Evaluation of cytotoxicity and antiviral activity by cell viability

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Sugars

In general, total sugars were abundant components compared to proteins, phenols, and flavonoids (Hao et al. 2019). In the last decades, sulfated polysaccharides from algae have attracted much attention as functional additives in the pharmaceutical field, food, and cosmetic industries. The major sulfated polysaccharides found in marine algae are ulvan (green algae), sulfated polysaccharide rich in fucose – fucan and fucoidan- (brown algae), and carrageenan (red algae) (Cunha and Grenha 2016).

Green seaweeds

In green algae, it has polymer assemblage cell walls similar to the cellulose, pectins, hemicelluloses, and exceptionally arabinogalactan proteins (AGPs). Ulvophyceae seaweeds have abundant fibrillar constituents’ cell wall components. Most may change from cellulose to β-mannans to β-xylans during different life cycle phases; these also produce complex sulfated polysaccharides, AGPs, and extensin. Chlorophyceae green algae had a wide array of walls ranging from cellulose–pectin complexes to ones made of hydroxyproline-rich glycoproteins (Domozych et al. 2012). The schematic diagram of the green seaweed (Lahaye and Robic, 2007) is shown in Fig. 9. Cell wall polymers common to green seaweed, particularly for Caulerpa and Ulva species is shown in Table V (Holdt and Kraan, 2011).

Red seaweeds

Carrageenans is a generic name for a family of galactans (Cunha and Grenha 2016), a family of linear sulfated polysaccharides. They are found and extracted from the cell wall of certain species of red seaweeds (Al Nahdi et al. 2019). Rhodophyta is characterized by their content on non-fibrillar and sulfated polysaccharides such as carrageenan which is up to 20-38% (Lalegerie et al. 2020), agars and complex sulfated galactans, which are the main components of their cell walls (Fenoradosoa et al. 2009), shown in Fig. 12 and Table VIII. These polysaccharides, well known for their gelling, stabilizing, and thickening properties in food, pharmaceutical, and cosmetic industry (Cunha and Grenha 2016), are made up of linear chains of alternating 3-linked-galactopyranosyl and 4-linked galactopyranosyl residues. The 3-linked units always belong to series, whereas the 4-linked ones may have the D or L configurations, often occurring as a 3,6-anhydrogalactopyranosyl moiety after enzymatic or alkaline treatment. Many rhodophycean galactans have been described depending on their structural features. Generally, the large carrageenan family is obtained from different species of Rhodophyta, including Gigartina, Chondrus crispus, Eucheuma, and Hypnea (Fenoradosoa et al. 2009). The source of carrageenan was the red seaweed Chondrus crispus which contains a mixture of kappa and lambda carrageenan that cannot be separated during commercial extraction. Today, most of the carrageenan is extracted from K. alvarezii and E. denticulatum, a predominant source of kappa- and iota-carrageenan, respectively (Cunha and Grenha 2016).

Lipids content and fatty acids

Generally, seaweeds are characterized by a relatively low content of saturated fatty acids (SFA) and substantial amounts of polyunsaturated fatty acids (PUFAs), which are considered beneficial for human health as long as they are not oxidized or converted into saturated lipids (Hamed et al. 2015).
Brown algae contain relatively lower contents of lipids having less than 5% of dry weight. Quantitative analysis revealed that the total lipid content of a major brown algae family, Sargassaceae, was higher in subarctic zones (approximately 5% of dry weight) than tropical zones (0.9-1.8% of dry weight). Despite their low total lipid content, brown algae are a rich source of polyunsaturated fatty acids (PUFAs) (van Ginneken et al. 2011). In red seaweed, lipids and fatty acids are present in low amounts, generally 1–5% of the dry weight (Torres et al. 2019). The mean total lipid content of Halymeniales species can vary (0.1-3.9% dw). Kumari et al. 2013 compiled the total lipid content and fatty acid distribution of different seaweeds and suggested that the variations observed between different species of the same genus were more likely to be due to the inter-specific/intra-generic variations rather than to geographical and environmental conditions as apparent from the minor variations found with the environmental parameters for the studied collection sites.
Table X shows the total lipid content in several species of brown and red seaweeds, including Sargassum and Halymenia species. Macroalgae also contain various other lipids and lipids like compounds such as sterols, phospholipids, and glycolipids, but red seaweeds have a high ω-3 fatty acids content, being a rich source of α-linolenic acid (ALA) [18:3(n-3)], AA, eicosapentaenoic acid (EPA) [20:5(n-3)], and docosahexaenoic acid (DHA) [22:6(n-3)]), and most species showed a nutritionally beneficial ω6/ω3 ratio (Torres et al. 2019) (Table IX).

Algae as vegetable in human consumption

Of the 221-seaweed species worldwide that have commercial value, only about ten are intensely cultivated, and use also as food such as brown seaweed (Phaeophyceae—Saccharina japonica, Undaria pinnatifida, and Sargassum fusiforme); red seaweed (Rhodophyta—Porphyra spp., Eucheuma ssp., Kappaphycus alvarezii, and Gracilaria spp.); and green seaweed (Chlorophyta— Ulva clathrata, Monostroma nitidum, and Caulerpa spp.) (Pereira 2018a). Seaweeds in East Asia have been documented to have been harvested as consumptive and non-consumptive for thousands of years (6000 BCE–300 BCE) in Japan, (600 BC) in China, (AD 918–1392) in Korea (Dicks and Doll, 1983; Delaney et al. 2016), and the rest of the Pacific islands. The introduction of seaweeds in Europe as human food began in the 15th century, especially by littoral populations suffering from famine. Now, seaweeds as human food is widely distributed throughout the world, especially as a resource for the production of food, food additives, and nutritional supplements in Asia, Europe, North and South America, Africa, and Pacific Islands nations (Fleurence 2016). Although nutritional values can vary and are widely affected by physical and environmental factors, edible macroalgae are mostly rich in bioactive antioxidants, soluble dietary fibers, proteins, minerals, vitamins, phytochemicals, and polyunsaturated fatty acids (Hamed et al. 2015; de Gaillande et al. 2017). Seaweeds are also very rich in essential minerals and trace elements required for the human diet compared to terrestrial foods due to their ability to maintain inorganic atoms from seawater (Hamed et al. 2015). Macroalgae have many uses; food for human consumption, ingredients in the manufacture of cosmetics and fertilizers, treated products for extracting gelling agents, or animal feed additives (de Gaillande et al. 2017). In brown algae, Sargassum fusiforme, also known as Hijiki is served as food, named as “vegetable for longevity” in food industries of Japan. It is a side dish prepared by soaking and boiling the dried algae with water and served with soy sauce (Liu et al. 2012). S. polycystum has been eaten as sea vegetables in Asian countries such as Malaysia and Vietnam (Hong et al. 2007; Matanjun et al. 2009). Macroalgae are mainly used as food in the form of fresh vegetables, and their popularity tends to be on the increase due to the general awareness of health-conscious consumers of the advantages of its natural products. Annual production of seaweed for direct (sea vegetables) and indirect (phycocolloids) human consumption is estimated at 2 million tonnes of dry matter, and 90% of this production is from China, Korea, Japan, Vietnam, Chile, the Philippines, Norway, France, Spain, and the United Kingdom (Fleurence, 2016).
The Philippines is the third-largest producer of seaweed in the world following China and Indonesia, where Eucheuma species is the most commercially significant. In 2015, 90% of the Philippines’ farmed output comprised Eucheuma species, such as E. cottonii (Kappaphycus) and E. denticulatum. Also, about 80% of the E. cottonii (Kappaphycus) production comes from the Philippines (777 963 tons), and about 30% traded in dry matter (FAO, 2018). Several edible species of the green, brown, and red seaweeds’ nutrient contents are shown in Table X.

Active ingredients (cosmetics and health)

Viral infections, as this present pandemic that the world is facing for COVID-19, count as one of the most predominant cause of death in human life and the economy worldwide. So far this year, COVID-19 has killed more than 1.5 million people and forecasters predict that by the end of the year 2020, the pandemic death toll could rise to 1.9 million. If that happens, COVID-19 would rank as the 6th-deadliest disease in the world (WHO, 2019). Seaweeds are known rich source Algues marines d’importance économique aux Philippines : évaluation de six espèces Rexie Magdugo 2020 for active antiviral metabolites. Extracted polysaccharides exhibit antiviral activity against a broad spectrum of viruses including; Human Immunodeficiency Virus (HIV), Herpes Simplex Virus (HSV), Vesicular stomatitis virus (VSV), Cytomegalovirus (CMV) (Bouhlal et al. 2011; Wang et al. 2014; Mahomoodally et al. 2019).
Algae proteins and their derivatives have anti-inflammatory, antioxidant, antitumor, anti-aging, and protective activity. They are also applied as a moisturizer to hair and skin. Amino acids are generally used as skin moisturizers since many are constituents of the natural moisturizing factor (NMF) in human skin (Pereira, 2018b). Xanthophylls (astaxanthin, fucoxanthin, loraxanthin, lutein, violaxanthin), and carotene (α-carotene, β-carotene, lycopene) are widely used as natural dyes and vigorous antioxidants with antitumor, anti-inflammatory and radical sequestering properties (Stengel et al. 2011; Pereira 2018a). Pro-vitamin A activity can also be found in carotenoids where it can remain active when standard vitamin C cannot, especially under low oxygen condition; β-carotene, protect the skin and eyes from photo-oxidation against UV radiation and prevent eye disease such as cataracts; Astaxanthin performs a variety of activities, e.g., UV-mediated photo-oxidation, tumors, and inflammatory processes; and fucoxanthin, a typical xanthophyll of brown algae with extraordinary antioxidant activity. Its main bioactivities are antimalarial, antitumor, anti-obesity, anti-inflammatory, antidiabetic, and antiangiogenic activities. It also has protective effects on the brain, skin, liver, blood vessels, bones, and eyes (Joshi et al. 2018; Pereira 2018a).
The fatty acid alpha-linolenic acid (ALA), the precursor to EPA and DHA were reported to inhibit the production of eicosanoids, lowering blood pressure, and blood triglyceride levels (Oomah and Mazza, 1999) while docosapentaenoic acid (DPA) was shown to inhibit platelet aggregation and has strong endothelial cell migration abilities (Kaur et al. 2011). Commercial ALA or hybrid liposomes of ALA ethyl ester (HL-ALAE) seemed to provide benefits against several health problems, e.g., decrease in blood cholesterol levels, reduction in the risk of heart attacks, and a growth reduction in breast, colon, and prostate cancer (Tanaka et al. 2008). Researchers have suggested that elevated blood pressure in adulthood may Algues marines d’importance économique aux Philippines : évaluation de six espèces Rexie Magdugo 2020 be associated with peri-natal omega-3 fatty acid deficiency and that early exposure to dietary, long-chain omega-3 played a critical role in supporting heart health and reducing CVD risk in later life (Papanikolaou et al. 2014). Given the situation above, then it is safe to assume that consumption of recommended amounts of omega-3s as ALA in food, or in a supplement, would be generally beneficial to consumers.

Caulerpa and Ulva species

Antioxidant compounds from macroalgae (Caulerpa and Ulva spp.) have earned a reputation for protecting healthy cells against various diseases, aging processes, and considerable commercial potential in medicine, food production, and cosmetics industry (de Gaillande et al. 2017; Olasehinde et al. 2019). Several studies isolating C. racemosa as cited by de Gaillande et al. (2017) in her paper that Liu et al. (2013) found caulerprenylols A and B (new prenylated para-xylenes) which have antifungal and antitumoral activities, and the β-sitosterol which is a plant stanol known to reduce blood levels of cholesterol as it inhibits cholesterol absorption in the intestine was also isolated from C. racemosa (Ragasa et al. 2015). A toxic constituent called caulerpicin (from C. racemosa) has anesthetizing sensation and causes numbness of the tongue, lips and cold sensation in the fingers and the feet (Kumar and Sharma, 2020). Another compound Caulerpin has painkilling and anti-inflammatory properties (C. racemosa, C. serrulata and C. sertularioides) (de Gaillande et al. 2017), however, it also produces mild anesthetic action, difficulty in breathing, sedation and loss of balance (Kumar and Sharma, 2020). Simultaneously, Caulerpenyne is also known as a repellant against other organisms (grazers, epiphytes, competitors) and exhibiting anti-neoplastic, antibacterial, and anti-proliferative activities (de Gaillande et al. 2017). Tropical species of Caulerpa are highly variable in their production of caulerpenyne; for  example, in Guam are higher than those from the Mediterranean alga C. sertularioides. However, caulerpenyne also represents the most abundant cytotoxic sesquiterpenoid in Caulerpa taxifolia, showed ichthyotoxic activity, which blocked cleavage of developing sea urchin eggs and can induce apoptosis in mammalian (neuroblastoma) cells. Caulerpenyne also shows neurological activity in invertebrate model organisms (leeches) by modifying the electrophysiological properties of touch mechanosensory cells. These electrophysiological effects on neurons might explain human poisoning incidents after the fish Sarpa salpa, which eats C. taxifolia, was consumed as having neurological symptoms of amnesia, vertigo, and hallucinations. These findings may have implications about the neuro-ecological effects of caulerpenyne on other marine consumers. Caulerpenyne’s cytotoxic activity is at the concentration of 60-90 μM leading to the destruction of human hematopoietic progenitors (melanocytes, keratinocytes and fibroblasts) but not toxic to normal melanocytes at <10.5 μM and normal keratinocytes at 12.6 μM (Kumar and Sharma, 2020). It is important to remember the importance of monitoring Caulerpa species’ toxicity before consumption (de Gaillande et al. 2017), as they exhibited significant increase in their toxic metabolites when eaten by predators, are then transferred to the marine food chain, resulting into toxicity to its predators (Kumar and Sharma, 2020).

Table of contents :

Chapter 1 Review of Literature
1 Definition
2 Taxonomy and classification
3 Biochemical composition
3.1 Minerals
3.2 Proteins
3.3 Sugars
3.3.1 Cell wall
3.3.1.1 Green seaweeds
3.3.1.2 Brown seaweeds
3.3.1.3 Red seaweeds
4 Lipids content and fatty acids
5 Applications
5.1 Algae as vegetable in human consumption
5.2 Algae as a source of phycocolloid
5.3 Active ingredients (cosmetics and health)
5.3.1 Caulerpa and Ulva species
5.3.2 Sargassum species
5.3.3 Halymenia species
Chapter 2 Materials and Methods
2.1 Sampling areas and abiotic factors
2.1.1 Overview of the studied location
2.1.2 Samples preparation, drying, and grinding
2.2 Biochemical analyses
2.2.1 Acid and water extraction for the charactrezation of the raw material
2.2.2 Proteins and amino-acids content
2.2.2.1 Amino-acids content
2.2.3 Total sugars content analysis
2.2.4 Uronic acid content analysis
2.2.5 Polyphenol content analysis
2.2.6 Sulfates group content analysis
2.2.7 Ash content analysis
2.2.8 Lipid and fatty acid methyl ester (FAME) analysis
2.2.8.1 Nutritional quality indexes
2.3 Characterization of polysaccharides
2.3.1 Caulerpa racemosa and Ulva lactuca cell wall polysaccharides
2.3.1.1 C. racemosa hot water extraction (HWE)
2.3.1.2 U. lactuca Ulvan extraction
2.3.2 Sargassum polycystum and Sargassum ilicifolium polysaccharides
2.3.3 Halymenia durvillei and Halymenia dilatata polysaccharides
2.3.4 Monosaccharides profile by HPAEC-PAD
2.3.5 Fourier transform infrared (FT-IR) spectroscopy
2.3.6 Minerals by flame atomic absorption spectrophotometry (AAS)
2.4 Analysis of pigments
2.5 Screening of biological activities
2.5.1 Evaluation of cytotoxicity and antiviral activity by cell viability
2.5.2 Antioxidant activity
2.5 Statistical analyses
Chapter 3 Results and Discussions
Results and Discussion
Green Seaweed (Caulerpa racemosa and Ulva lactuca)
3.1 Cell wall polysaccharides purification
3.1.1 First treatment
3.1.2 Polysaccharide extraction
3.1.3 Biochemical composition analysis of extracts
3.1.4 Monosaccharide composition analysis
3.1.5 Fourier Transformed Infra-Red (FT-IR) spectroscopy
3.2 Biological activity
3.2.1 Cytotoxicity and antiviral activity evaluation
3.2.2 Antioxidant activity, DPPH radical scavenging activity
4 Brown Seaweed (Sargassum polycystum and Sargassum ilicifolium)
4.1 Biochemical composition analysis of raw material
4.2 Cell wall polysaccharides purification
4.2.1 First treatment
4.2.2 Polysaccharide extraction
4.2.3 Biochemical composition analysis of extracts
4.2.4 Monosaccharide composition analysis
4.2.5 Fourier Transformed Infra-Red (FT-IR) spectroscopy
4.3 Mineral analysis
4.4 Pigments
4.5 Biological activity
4.5.1 Cytotoxicity and antiviral activity evaluation
4.5.2 Antioxidant activity, DPPH radical scavenging activity
5 Red Seaweed (Halymenia durvillei and Halymenia dilatata)
5.1 Biochemical composition analysis of raw material
5.2 Amino acids composition of raw material
5.3 Lipid composition
5.4 Cell wall polysaccharides purification
5.4.1 First treatment
5.4.2 Polysaccharide extraction
5.4.3 Biochemical composition analysis of extracts
5.4.4 Monosaccharide composition analysis
5.4.5 Fourier Transformed Infra-Red (FT-IR) spectroscopy
5.5 Mineral analysis
5.6 Pigments
5.7 Biological activity
5.7.1 Cytotoxicity and antiviral activity evaluation
5.7.2 Antioxidant activity, DPPH radical scavenging activity
6 Conclusion
References

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