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Pharmaceutical

Applications of Eukaryotic Microalgae

Angeliki Tsianta SCHOOL OF ECONOMICS, BUSINESS ADMINISTRATION & LEGAL STUDIES A thesis submitted for the degree of Master of Science (MSc) in Bioeconomy: Biotechnology and Law

December, 2019 Thessaloniki – Greece

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Student Name: Angeliki Tsianta

SID: 4402180006.

Supervisor: Dr. Savvas Genitsaris I hereby declare that the work submitted is mine and that where I have made use of another person’s work, I have attributed the source(s) according to the Regulations set in the Student’s Handbook.

December 2019

Thessaloniki - Greece

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Abstract

This dissertation was written as a part of my MSc in Bioeconomy: Biotechnology and

Law at the International Hellenic University.

The first section of this study deals with the applications of microalgae in the

pharmaceutical sector. Eukaryotic microalgae are photoautotrophic micro-organisms

and constitute the foundation of most aquatic environments due to their rich

biodiversity and possibly high growth efficiency; nevertheless, they represent a vast and

underexplored bioresource. About the safety and the prevention of diseases,

microalgae have been reported as potential sources of new natural antimicrobials. Such

species develop various secondary bioactive metabolites with antibacterial activities

such as free saturated or unsaturated fatty acids, carbohydrates, proteins, and lipids,

and work is underway to find natural molecules to restrict the misuse of industrial

antibiotics. Therefore, microalgae consist of one major promising commercial

opportunity for these substances in the field of pharmaceuticals, antibiotics, anticancer,

antibacterial, antiviral, anti-fungal functions, and the treatment of other human

diseases.

The second part includes a SWOT analysis of a start-up pharmaceutical company using

microalgae, with emphasis on European regulations for new bioproducts, as well as

initiatives for public and private funding. The pharmaceutical industry has had an

accelerating rate of costs increase in new drug production while the number of new

drugs authorized and released in the market has been very small due to increased

public regulation. Partnership with the biotechnology industry is one of the key choices

to improve this situation. If the pharmaceutical industry succeeds in addressing the

issues responsible for high cost in the sector with a new marketing strategy, it might

prove very beneficial for both of them as a partner of the biotechnology industry.

Finally, this sustainable business model for a start-up pharmaceutical company is

recommended in accordance with European strategies for a sustainable Bioeconomy.

Keywords: Microalgae; Pharmaceutical applications; European regulations; Business

model

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Acknowledgements

This thesis could not be carried out without valuable suggestions and support in the

context of planning and research progress from my supervisor Dr. Savvas Genitsaris,

who I am extremely thankful for his contribution and mentoring throughout the Master

program and in every area of my education at the International Hellenic University.

I would finally like to express my thanks, love, and respect to my family and my

husband for their tolerance and support for my projects.

Angeliki Tsianta 31st December 2019

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Contents Abstract .................................................................................................................................................... iii Acknowledgements ............................................................................................................................. iv 1. Introduction ....................................................................................................................................... 2

1.1. General Characteristics of Microalgae ............................................................................. 2 1.2. Microalgae as Organisms with Particular Biotechnological Interest................... 3 1.3. Macromolecules of microalgae useful for pharmaceutical applications ............ 5 1.4. Aim of the study........................................................................................................................ 7

2. Methodology ...................................................................................................................................... 9 2.1. Results .......................................................................................................................................... 9

3. Pharmaceutical uses of microalgae ........................................................................................ 12 3.1. Anticancer activity ................................................................................................................ 12 3.2. Antiviral activity .................................................................................................................... 16 3.3. Antibacterial activity ........................................................................................................... 20 3.4. Antifungal activity ................................................................................................................ 23 3.5. Potential of Microalgae in Cardiovascular Disease Prevention .......................... 26 3.6. Metabolism .............................................................................................................................. 30 3.7. Respiratory .............................................................................................................................. 31 3.8. Inflammation-Oxidative Stress-Allergies .................................................................... 31 3.9. Immunology (various & infectious diseases) ............................................................. 33 3.10 Hepatology ............................................................................................................................. 35 3.11 Dermatology .......................................................................................................................... 36 3.12 Neuroscience......................................................................................................................... 39 3.13 Ophthalmology ..................................................................................................................... 40

4. European Regulations concerning Microalgae .................................................................. 41 5. Swot analysis – Case study of a fictional microalgae-based carotenoid production company ................................................................................................................................................ 43 6. Conclusions ...................................................................................................................................... 47 7. Future perspectives ...................................................................................................................... 49 Bibliography......................................................................................................................................... 50

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1. Introduction

1.1. General Characteristics of Microalgae

Certainly, “our life depends on microalgae” since they compose 50 percent of overall

oxygen production (Chapman, 2013). As autotrophs support aquatic ecosystems, and in

general all land plants and animals lean on microalgae directly or indirectly. Firstly, the

primary production of marine and freshwater bodies relies on microalgae (Borowitzka

et al, 2016). Secondly, they are situated in the base of food chains having an essential

role in the world’s aquaculture industries for their protein needs (Griffiths, 2013).

Based on the size and morphology, algae may be subdivided into 2 main categories:

macroalgae and microalgae. Macroalgae are composed of multiple cells, organized into

structures that resemble higher plant roots, stems, and leaves (e.g. kelp). Microalgae

are mostly unicellular, a diverse group of primary producers (autotrophs) organisms

capable of forming organic compounds from pure inorganic materials like atmospheric

or marine carbon dioxide (Sigamari et al, 2016). They are microscopic in comparison to

algae which are simpler autotrophic organisms with a large variety (Ranganathan and

Yaakob, 2013). Today's advanced plant life is believed to have been developed from

these basic plant-like microscopic organisms (Mutanda, 2013).

Around 41,000 species of macroalgae and microalgae were described. The

microalgae are categorized according to the quality of photosynthetic pigments, the

food reserve for carbohydrates, the cell walls, the structure and orientation of flagella

1(Levine and Fleurence, 2018). Also, the microalgae are described in the generally

accepted algae phyla (divisions), meaning:

1 A flagellum (plural: flagella) is a long, whip-like structure which allows to move certain single organisms https://simple.wikipedia.org/wiki/Flagellum.

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Figure 1. Microalgae Divisions. Modified from: Griffiths, 2013.

Prokaryotic cyanobacteria are often included in this term as well, but some scientists

exclude the prokaryotic blue-green algae (=cyanobacteria) and limit the definition of

microalgae to include solely eukaryotic cells (Stanier et al., 1971). This thesis is

restricted to eukaryotic microalgae only.

1.2. Microalgae as Organisms with Particular Biotechnological Interest

There are several reasons why interest in microalgae growth is now thriving globally.

Micro-algae are not particularly complex micro-organisms and are present in almost all

earth's habitats from the sea, freshwater, desert sands, and hot springs, to snow and

ice (Williams & Laurens, 2010). These many different environmental niches are,

planktonic, benthic (sand, water, soil), epilithic, epiphytic, (symbiosis with the fungus

and invertebrates), aquatic and in rare occasions parasitic relationships. Other classes

of microalgae may form biofilms, colonial structures, mats and grasses.

Although the total amount of microalgae listed is vast, only a few dozen are genuinely

elucidated in their nutritional, environmental, pathogenic or business potential (Levine

and Fleurence, 2018). Additionally, only a small percentage, i.e. approximately 20

microalgae, are used economically (Posten and Chen, 2016).

Furthermore, these microalgae contain proteins, pigments, essential fatty acids,

vitamins, polysaccharides and minerals (Parsaeimehr, 2017). Such high-value products

Microalgae Divisions (phyla)

Chlorophyta (green algae)

Chrysophyta

Cryptophyta

Dinophyta (dinoflagellates)

Haptophyta (coccolithophorids)

Euglenophyta (euglenoids)

Glaucophyta

Rhodophyta (red algae)

ochrophyta (diatoms of

bacillariophyta)

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may be proven particularly important and beneficial to the pharmaceutical industry by

giving new choices to treat illnesses like inflammation and immunomodulating actions

(Ahmadi et al, 2015). Eventually, the microalgae that provide us with oxygen, foods,

and car fuel, also provide us with effective medical substances against virus diseases

(Herpes simplex and AIDS), cancer and drug-resisting bacterial strains. Pharmaceutical

products of quality and their industrial marketing are still developed and can be viewed

as a key to a multimillion-dollar industry. Scientists have just begun to explore the huge

biological resource and physiological perspective of microalgal species as growing in

every environmental niche (Bhattacharjee, 2016).

The evolution of algae over 2,500 million years, its abundance and ability to

withstand extreme environments have contributed to novel strategies of protection,

interaction, and survival, leading to their human drug and nutraceutical potential.

Nevertheless, one cautionary note is that the discovery of drugs in microalgae depends

on environmental regimes that induce metabolic plasticity (Lauritano et al., 2016). The

rates of metabolites and bioactivity depend on habitat and seasonal conditions.

Production and concentration of primary and secondary metabolites are based on a

process of development (Vidoudez and Pohner, 2012), different clones (Gerecht et al.,

2011), kind of light (Depauw et al., 2012), control of temperature (Huseby et al., 2013),

media for seed (Alkhamis and Qin, 2015), weeding control procedure (Pohnert, 2002), a

technique for extraction (Juttner, 2001) and various elements (Chen et al., 2011).

The nutritional needs of some microalgal strains are recognized and microalgal

growing technology is evolving quickly. Also, the development of protocols in genetic

engineering has brought new approaches to molecular algal systems. The general study

recently carried out in genomics and molecular biology methods of microalgae have

achieved remarkable dimensions, with huge numbers of undiscovered algae strains

from extreme environments (Brodie and Lewis, 2007).

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1.3. Macromolecules of microalgae useful for pharmaceutical applications

A large quantity of various components is produced by microalgae, such as:

Lipids

Microalgae contain a large number of lipids, around 30–50% of their total weight. In

comparison to other lipid-based crops, microalgae lipids can assemble a higher

percentage which makes them more appealing to produce biodiesel and health food

supplements (Yeh and Chang, 2012).

Proteins

Proteins are part of the main components of microalgae, representing between 50

and 70% of their composition. Besides, protein is can be consumed by humans or

animals (Chew et al., 2017). Even though some microalgae comprise toxic proteins,

testing can be carried out to discover the safe proteins for use.

Carbohydrates

Microalgae usually have a high carbohydrate amount that is around 50% greater

than their dry weight because they have good effectiveness in photo-conversion and

easy storage of carbohydrates (Yen et al., 2013). Algal carbohydrates are composed

mainly of glucose, starch, cellulose, and polysaccharide of several kinds. Microalgae

polysaccharides can regulate the immune system and inflammatory reactions, and

produce cosmetic chemicals, food ingredients, and natural therapeutic agents.

Pigments

It is important to highlight three basic natural pigment classes in microalgae:

carotenoids, chlorophyll, and phycobiliproteins. These pigments were used as vitamins

in food and feed, additives, cosmetics, pharmaceutical industries, agents to color food

and biomaterials (Nobre et al., 2013; Tamiaki et al., 2014, Zhou et al, 2015). First of all,

carotenoids are fat-soluble coloring agents, which dye plants, and secondly

chlorophylls are lipid-soluble pigments existing in vegetables and fruit during

photosynthesis. Another carotenoid from microalgae is astaxanthin, an important

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antioxidant agent, as carotenoids have strong anti-aging, sun protection, and anti-

inflammatory impact in the immune system (Cheng et al., 2016).

Vitamins

Compared with common food, microalgae have high levels of vital vitamins, like

riboflavin a very important vitamin for maricultured animals (Brown and Farmer, 1994).

Also, more studies must be performed to test the safety of these vitamins after

consumption (Bonnet et al., 2010).

Polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFA) are commonly known as important food

elements that help prevent multiple cardiovascular diseases. The fast diminution of

marine resources has hampered the acquisition of these ingredients from ocean fish,

where augmented PUFAs demand has been responsible for the revealing of substitute

sources for EPA and DHA (fatty acids). Microalgae can produce PUFAs that are

important to human health and nutrition (Wang et al., 2015b).

These previous components may have different applications as table 1 demonstrates.

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Table 1. Potential uses for bioproducts obtained from microalgal biorefineries. Modified from: Chew et al., 2017.

Activity Application Nutraceutical, antimicrobial, anti-

inflammatory Nutritional supplement,

antiproliferative, capacity to resist

infections and diseases Antioxidant, natural pigment Supplement and food ingredient for

humans, feeding of fish and shellfish Biofuels Fermentation of natural gas via

biomass digestion to produce biodiesel

Fertilizers Utilization of biomass in arable land for

production of nitrogen and phosphorus High-value molecules

Chlorophyll-a, phycocyanin, b-carotene,

γ-linolenic acid, eicosapentaenoic acid,

and stable biochemical isotopes

Anticancer and antitumor Antiproliferative. Inducing G1 inhibition

in post-gastric carcinoma cells Chemical industry Evaporative organic compounds

1.4. Aim of the study

The current study΄s aim is to discuss on a global scale the latest developments in

pharmaceutical research with the use of microalgae and to highlight the recent

discoveries in the field.

The study's first section attempts more explicitly to summarize their potential

pharmaceutical innovations in international literature as well as the different

applications in the areas of human health. This section discusses the understanding of

the microalgae as the origin and ingredients in bioactive molecules, i.e. in cancer

treatment, metabolism, oxidative stress, inflammation, immunology, cardiovascular-

renal-respiratory-hepatology-dermatology-neuroscience-ophthalmology, and rare

diseases and accidents which consist of basic domains in the pharma industry (www.

novartis.com).

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The second section includes the regulations and the restrictions as far as the

pharmaceutical use of microalgae is concerned. An attempt to link the pharmaceutical

uses of microalgae with the concepts of Bioeconomy, circular economy, and

sustainability on the European Union level was made. In this chapter also, a swot

analysis of a start-up biotechnological company using microalgae is presented. Swot

analysis is essential to coordinate the different departments of the company by

defining the internal planned factors-strengths and weaknesses, and the external

planned factors-opportunities and threats. With the practical application of the above

principles, a sustainable business model is introduced which illustrates the high level of

applicability and inclusion into the EU project.

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2. Methodology

The Elsevier Scopus and ScienceDirect databases simultaneously with Google

Scholar and PubMed were thoroughly searched by applying the following keywords

[pharmaceutical uses of microalgae, anticancer-antiviral-antibacterial – antifungal –

cardiovascular – renal – metabolism – respiratory – inflammation – immunology –

hepatology – dermatology – neuroscience -ophthalmology – rare diseases – wounds

(alone or in combination with microalgae)] in the "Article title, abstract, keywords"

section in Scopus of ScienceDirect, without any constraint of the date or report form.

From 1960 to 2019 the search provided thousands of documents. The findings have

been categorized and checked, excluding those that did not match the study criteria.

2.1. Results

This research reveals the augmentation of publications about the pharmaceutical

use of microalgae during the last 3 decades, as demonstrated in Fig.2, which follows

the growth of technologically advanced equipment and the marketing of microalgae-

based products. Especially the last decade from 2010 until 2019 the rise of publications

is spectacular:

Figure 2: Graphical representation of researches on microalgae pharmaceuticals from 1990 to

2019.

0

200

400

600

800

1000

1200

Publications

Publications

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As far as pharmaceutical uses of microalgae are concerned, we ascertain that the publications about anticancer, antiviral, antibacterial, and

antifungal drugs from microalgae have risen especially since 2010. This is due to the growing interest in finding new substances that will defeat

various diseases. In graph 1, we observe that microalgae substances for metabolism show a big development. Moreover, pharmaceutical use of

microalgae in cardiovascular, dermatology, inflammation, and neuroscience present also, a significant rise during the last decade. On the other

hand, renal and respiratory drugs for microalgae have not been studied satisfactorily yet, as well as hepatology drugs. Finally, microalgae

research for accident wounds augments especially the last years, while publications for ophthalmology and rare diseases are also limited.

Graph 1: Publications on microalgae per human diseases from 1990 to 2019.

0

200

400

600

800

1000

1200

1400

1600

1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019

Publications per are of interest

Anticancer Antiviral Antibacterial Antifungal Cardiovascular renal metabolism respiratory

inflammation immunology hepatology dermatology neuroscience opthalmology rare diseases wounds

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On figures below, we see the top 15 countries in microalgae biotechnology and

research such as United States, Russia, Iran, China, Japan, Germany, India, France,

Canada, Australia, Brazil, Spain, Algeria, United Kingdom, South Korea. The United

States, Russia, and China are the three leading counties in publications.

Figure 3: World map with main countries and their number of scientific publications on

microalgae. The red-orange color indicates a greater number of publications, the blue-green color

indicates the smaller, and white when it does not exist. Modified by Garrido-Gardenas et al.,

2018.

Figure 4: Graph on publications concerning the use of microalgae in pharmaceuticals per country.

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3. Pharmaceutical uses of microalgae

Physicochemical and pharmacological studies have given particular attention to

extracting and generating new bioactive metabolites for therapeutic purposes from

marine micro-algae. On the other hand, microalgae are a healthy source of human

dietary due to macro and micronutrients. These bioactive molecules may have the

ability to improve human health (Nazih and Bard, 2018). Hence, microalgae can be

utilized as a functional food (supplements) as well as pharmaceutical.

In this coursework, eukaryotic microalgae products are discussed in terms of

pharmaceutical use together with their use as functional food (nutraceuticals).

3.1. Anticancer activity

In 2015, cancer, worldwide the second most important cause of death, killed 8.8

million people, mainly: Cancer of the liver (788,000 deaths); cancer of the colon

(774,000 deaths); cancer of the stomach (754,000 deaths); cancer of the breast

(571,000 deaths) (OMS data). It is predicted that the number of new cases will increase

by 70% over the next two decades. Also, the increasing economic effect of cancer is

significant: in 2010, the total costs per year of the disease were projected to be around

US$ 1160 billion (US$) (Dewi et al., 2018).

Cancer is the failure or dysfunction of precisely regulated processes of a cell, such as

a cell division, proliferation, and programmed cell deaths (apoptosis), as well as the

mechanisms of controlled feedback (Klaunig and Kamendulis 2004). This contributes to

the rapid growth and metastases of the cancer cell (Abel et al. 2014).

Chemotherapy, one of the key therapies currently available, has little efficacy by some

life-threatening negative results and the formation of therapeutic resistant cell lines. The

use of natural products in the therapy of various diseases consists of a significant field of

research for new drugs. Microalgae can generate biologically active molecules for the

pharmaceutical industry because they can be conveniently grown, short-produced, and

environmentally friendly. The majority of published research on anticancer properties

from microalgal strains studied pure ingredients rather than extracts to diminish the

danger of patient side effects (Dewi et al., 2018). Bioactive microalgae molecules with

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anticancer applications are carotenoids, glycolipids, polysaccharides, and proteins (Talero

et al., 2015).

Carotenoids and Cancer

Five carotenoids with important anti-cancer activities are reported: β-carotene,

lutein, astaxanthin, violaxanthin, and fucoxanthin. Since the majority of organisms

cannot generate these compounds, they depend on nutrition as a source.

β-carotene

Microalgae are a rich carotenoid origin (Varela et al., 2015), with Dunaliella for β-

carotene and Haematococcus for astaxanthin being the major generators. Palozza et al.

(2005) therefore, found out that the development of human colon cancer cell lines was

significantly limited by β-carotene pigments.

Astaxanthin

Haematococcus pluvialis, Chlorella zofingiensis, and Chlorococcum sp. are the major

microalgal sources of astaxanthin (Liu et al., 2014). Palozza et al. (2009) have

investigated the impact of astaxanthin on the development of cell lines and have

demonstrated the ability of this carotenoid to impede the proliferation of human CRC

(colorectal cancer) cell lines. Nonetheless, no astaxanthin clinical trials have been

published yet. The strong association between astaxanthin concentrations and

antiproliferative impact on the lines of hepatoma, breast, and pulmonary cancer has

been stated by Song et al. (2011).

Zhang et al. (2011) found that astaxanthin was the most powerful of the four types of

carotenoids to impede cell proliferation. Some studies have documented the impact of

astaxanthin on apoptosis (programmed cell death). Astaxanthin from microalgae

should, therefore, be a perfect candidate, not only for the control of cancer but also as

a chemotherapy tool. However, to prove this hypothesis, clinical research is required

(Nazih and Bard, 2018).

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Lutein

An inverse connection between nutritional carotenoid, particularly lutein intake, and

various types of cancers (colon, lung, breast ) is generally proven (Dorgan et al., 1998).

Lutein is primarily developed by Dunaliella salina (Fu et al., 2013), Chlorella sorokinian

(Pasquet et al., 2011) and Chlorella prothelkoides (Shi and Chen, 2002), and has anti-

proliferative impact on human colon cell line HCT-116 (Cha et al., 2008). Also, low

levels of dietary lutein (0.002% and 0.02%) have been shown to prevent the occurrence

and development of mammary tumors (Park et al., 1998).

Violaxanthin

Violaxanthin isolated from D. salina (Pasquet et al., 2011) and Chlorella ellipsoidea

(Talero et al., 2015) has antigrowth and proapoptotic effects against human colon

cancer cell line HCT-116.

Fucoxanthin

Fucoxanthin is the most promising carotenoid against cancer expansion. Their anti-

proliferative results on SK-hepatoma, BNL CL.2(embryonic liver) cells, cancer cells

(Caco-2, HT29, and DLD-1), the cells of prostate cancer PC-3, and HL-60 leukemia, were

described by Kumar and al. (2013). Fucoxanthin, therefore, is demonstrating great

potential in cancer as a chemotherapeutic product.

Lipids and Cancer

Human nutrition includes low amounts of a fatty acid group, ω-3 polyunsaturated

fatty acids (ω-3 PUFAs), and high quantities of ω-6 PUFAs, which lead to cancer

incidence. PUFAs and glycolipids are known to be effective anticancer factors by

activating apoptosis and also by increasing the responsiveness of tumors to anticancer

drugs (Nazih and Bard, 2018). Fish oils are the main sources of ω-3 PUFAs. Over the last

years, microalgae have been turned into a good option for ω-3 PUFAs (DHA2, EPA3).

The fact that the high consumption of ~ 3 PUFAs, mainly DHA and EPA, minimizes the

2 Docosahexaenoic acid 3 Eicosapentaenoic acid

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risk of breast, colon, prostate, pancreatic and endometrial cancer has been indicated

by epidemiological studies (Arem et al., 2013).

A lot of in vitro studies have presented that DHA shows anticancer activity by

provoking oxidative stress and oxidative DNA damages to cancer cells. Indeed, both

EPA and DHA cause ROS (reactive oxygen species) concentration and caspase-84-

dependent apoptosis in breast cancer, pancreatic cancer (Fukui et al., 2013; Kang et al.,

2010), and prostate cancer cells. Clinical trials have also revealed that the efficiency of

chemotherapy increases with ω 3-PUFAs, particularly DHA (Bougnoux et al., 2009).

Glycolipids, these variant lipids, are located at high amounts in thylakoid

membranes of microalgae chloroplasts. Monogalactosyldiacylglycerol (MGDG) shrunk

the HT-29 human colon adenocarcinoma tumors (Mizushina et al., 2012) and also

digalactosyldiacylglycerol (DGDG) created apoptosis in Caco-2 colon cells (Hossain et

al., 2005).

Polysaccharides and Cancer

Diatoms, chlorophytes, prasinophytes, haptophytes, rhodophytes, and dinoflagellates

(Raposo et al., 2015) produce a large number of polysaccharides. One important

anticancer substance is GA3P (d-galactan sulfate, associated with l-(+)- lactic acid), an

extracellular polysaccharide, refined from Gymnodinium sp., which hampered the

proliferation of different cancer cell lines, especially some colon cancer cell lines

(Umemura et al., 2003). It has been documented that the same polysaccharide induces

apoptosis in lines of human leukemia (Sogawa et al., 1998). Homopolysaccharide from G.

impudicum in vitro and in vivo, suppresses the development of tumor cells, activating the

immune system.

Proteins, Peptides, and Cancer

As far as proteinic substances are concerned, a large number of microalgae can

produce therapeutic proteins and peptides (Talero et al., 2015). It has been reported

that phycobiliproteins from red-algae Porphyridium sp. demonstrate anti-cancer,

4 Caspase-8 is a cysteine proteases (proteins), which are involving in apoptosis and cytokine processing.

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antioxidant and anti-inflammatory properties (Romay et al., 2003). Therefore, multiple

microalgae have shown signs of anticancer peptides (Kang and Kim, 2013).

Among them, we can identify a polypeptide from Chlorella pyrenoidosa, Chlorella

vulgaris (Wang and Zhang, 2013). Such compounds have expressed cytotoxic properties

along with antimitotic or proapoptosis function and, in some cases, inhibitory impact

on proteasome5. It can thus be proposed that proteins and peptides produced by

microalgae can aid in preventing and/or treating human cancer. In vivo studies would,

though, be necessary to bring valid conclusions, particularly on humans (Nazih and

Bard, 2018).

3.2. Antiviral activity

Unfortunately, there is no specific vaccine for many viral infections, some

respiratory tract viruses, Herpes Simplex Viruses (HSV-1 and HSV-2), or dengue viruses.

Also, drug resistance by multiple viruses such as HIV (Human Immunodeficiency Virus)

type 1 or HSV-1 to usable antiviral agents has always been a significant barrier to the

treatment of viral infections, prompting the search for new effective molecules

(Ahmadi et al., 2015).

Therefore, methanolic or ethanolic extracts have been used to identify antiviral

agents in microalgae for many years, with success rates in study and implementations

(Lau et al., 1993; Ohta et al., 1998; Fabregas et al., 1999; Bergé et al., 1999; Abdo et al.,

2012; Sanmukh et al., 2014; Ahmadi et al., 2015). These various solvents isolate

antiviral components with environmentally sustainable approaches. For example,

Santoyo et al. (2010) examined the antiherpetic properties of Pressurized Liquid

Extraction (PLE) extracts (hexane, ethanol, and water) from Haematococcus pluvialis

and Dunaliella salina. Pretreatment of Vero cells with 75 μg/mL of H. pluvialis ethanol

extract stopped virus infection by about 85%, whilst the same water and hexane

extract levels diminished the virus infection by 75% and 50%, accordingly. The antiviral

function of PLE water extracts was shown to interact with polysaccharide

concentration; nevertheless, other components, particularly fatty acids, could be

essential for this antiviral activity (Santoyo et al., 2012).

5 Proteasome is an extensive protein complex that causes intracellular protein degradation

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Purified Molecules

Comparing certain types of molecules, lipids, proteins, or pigments, a broad

spectrum of literature concerns antiviral algal polysaccharides. Ginsberg et al. (1947)

and Gerber et al. (1958) first showed potential antiviral activity for algal

polysaccharides, finding that polysaccharides isolated from macroalgae protected

embryo eggs from influenza B and mump viruses. In general, polysaccharides are

widely cited as having several benefits, such as reduced cost of production, a broad

range of antiviral effects, low cytotoxicity, safety, and strong approval. But such

complex molecules, together with the enzyme's failure to absorb them, aid in the

body's cytotoxic results, which limits their clinical development. Recently, interest in

microorganisms as sources of high molecular weight polysaccharides has grown, as

these biopolymers often have advantages over the commonly produced

polysaccharides (Dewi et al., 2018).

Polysaccharides

As regards red macroalgae, extracellular polysaccharides were the focus of active

research (Geresh et al., 1990, 2002a,b; Huleihel et al., 2001, 2002; Lupescu et al., 1991;

Raposo et al., 2013, 2014a; Shrestha et al., 2004; You and Barnett, 2004). This table

shows various purified polysaccharides of microalgae showing antiviral activities.

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Table 2: Several purified polysaccharides of microalgae with antiviral activities:

Modified by Dewi et al., 2018.

Compounds Microorganisms Viruses References Rhodophyta Highly sulfated polysaccharide

Porphyridium cruentum

HSV-16, HSV-2, Vaccina

Huang et al., (2005) Raposo et al., 2014a,b

Sulfated exopolysaccharide

P. purpureum Porphyridium sp.

Vaccina HSV-1, HSV-2

Radonic et al., (2010)

Sulfated polysaccharide

Rhodella reticulata

Varicella zoster virus7

Huleihel et al., (2001)

exopolysaccharides Murine sarcoma and leukemia viruses

Radonic et al., (2010) Talyshinsky et al., (2002)

Bacillariophyta Sulfated polysaccharide

Navicula directa HSV-1, HSV-2, Influenza-A; HIV-1

Lee et al., (2006) Ahmadi et al., (2015)

Naviculan Dinoflagellates Extracellular polysaccharide

Gyrodinium impudicum

EMCV 8 , influenza A virus

Yim et al., (2004) Kim et al., (2012)

p-KG03 Extracellular sulfated polysaccharide A1 and A2

Cochlodinium polykrikoides

Influenza A and B viruses, RSV-A9, RSV-B, parainfluenza-2

Hasui et al.,(1995)

Compounds Microorganisms Viruses References Chlorophyta Sulfated polysaccharide

Chlorella vulgaris Dunaliella salina C.autotrophica (chlorella), Ellipsoidon sp. Haematococcus pluvialis

HSV-1 VHSV10, ASFV11 HSV-1

Santoyo et al. (2012) Fabregas et al. (1999) Santoyo et al. (2012)

Sulfated exopolysaccharides from marine microalgae are believed to interact with

the first phase of the development of some enveloped viruses (Batinic and Robey,

1992), therefore blocking the virus from entering the host cells and providing

competitive advantages due to their wide antiviral range, such as against HSV and HIV-

1 (Amaro et al., 2011; Damonte et al., 2004). The sulfated polysaccharide by a cell wall

of the red microalga Porphyridium sp. demonstrated remarkable antiviral capacity

6 Herpes simplex virus 1 and 2 7 is one of eight herpesviruses 8 encephalomyocarditis virus 9 Respiratory Syncytial Virus Subtypes A and B 10 Viral hemorrhagic septicemia 11 African swine fever virus

19

versus HSV type 1 and 2, both in vitro by culturing cells and in vivo by experimenting in

rats and rabbits (Huleihel et al., 2002).

Certain microalgae from different taxonomic categories generate sulfated

polysaccharides with antiviral activity. For example, the diatom Navicula directa creates

an extracellular sulfated polysaccharide, naviculan, consisting of galactose, xylose,

rhamnose, fucose, mannose, and sulfate. Naviculan has a strong antiviral activity

against HSV-1 and HSV-2 and influenza viruses by impeding the early stages of

viral proliferation and may block viral incorporation in host cells (Lee et al., 2006).

The Cochlodinium polykrikoides dinoflagellate provides extracellular sulfated

polysaccharides consisting of glucose, galactose, mannose, uronic acid, and sulfate.

The development of HIV-1, flu A and B viruses, and respiratory syncytial viruses A and

B have been impaired by these polysaccharides (Hasui et al., 1995).

Proteins, Lipids, Pigments

The application of microalgal extracts in antiviral therapy appears to be a

particularly attractive choice due to the large chemical diversity of their bioactive

compounds which are less likely to product resistant mutants than other compounds

during the viral life cycle (Fábregas et al., 1999; Falaise et al., 2016). Firstly, various

microalgae organisms generate antiviral proteins, such as lectins which are

carbohydrate-binding proteins or glycoproteins. During the last decade, the

researchers found that numerous lectins creating anti-HIV activity by attaching directly

and closely to carbohydrate parts at the glycosylated envelope of HIV. Secondly, many

algal lipids have the antiviral activity to a smaller extent relative to polysaccharides and

proteins (Dewi et al., 2018).

The most common are sulfolipids and glycolipids, such as

sulfoquinovosyldiacylglycerides (SQDG), monogalactosyldiacylglycerides (MGDG).

Finally, microalgae formed specific pigments demonstrating similar biological anti-viral

activities. Chlorophyll substances from Dunaliella primolecta have shown anti-HSV

(Herpes Simplex) function (Ohta et al., 1998). Moreover, a water-soluble component

containing marennine, the blue pigment formed by the Haslea ostrearia marine

diatom, was able to block the replication of HSV-1 in vitro (Bergé et al., 1999).

20

3.3. Antibacterial activity

After Pratt et al. pioneering work in the 1940s, which revealed chlorellin's

antibacterial activity, the quest for antibacterial components from microalgae (Falaise

et al., 2016) has created growing interest. This antibiotic, a synthesis of the green fatty

acids Chlorella sp., was demonstrated to be efficient for various Gram positive (G+) and

Gram negative (G−) bacteria (Pratt et al., 1944).

Fatty acids (Desbois et al., 2009; Khoeyi et al., 2012; Sanabria-Ríos et al., 2014),

polyphenols (López et al., 2015), biphenyls (Volk and Furkert, 2006), polysaccharides

(Geresh and Malis, 1991; Raposo et al., 2015), and nanoparticles12 (Garcıa et al., 2014;

Merin et al., 2010; Patel et al., 2015) consist of various antibacterial components from

microalgae. Those substances have different mechanisms to prevent the growth of

bacteria. Fatty acids can affect the permeability of the bacterial cell wall by penetrating

its long carbon chains (Galbraith and Milter, 1973), they can also impede the

development of some bacterial enzymes (Kurihara et al., 1999) and restrict nutrient

uptake from the atmosphere, blocking bacterial growing (Desbois and Smith, 2010).

Polyphenol and biphenyl compounds from microalgae induce bacterial cell lysis by

interfering with membrane permeability and solidity of the phospholipid layer (Daglia,

2012; Jain et al., 2013). Moreover, multiple antibacterial modes of action remain to be

examined, such as nanoparticles limiting bacterial growth (Shankar et al., 2016).

Therefore, screening studies discovered that microalgae are a source of various

antimicrobial agents, due to their species chemodiversity (e.g., Mudimu et al., 2014).

Lastly, a compound's antibacterial efficiency can vary depending on the bacteria's Gram

type. The bacteria (G−) tended to be more tolerant than the bacteria (G+) to several

antibiotic agents. An exception is the appearance of lipopolysaccharides of microalgae

in (G−) bacteria which tends to inhibit the penetration of extracellular compounds into

the bacterial cells (Takeuchi et al., 1999).

Green Microalgae

Recently, microalgae antibacterial compounds have been examined, attesting the

range of compounds being analyzed with biological activities and the abundance of

12 nano-sized materials

21

microalgae organisms (Amaro et al., 2011; Falaise et al., 2016; Pradhan et al., 2014).

Table 3 presents several known compounds with antibacterial efficacy extracted from

microalgae.

Table 3: Antibacterial compound from eukaryotic microalgae: Modified by Dewi et al., 2018

Microalgae Antibacterial Compound Examples of Target Bacteria

References

Green Microalgae Chlorella sp. Mixture of fatty acids Bacillus subtilis,

Staphylococcus aureus, Streptococcus pyogenes, Escherichia coli, Pseudomonas aeruginosa

Pratt et al. (1944)

C. vulgaris Phenolic compounds E. coli, Klebsiella sp., Bacillus sp., Pseudomonas sp.

Syed et al. (2015)

C. ellipsoidea Saturated and unsaturated fatty acids

Propionibacterium acnes Sibi (2015)

C. protothecoides C. pyrenoidosa Chlorococcum HS-101

Alpha-linolenic polyunsaturated fatty acid

B. subtilis, B. cereus, S. aureus, MRSA13, Enterobacter aerogenes

Ohta et al. (1995)

C. humicola Pigments (carotenoid, chlorophyll)

B. subtilis, S. aureus, E. coli, P. aeruginosa, Salmonella typhimurium, Klebsiella pneumoniae, Vibrio cholerae

Bhagavathy et al. (2011)

Dunaliella primolecta

Alpha-linolenic polyunsaturated fatty acid

B. cereus, B. subtilis, S. aureus, MRSA, E. aerogenes

Ohta et al. (1995)

D. salina Indolic derivative, polyunsaturated fatty acids, beta-ionone and neophytadiene

S. aureus, E. coli, P. aeruginosa

Herrero et al. (2006)

Mendiola et al. (2008) Pane et al. (2015) Microalgae Antibacterial Compound Examples of

Target Bacteria References

Haematococcus pluvialis

Short-chain fatty acid S. aureus, E. coli Santoyo et al. (2009)

Scenedesmus obliquus

Long-chain fatty acid S. aureus, E. coli, P. aeruginosa, Salmonella sp.

Guedes et al. (2011)

Tetraselmis suecica Fatty acid Proteus sp., S. pyogene Bai and Krishnakumar (2013)

Red microalgae Porphyridium aerugineum, P. cruentum

Phycobiliprotein S. aureus, S. pyogenes Najdenski et al. (2013)

Salmonella typhimurium P. cruentum Extracellular sulfated

polysaccharides S. enteritidis Raposo et al. (2014b)

Rhodella reticulata S. aureus, S. pyogenes, B. cereus, S. typhimurium

Najdenski et al. (2013)

Diatoms Chaetoceros muelleri

Unsaturated fatty acid B. subtilis, S. aureus, E. coli

Mendiola et al. (2008)

Phaeodactylum tricornutum

Fatty acid B. cereus, S. aureus, S. epidermidis, MRSA

Desbois et al. (2009)

Haslea karadagensis

Marennine-like pigment Polaribacter irgensii, Pseudoalteromonas elyakowii, V. aestuarianus

Gastineau et al. (2012b)

Haslea ostrearia Marennine P. irgensii, P. elyakowii, V. coralliilyticus, V. tubiashii, V. aestuarianus

Gastineau et al. (2012a)

Gastineau et al. (2014)

Falaise et al. (2016)

13 Methicillin-resistant Staphylococcus aureus

22

The green microalgae of the Chlorella group contain many antimicrobial compounds,

such as fatty acids, pigments, and phenolic compounds (Jorgensen, 1962). Bacteria like

Escherichia coli, Klebsiella sp., Bacillus sp., and Pseudomonas sp. can be inhibited by

phenolic ingredients such as flavonoids and tannins from methanolic extracts of

Chlorella vulgaris (Syed et al., 2015). Lipid derivatives of Chlorella ellipsoidea, Chlorella

protothecoides, and Chlorella pyrenoidosa show a lipase activity reduction of

Propionibacterium acnes (Sibi, 2015), a (G+) anaerobic bacteria. Furthermore, Ferreira

et al. (2016) analyzed a silver nanoparticle, biosynthesized from C. vulgaris, as an

antibacterial component against Staphylococcus aureus and Klebsiella pneumoniae.

Results indicate that silver nanoparticles blocked both bacteria's activity by 98 percent,

at levels as low as 10 μg /mL. Dunaliella primolecta methanolic samples indicated

significant activity against methicillin-resistant S. aureus (MRSA) (Ohta et al., 1995).

Additionally, methanolic compounds from Dunaliella tertiolecta showed in vitro

antibacterial function towards S. Aureus and P. aeruginosa, bacteria that can create

external otitis (Pane et al., 2015). D. Salina contains various exopolysaccharides, which

are recognized as strong antibacterial agents against E. Coli and S. aureus (Mendiola et

al., 2008). The green microalga Tetraselmis suecica was also confirmed to inhibit the

growth of bacteria. In one study a combination of chloroform and ethanol compounds

is powerful versus Proteus sp. and S. pyogenes bacteria. The fatty acids were identified

as the bioactive compounds in T. suecica (Bai and Krishnakumar, 2013).

Red microalgae

Porphyridium aerugineum phycobiliproteins contain phycocyanin that is

documented to impede the production of the (G+) S. aureus14. In Porphyridium

cruentum, phycoerythrin was described as an antibacterial ingredient against S. aureus

(Najdenski et al., 2013), and 1% of extracellular sulfated polysaccharides (EPS)

demonstrated activity against E. coli and S. aureus (Raposo et al., 2014b). Liberman et

al. (2016) assessed the antibacterial behavior of the Porphyridium sp. polysaccharide

combination with ion Zn2+. In addition to the Porphyridium species, in vitro

experiments with the red microalga Rhodella reticulata using exopolysaccharides

14 Staphylococcus aureus is a Gram-positive, round-shaped bacterium

23

observed antibacterial activity against S. Auroreus, S. Pyogenes, Cereus Bacillus, and

Typhimurium Salmonella (Najdenski et al., 2013).

Diatoms

It is also recognized that several diatom microorganisms have an antibacterial

function. Seraspe et al. (2012) analyzed N-hexane, methanol, dichloromethane, and

ethyl acetate extracts of Chaetoceros calcitrans and this in vitro assessment proved a

growth inhibition of S. aureus, B. subtilis and E. coli. Purified by the diatom

Phaeodactylum tricornutum, the fatty acid eicosapentaenoic acid (EPA) demonstrated

important in vitro activity against S. Aureus (Desbois et al.,2009).

Methanol extracts of Skeletonema costatum showed within vitro assessment

effectiveness against the pathogenic bacteria Proteus mirabilis, Proteus vulgaris,

Proteus aeruginosa, Salmonella paratyphi B, S. aureus, and Vibrio cholerae. (Daniel,

2016). Unpurified compounds of the diatom S. marinoi, under phosphate culture and

nitrogen deprivation, impede S. aureus growth (Lauritano et al., 2016). A concentration

of 100 μg/mL, crude extracts from Amphora cf capitalata, and Nitzschia comunis

stopped S. aureus production by 83% and 100%, respectively (Montalvão et al., 2016).

3.4. Antifungal activity

Many fungal species have reported an invasive infection that can often damage

serious organs such as the skin and the nails, lungs, brain, and all the parts of the oral

and genitals (Husain et al., 2001; Tyagi, 2016). The genera Candida, Cryptococcus,

Aspergillus, and Pneumocystis of fungi are blamed for more than 90 percent of all

fungal-related deaths in persons with reduced immunity (Brown et al., 2012). For

example, on the one hand, dermatophytes are responsible for superficial skin and nails

infections, on the other hand, Candida species causes candidiasis, a very frequent

fungal infection (Tong and Tang, 2017). Candidiasis generally affects the digestive and

respiratory system, bloodstream, and vagina. Cryptococcus neoformans, also causes

another life-threatening infection, cryptococcis. This uncapped yeast can influence the

central nervous system (CNS) and causes brain and meninges inflammation (Chrétien et

al., 2002), but also inflammation of lungs (Fang et al. 2015). Aspergillosis is also a very

24

dangerous fungal infection, mainly due to Aspergillus fumigatus and A. Flavus which

creates pneumonia and weakens the immune system (Latge, 1999). Moreover, these

aggressive fungi, creates nosocomial infections which lead to an increased death rate in

intensive care units (Majumdar and Padiglione, 2012). Pneumocystis infections consist

of another usual respiratory disease, life-threatening when HIV or prolonged

immunosuppressive therapy damages immunity (Brown et al., 2012).

Generally, fungal infection therapy has become increasingly difficult because of

strain resistance (Ghannoum and Rice, 1999). Currently, a preferred approach to deal

with invasive fungal infection is a combination of antifungal medications (Carrillo-

Munoz et al., 2014). Nevertheless, due to the fast and significant spread of resistant

fungi, other therapeutic approaches may need to be developed that require new

antifungal drugs. Specific research on the availability of new sources of antifungal

agents has been performed. Microalgae because of their richness in bioactive

compounds and their versatility, become one of the most valuable candidates of new

antifungal medicines (Skulberg, 2000).

Green and Red Microalgae

Many green microalgae contain organically active compounds, for example, the

Chlorella and Scenedesmus genera, which may be antimicrobial agents. For instance,

the culture liquids from C. vulgaris and C. ellipsoidea had antifungal activity against

Candida kefyr, Aspergillus fumigatus, and Aspergillus niger (Ghasemi et al., 2007).

Often these bioactive substances from microalgae are diffused in organic solvents.

Thus, extracts with various organic solvents (acetone, benzene, chloroform,

diethylether, ethylacetate, ethanol, hexane, and methanol) of the freshwater

Chlorococcum humicola demonstrated antifungal function versus Candida albicans, A.

niger, and A. flavus, and the most powerful from benzene extracts (Bhagavathy et al.,

2011).

Abedin and Taha (2008) also measured the antifungal activity of solvents like

acetone, ethanol, diethylether, and methanol extracts of Chlorella pyrenoidosa and

Scenedesmus quadricauda against A. niger, A. flavus, Penicillium herquei, Fusarium

moniliforme, Alternaria brassicae, Helminthosporium sp., Saccharomyces cerevisiae, C.

25

albicans. Algal extracts showed different inhibitory effects of the fungi tested, with

ethanol extracts to be the most effective.

Because of the low number of available red microalgae, only a few experiments

have taken place, compared to other classes. This explains why Mudimu et al. (2014)

noticed an inhibition activity against C. albicans only in 2 out of 88 microalgal strains,

Heterochlorella luteoviridis and the rhodophyte Porphyridium purpureum.

For example, phycobiliproteins removed from Porphyridium aerugineum and P.

cruentum presented antifungal action against C. albicans, as well as the

exopolysaccharides of Rhodella reticulata (Najdenski et al., 2013).

Diatoms and Dinoflagellates

A large number of diatoms antimicrobials are also a powerful antifungal.

Polysaccharides from Asterionella glaciallis, Chaetoceros lauderi, and Chaetoceros

diadema have strong antifungal function against Candida pseudotropicalis,

Trichophyton rubrum, Fusarium fuhum, Fusarium oxysporum, and Colletotrichum

acutatum (Viso et al., 1987). Besides, since the antimicrobial impact depends on the

emitter as well as on the target organism, Chaetoceros Lauderi show the highest

efficiency towards Fusarium oxysporum.

The efficacy of the antifungal agent also depends on the extraction solvent. Walter

and Mahesh (2000) tested 11 marine diatoms for their antimicrobial function towards 8

pathogenic fungi. Eight of the 11 diatom species examined showed

important antifungal behavior, which differed in intensity accordingly to the solvent

and the target species. The strongest antifungal activity was of Thalassiothrix

frauenfeldii and Navicula sigma, and Aspergillus niger and Cryptococcus neoformans

were the most vulnerable fungi. Acetone extracts seemed to have the lowest antifungal

efficacy, the lowest chloroform-methanol, and methanol-water proportions.

Finally, several dinoflagellates can produce toxins, but some of these toxins are

considered antimicrobial (Camacho et al., 2007; Gallardo-Rodríguez et al., 2012).

Consequently, the dinoflagellate Gambierdiscus toxicus produces polyethers,

gambierdic acids with antifungal effectiveness (Nagai et al., 1993). Gambierdic acid A

and B impeded fungal growth of A. niger and A. fumigatus, as the most sensitive

26

species. Antifungal agents are extracted from Amphidinium sp. dinoflagellates such as

polyol compounds (karatungiols A and B) which block A. niger (Washida et al., 2006).

3.5. Potential of Microalgae in Cardiovascular Disease Prevention

The primary cause of mortality and premature death is cardiovascular disease (CVD).

It covers coronary heart problems, stroke, peripheral artery disease, and heart failure.

Hypertension, hyperlipidemia, and diabetes are the major risk conditions of

atherosclerosis. Different results show that in addition to their specific fatty acid

composition, microalgae may also minimize some of these risk factors and provide a

dietary preventive strategy intrinsically (He et al., 2004). The plasma cholesterol level in

rats has been decreased with the use of red microalga Porphyridium sp. (Dvir et al.,

2000). This effect is mainly due to the microalgae's polysaccharide component, but

other components such as phycocyanin can influence lipid metabolism (Nagaoka et al.,

2005). In addition to these animal studies, microalgae's hypolipidemic activity was used

in human clinical studies (Nazih and Bard, 2018).

The Omega-3 Index was introduced in 2004 as a modern measure of risk concerning

cardiovascular mortality. Though initially synthesized by microorganisms in the seas,

the long-chain n–3 (omega-3) fatty acids eicosapentaenoic acid (EPA) and

docosahexaenoic acid (DHA) are mainly produced from fish consumption (Harris, 2014).

Fish and fish oil are indeed the main sources of polyunsaturated fatty acids (LC-PUFA)

in the omega-3 long-chain, however, contaminants or toxins have been increased in

fish. Also, using fish oil is relatively poor in part owing to odor, taste, and oxidation

issues (Mimouni et al, 2015). Because of these drawbacks, studies have been published

for an application of microalgae (marine or freshwater) in human aliment, as a new

source of fatty acids, according to their versatility of lipids and fatty acids. Even if still in

its development, a near-infinite supply of these essential nutrients will be generated by

commercial production of EPA and DHA from nonfish origins (Adarm-Vega et al, 2012).

Freshwater microalga Chlorella vulgaris produces oleic, palmitic, and linolenic acids

(Mendes et al., 1995). Dunaliella salina generates fatty acids in a percentage of about

80% of the overall fatty acids (Herrero et al., 2006). Porphyridium species can also

extract gamma-linolenic and arachidonic acids. Other species of microalgae synthesized

27

omega-3 fatty acids, such as EPA in genus Nannochloropsis, Phaeodactylum, Nitzschia,

Odontella, Isochrysis, and Pavlova species, although DHA can be found in the

Crypthecodinium, Pavlova and Schizochytrium ones. Additionally, few species of these

microalgae are approved for commercial use as nutritional supplements, Chlorella,

Crypthecodinium, Dunaliella, and Odontella (Rodriguez-Meizoso et al., 2010).

In the latest decades from the advent of healthy supplements with probiotics, the

health benefits of microalgae have been gradually recognized and accepted, especially

in cardiovascular diseases. Another freshwater microalga, Chlorella sp., has had many

health benefits, such as a reduction of glycemia and cholesterolemia. Such organisms

could also be used to improve the development of cytokines to enhance the response

of the immune system (Barrow and Shahidi, 2008).

Although few strains of microalgae have been approved for human consumption,

animal models have performed several trials with several microalgae. Also,

Porphyridium cruentum lyophylisate was utilized for the Syrian golden hamster as a

food additive. This test showed the reduction in cholestine distribution (dose-

dependent) and body fat (percentage-expressed) in hypercholesterolaemic animals

with the use of this microalgae biomass (Harding et al., 2009).

Retinoids, the metabolic components of carotenoids, are significant in the growth of

the cardiovascular system during embryogenesis. This function is regulated by

triggering the RAR (retinoic acid receptor) in the signal transduction pathways

controlling the growth of embryos, tissue homeostasis, and cell division and

multiplication. Nonetheless, earlier studies have shown that retinoids play a significant

role in cardiac repair after myocardial infarction in hypertensive rats (Paiva et al, 2005).

Carotenoids, these plant pigments synthesized first retinol then retinoic acid (RA) in the

intestine, the liver, and eventually in target cells. RA is important to modify several

biological processes such as cell replication and growth, vision, bone formation,

metabolism, and immunology. The medical research of retinoid derivatives as

medicines for the treatment of many diseases is, therefore, very promising (Das et al,

2014).

Dunaliella Salina which is unicellular and part of the phylum chlorophylta, is known to

be the largest natural source of large carotenoids (Phadwal et al, 2003). Zeaxanthin,

one of nature's most abundant carotenoids, is an antioxidant. Consequently,

28

Zeaxanthin isolated from D.salina can be used as a natural medicinal agent by

activating retinoid receptors in cardiac tissue to improve heart damage (El-Baz et

al,2019), while it is proven a promising curative and prophylactic activity of D. salina

against cardiac dysfunction in aging rats (El-Baz et al, 2018).

Astaxanthin is a carotenoid of xanthophyll contained in microalgae. It is an

antioxidant with anti-inflammatory effects and therefore has potential as a therapeutic

drug in coronary atherosclerotic diseases. A small number of clinical trials have

examined the efficacy of astaxanthin on oxidative stress and inflammation that are

important to the pathophysiology of cardiovascular diseases. Laboratory research of

multiple species using a myocardial ischaemia-reperfusion model has shown that

astaxanthin supports myocardium both orally and intravenously before the ischaemic

event is inducted (Fassett and Coombes, 2011).

Astaxanthin has a special molecular structure, which controls its intense antioxidant

activities by purifying singlet oxygen15 and free radicals. Astaxanthin was indicated to

reduce oxidation of low-density lipoprotein (LDL) (bad cholesterol) and to improve

clinical trial levels of high-density lipoprotein (HDL)-cholesterol and

adiponectin (peptide) (good cholesterol particle). Current evidence indicates that the

potential of astaxanthin for improving oxidative stress, inflammation, lipid metabolism,

and glycosemia may contribute to preventative measures against atherosclerotic

cardiovascular disease (Kishimoto et al, 2016).

The largest amount identified of astaxanthin in nature is Haematocaccus pluvialis, a

chlorophyte alga that has to turn into a major source of astaxanthin in the food field

(Lorenz and Cysewski, 2000; Yuan et al, 2011). In 1987, the United States FDA

permitted astaxanthin as a food supplement for the aquaculture sector and in 1999

authorized astaxanthin as a nutritional supplement (Guerin et al, 2003).

15 gaseous inorganic chemical

29

Table 4. Summary of the anti-atherosclerotic activity of astaxanthin:

Modified by Kishimoto et al, 2016.

Anti-Oxidation

Iwamoto et al. (2000)

Healthy volunteers (n = 24)

Open labeled; 2 weeks; 1.8, 3.6, 14.4 or 21.6 mg/day

 (decrease)LDL oxidation

Nakagawa et al. (2011)

Middle-aged subjects (n = 30)

Randomized, double-blind, placebo controlled; 12 weeks; 6 or 12 mg/day

 (decrease) phospholipid peroxidation 16 in erythrocytes

Karppi et al. (2007)

Healthy non-smoking males (n = 40)

Randomized, double-blind, placebo controlled; 12 weeks; 8 mg/day

 (decrease) plasma 12- and 15-hydroxy fatty acids

Choi et al. (2011)

Overweight adults (n = 23)

Randomized, double-blind; 3 weeks; 5 or 20 mg/day

  (decrease) plasma MDA17(marker of oxidative stress), isoprastane 18 �(increase) SOD19, TAC 20

Anti-Inflammation

Park et al. (2010)

Healthy female students (n = 42)

Randomized, double-blind, placebo controlled; 8 weeks; 0, 2 or 8 mg/day

 (decrease)plasma 8-hydroxy-2- deoxyguanosine21 , CRP22

Lipid Metabolism-Modulating

Yoshida et al. (2010)

Non-obese subjects with mild hypertriglycemia (n = 61)

Randomized, placebo-controlled study; 12 weeks; 0, 6, 12 or18 mg/day

 (decrease) serum TG, �(increase) HDL-C, adiponectin23

Ursoniu et al. (2015)

Meta-analysis of seven randomized controlled studies

No important effect on plasma lipid profile (LDL-C, HDL-C, TG24)

Glucose Lowering

Ursoniu et al. (2015)

Meta-analysis of seven randomized controlled studies

Slight reducing reaction to plasma glucose

For clinical settings, the current data may be positive, but the medicinal efficacy of

this natural compound needs to be identified for human beings.

16 Oxidative degradation of lipids 17 Malondialdehyde (MDA) is one of the final products of polyunsaturated fatty acids peroxidation in the cells. 18 Biomarkers serve as indicators of oxidative stress in cardiovascular diseases. 19 Antioxidant enzyme that reduces harmful free radicals 20 total antioxidant capacity 21 products of DNA oxidation 22 protein, marker of inflammation 23 a protein hormone that is produced by fat cells. 24 Thyroglobulin, glycoprotein produced by the follicular cells of the thyroid

30

3.6. Metabolism

Metabolism defines all chemical reactions involved in the preservation of the cell

and the organism's living state. The major public health concern is metabolic syndrome

causing dyslipidemia, obesity, and insulin resistance which leads to cardiovascular

disease and death. Omega-3 PUFA in fish oil is well proven to lower the frequency of

metabolic syndrome risk elements (Poudyal et al., 2011). An additional source of

Omega-3 is from Odontellla aurita marine diatom which synthesizes high levels of

eicosapentaenoic acid (EPA, 25 to 26% of total fatty acids) which is known to obstruct

cardiovascular risks. O. aurita's efficiency as a food supplement is proved by Mimouni

et al (2015), in controlling physiological and biochemical parameters involved in

metabolic syndrome deployment, platelet aggregation,25 oxidative stress which leads

to cardiovascular health problems.

Another serious health problem due to the modern lifestyle is obesity. The anti-

obesity activity of refined FX powder (Phaeodactylum extract (PE)) from microalgae

Phaeodactylum tricornutum as a functional food has been explored in the other

research (Koo et al, 2019). PE containing FX exercises anti-obesity functions and

facilitates lipolysis via lipogenesis inhibition. It is, therefore, a good candidate in the

production of antiobesity foods and integrated health foods produced by modern

marine microalgae.

Diabetes mellitus (DM) is a category of metabolic disorders with a high level of blood

sugar for a long time. Serious long-term problems from diabetes include heart attack,

stroke, chronic kidney disease, foot ulcer, and eye injury (www.wikipedia).

An assessment of antihyperglycemic and antihyperlipidemic behaviors and weight

monitoring of Nannochloropsis oculata microalgae (NOM) in Streptozotocin26-induced

diabetic male rats were performed by Nasirian et al., in 2019. Other studies have

shown that the treatment of chlorella microalgae in diabetic rats has lowered glucose

levels and increased insulin levels (Mokady and Sukenik, 1995). In this test, NOM

therapy significantly decreased plasma TG27, total cholesterol and LDL-C associated

25 Aggregation of platelets is part of the sequence of events that contribute to thrombal formation (clot). 26 is consisting an alkylating antineoplastic agent founded in the nature which is very toxic to the insulin-producing beta cells of the pancreas in mammals. 27 triglyceride levels

31

with a significant increase in HDL-C rates in diabetic rats in three weeks, showing its

strong antihyperlipidemic and anti-atherogenic action.

Markovits et al. (1992) also stated that dietary fibers existing in Nannochloropsis, in

particular, insoluble fibers, inhibit intestinal absorption of cholesterol (Kagan et al,

2014) and have an anti-hypercholestromic effect. Diabetic rats (control group) display

less body weight than healthy rats. NOM can retain weight in diabetic rats and improve

metabolic disorders by increasing insulin and reducing the level of glucose. However,

the antioxidant pigments and EPA28 present in the NOM will retain glucose, insulin, and

lipid profile values, and can boost body metabolism and therefore maintain weight.

These findings suggest that NOM can be beneficial for diabetes mellitus care. Further

research will be needed in the relationship between NOM and diabetes.

Moreover, diabetic rats have seen decreased values of glucose and lipid

(triacylglycerol and cholesterol) and it is a proven loss of weight in groups fed with

microalgae Isochrisis Galbana. About the synthesis of lipoproteins, this microalga raises

law lipoprotein content and reduces the concentrations of large lipoproteins (Nuno et

al. 2013).

3.7. Respiratory

Acute respiratory distress syndrome (ARCS) is considered an immediate start of

noncardiogenic edema and as a result of severe inflammatory processes, an obstacle of

gaz-exchange. Ω-3 fatty acids have been suggested to modulate the inflammatory

response in ARDS, even though the data so far collected is limited (García de Acilu et al,

2015).

3.8. Inflammation-Oxidative Stress-Allergies

The incidence of oxidative stress and inflammatory disorders in developed countries

is steadily growing (Bonomini et al, 2008; Molodecky et al, 2012). The development of

these conditions may also be correlated with cancer (Reuter et al, 2010; Siegel et al,

2016). Studies are, therefore, trying to trace new compounds that can be applied to

decrease inflammation, oxidative stress and cancer cell activity. A lot of proves are

32

available in the literature, indicating that certain microalgae extracts have an anti-

inflammatory effect. Nevertheless, aqueous extracts of both microalgae decreased paw

edema in rats caused by carrageenan (Guzman et al., 2001).29 The inflammation was

decreased by 90 percent with Chlorella stigmatophora at a dosage of 250 mg/kg and

with Phaeodactylum tricornutum by 87%. The anti-inflammatory activity of coarse

extracts was 25%-30% higher than indomethacin30, the pharmacological equivalent

(Nazih and Bard, 2018).

The researchers proposed that these results could be due to phycocyanin because

both oxidative stress and edema were prevented by this molecule; this anti-

inflammatory activity may also be due to the presence in microalgae of sulfated

polysaccharides.

In a test utilizing polysaccharides obtained from Porphyridium microalgae, it was

shown in vitro that these polysaccharides prevented leucocyte chemotaxis and

endothelial cell adherence. Such compounds have also been evaluated in human

volunteers with a background of skin allergy to balsam of Peru (Matsui et al., 2003).

Microalgae-based polysaccharide fractions reduced skin redness even more than an

active anti-inflammatory compound derived from a cola extract. Such results indicate

that these extracts might be utilized for external use as effective anti-inflammatory

drugs.

Moreover, Fucoxanthin, this oxygenated carotenoid from diatom Phaeodactylum

tricornutum, may be used as a new nutraceutical if it also has the positive health

effects that have already been shown for microalgae carotenoids. In cell-free and cell-

based assays, Fucoxanthin had powerful antioxidant effects. Fucoxanthin's antioxidants

extracts from P. tricornutum do not vary greatly from astaxanthin, a carotenoid that

has powerful antioxidant effects produced from the already commercially marketed

red algae Haematococcus pluvialis. High amounts of fucoxanthin could be found in the

diet to mitigate the effects of oxidative stress-related diseases. However, because of its

protective health effect, fucoxanthin may assist in conventional cancer treatment. To

28 eicosapentaenoic acid, fatty acid 29 Carrageenans or carrageenins are sulfated polysaccharides derived from red edible seaweeds. 30 An anti-inflammatory medicine

33

further endorse these observations, human trials are needed in the future (Newman et

al, 2019).

Finally, several microalgae species have been identified as anti-allergic. The

existence of an antiallergic substance in the ethanol-insoluble fraction of Dunaliella

salina is strongly suggested (Fujitani et al., 2001). For their anti-allergy characteristics,

other species such as Chlorella vulgaris or Chlorella pyrenoidosa (Chlorophyta) have

also been presented (Vo et al., 2012).

3.9. Immunology (various & infectious diseases)

It is difficult to differentiate the immunomodulative effect of microalgae from the

anti-inflammatory effect. The reported capacity of microalgae polysaccharides to

suppress leucocyte migration may be correlated with their anti-inflammatory function

(Matsui et al., 2003). Nevertheless, certain microalgae have been shown to have a

strong stimulating influence on immune cells.

This was the example of Phaeodactylum tricornutum, which was demonstrated to

trigger phagocytosis and the sulfated polysaccharide from Chlorella stigmatophora,

which indicated immunosuppressant functions (Guzman et al., 2003). Gymnodinium

impudicum sulfated polysaccharides also enable nitric oxide synthesis and induce

cytokine development in macrophages (Bae et al., 2006). However, in contrast to these

promising in vitro and animal findings, there is a distinct deficiency of clinical studies to

suggest that microalgae are of significant interest as a source of compounds for the

treatment of human inflammation or immunomodulation (Nazih and Bard, 2018).

34

Table 5. Marine microalgae producing immunomodulatory compounds or displaying

immunomodulatory properties. IL is for interleukin, PBMC is for human peripheral

blood mononuclear cells. Modified by Riccio and Lauritano, 2019.

Microalgae Extract/Fraction/ Compound

Mechanism/ Organism and Target Cells (or Model)

Reference

Alexandrium tamarense

Total Extract/Fractions

Activation of IL-6/Human PBMC

Cutignano et al, 2015

Chaetoceros calcitrans

Fractions

Activation of IL-6/Human PBMC

Cutignano et al, 2015

Chaetoceros socialis

Total extract

Activation of IL-6/Human PBMC

Cutignano et al, 2015

Chlorella stigmatophora

Crude polysaccharide extracts

Activation of phagocytic activity - SRBC/Mouse

Guzmán et al, 2003

Chlorella vulgaris

Diet supplementation/ commercially available pills

Improve of NK activity and serum level of INF- , IL-1 and IL-12/Human trials

Kwak et al, 2012

Dunaliella salina

Diet supplementation of commercially available spray-dried preparations

MiceNK and Macrophage activation/In vivo mice model

Chuang et al, 2014 Cutignano et al, 2015

Dunaliella salina

Fractions

Activation of IL-6/Human PBMC

Cutignano et al, 2015

Euglena gracilis

β-Glucans

Activation of NK cells and improve in inflammatory mediator/Human PBMC

Barsanti and Gualtieri, 2019 Russo et al., 2017

Gyrodinium impudicum

Sulfated exopolysaccharides

Macrophage activation/Murine

Bae et al, 2006 Yim et al, 2004

Skeletonema costatum

Total Extract/Fractions

Activation of IL-6/Human PBMC

Cutignano et al, 2015

Skeletonema dohrnii

Total Extract/Fractions

Activation of IL-6/Human PBMC

Cutignano et al, 2015

Skeletonema marinoi

Total Extract

Activation of IL-6/Human PBMC

Cutignano et al, 2015

Tetraselmis chuii

Orally administration of lyophilized microalgae

Increase in hemolytic complement activity, phagocytic capacity and expression levels of β-defensin, major histocompatibility complex II a and colony-stimulating factor receptor-1/ Gilthead sea bream

Cerezuela et al, 2012

Thalassiosira weissflogii

Fractions

Activation of IL-6/Human PBMC

Cutignano et al, 2015

Tribonema sp.

Sulfated polysaccharides

Macrophage proliferation and improved expression of cytokines/Mouse

Chen et al, 2019

35

As stated in Table 5, dried algae and crude extracts have been effective versus many

cells of the immune system. Cutignano et al., (2015) examined unrefined methanol

fragments and amounts of different species of microalgae. Fractionation also managed

to distinguish active fractions for other organisms whose raw methanol extracts were

not effective (Riccio and Lauritano, 2019). To determine their immunostimulatory

function, other compounds extracted from microalgae have been studied. Chlorella

stigmatophora polysaccharide aqueous compounds have been analyzed in vitro and in

vivo mouse models (Guzmán et al, 2003). Supplementation of microalgae food was also

correlated with immunostimulatory function. In fact, diet supplements of commercially

accessible spray-dried products of Dunaliela salina in mice boosted NK (natural killers)

and macrophage activation, leading to the survival of leukemic mice (Chuang et al,

2014).

Table 5 lists the algae with immunostimulatory function.

3.10 Hepatology

Chlorella vulgaris has been recorded for reducing dyslipidemia and high blood

pressure; Its impact on inflammatory and insulin-resistant biomarkers, however, has so

far not been discovered. Non-alcoholic fatty liver disease (NAFLD) is strongly associated

with insulin-resistance and inflammation as a hepatic complication of metabolic

syndrome. C. vulgaris supplementation may have beneficial effects as far as glucose

homeostasis, insulin condition, and biomarkers related to inflammation in patients with

NAFLD. C. vulgaris supplementation could be considered as a part of treatment for

weight loss and improvement of glycemic situation and reduction of hs-CRP31 together

with the improvement of the liver condition in NAFLD as stated Ebrahimi- Memeghani

et al. (2017).

31 The increased risk of heart attack is associated with high levels of hs-CRP (high sensitivity C-reactive protein in our blood)

36

3.11 Dermatology

Astaxanthin, a secondary metabolite produced in nature by a series of bacteria,

microalgae, and yeasts, is xanthophyll carotenoid. Traditionally, chemical synthesis has

been used for commercial production, but the Haematococcus pluvialis microalgae

seem to be the most important path for its commercial biological supply. Because of its

common and complex function in skin biology, astaxanthin has various health benefits

and significant nutraceutical uses in dermatology (Davinelli et al, 2018). The

Haematococcus pluvialis chlorophyte algae have the largest potential for the

concentration of ASX (Boussiba, 2000). The strong antioxidant function and special

molecular and biochemical properties of ASX have recently drawn researchers' interest,

for the treatment and prevention of skin disease. Comparative studies studying

carotenoid photoprotective activity have found ASX to be a supreme antioxidant and

more antioxidant compared to canthaxanthin and carotene in human epidermic

fibroblasting (Camera et al, 2009).

Therapy also, with ASX prevents deleterious effects of UV by reducing the

production of UV-induced reactive nitrogen, inflammatory cytokine, and keratinocyte

apoptosis. Inhibitory properties of ASX on iNOS32 are important in developing anti-

inflammatory medicines for skin inflammatory conditions like psoriasis and atopic

dermatitis (AD) (Park et al, 2017). As far as immune effects are concerned, ASX greatly

affects immune function across in vitro and in vivo assays (Lin et al, 2015). In several

studies, ASX increased its natural killer activity (NK) and indicated that ASX may control

NK cells, which serve as a tumor and virus-cell immunosurveillance system (Chew et al,

2011). Moreover, ASX effectively eliminates in vitro cell damage from free radicals and

MMP-133 entry into the skin after UV radiation (Suganuma et al, 2010). In specific, ASX

was able to minimize the damage of DNA and influence DNA repair kinetics (Santocono

et al, 2006). To fully understand ASX skin activities, further, systematic research will be

required.

32 Nitric oxide synthases (NOSs) are a family of enzymes catalyzing the production of nitric oxide (NO) from L-arginine. 33 Matrix metalloproteinases (MMPs), the main extracellular matrix (ECM) enzymes in collagen degradation,

37

38

Table 6: Studies on skin and astaxanthin. Modified by: Davinelli et al, 2018.

Intervention

Study Design

Outcomes

Author, Year

Administration of ASX capsules

Randomized double-blind, controlled study

p DNA damage biomarkers; n of NK cells, T cells, B cells, and IL-6

Park, 2010

Administration of ASX capsules

Monitoring of oxidative stress and skin aging

p MDA; pRSSC

Chalyk, 2017

Administration of ASX capsules

Randomized, double-blind, parallel-group, placebo-controlled

pWrinkle parameters; p IL-↵

Tominaga, 2017

Administration of ASX cream

Pilot study 34

pWrinkle parameters

Seki, 2001

Topical application of ASX

Pilot study perythema Yamashita, 1995

Administration of ASX capsules

Randomized, single-blind, placebo-controlled

pWrinkle parameters

Yamashita, 2006

Oral and topical treatment with ASX

N/S

pWrinkle parameters

Tominaga, 2009

Two oral forms (ASX capsules; tablets collagen)

Randomized, double-blind placebo-controlled

nviscoelastic parameters; pTEWL;" procollagen type I; nMMP-1 and pMMP-12

Yoon, 2014

Capsules of ASX combined with topical application of ASX

Open-label noncontrolled

pwrinkles; page spot size; n"elasticity; n"skin texture

Tominaga, 2012

Administration of ASX capsules

Randomized double-blind controlled

pwrinkles; n"elasticity; pTEWL; n"moisture content; psebum oil

Tominaga, 2012

Abbreviations: "nincrease;pdecrease; ASX, astaxanthin; NK, natural killer; IL-6, interleukin-6; MDA, malondialdehyde;

RSSC, residual skin surface components; N/S, not specified; TEWL, transepidermal water loss; MMP, matrix

metalloproteinase.

34 A pilot study is a small scale preliminary study

39

3.12 Neuroscience

The disorder of Parkinson is mainly characterized by dopaminergic 35 neuron

degradation. Neuroinflammation is an important element that damages cells during

disease progression, and experimental settings have been effective in reducing the

inflammatory process. Fractalkine36 signals have proved to be a crucial path in

inflammatory cell death in animal PD models. Astaxanthin has several biological

functions, many of which seem to be specifically contrasting with the pathological

mechanisms involved in SN37 neurodegeneration. The two promising new therapeutic

agents for PD diagnosis and management include both fractalkine and astaxanthin

(Grimming et al., 2016).

Alzheimer’s Disease (AD) is a neurological dysfunction occurring through complex

mechanisms of pathology and physiology. AD leads to dementia, cognitive decline, loss

of synapses and cholinergic neurons, and death. Increasing numbers of AD patients

have centered research into new drugs with minimal or no side effects relative to

conventional pharmaceutical goods (Rajakumar, 2018). With the expectation that

experts will soon be able to reverse AD, prevention measures such as lifestyle changes,

exercise and the use of antioxidants can be taken to delay the progression of AD or to

mitigate it.

So how can microalgae lead to AD treatment? Microalgae are important sources of

many bioactive materials such as carotenoids, polysaccharides, PUFA's, amino acids

and proteins, sterols and polyphenols (Olasehinde et al., 2017). A major cause of a

cognitive decline in patients with AD is accelerated deterioration of the function of

acetylcholinestrase (AChE).38 ACh, which eventually causes cholineergic damage and

memory loss, is impaired due to high cholinesterase concentrations (ChE). The AChE

inhibitory behaviors were contained in extracts from Nannochloropsis oculata,

Chlorella minutissima, Tetraselmis chuii and Rhodomonas salina (Custódio et al., 2012).

35 Dopamine is one of the chemicals that is responsible for signal processing between the neurons of the brain and between nerve cells. 36 anti-inflammatory cytokine (protein) 37 The substantia nigra (SN) is a midbrain-located basal ganglia system that plays a significant role in reward and mobility. 38 The major enzyme of neurotransmitter acetylcholine (ACh) is responsible for its hydrolytic synthesis.

40

Extracts from Nannochloropsis sp., Picochlorum sp., and Desmochloris sp. have been

reported to exhibit BChE39 inhibitory activities (Pereira et al., 2015).

3.13 Ophthalmology

Zeaxanthin, one of nature's most abundant carotenoids, is an antioxidant that

accumulates in the human eye's retina and protects the retinal system against light-

induced harm. Eating diets containing higher levels of zeaxanthin were found to be

followed by a low incidence of eye disorders such as age-related macular diseases, and

cataracts (Madhavan et al, 2018).

39 is a nonspecific cholinesterase enzyme

41

4. European Regulations concerning Microalgae

First and foremost, the EU legal framework for microalgae-based medicinal products

provides certain requirements to secure a high level of public health safety and the

quality, consistency, and effectiveness of licensed medicinal products. Also, not only

can it promote the pure functioning of the internal market but also encourage

innovation progress (Europa, 2019). In Europe, the EFSA (European Food Safety

Authority) is the competent authority ordered to evaluate the safety of any new

functional food and supplement ingredient before they are licensed for production and

market introduction (EFSA, 2019).

Three-pillar European legislation applies to the regulation of microalgae or its

components. The first pillar focuses on general supplement safety, the second one on

novel ingredients, and the last one on functional nutrition and health claims

(Novoveská et al, 2019).

The safety pillar for microalgae nutraceutical products is mainly combined by the

European Commission (EC) No 178/200240 and 852/200441 regulations. The EC No

178/2002 regulation provides a general framework in the development of any

supplement legislation and determines the general definitions, regulations, and

responsibilities during the process of production, distribution, and safety at the

Community level. It also establishes the European Food Safety Authority (EFSA) and

refers to the commercialization of supplement products using either whole microalgae

organisms or cells, such as Spirulina and Chlorella, or products that incorporate the

micro-algae such as the green algae color pasta. Both EC No 178/2002 and 852/2004

state that all these products based on microalgae are subject to regulations on food

safety which apply to all supplementary products. Experts issue that safety is an

important factor in algae-technology especially when algae are generated in open-air

systems because they have proved to be easily contaminated by other microorganisms

(Enzing et al, 2014). The safety of the supplement must be checked in extended use

40 https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32002R0178&from=EL

42

because products containing microalgae can not be approved in the European market

without health evaluation being carried out first (Serafini et al, 2010).

As far as pharmaceuticals are concerned the European Medicines Agency (EMEA) is

responsible for securing a commercial authorization application for the EU under the

“centralized” process. Authorization for use is generally required for 1) all prescription

biotech products, 2) pharmaceuticals with a newly formed active ingredient used for

the treatment of HIV, cancer, neurodegenerative disorders, and diabetes, and 3)

medicinal drugs known as "orphans42". In the sense of "decentralized practice," the

“mutual recognition” concept extends to the Member States at a national level.

According to Article 8 of Directive 2001/83/EC43, an authorization may only extend to

organizations set up in an EU country. Whether the trials are carried out or not within

the EU, the clinical studies have been morally implemented and protected the privacy

of individuals. In the appendix of directive 2003/63/EC44, the specific guidelines for

clinical studies are imposed. The Commission regulations set strict deadlines to

approve or deny a request for marketing authorization. The EMEA or the Member State

authorities shall decide in 210 days, in compliance with both decentralized and

centralized policies.

The present European policy on data protection offers a period of “8+2+1” years:

Generic manufacturers may apply for a marketing license of the provided medicine

eight years after a marketing authorization of the original product, but only two years

later could the genetic product in question become available on the market. In case of

the discovery of new therapeutic benefits in the first 8 years data security can be

extended by one year. Data and ads on prescription drugs are severely restricted (EU,

Pharma, legislation, 2019).

41 https://eur-lex.europa.eu/legal-content/EL/TXT/?uri=CELEX%3A02004R0852-20090420 42 The aim of ' orphan drugs ' is to treat diseases that are so rare. 43 https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:02001L0083-20121116&qid=1472567249742&from=EN 44 https://eur-lex.europa.eu/legal-content/EN/ALL/?uri=CELEX%3A32003L0063

43

5. Swot analysis – A case study of a fictional microalgae-based carotenoid production company

In this section, the strengths, weaknesses, opportunities, and threats (SWOT

analysis) of a fictional microalgae-based carotenoid production company are

presented.

Strengths

x One of the major strengths of the industry is that there is a rich diversity in terms of

microalgae and carotenoids. As such, carotenoids based on their antioxidant

properties continue to increase interest in multiple markets, including food and feed

products, nutritional products, and cosmetics.

x Furthermore, the fact that carotenoid’s source is natural, adds an advantage for use

of the specific products.

x The further benefits ensuing from other compounds existent in microalgae are

adding forces to the industry.

x The ongoing increasing awareness and enthusiasm of consumers for green chemistry

is another advantage towards future expansion of the specific markets.

Weaknesses

x One of the major weaknesses of microalgal carotenoids is undoubtedly their

currently increased costs of production as it can be exhaustive labor and costly

operation. It is imperative that new methods need to be developed in order to drive

costs further down. Microalgae are cultivated in open ponds or closed systems

requiring procedures of mixture and concentration (Chew et al., 2017). These

procedures are energy consuming and the major concern is minimum energy in

combination with maximum exploitation of microalgae biomass (Vanthoor-Koopmans

et al., 2013). Although open structures for cultivation, like open ponds or raceway

ponds, are more commercially viable for large-scale production, they may not be

appropriate for cultivating microalgae which would be employed on medicinal or

pharmaceutical purposes (Chisti, 2007; Milano et al., 2016). On the contrary, closed

culture systems (e.g. photobioreactors) (Costa and De Morais, 2011; Khoo et al.,

44

2011) are more practicable by monitoring the cultivation conditions (e.g.,

temperature, pH and CO2 concentration, and so on), even if capital and operating

costs rise.

x The biological systems entail some inherent challenges that constitute another

weakness of the industry. The microorganisms that manufacture carotenoids typically

have a low rate and target productivity of carotenoids in accordance with

recalcitration, resulting in inefficiencies in the extraction of the carotenoid. The

biomass must be collected after harvesting in a system separating the solid material

(biomass) from the liquid (cultural medium). The biorefinery process uses microalgae

biomass to create renewable carotenoid products with many advantages like quick

maturation, increased demand on the food industry, and selective composition

(González-Delgado and Kafarov, 2011).

x Downstream processing and the associated weaknesses of the specific methods

constitute further disadvantages.

x Climate limitations for open systems pose further barriers to the industry.

Opportunities

x There is an increase in end-user applications for carotenoid products formulating

niche markets. Consequently, the advance of the aquaculture industry, the medicinal

applications spread, and the increasing preference of consumers for natural

products, support carotenoids from microalgae potential for future revenue growth

(Ambati et al, 2019)

x Improvements in cultivation and downstream processing. The microalgal cultivation

systems (smart microalgae screening and strain enhancement included) are steadily

being developed, while carotenoid extraction methods become more efficient and

environmentally friendly.

x Increasing end-user awareness is apparent. The end-user numbers are constantly

increasing with evident interest in health and sustainability-oriented lifestyles that

encourage the development of sustainable natural carotenoids.

x Future research (a combination of natural and synthetic production). In future

research, there are numerous opportunities, including evolving carotenoids, new

45

applications, and innovations. The development of markets (both niche and

commodity) will expedite the research as well.

Threats

x Stringent regulations are one of the main concerns of the industry and adaptation to

the strict requirements undermines the rapid development of the industry.

x Low-cost competition from Asian countries (due to lower labor and production costs

in such labor-intensive industry) creates concerns for the western carotenoid

markets. On the other hand, such Asian markets provide a huge number of

consumers with inexpensive carotenoids increasing in such a way the total market.

x Very low cost of synthetic production consists of a threat as in the laboratory it is

hard to differentiate natural and synthetic carotenoids unless specialized techniques

such as isotope ratio mass spectroscopy are applied (Kroll et al, 2008). Therefore,

manufactured pigments will quickly enter the market, pretending to be falsely

natural products.

x The potential of contamination of biomass in an open system renders a necessity to

manage the process with extravagant care.

x Impact on Algae Production Due to Climatic Conditions (HTF, 2019)

x As the process is not well-established yet it is a possible threat for any company

entering the specific industry.

46

Table 7: A Swot analysis for microalgae-based carotenoid production companies

Strengths Weaknesses x Rich diversity in terms of microalgae and

carotenoids applicable in multiple markets.

x Natural source of carotenoids. x Benefits from other compounds existent

in microalgae. x Consumer’s awareness and enthusiasm

for green chemistry.

x Increased costs of production. x Inherent challenges of biological

systems. x Downstream processing weaknesses. x Climate limitations for open systems.

Opportunities Threats x Increase in end-user applications for

carotenoid products and for natural products.

x Improvements in cultivation and downstream processing.

x Increasing end-user awareness. x Future research (combination of natural

and synthetic production).

x Stringent regulations. x Low-cost competition from Asian

countries creates concerns to the western markets.

x Very low cost of synthetic production. x Potential of contamination of biomass

in open system. x Impact on Algae Production Due to

Climatic Conditions. x Not yet well-estabished process poses

threats to any incoming company.

47

6. Conclusions

The analysis of scientific data reveals that microalgae were commonly used for the

development of different bioactive compounds as a renewable resource.

The following development limitations could be regarded to optimize possible future

success and restricting the limitations of microalgae biotechnology applications:

x Therefore, it is critical to assure the quality and safety of the microalgal biomass

and its ingredients for the consumption of human beings (as per the FDA and

other regulatory agencies, EFSA, EMEA). On the other hand, the strict public

European regulations for pharmaceutical and nutraceutical products delay the

emergence of new drugs with potential health benefits. A middle way must be

found so that regulations could be respected protecting humans without impeding

the production of new promising medicines.

x Research on the clinical validity of the nutraceutical and medicinal benefits of the

bioactive products extracted from microalgae shall further enhance their

acceptance.

x The lack of clinical studies with humans is the main obstacle that postpones the

significant research to proceed. In a lot of studies, the conclusion was that human

clinical researches must be conducted in order to evaluate the scientific

conclusions or tests on animals.

x Many positive health effects will likely be found with increasing microalgae

consumption as food supplements, nutraceutical, pharma, and cosmetic products.

x Intensive bioprospecting should be pursued by the newer bioactive microalgal

species (Nethravathy et al, 2019).

x A comprehensive attempt should be made to evaluate the sustainability process

through the techno-economic study, environmental efficiency, and life cycle

assessment for the production of high-value microalgae metabolites.

x To improve the process chain and business portfolio, the identification of the

important factors in the manufacture, refining, storage, marketing, and

distribution of algal products can help.

48

x A productive microalgal industry would include a biorefinery design, a zero-waste

production strategy, and plant incorporation and intensification, meaning each

advancement of chemical engineering, contributing to significantly reduced,

healthier, safer, and more energy-efficient technologies.

49

7. Future perspectives

Microalgae are abundant and economically important bio-resources with various

bioactive metabolites. A variety of microalgae species are yet to be studied. Such

organisms require extensive scientific research to uncover their ability to benefit

people. This will add more species to just a few existing industrial-scale microalgae. The

food, feed, health, and cosmetic industries are covered by many high-value bioactive

components from microalgae such as essential long-chain PUFAs (EPA, DHA), pigments,

single-cell proteins, and mineral derived vitamines. Sustainable development and the

growing market will likely help commercialize microalgae-based products by knowledge

and clarity on the health of the use of microalgae and regulatory issues related to

genetically engineered microalgae use. Research on the antibacterial activity of

nanosized silver colloidal solutions against human pathogens has shown strong efficacy

as a potent antimicrobial agent in the use of silver nanoparticles in drugs (Merin et al,

2010).

Additionally, multi–omics (genomics, transcriptomics, proteomics, and metabolomics)

combined with data analysis have rendered biological processes easier to understand

and require easy access to a variety of metabolic pathways for genetically modified

microorganisms. Finally, the –omics resources will create future perspectives opening

wider doors into microalgae research applications (Mishra et al, 2019).

Therefore, given the different aspects of microalgae examined, it is clear that

microalgae with a large existence around the world will satisfy the nutrition, feed, fuel

as well as human health needs. Marine microalgae provide a simple and effective route

for nanoparticle syntheses with controllable, particle-sized optical properties.

50

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