Original Article J. Clin. Biochem. Nutr.

, 44, 280–284, May 2009

Journal
JCBN
the
1880-5086
0912-0009
Kyoto,
Original
10.3164/jcbn.08-238
jcbn08-238
Society
Japan
ofArticle
Preliminary Clinical
for FreeBiochemistry
Radical
ClinicalResearch
and Nutrition
Japan
Evaluation of Toxicity and Efficacy
of A New Astaxanthin-rich Haematococcus pluvialis Extract

Akira Satoh1, Shinji Tsuji1, Yumika Okada1, Nagisa Murakami1, Maki Urami1,
Keisuke Nakagawa1, Masaharu Ishikura1,*, Mikiyuki Katagiri2, Yoshihiko Koga3,
and Takuji Shirasawa2
1
Life Science Institute, Yamaha Motor Co., Ltd., 3001-10 Kuno, Fukuroi, Shizuoka 437-0061, Japan
2
Department of Aging Control, Graduate School of Medicine, Juntendo University, 3-3-10-201 Hongo,
Bunkyo-ku, Tokyo 113-0033, Japan
3
Department of Neuropsychiatry, Kyorin University School of Medicine,
6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan

580
4
30
2
3
44
Received
;284
009
accepted27.10.2008
2.12.2008

Received 27 October, 2008; Accepted 2 December, 2008

Copyright © 2009 Astaxanthin

Summary JCBN (Ax), a carotenoid ubiquitously distributed in microorganisms, fish,
and crustaceans, has been known to be a potent antioxidant and hence exhibit various
physiological effects. We attempted in these studies to evaluate clinical toxicity and efficacy of
long-term administration of a new Ax product, by measuring biochemical and hematological
blood parameters and by analyzing brain function (using CogHealth and P300 measures).
Ax-rich Haematococcus pluvialis extracts equivalent to 4, 8, 20 mg of Ax dialcohol were
administered to 73, 38, and 16 healthy adult volunteers, respectively, once daily for 4 weeks to
evaluate safety. Ten subjects with age-related forgetfulness received an extract equivalent to
12 mg in a daily dosing regimen for 12 weeks to evaluate efficacy. As a result, no abnormality
was observed and efficacy for age-related decline in cognitive and psychomotor functions was
suggested.

Key Words: astaxanthin, bioreactor, brain function, clinical efficacy, clinical safety

chemically-synthesized version, has therefore been used in

Introduction feed additives as a color enhancer in aquaculture and
chicken egg farms. Recently, attention has been focused on
Astaxanthin (Ax) is a fat-soluble compound classified into the possible use of Ax in human health management,
xanthophylls that are oxygenated derivatives of carotenoids. because of its unique structural and chemical properties,
In nature, Ax is a naturally-occurring red pigment and is including strong antioxidant activity [1–8]. For this market,
widely found in microorganisms (e.g., bacteria, microalgae, a green microalga Haematococcus pluvialis, a red yeast
yeast), crustaceans (e.g., lobster, krill, shrimp), fish (e.g., Phaffia rhodozyma and crustacean byproducts are commer-
salmon, trout) and some birds (e.g., flamingo, quail) [1–4]. cially available as natural sources of the pigment. Of these
Despite the ubiquitous distribution of Ax, animals cannot three sources, H. pluvialis is known to be the richest source
synthesize the carotenoid de novo, while microorganisms of natural Ax (1.5–3.0% of the dry biomass) and various
and plants are able to produce it. Ax, particularly the algal cultivation systems (mainly outdoor systems) have
been developed at an industrial scale [2, 4, 5, 9–11]. As
*To whom correspondence should be addressed.
pointed out by Margalith [12], light intensity and tempera-
Tel: +81-538-32-2407 Fax: +81-538-44-0910 ture are the most critical factors affecting both algal growth
E-mail: ishikuram@yamaha-motor.co.jp and Ax accumulation. He also noted that a selective culture

280
 Toxicity and Efficacy of an Astaxanthin-Rich Algal Extract 281

environment to prevent bacterial and/or protozoal over- Study 2

growth is a key factor to successful algal cultivation, but no An open-label clinical study was conducted using 10
such environment is currently available for this alga. A otherwise healthy male subjects (50–69 years of age) who
culturing technology which enables both higher Ax produc- complained of age-related forgetfulness. The subjects
tion and greater sanitary control is therefore desired. ingested Ax-rich H. pluvialis oil (Puresta®, Yamaha Motor
We have recently developed an indoor cultivation system, Co., Ltd.) equivalent to 12 mg of Ax dialcohol contained in
namely the “YAMAHA High-efficiency Photobioreactor” to soft capsules once daily for 12 weeks. Cognitive function
manufacture Ax-containing H. pluvialis algal biomass. The was evaluated before administration (at baseline) and again
YAMAHA bioreactor is a vertical flat-plate type reactor every 6 weeks during the study, using either the CogHealth
known to have excellent light illumination efficiency, tool (CogState; Melbourne, Australia) or the event-related
leading to superior algal growth [13]. The bioreactors are P300 recognition response elicited by an auditory task [14].
illuminated continuously with strong synthetic light and Differences between baseline and post-baseline values were
maintained at a desired temperature to maximize Ax analyzed using Dunnett’s multiple comparison test, where
accumulation in algal cells. In order to minimize the risk of p<0.05 was considered statistically significant and p<0.1
contamination, the reactors are isolated in a Class 10,000 was considered as tendency toward significance.
cleanroom; cultivation and all operations are performed in
this environment. High-quality and high-quantity Ax- Results
containing algal oil has thus been produced using these
bioreactors since October 2006, and the manufacturing Study 1
process was given GMP certification by the Japanese The results of the blood pressure and hematological
Institute for Health Food Standards (JIHFS) in July 2007. examinations are shown in Table 1. Findings from the blood
In the present study, we investigated the clinical safety of biochemical examinations are shown in Table 2. Among
this new Ax-rich oil, since it is qualitatively and quantita- vital signs and laboratory analysis (hematology, hepatic, and
tively different from other existing Ax-containing H. pluvialis renal function tests) changes from baseline, no statistically
oil products. Possible efficacy in the treatment of age-related significant changes were noted for any of the dose groups.
forgetfulness was also studied. There were no adverse effects or laboratory abnormalities
observed for any dose group. In addition, no adverse event
Methods attributed to the administration of the test material was
reported in the subject interviews expect for ‘red stool’,
Clinical study design which was associated with the color of the test material.
All study protocols and informed consent documents We then attempted to predict clinical efficacy of Ax in the
were reviewed and approved by the ethics committee of treatment of metabolic syndrome. Subjects whose starting
the Yamaha Motor Co., Ltd. and Anti-Aging Science. All systolic blood pressure (SBP), diastolic blood pressure
study subjects provided written informed consent prior to (DBP), triglyceride (TG) and fasting glucose (FG) values
the start of study participation. The protocols were carried meet the diagnosis of metabolic syndrome (SBP≥130
out under the provisions of the Declaration of Helsinki. mmHg, DBP≥85 mmHg, TG≥150 mg/dl, and FG≥100 mg/dl;
[15]) were selected from Group A. Mean baseline values
Study 1 were calculated and compared to those after treatment
An open-label clinical study was conducted in 127 (Fig. 1). As shown in Fig. 1A, a significant (p<0.01)
subjects between 20 to 60 years of age. The subjects were decrease in SBP was observed when subjects were admin-
divided into three groups, low-dose (Group A: 51 men, 22 istered 4 mg Ax orally once daily for 4 weeks. No such
women), middle-dose (Group B: 21 men, 17 women), and decrease was apparent in DBP (Fig. 1B). The mean TG
high-dose (Group C: 12 men, 4 women). The subjects value after treatment (218 mg/dl) was much lower than the
ingested Ax-rich H. pluvialis oil (Puresta®, Yamaha Motor baseline value (292 mg/dl), although there was no statistical
Co., Ltd.; Iwata, Shizuoka, Japan) equivalent to 4 mg significance between them (Fig. 1C). Fasting glucose
(Group A), 8 mg (Group B), or 20 mg (Group C) of Ax decreased with a tendency toward significance (p<0.1) after
dialcohol contained in soft capsules, once daily for 4 weeks. treatment (Fig. 1D).
Blood biochemical and hematological examinations were
completed before (baseline) and after 4 weeks of dosing. Study 2
Differences between baseline and post-dosing values were In the present study, we investigated the effect of Ax on
tested using Wilcoxon’s Signed Rank test (using SPSS for brain function in healthy older adults with age-related
Windows; SAS Institute, Cary, NC), where p<0.1 was forgetfulness using the CogHealth test, since CogHealth is a
considered statistically significant. cognitive function test specifically designed to detect changes

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282 A. Satoh et al.

Table 1. Changes in blood pressure and hematological para- Table 2. Changes in blood biochemical parameters
meters (mean ± SD) before and after administration of (mean ± SD) before and after administration of Pur-
Puresta® esta®
Parameter Group A Group B Group C Parameter Group A Group B Group C
HBP (mm Hg) BUN (mg/dl)
before 121 ± 14 118 ± 14 119 ± 17 before 12.7 ± 3.1 12.2 ± 2.5 12.1 ± 1.3
after 125 ± 12 122 ± 15 116 ± 13 after 13.3 ± 3.1 12.6 ± 2.7 12.6 ± 1.7
LBP (mm Hg) Creatinine (mg/dl)
before 76 ± 12 74 ± 12 72 ± 14 before 0.8 ± 0.1 0.8 ± 0.2 0.8 ± 0.1
after 78 ± 12 74 ± 13 68 ± 11 after 0.9 ± 0.1 0.9 ± 0.2 0.9 ± 0.1
WBC (×109/liter) Fasting glucose (mg/dl)
before 5.7 ± 1.7 6.2 ± 1.6 5.5 ± 1.6 before 92 ± 13 95 ± 19 85 ± 12
after 6.2 ± 2.1 6.0 ± 1.7 5.3 ± 0.8 after 93 ± 9 95 ± 15 91 ± 9
RBC (×1012/liter) AST (U/liter)
before 4.7 ± 0.4 4.8 ± 0.6 4.8 ± 0.4 before 23 ± 8 22 ± 5 24 ± 8
after 4.8 ± 0.6 4.9 ± 0.5 4.9 ± 0.5 after 22 ± 8 22 ± 7 22 ± 8
Hb (g/liter) ALT (U/liter)
before 144 ± 15 137 ± 21 148 ± 12 before 25 ± 14 24 ± 13 32 ± 17
after 146 ± 15 139 ± 21 150 ± 13 after 25 ± 13 23 ± 12 31 ± 21
Hct (%) γ-GTP (U/liter)
before 45 ± 4 43 ± 5 45 ± 3 before 43 ± 33 34 ± 26 37 ± 23
after 46 ± 4 44 ± 5 46 ± 3 after 46 ± 38 33 ± 22 33 ± 20
MCV (fL) Group A = Puresta dose equivalent to 4 mg Ax/day.
before 94 ± 6 90 ± 7 95 ± 4 Group B = Puresta dose equivalent to 8 mg Ax/day.
after 95 ± 6 91 ± 7 95 ± 4 Group C = Puresta dose equivalent to 20 mg Ax/day.
MCH (pg) No significant differences noted from baseline to end of treatment
before 30.5 ± 2.4 28.9 ± 3.2 30.7 ± 1.6 for any of the treatment groups.
after 30.2 ± 2.4 28.7 ± 3.3 30.6 ± 1.7
MCHC (%)
before 32 ± 1 32 ± 2 33 ± 1
after 32 ± 1 32 ± 2 32 ± 1 in the table. Significant reduction in response time was
Platelets (×109/liter) apparent for the ‘divided attention’ task after 6 weeks and on
before 258 ± 50 279 ± 59 237 ± 52
all tasks after 12 weeks. The accuracy on the ‘working
memory’ task was improved significantly after 12 weeks of
after 256 ± 51 280 ± 58 239 ± 56
treatment, while no such effect was observed for the
Group A = Puresta dose equivalent to 4 mg Ax/day, Group ‘delayed recall’ task.
B = Puresta dose equivalent to 8 mg Ax/day, Group C = Puresta We then evaluated cognitive function using an event-
dose equivalent to 20 mg Ax/day. related potential, the P300, which reflects processes
No significant differences noted from baseline to end of treatment
involved in memory, response modulation, and contextual
for any of the treatment groups.
updating, and has been used to evaluate mental workload
[17, 18]. The mean P300 latency and amplitude values at
baseline and after 12 weeks of treatment are shown in
in healthy or mildly-impaired subjects at an early date [16]. Table 4. The P300 peak amplitude tended to increase (p<0.1)
The test evaluates five cognitive domains—psychomotor after 12 weeks, while no such change was apparent in the
speed, impulse control, working memory, episodic learning, latency. It is thought that the amplitude and latency reflect
and attention—by measuring the subject’s response times capacity and speed, respectively, of cognitive information
(RTs) and accuracy (i.e., the hit rate percentage). Table 3 processing [19], and that both parameters closely relate to
represents mean response time on the following CogHealth selective attention [20]. The improving effect of Ax on the
tasks—simple reaction, choice reaction, working memory, amplitude of P300 therefore indicates that this carotenoid
delayed recall, and divided attention—at baseline, and after might increase patient information processing capacity and
6 and 12 weeks of Ax treatment. The mean accuracy of the selective attention.
‘working memory’ and ‘delayed recall’ tasks is also shown

J. Clin. Biochem. Nutr.

 Toxicity and Efficacy of an Astaxanthin-Rich Algal Extract 283

Discussion

In the present study, we found that no abnormality was

apparent when adult subjects ingested Ax-containing H.
pluvialis oil product, Puresta®, up to 20 mg (equivalent to
Ax dialcohol) once daily for 4 weeks. The safety of Puresta®
has been investigated previously in a clinical study where 17
subjects were administered Puresta® amounts equivalent to
8 mg of Ax dialcohol twice daily for 12 weeks [21]. Our
current findings further support the safety of this product at
a dose of 20 mg/day, with no negative effects on blood
chemistry or hematology results over time, and no subject
reports of adverse experiences during treatment. We also
found positive effects of this product on metabolic syndrome
and cognitive function.
Metabolic syndrome is a common growing problem in
human health in industrialized countries. Hussen reported
that administration of astaxanthin decreased the blood
pressure of the SHR rat (a hypertensive rat model) [22].
However our study is the first instance to show efficacy in
humans. We observed previously that HbA1c and TNF-α
levels decreased significantly, while adiponectin levels
increased significantly when volunteers with borderline
metabolic syndrome were administered Puresta® equivalent
to 8 mg of Ax dialcohol twice daily for 12 weeks [21]. The
decreases in systolic blood pressure, triglyceride, and fasting
Fig. 1. Changes in systolic blood pressure (A), diastolic blood
pressure (B), triglyceride (C), and fasting glucose (D)
Table 4. Mean ( ± SD) P300 latency and amplitude values at
values before and after oral administration of Puresta
baseline and after 12 weeks of Ax treatment.
equivalent to 4 mg of Ax dialcohol once daily for 4
weeks. The number of subjects per group was 20, 16, 12, Mean ± SD
Parameter
and 10 in Groups A, B, C, and D, respectively. Error Baseline 12 weeks
bars represents standard deviations. *** and * represent Latency (ms) 359.40 ± 16.49 363.10 ± 29.22
p<0.01 and p<0.1, respectively.
Amplitude (μV) 7.60 ± 4.05 10.54 ± 3.39*
* p<0.1 vs baseline

Table 3. Mean response times and accuracies (±SD) on CogHealth tasks at baseline, and after 6 and 12 weeks of Ax treatment.
Mean ± SD
Tasks
Baseline 6 weeks 12 weeks
Response time (ms)
Simple Reaction 341.68 ± 94.41 303.31 ± 33.80 281.76 ± 33.56**
Choice Reaction 504.53 ± 56.84 480.63 ± 39.87 463.63 ± 26.49**
Divided Attention 494.13 ± 135.57 419.52 ± 59.32** 412.07 ± 51.97**
Working Memory 762.94 ± 141.65 732.95 ± 174.83 654.83 ± 128.42**
Delayed Recall 1008.19 ± 153.37 975.40 ± 190.75 916.77 ± 151.04**
Accuracy (%)
Working Memory 90.46 ± 7.18 95.22 ± 5.37 96.30 ± 3.94**
Delayed Recall 70.95 ± 6.42 71.19 ± 5.98 70.71 ± 8.91
** p<0.05 vs baseline.

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284 A. Satoh et al.

glucose values observed in the present study may support Outdoor cultivation of microalgae for carotenoid production:
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J. Clin. Biochem. Nutr.