POWER OF ASTAXANTHIN

ALGAETECH INTERNATIONAL SDN BHD, MALAYSIA“T H E F U T U R E I S N O W”

Frost & Sullivan 2011 Best Practice Award

“Asia Pacific Green Excellence Award for Service Innovation in Algae Technology”

BIONEXUS Status Award

Astaxanthin is a natural antioxidant with capability to scavenge free radicals

Astaxanthin is a keto carotenoid, classified under Xanthophylls. It is a metabolite of Zeathanthin and / or Canthaxanthin. Astaxanthin is a colourful lipid soluble pigment

Astaxanthin has two chiral centers/stereoisomers at the 3 and 3’ positions. There are three stereoisomers recorded so far in Astaxanthin3R, 3’R, 2. 3R, 3’R and 3. 3S, 3’S

What is Astaxanthin?

Astaxanthin Benefits

Strong Antioxidant Anti-Inflammatory Provides Eye Fatigue Relief Increase Muscle Endurance Supports Healthy Blood Lipids Aids in Wrinkle Reduction

Astaxanthin is assimilated with lipoprotein and transported into the

tissues. Of several naturally occurring carotenoids, Astaxanthin is

considered as one of the best carotenoids being able to protect cells

lipids and membrane lipid proteins against oxidative damages

(Olson, J.A., 2004. Pure Appl. Chem. 66: 1011 – 1016)

• The colour of Astaxanthin is due to the extended chain of conjugated – alternating double and single bonds at the centre. The double bonds of hydroxyl and keto groups are responsible for antioxidant function of Astaxanthin.

• Astaxanthin protects body tissues from oxidative and Ultra-violet damage through its suppression of NF-kB activation

(Lennikov, A., Nobyoshi, K. and Risa Fukase, 2012. Molecular vision 18: 455 – 464)

• The yeast, Xanthophyllomyces dendrorhous produces 3R, 3’R stereoisomers for the synthesis of Astaxanthin

• Synthetic Astaxanthin obtained from petroleum products contains a mixture of all three stereoisomers

• Astaxanthin was approved by US, Food and Drug Administration (FDA) as a New Dietary ingredient in 1999

(Lennikov, A., Nobyoshi, K. and Risa Fukase, 2012. Molecular vision 18: 455 – 464)

Production

Haematococcus pluvialis cultivationVegetative cell Immature Aplanospore Aplanospore

４０－８０μｍ

Haematococcus pluvialisHaemato (Blood) Coccus (Seed) Pluvialis (Fresh Water)

Microalgae family – includes Chlorella and Spirulina

Single cell fresh water Microalgae (Plant source)

Found in standing pools and ponds world wide (non-GMO)

During harsh environment conditions – Hibernates

Why we choose Haematococcus pluvialis for Astaxanthin:

The freshwater unicellular microalga Haematococcus pluvialissynthesizes and accumulates the highest levels of Astaxanthincompared to the other Astaxanthin producer organisms.

Commercially grown Haematococcus pluvialis can accumulate morethan 40g of Astaxanthin per kg of dry biomass.

Studies showed Astaxanthin’s health-beneficial effects in humans wereperformed on the stereoisomer found in Haematococcus, 3S-3’S.

In nutraceutical applications , scientists have proven that one of themain advantages of natural Astaxanthin esters is that the esterifiedform is inherently more stable than the free form, providing for asignificantly longer shelf life without being oxidized.

The advanced commercial production of natural Astaxanthin from themicroalga Haematococcus pluvialis supplies the market with high-qualityproducts rich in Astaxanthin, suitable for human applications.

Free Astaxanthin from H. pluvialis has 4.4 fold higher free radicalscavenging activity (IC50 value of 8.1 microgram/ml) when compared tothat of Astaxanthin esters (Kamath, 2007).

Natural sources of Astaxanthin

Astaxanthin has shown both lipophilic and

hydrophilic properties. It acts as a strong

antioxidant by donating the electrons and

reacting free radicals to convert them to be

more stable products and terminate free

radical chain reaction.

Astaxanthin has a unique structure which

enables to stay both in and outside cell

membrane. It gives better protection than

beta carotene and vitamin C since, it could

link with cell membrane from inside to

outside.

Functional properties of Astaxanthin and other compounds

Its unique lipophilic and hydrophilic properties allow it to span the entire cell, with one end

of Astaxanthin molecule protecting the fat soluble part and another end protecting water

soluble part of cells.

(Yamashita, 2013. Funct. Foods Health Dis. 3: 254 – 258)

This Red Color in Nature is Astaxanthin

Health Benefits of Astaxanthin

• For humans, Astaxanthin is a powerful antioxidant with broad health implications and unlike other antioxidants such as beta carotene, zeaxanthin, vitamin E, C, D. and selenium, Astaxanthin never becomes pro-oxidant in the body

(Pearson, P., et al., 2006. BioDrugs 20: 271 – 273)

• Haematococcus pluvialis extract is known to inhibit the growth of human colon cancer cells by arresting cell cycle progression and promoting apoptosis

(Palozza, P. et al., 2009. Cancer Lett. 283: 108 – 117)

• Astaxanthin has shown excellent results for helping with skin moisture levels, smoothness and elasticity and preventing fine wrinkles and spots.

(Camera, E., et. al., 2009. Exp. Dermotol. 18: 222 – 231)

• Bioavailability of Astaxanthin in human plasma was confirmed with single dosage of 100 mg

(Osterlie, M., Bjerkeng, B. and Liaaen-Jensen, 2000. J. Nutr. Biochem. 11: 482 – 492)

Health Benefits of Astaxanthin

• Supplementation of Astaxanthin in human for eight weeks resulted in increased blood levels and improved the activity of killer cells which targeted to destroy the cells infected with viruses

(Park, J.S. et al., 2010. Nutr. Metab. 7: 1 – 10)

• A clinical trial made on humans revealed that Astaxanthinhelps diabetic retinopathy, macular degeneration, eye strain and fatigue and seeing in fine details

(Sun, Z., et al., 2011. Food Funct. 2: 251 – 218)

Health Benefits of Astaxanthin

Oxygen Radical Absorbance Capacity (ORAC) values for H. pluvialis made Axtaxanthin isabout 3 times more than that of the synthetic product and about a third more than theyeast- made version (Nguyen, 2013).

Table: Sustainability comparison on economical, environmental and societal impact of Astaxanthin production via chemical synthesis,yeast fermentation (Phaffia rhodozyma) and algal induction (H. pluvialis):

Per kg of Astaxanthin

Economical Environmental Societal

Production method

Raw material cost ($)

Land usage(Sq.km)

Energy cost ($)

Waste water

Emissions (Air)

Cost ($) Oxygen Radical Absorbance Capacity (ORAC)

Human consumption?

Chemical synthesis

40 10 26 0 2 2,000 33% NO

Yeastfermentation

140 20 160 9 5 2,500 66% Most

H. pluvialissynthesis

164 25 120 2 9 >7,000 100% YES

Note: Green indicates the best option, yellow medium and red is the worse.

Organism Content% W/W dry wt

Reference

Green algae

Haematocus pluvialis 2.0-3.0 Lorenz and Cysewski , 2000

Neochloris wimmeri 0.6 Orsa et al.2000

Chloroccum <0.2 Zhang et al. 1997

Nannochloropsis gaditana <0.3 Lubian et al. 2000

Scenedesmus vacuolatus 0.01 Orsa et al. 2000

Chlorella zofingiensis <0.01 Ip and Chen 2005

Chlamydomonus nivalis 0.04 Bidigare et al. 1993

Fungi

Phaffia rhodozyma 0.4 Jacobson et al. 2000

Yeast-Candida utilis 0.04 Miura et al. 1998

Bacteria

Agrobacterium aurantiacum 0.01 Yokoyama et al. 1995

Animals

Shrimp –Pandalus clarkii 0.015 Mayers and Bligh 1981

Back snow crab-Chinoecetes opilio 0.011 Shahidi and Synowiecki,1991

Comparisons of Astaxanthin content from Natural sources :

Cultivation system of H. pluvialis:

Astaxanthin production from H. pluvialis was developed on the industrial scale aroundthe late 1990s. Many different system have been developed for large scale productionof H. pluvialis.

First growth stage (Green stage):Usually facilitated by either closed indoor photo-bioreactors or enclosed outdoorsystems.Closed system:

-Environmentally controlled process with less biological and chemicalcontamination.

-Much more popular and optimal growth conditions can be achieved.

The cyst induction ‘reddening phase’ :Conducted via open raceway ponds, where nutrient deprivation and cellular stress areactually preferred.

Industrial systems have utilized nutrient deprivation, high irradiance, and/or hightemperature. Yields have hovered around 1-3% Astaxanthin harvested from cells.

17

Excess of light

Generation of ROS

Cellular sensing, mediators

Activation of cell response

+ LIGHT

Environmental Stresses: Nutrient deprivation Salt stress High light Low temperature Drought Aging

Cell response to stress in the green alga Haematococcus

Mode of action

Slowdown of cell division

1. Xanthophyll cycle 2. ROS quenching enzymes3. antioxidants

Change in cellanabolism(lipids)

PalmelloidMotile cell Astaxanthin accumulation

Encystment

Red cyst

18

Synchronized cultures and Life Cycle

0 10 20 30 40 50 60 70 80

Time (h)

1

10

1

10

40

0 20 40 60 80 100

Time (h)

Lag enhanced productivity

High LightNormal Light

Very few companies commercially produce Astaxanthin from Haematococcus pluvialis. TheHawaiian companies Cyanotech Corporation and Mera Pharmaceuticals cultivate the algaeusing an open pond system for the “Red Stage.”

The Japanese company Fuji Chemicals operates an indoor facility in Sweden and its“domeshaped” bioreactors in Hawaii. The Israeli company Algatechnologies uses tubularsolar-powered photobioreactors for both the “Green and Red stages” in closed, strictlycontrolled systems

The major parameters used to assess high-quality commercial Haematococcus biomassand oleoresins are high Astaxanthin content in the product, low levels of biological andchemical contamination, and excellent stability of the Astaxanthin in the product.

Producing Astaxanthin in a closed system throughout the entire process (“Green” and“Red”) in an area with high solar-radiation intensity year-round, as in the case ofAlgatechnologies, yields high-quality Astaxanthin products

Company LocationAlga Technologies IsraelCyanotech HawaiiJingzhou Natural Astaxanthin Inc China

Algaetech International Malaysia

Parry Nutraceuticals IndiaMera Pharmaceuticals Inc., Hawaii

Fuji Chemicals Japan, Sweden

Valensa International Florida

Prominent Players in the Astaxanthin Market

(http://www.oilgae.com/non_fuel_products/astaxanthin.html#sthash.FVwemMuu.dpuf)

The demand for natural astaxanthin is now emerging in the multi-billion dollarnutraceutical market.

Astaxanthin is principally consumed by the salmon feed industry. The annualworldwide aquaculture market of this pigment is estimated at US$ 200 million withan average price of US$ 2500/kg.

The global astaxanthin market is estimated at about $257 million, most of which isused in fish coloration .

The human uses market is growing and estimated at about $27-$40 million. Mostastaxanthin is derived from the H pluvialis, which is commonly consumed by fishand crustaceans – like salmon and lobster – and is responsible for their pinkcoloration.

(http://www.oilgae.com/non_fuel_products/astaxanthin.html#sthash.vyvgtBKR.dpuf)

Market Scope of Astaxanthin

Market Sectors Market Size ( as of 2009) ( Million USD)

Potential Market(2020)( Million USD)

Animal feed colouring agents 300 800Antioxidant nutraceuticals 30 300Pharmaceuticals Emerging 500Cosmetics Emerging 30

(http://www.oilgae.com/non_fuel_products/astaxanthin.html#sthash.FVwemMuu.dpuf)

Market Scope of Astaxanthin

Challenges in Astaxanthin Production

Although natural sources have long been exploited for astaxanthin production, it is stilluncertain if natural astaxanthin can be produced at lower cost than that of syntheticastaxanthin or not.

One of the major limitations with the H pluvialis production system is that the astaxanthingets trapped behind thick cells walls, thus complicating the extraction process and theproduction yields.

Production capacity of H pluvialis is constrained by its intrinsic slow growth, low cell yield,ease of contamination by bacteria and protozoa, and susceptibility to adverse weatherconditions. These challenges are magnified as processes are scaled up, and thereforerequire advanced technology to control

H pluvialis cannot be efficiently cultivated in dark heterotrophic mode, which requires highlevels of irradiance, making the process economically less reasonable.

(http://www.oilgae.com/non_fuel_products/astaxanthin.html#sthash.vyvgtBKR.dpuf)