosis [53]. In triple-negative breast cancer cells (MDA-MB-231), morin exerted anti-proliferative activity by arresting cells at S and G2/M phase, via upregulation of the ERK/p21 signaling pathway and downregulation of the FOXM1 signaling, which reduced cyclin A2 and cyclin B1 [54]. Another anticancer activity of morin is the downregulation of Glut 1 expression that restricts the entry of glucose into the cells. As glucose is the main nutrient, blocking its uptake leads to mitochondria-mediated apoptosis [55].

Catechin
Catechin, a flavan-3-ol, is mainly found in Camellia sinensis and C. assumica. Catechin is divided into eight different classes [28]. Catechin has been detected in Euglena cantabrica (71.4 µg/g of dry weight) and these flavonoid-containing extracts have radical scavenging activity [29]. It was also found in Dunaliella tertiolecta, even when exposed to low copper concentration, up to 315 nmol/L [19]. Seaweed, Porphyra tenera produced a high amount of catechin than the microalgae, which was measured as 128.8 ± 2.9 µg/g [32].
Catechin can kill human glioma (U87MG) cells in a concentration-dependent manner while showing lower cytotoxicity in normal astrocytes. It can inhibit cell proliferation by blocking cells in G2/M phase of the cell cycle and also by inhibiting MAPK/ERK signaling pathway. Moreover, catechin can cause autophagy-induced glioma cell death by forming autophagosomes and autophagic vacuoles and triggering increased expression of LC3II and decreased expression of p62 [56]. Nanohybrid formulation with catechin (50 µg/mL) inhibited human melanoma (WM266-4) cells proliferation and new blood vessel formation in zebrafish xenotransplants [57].

Epicatechin
Epicatechin, another flavanol, is subclassed into catechin and was isolated from the pith of the palm Metroxylon sagu. Epicatechin is abundant in green tea and black tea, berries, red wine and cacao. Epicatechin is also found in algae, such as Euglena cantabrica but in small amounts (7.1 µg/g of dry weight) [19]. In addition, epicatechin was found in edible macroalgae, Porphyra tenera but the highest amount was found in Spirulina platensis, at concentration of 27.5 ± 1.3 µg/g [32]. Along with other bioactivities, epicatechin exerts anticancer activity. For example, epicatechin can kill breast cancer (MDA-MB-231 and MCF-7 cells,) cells in a concentration-dependent manner with an IC 50 of 350 µM. It can induce apoptosis through DNA fragmentation, augmentation of the expression of proapoptotic proteins (Bad and Bax) and an increased level of ROS [58]. Epicatechin can act as a restorative agent which is assumed to mitigate negative side effects of chemotherapy drug, bleomycin and helps improving lung damage which in turn enhances the quality of life of the patient. In the animal model, this flavanol reduced the negative effects of bleomycin by alleviating oxidative stress, inflammation and fibrosis [59].

Apigenin
Apigenin (4 ,5,7-trihydroxyflavone) is a flavone and widespread in different plant species. A high amount of apigenin was found in the biomass methanol extract of cyanobacteria Leptolyngbya sp., which was 0.4 ± 0.02 mg/g and this methanol extract showed the highest radical scavenging activity [27].
Apigenin can kill different kinds of cancer cells. In breast cancer cell line MCF-7, apigenin can induce apoptosis through increased ROS production and DNA fragmentation. In addition, it can enhance the expression of p53, Bax/Bcl-2 ratio, caspase proteins and thus, influencing the cleavage of PARP. Apigenin can arrest cells at G2/M phase as well [63]. In cisplatin-resistant colon cancer cells HT-29, apigenin can augment the expression of Beclin-1 and LC3-II and downregulate the expression of p62 which leads to autophagic death. It can also induce apoptosis by increasing Bax expression while downregulating Bcl-2 expression in the same cell line [64].
In a study, dimethoxyflavone induced apoptosis in endometrioma cells by inhibiting PI3K/AKT and ERK1/2 signaling pathways, activating ER-stress response proteins and MAPK proteins, JNK and p38, increasing the production of ROS and calcium in high levels which led to disruption of mitochondrial membrane potential while inactivating the PI3K/MAPK pathways [65].
Cancer cells are affected by genistein via apoptosis induction, cell cycle arrest, inhibition of metastasis and angiogenesis. To induce apoptotic death, genistein hindered NF-κB pathways and upregulates pro-apoptotic proteins like, Bax, Bad and Bak, as well as cyt c release which in turn caused caspase-dependent apoptosis. Genistein also induced calpain which is a Ca 2+ dependent protein that is responsible for cleaving Bax and Bid, rendering apoptotic cell death. Genistein can impede the cell cycle by modulating Ras/MAPK/activator protein-1 and downregulating the expression of Cdk1, cyclin B1 and Cdc25C. Moreover, genistein inhibited angiogenesis through reduced expression of VEGF, MMP-2/9 and JNK, p38, PTK/MAPK pathways [37].
Hesperidin exerts its anticancer activity against different cancer cells. In MDA-MB231 breast cancer cells, it can inhibit metastasis of these cells by downregulation of the expression of programmed death-ligand 1 (PD-L1) with reduced expression of Akt and NF-κB signaling and also decrease in the expression of MMP-9 and MMP-2 [66]. A study showed that co-administration with imatinib mesylate, hesperidin potentiated the drug action on the imatinib-resistant breast cancer cells and also with less negative effects. It downregulated the expression of the multidrug-resistant (MDR-1) gene, thus, overcome the drug resistance. Hesperidin upregulated Bax/Bcl-2 and caspase-3 expression to induce apoptosis. In addition, it protected heart tissue which was revealed through the reduced serum enzymes LDH and SGOT [67].

Prospects and Limitations
Flavonoids are an excellent reservoir of biological activities that can be exploited to treat different medical conditions, for instance, diabetes, cardiovascular dysfunction, ocular diseases, aging problem and neurological complications. Some clinical trials also proved the efficiency of using flavonoids in different kinds of health-related problems. However, utilizing flavonoids in the drug discovery process have some major concerns. Flavonoids present in a very small amount in plants or algae, from µg to mg per kg of biomass. Moreover, this small amount is mostly found in complex with other bioactive compounds, which renders it difficult to determine the actual source of the pharmacological effect. Purification and identification of these flavonoids are a multistage, expensive and time-consuming process. In addition, their high lability and being prone to chemical alteration can lead to degradation during the purification process. Poor bioavailability is another problem for flavonoids as a drug. However, with novel optimization techniques, multiplex purification systems and metabolic engineering, flavonoids can be extracted in a high amount from natural sources. However, another most important problem for plant flavonoids is that a regular and high amount of extraction from plant species may cause the elimination of useful plant species or may risk food security [68]. Algae, as a potential source of flavonoids, offer a suitable solution for this problem.
Microalgae give several benefits over higher plants in the production of high-value products. The fast and foremost benefit of microalgae is their high photosynthetic efficiency than terrestrial plants, which is linked to their fast growth rate and high yield per unit dry biomass than plants. Moreover, microalgae can be grown in ranges of water media; saltwater, freshwater and even wastewater. Industrial effluent or aquaculture wastewater can be an alternative source of media for the economic production of microalgae [69]. Moreover, microalgae are now grown in large photobioreactors which limits vast land-use and contamination risk. Microalgae and seaweed farming don't have any impact on food security and the environment, as no chemical fertilizer is used in the cultivation process. Specialized photobioreactors provide optimum and evenly distributed illumination to the microalgae culture which is important for a high production rate. Though this sophisticated bioreactor is a bit expensive. Efficient and low-cost harvesting is another challenge to get high-value compounds from microalgae. In recent years, membrane bioreactors are in use for harvesting microalgae which is a cheaper but efficient harvesting alternative. The system called magnetically induced membrane vibration makes this harvesting process more easier and cost-effective [70]. Therefore, to get benefit from these excellent species, a suitable and effective separation technique along with extensive anticancer study is warranted for algal flavonoids.

Conclusions
Microalgae and seaweeds are both suitable sources of different classes of flavonoids. However, the anticancer study from such valuable sources is overlooked. Though studies predicted that the total flavonoid contents found in the microalgae species is responsible for the antitumor activity in various cell lines, further purification and identification have not been done yet. More emphasis should be put on the isolation of specific flavonoids from algal sources and their bioactive properties. Moreover, seaweed and marine microalgae may possess unique flavonoid types, which can further be revealed through marine bioprospecting. Thus, algae can become an alternative source of flavonoids with anticancer activity.