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Potential of Chlorella as a Dietary Supplement to Promote Human Health

MDPI
Nutrients
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Chlorella is a green unicellular alga that is commercially produced and distributed worldwide as a dietary supplement. Chlorella products contain numerous nutrients and vitamins, including D and B12, that are absent in plant-derived food sources. Chlorella contains larger amounts of folate and iron than other plant-derived foods. Chlorella supplementation to mammals, including humans, has been reported to exhibit various pharmacological activities, including immunomodulatory, antioxidant, antidiabetic, antihypertensive, and antihyperlipidemic activities. Meta-analysis on the effects of Chlorella supplementation on cardiovascular risk factors have suggested that it improves total cholesterol levels, low-density lipoprotein cholesterol levels, systolic blood pressure, diastolic blood pressure, and fasting blood glucose levels but not triglycerides and high-density lipoprotein cholesterol levels. These beneficial effects of Chlorella might be due to synergism between multiple nutrient and antioxidant compounds. However, information regarding the bioactive compounds in Chlorella is limited.
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nutrients
Review
Potential of Chlorella as a Dietary Supplement to
Promote Human Health
Tomohiro Bito 1, Eri Okumura 2, Masaki Fujishima 2and Fumio Watanabe 1,*
1Department of Agricultural, Life and Environmental Sciences, Faculty of Agriculture, Tottori University,
Tottori 680-8553, Japan; bito@tottori-u.ac.jp
2Sun Chlorella Corporation, Kyoto 600-8177, Japan; okumura@sunchlorella.co.jp (E.O.);
mfujishima@sunchlorella.co.jp (M.F.)
*Correspondence: watanabe@tottori-u.ac.jp; Tel.: +81-857-31-5412
Received: 26 May 2020; Accepted: 17 August 2020; Published: 20 August 2020


Abstract:
Chlorella is a green unicellular alga that is commercially produced and distributed worldwide
as a dietary supplement. Chlorella products contain numerous nutrients and vitamins, including D
and B
12
, that are absent in plant-derived food sources. Chlorella contains larger amounts of folate
and iron than other plant-derived foods. Chlorella supplementation to mammals, including humans,
has been reported to exhibit various pharmacological activities, including immunomodulatory,
antioxidant, antidiabetic, antihypertensive, and antihyperlipidemic activities. Meta-analysis on the
eects of Chlorella supplementation on cardiovascular risk factors have suggested that it improves total
cholesterol levels, low-density lipoprotein cholesterol levels, systolic blood pressure,
diastolic blood
pressure, and fasting blood glucose levels but not triglycerides and high-density lipoprotein cholesterol
levels. These beneficial eects of Chlorella might be due to synergism between multiple nutrient
and antioxidant compounds. However, information regarding the bioactive compounds in Chlorella
is limited.
Keywords:
antioxidants; Chlorella; dietary fibers; dietary supplements; folate; lutein; vitamin B
12
;
vitamin D2
1. Introduction
Microalgae are primarily found in aquatic ecosystems, living in both seawater and freshwater,
and are photosynthetic eukaryotic organisms that contain chloroplasts and nuclei,
similar to plants
.
Microalgae more eciently yield biomass than land-based plants owing to their higher performance
in utilizing sunlight and CO
2
, leading to their extremely high growth rates [
1
]. Therefore,
microalgae have
been used in the food, pharmaceutical, and cosmetic industries,
and their
pigments,
nutrients,
bioactive compounds
and whole biomass are already in use worldwide. Recently,
various bioactive compounds and nutrients have been detected in both seawater and freshwater
microalgae,
including cyanobacteria
. These compounds and nutrients have been reported to promote
human health [
1
,
2
]. However, there is limited information regarding the bioactive compounds of
freshwater-living Chlorella species, which are classified as green algae.
Chlorella species can be mass-cultured, and their dietary supplement products are commercially
available worldwide. However, the commercial cultivation of their biomass has started only several
years ago. Chlorella vulgaris was discovered and reported in 1890 by Dr. Martinus Willem Beijerinck,
a famous microbiologist and botanist [
3
]. Another Chlorella species, distinguished by the presence
pyrenoids in chloroplasts, was identified and accordingly named C. pyrenoidosa in 1903 [
4
]. Since then,
more than 20 Chlorella species have been characterized, with over 100 strains described [
5
]. At present,
Chlorella species are divided into three varieties: C. vulgaris, C. lobophora, and C. sorokiniana [
6
].
Nutrients 2020,12, 2524; doi:10.3390/nu12092524 www.mdpi.com/journal/nutrients
Nutrients 2020,12, 2524 2 of 22
C. sorokiniana
is a sub-species first isolated in 1953 by Sorokin and originally thought to be a
thermotolerant mutant of C. pyrenoidosa [
7
,
8
]. C. pyrenoidosa, the subject of many scientific studies,
is now called C. sorokiniana.
Investigations of the dietary value of Chlorella in human health began in the early 1950s,
when the
use of Chlorella as a food source was initiated in the midst of a global food crisis [
9
]. Chlorella was
first produced and consumed in Asia, mainly in Japan, and then used as a dietary supplement
worldwide [
10
]. Chlorella is produced commercially for use in foods and as a source of its intrinsic
compounds. Using large-scale cultivation technology, C. vulgaris and C. pyrenoidosa are prepared as
commercial sources for dietary supplements [
11
]. Studies have shown that Chlorella cells contain
a variety of nutrients and bioactive compounds that promote human health and prevent certain
diseases [
10
,
12
], suggesting that Chlorella-derived natural compounds might provide substitutes for
synthetic compounds or drugs. The content of natural compounds in Chlorella diers greatly between
culture conditions and Chlorella species [13,14].
Here, we present updated information on the Chlorella content of nutrients and bioactive
compounds that promote human health. However, at present, there is limited information available
regarding the bioactive compounds responsible for its pharmacological activities, which might be due
to the synergistic eects of various nutrients and antioxidant compounds in Chlorella.
2. Nutrients in Commercial Chlorella Products
2.1. Macronutrients
The macronutrient content of 13 commercially available Chlorella products, based on information
provided on the packaging label, are summarized in Table 1. Humans cannot digest Chlorella cells
in their natural state because their cell walls are made of cellulose. Therefore, Chlorella cell walls are
mechanically broken down in most dietary supplements. An animal study has shown that more than
80% of Chlorella proteins are digestible [15].
Table 1. Nutrient content of 13 commercially available Chlorella products.
Macronutrients (Per
100 g Dry Weight) A B C D E F G H I J K L M
Proteins (g)
50–65
61 63 65 57
50
56–72 50–67 50–70 62 60 58 57
Fats (g) 7–14 10 13 12 11 7–20 5–15 8–15 11 10 10 12
Carbohydrates (g) 15 11 5–23 8–42 8–20 20 18
Sugars (g) 5–21 7 5
0–1
0–5 2–23
1–10
11
Dietary fibers (g) 7–14 11 10 11 11 5–18 7–18
8–16
10
Remarks
*1
*2
78%
*1
*4*2*2
*1
*2
77–82%
*3
*2
75–85%
*3
*1
*283%
*3
*2
82%
*3*4*1
*3
*4
*1Cell walls disrupted; *2digestibility of proteins; *3contains Chlorella extract; *4C. pyrenoidosa.
These Chlorella products contain a large amount of proteins (approximately 59% based on dry
weight), coinciding with the analytical data of the protein contents of C. pyrenoidosa (57%) [
16
] and
C. vulgaris
(51–58%) [
17
]. This protein content is higher than that of soybeans (approximately 33%,
dry weight
). The amino acid composition of Chlorella products C and M are shown in Table 2.
These amino
acid profiles indicate that all essential amino acids for humans (isoleucine, leucine, lysine,
methionine, phenylalanine, threonine, tryptophan, valine, and histidine) are present in substantial
concentrations in these products. According to the essential amino acid index (EAAI) used to
evaluate protein quality for human nutrition, the quality of C. pyrenoidosa (EAAI, 1.35) [
18
] and a
commercially available Chlorella product (EAAI, 0.92) [
19
] are higher than that of soybean protein
(EAAI, 0.66) [
18
]. These results indicate that proteins in Chlorella products are of high or good quality.
Nutrients 2020,12, 2524 3 of 22
Notably,
Chlorella products
contain a considerable amount of arginine (approximately 3200 mg/100
g dry weight), which serves as a substrate for the production of NO, a potent intracellular signaling
molecule that influences every mammalian system [
20
]. Arginine also serves as a potent modulator of
immune functions [21].
Table 2. Amino acid content of commercially available Chlorella products C and M.
Amino Acids (mg/100 g Dry Weight) C M
Essential
Isoleucine 1820 2030
Leucine 4180 4480
Lysine 4659 3140
Methionine 1009 1240
Phenylalanine 2230 2580
Threonine 2209 2490
Tryptophan 1030 1090
Valine 2780 3090
Histidine 1141 1040
Non-essential
Tyrosine 1720 1940
Cystine 659 650
Aspartic acid 4469 4710
Serine 1930 2120
Glutamic acid 6209 6030
Proline 2320 2560
Glycine 2859 2990
Alanine 4009 4170
Arginine 3109 3260
Approximately 17% (dry weight) of carbohydrates are found in the commercially available
Chlorella products. Similar results have been reported for C. vulgaris [
17
]. As shown in Table 1,
more than
65% of the carbohydrate is dietary fiber, which appears to be derived from the Chlorella cell
wall. Various polysaccharides have been extracted and characterized [
22
25
]. Chlorella polysaccharides
exhibited a variety of biologically active compounds, including antioxidants [
24
] and stimulators of
plant growth [
25
]. Tabarsa et al. [
26
] characterized an immune-enhancing water-soluble
α
-glucan
prepared from C. vulgaris.
Commercially available Chlorella products contain a small amount of fats (approximately 11%,
dry weight
) (Table 1), which coincides with the analytical data of the fat content of C. vulgaris
(14–22%) [
17
]. Chlorella products contain
α
-linolenic acid (approximately 10–16% of total fatty acids)
and linoleic acid (approximately 18% of total fatty acids) but not eicosapentaenoic acid, docosahexenoic
acid, or arachidonic acid [
19
,
27
]. Approximately 65–70% of the total fatty acids found in commercially
available Chlorella products are derived from polyunsaturated fatty acids [19,27].
Dierent growth conditions, such as temperature, nutrient composition, and light availability,
can readily
alter the levels of biomass, macro- and micronutrients, and other valuable bioactive
compounds, including antioxidants, in Chlorella cells [2830].
2.2. Micronutrients
2.2.1. Vitamins
As shown in Table 3, commercially available Chlorella products contain all the vitamins required
by humans, i.e., B
1
, B
2
, B
6
, B
12
, niacin, folate, biotin, pantothenic acid, C, D
2
, E, and K, and
α
- and
β
-carotenes. Chlorella products contain substantial amounts of vitamins D
2
and B
12
, both of which
are well known to be absent in plants. Commercially available Chlorella (C. vulgaris) products contain
Nutrients 2020,12, 2524 4 of 22
higher amounts of folate (approximately 2.5 mg/100 g dry weight) than spinach [
31
]. Vitamin B
12
and
folate deficiencies induce the accumulation of serum homocysteine, which is involved in cardiovascular
diseases. In this section, we discuss vitamin D2, vitamin B12, and folate.
Table 3. Content of vitamins and related compounds in 13 commercially available Chlorella products.
Vitamins (Per 100 g
Dry Weight) A B C D E F G H I J K L M
Vitamin B1(mg) 1.9 2.5 6.5 1.0–3.0 1.0–3.0 1.8 1.6
Vitamin B2(mg) 3–8 5.6 5.0 5.7 5.5 2.0–9.0
4.0–9.0
4.0–8.0 5.0
5.0
4.8
Vitamin B6(mg) 0.9 2.5 1.7 1.0–3.0 1.0–3.0 1.0–3.0 1.8
Vitamin B12 (µg) 20.0 6.0–30.0
200.0–500.0
230.0
Niacin (mg) 20.4 50.0 40.0–80.0
20.0–50.0
10.0–40.0 45.9
Folate (mg) 0.3 2.0 1.2–3.6 1.4
Biotin (µg) 227.0
Pantothenic acid (mg) 1.0–6.0 1.8
Vitamin C (mg) 7.0 50.0 30.0 10.0–200.0 14.0
Vitamin D2(mg) 1.4
Vitamin E (mg) 3.0 25.0 10.0–45.0 6.2
Vitamin K (mg) 1.4 1.1 0.3 0.5–3.5 *11.2 *1
Carotenoids (mg) 25.0 *236.0–150.0 *2
100.0–500.0
31.5 *3
*1Vitamin K1(mg), *2β-Carotene (mg), *3α-Carotene +β-Carotene (mg).
Vitamin D, a major regulator of calcium absorption, reduces the risk of osteomalacia in adults
and rickets in children [
32
]. The two main dietary forms of vitamin D are vitamin D
2
and D
3
,
which are found in fungi such as mushrooms [
33
,
34
] and animal-derived foods such as fish and fish
products [
35
], respectively. Mushrooms have the ability to synthesize ergosterol (known as provitamin
D
2
), which is converted into ergocalciferol as vitamin D
2
upon ultraviolet irradiation [
34
,
36
]. Thus,
ultraviolet-irradiated mushrooms are suitable for use as vitamin D
2
sources in strict vegetarians [
36
].
Cell walls of mushrooms contain high concentrations of ergosterol, which plays a physiological role
in modulating cell membrane strength and fluidity similar to cholesterol in animals [
37
]. Sun-dried,
commercially available mushrooms reportedly contain approximately 17
µ
g of vitamin D
2
per g dry
weight [38]. The bioavailability of vitamin D2from mushrooms has been studied in humans [39,40].
Ergosterol was first reported as the main sterol compound in C. pyrenoidosa in the early 1950
S
[
41
].
C. vulgaris also contains a substantial amount of ergosterol [
42
,
43
]. Our unpublished data show that one
commercially available Chlorella product contains both ergosterol (1.68 mg/g dry weight) and vitamin
D
2
(15.2
µ
g/g dry weight), similar in amounts to those in sun-dried mushrooms. The vitamin D
2
in this
Chlorella product is synthesized from ergosterol upon exposure to sunlight during cultivation (Figure 1).
Although it has been reported that vitamin D
3
is more eective than vitamin D
2
at increasing the
concentration of circulating 25-hydroxyvitamin D [
44
], Chlorella products and sun-dried mushrooms
could become sources of vitamin D for vegetarians.
Nutrients 2020, 12, x FOR PEER REVIEW 4 of 21
Table 3. Content of vitamins and related compounds in 13 commercially available Chlorella products.
Vitamins
(Per 100 g Dry Weight) A B C D E F G H I J K L M
Vitamin B
1
(mg) 1.9 2.5 6.5 1.0–3.0 1.0–3.0 1.8 1.6
Vitamin B
2
(mg) 3–8 5.6 5.0 5.7 5.5 2.0–9.0 4.0–9.0 4.0–8.0 5.0 5.0 4.8
Vitamin B
6
(mg) 0.9 2.5 1.7 1.0–3.0 1.0–3.0 1.0–3.0 1.8
Vitamin B
12
(μg) 20.0 6.0–30.0 200.0–500.0 230.0
Niacin (mg) 20.4 50.0 40.0–80.0 20.0–50.0 10.0–40.0 45.9
Folate (mg) 0.3 2.0 1.2–3.6 1.4
Biotin (μg) 227.0
Pantothenic acid (mg) 1.0–6.0 1.8
Vitamin C (mg) 7.0 50.0 30.0 10.0–200.0 14.0
Vitamin D
2
(mg) 1.4
Vitamin E (mg) 3.0 25.0 10.0–45.0 6.2
Vitamin K (mg) 1.4 1.1 0.3 0.5–3.5 *
1
1.2 *
1
Carotenoids (mg) 25.0 *
2
36.0–150.0 *
2
100.0–500.0 31.5 *
3
*
1
Vitamin K
1
(mg), *
2
β-Carotene (mg), *
3
α-Carotene + β-Carotene (mg).
Vitamin D, a major regulator of calcium absorption, reduces the risk of osteomalacia in adults
and rickets in children [32]. The two main dietary forms of vitamin D are vitamin D
2
and D
3
, which
are found in fungi such as mushrooms [33,34] and animal-derived foods such as fish and fish
products [35], respectively. Mushrooms have the ability to synthesize ergosterol (known as
provitamin D
2
), which is converted into ergocalciferol as vitamin D
2
upon ultraviolet irradiation
[34,36]. Thus, ultraviolet-irradiated mushrooms are suitable for use as vitamin D
2
sources in strict
vegetarians [36]. Cell walls of mushrooms contain high concentrations of ergosterol, which plays a
physiological role in modulating cell membrane strength and fluidity similar to cholesterol in animals
[37]. Sun-dried, commercially available mushrooms reportedly contain approximately 17 μg of
vitamin D
2
per g dry weight [38]. The bioavailability of vitamin D
2
from mushrooms has been studied
in humans [39,40].
Ergosterol was first reported as the main sterol compound in C. pyrenoidosa in the early 1950
S
[41]. C. vulgaris also contains a substantial amount of ergosterol [42,43]. Our unpublished data show
that one commercially available Chlorella product contains both ergosterol (1.68 mg/g dry weight)
and vitamin D
2
(15.2 μg/g dry weight), similar in amounts to those in sun-dried mushrooms. The
vitamin D
2
in this Chlorella product is synthesized from ergosterol upon exposure to sunlight during
cultivation (Figure 1). Although it has been reported that vitamin D
3
is more effective than vitamin
D
2
at increasing the concentration of circulating 25-hydroxyvitamin D [44], Chlorella products and
sun-dried mushrooms could become sources of vitamin D for vegetarians.
Figure 1. Structures of provitamin D
2
and vitamin D
2
found in commercially available Chlorella
products.
Serum homocysteine (Hcy) is an established biomarker of cardiovascular disease in humans
[45,46]. Hcy is a non-protein forming amino acid (Figure 2) produced as an intermediate compound
of methionine metabolism and is further metabolized to cystathionine via cystathionine β-synthetase,
a vitamin B
6
-dependent enzyme [46]. Alternatively, Hcy can be remethylated back to methionine by
Figure 1.
Structures of provitamin D
2
and vitamin D
2
found in commercially available Chlorella products.
Serum homocysteine (Hcy) is an established biomarker of cardiovascular disease in humans [
45
,
46
].
Hcy is a non-protein forming amino acid (Figure 2) produced as an intermediate compound of
methionine metabolism and is further metabolized to cystathionine via cystathionine
β
-synthetase,
a vitamin B6-dependent
enzyme [
46
]. Alternatively, Hcy can be remethylated back to methionine by
Nutrients 2020,12, 2524 5 of 22
methionine synthase, a vitamin B
12
-dependent enzyme. Folate is also required for the remethylation
of Hcy by providing 5-methyltetrahydrofolate. Deficiencies in vitamin B
12
[
47
], vitamin B
6
[
48
],
and folate [
49
] cause hyper-homocysteinemia. Several clinical studies report a correlation between
atherosclerosis and deficiencies in vitamin B
12
and folate [
50
,
51
]. Folate deficiency in women before
and during pregnancy is associated with neural tube defects in newborns [
52
]. Plants can synthesize
folate compounds de novo, but animals cannot [53]. Thus, plant-derived foods are sources of dietary
folates for humans. High concentrations of folate (approximately 1.69–2.45 mg/100 g dry weight) are
reported in commercially available Chlorella (C. vulgaris) products [
31
], with concentrations similar to
those of the products shown in Table 3(0.3–3.6 mg/100 g dry weight). The main folate compounds
identified in Chlorella products are 5-CHO-H
4
folate (60–62%) and 5-CH
3
-H
4
folate (24–26%) and
the minor folate compounds are 10-CHO-folate (5–7%), H
4
folate (4%), and fully oxidized folate
(3–6%) [
31
].
The chemical
structures of the Chlorella folate compounds are shown in Figure 3. The main
dietary sources of folates are vegetables (25%), bread and cereal products (22%), dairy products (10%),
fruit (10%)
, and oils and fats (5%) [
31
]. Spinach has high folate content (165
µ
g/100 g fresh weight;
1.7 mg/100 g
dry weight) [
31
,
54
], which is similar to that of Chlorella products. Thus, Chlorella products
are an excellent source of folate for humans.
Nutrients 2020, 12, x FOR PEER REVIEW 5 of 21
methionine synthase, a vitamin B
12
-dependent enzyme. Folate is also required for the remethylation
of Hcy by providing 5-methyltetrahydrofolate. Deficiencies in vitamin B
12
[47], vitamin B
6
[48], and
folate [49] cause hyper-homocysteinemia. Several clinical studies report a correlation between
atherosclerosis and deficiencies in vitamin B
12
and folate [50,51]. Folate deficiency in women before
and during pregnancy is associated with neural tube defects in newborns [52]. Plants can synthesize
folate compounds de novo, but animals cannot [53]. Thus, plant-derived foods are sources of dietary
folates for humans. High concentrations of folate (approximately 1.692.45 mg/100 g dry weight) are
reported in commercially available Chlorella (C. vulgaris) products [31], with concentrations similar to
those of the products shown in Table 3 (0.3–3.6 mg/100 g dry weight). The main folate compounds
identified in Chlorella products are 5-CHO-H
4
folate (60–62%) and 5-CH
3
-H
4
folate (2426%) and the
minor folate compounds are 10-CHO-folate (5–7%), H
4
folate (4%), and fully oxidized folate (3–6%)
[31]. The chemical structures of the Chlorella folate compounds are shown in Figure 3. The main
dietary sources of folates are vegetables (25%), bread and cereal products (22%), dairy products
(10%), fruit (10%), and oils and fats (5%) [31]. Spinach has high folate content (165 μg/100 g fresh
weight; 1.7 mg/100 g dry weight) [31,54], which is similar to that of Chlorella products. Thus, Chlorella
products are an excellent source of folate for humans.
Figure 2. Homocysteine metabolic pathway in mammals. Abbreviations: B
6
, vitamin B
6
; B
12
, vitamin
B
12
; CBS, cystathionine β-synthetase; DHF, dihydrofolate; MS, cobalamin-dependent methionine
synthase; SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine; THF, tetrahydrofolate.
Figure 3. Chemical structures of folate compounds found in commercially available Chlorella
products.
Vitamin B
12
(B
12
) is synthesized by certain bacteria and archaea but not by plants [55]. Animal-
derived foods, such as meats, milk, fish, and shellfish, are the major dietary sources of B
12
for humans
Figure 2. Homocysteine metabolic pathway in mammals. Abbreviations: B6, vitamin B6; B12, vitamin B12;
CBS, cystathionine
β
-synthetase; DHF, dihydrofolate; MS, cobalamin-dependent methionine synthase;
SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine; THF, tetrahydrofolate.
Nutrients 2020, 12, x FOR PEER REVIEW 5 of 21
methionine synthase, a vitamin B
12
-dependent enzyme. Folate is also required for the remethylation
of Hcy by providing 5-methyltetrahydrofolate. Deficiencies in vitamin B
12
[47], vitamin B
6
[48], and
folate [49] cause hyper-homocysteinemia. Several clinical studies report a correlation between
atherosclerosis and deficiencies in vitamin B
12
and folate [50,51]. Folate deficiency in women before
and during pregnancy is associated with neural tube defects in newborns [52]. Plants can synthesize
folate compounds de novo, but animals cannot [53]. Thus, plant-derived foods are sources of dietary
folates for humans. High concentrations of folate (approximately 1.692.45 mg/100 g dry weight) are
reported in commercially available Chlorella (C. vulgaris) products [31], with concentrations similar to
those of the products shown in Table 3 (0.3–3.6 mg/100 g dry weight). The main folate compounds
identified in Chlorella products are 5-CHO-H
4
folate (60–62%) and 5-CH
3
-H
4
folate (2426%) and the
minor folate compounds are 10-CHO-folate (5–7%), H
4
folate (4%), and fully oxidized folate (3–6%)
[31]. The chemical structures of the Chlorella folate compounds are shown in Figure 3. The main
dietary sources of folates are vegetables (25%), bread and cereal products (22%), dairy products
(10%), fruit (10%), and oils and fats (5%) [31]. Spinach has high folate content (165 μg/100 g fresh
weight; 1.7 mg/100 g dry weight) [31,54], which is similar to that of Chlorella products. Thus, Chlorella
products are an excellent source of folate for humans.
Figure 2. Homocysteine metabolic pathway in mammals. Abbreviations: B
6
, vitamin B
6
; B
12
, vitamin
B
12
; CBS, cystathionine β-synthetase; DHF, dihydrofolate; MS, cobalamin-dependent methionine
synthase; SAM, S-adenosyl methionine; SAH, S-adenosyl homocysteine; THF, tetrahydrofolate.
Figure 3. Chemical structures of folate compounds found in commercially available Chlorella
products.
Vitamin B
12
(B
12
) is synthesized by certain bacteria and archaea but not by plants [55]. Animal-
derived foods, such as meats, milk, fish, and shellfish, are the major dietary sources of B
12
for humans
Figure 3.
Chemical structures of folate compounds found in commercially available Chlorella products.
Vitamin B
12
(B
12
) is synthesized by certain bacteria and archaea but not by plants [
55
].
Animal-derived foods, such as meats, milk, fish, and shellfish, are the major dietary sources
Nutrients 2020,12, 2524 6 of 22
of B
12
for humans [
56
]. B
12
content is high in beef, pork, and chicken livers (approximately
25–53 µg/100 g
fresh weight) [
56
] and in edible bivalves such as clams (approximately
60 µg/100 g
fresh weight) [
57
]. The reported B
12
content of Chlorella products varies from <0.1 to 400
µ
g per
100 g of dry weight [58,59], consistent with that of the products shown in Table 3(6–500 µg/100 g dry
weight). Among Chlorella species, the B
12
content is much higher in C. pyrenoidosa than in C. vulgaris
when grown under open culture conditions [
59
]. B
12
is not essential for the growth of these Chlorella
species [
59
,
60
], suggesting that Chlorella cells absorb and accumulate large amounts of exogenous
B
12
. Some of the high B
12
-containing Chlorella products contain inactive corrinoid compounds such as
5-methoxybenzimidazolylcobamide and cobalt-free corrinoid (Figure 4). Thus, if Chlorella products
with high B
12
are consumed as a sole B
12
source, accurate content estimation requires the identification
of B12 compounds using liquid chromatography–tandem mass spectrometry [59].
Nutrients 2020, 12, x FOR PEER REVIEW 6 of 21
[56]. B
12
content is high in beef, pork, and chicken livers (approximately 2553 μg/100 g fresh weight)
[56] and in edible bivalves such as clams (approximately 60 μg/100 g fresh weight) [57]. The reported
B
12
content of Chlorella products varies from <0.1 to 400 μg per 100 g of dry weight [58,59], consistent
with that of the products shown in Table 3 (6–500 μg/100 g dry weight). Among Chlorella species, the
B
12
content is much higher in C. pyrenoidosa than in C. vulgaris when grown under open culture
conditions [59]. B
12
is not essential for the growth of these Chlorella species [59,60], suggesting that
Chlorella cells absorb and accumulate large amounts of exogenous B
12
. Some of the high B
12
-containing
Chlorella products contain inactive corrinoid compounds such as 5-methoxybenzimidazolylcobamide
and cobalt-free corrinoid (Figure 4). Thus, if Chlorella products with high B
12
are consumed as a sole
B
12
source, accurate content estimation requires the identification of B
12
compounds using liquid
chromatography–tandem mass spectrometry [59].
Rauma et al. [61] demonstrated that substantial consumption of Chlorella products can supply
adequate amounts of B
12
. Another study of strict vegetarians (vegans) with an elevated baseline of
serum methylmalonic acid (as an index of B
12
deficiency) showed that ingestion of 9 g of C. pyrenoidosa
daily for 60 days resulted in significant decreases in serum methylmalonic acid in 88% of the subjects
[62]; serum Hcy decreased and serum B
12
tended to increase, although the mean corpuscular volume,
hemoglobin, and hematocrit levels were unchanged. These results suggest that Chlorella products
with high B
12
and without inactive corrinoid compounds are suitable for use as B
12
sources in humans,
particularly vegans.
Figure 4. Chemical structures of vitamin B
12
and related compounds found in commercially available
Chlorella products. Abbreviations: Factor IIIm, 5-methoxybenzimidazolylcobamide.
2.2.2. Minerals
As shown in Table 4, commercially available Chlorella products contain a variety of minerals that
are required in humans. In particular, Chlorella products contain substantial amounts of iron (104
mg/100 g dry weight) and potassium (986 mg/100 g dry weight), of which adequate intake prevents
anemia [63] and hypertension [64], respectively. Iron plays physiological roles in respiration, energy
production, DNA synthesis, and cell proliferation [65]. The phytates in grains potently inhibit the
intestinal absorption of iron because they chelate iron to form an insoluble complex [66]. Thus, people
on vegan and vegetarian diets may be at risk for iron-deficiency anemia [63]. Studies in rats and
humans have investigated whether Chlorella supplementation can prevent iron-deficiency anemia
[67,68]. In a cohort of 32 women in the second and third trimester of pregnancy, oral Chlorella
supplementation (6 g/day) for 12–18 weeks decreased markers of anemia as compared to the control
group [68], suggesting that Chlorella supplementation significantly reduces the risk of pregnancy-
associated anemia.
Figure 4.
Chemical structures of vitamin B
12
and related compounds found in commercially available
Chlorella products. Abbreviations: Factor IIIm, 5-methoxybenzimidazolylcobamide.
Rauma et al. [
61
] demonstrated that substantial consumption of Chlorella products can supply
adequate amounts of B
12
. Another study of strict vegetarians (vegans) with an elevated baseline of
serum methylmalonic acid (as an index of B
12
deficiency) showed that ingestion of 9 g of
C. pyrenoidosa
daily for 60 days resulted in significant decreases in serum methylmalonic acid in 88% of the subjects [
62
];
serum Hcy decreased and serum B
12
tended to increase, although the mean corpuscular volume,
hemoglobin, and hematocrit levels were unchanged. These results suggest that Chlorella products with
high B
12
and without inactive corrinoid compounds are suitable for use as B
12
sources in humans,
particularly vegans.
2.2.2. Minerals
As shown in Table 4, commercially available Chlorella products contain a variety of minerals that are
required in humans. In particular, Chlorella products contain substantial amounts of iron (
104 mg/100 g
dry weight) and potassium (986 mg/100 g dry weight), of which adequate intake prevents anemia [
63
]
and hypertension [
64
], respectively. Iron plays physiological roles in respiration,
energy production
,
DNA synthesis, and cell proliferation [
65
]. The phytates in grains potently inhibit the intestinal
absorption of iron because they chelate iron to form an insoluble complex [
66
]. Thus, people on vegan
and vegetarian diets may be at risk for iron-deficiency anemia [
63
]. Studies in rats and humans have
investigated whether Chlorella supplementation can prevent iron-deficiency anemia [
67
,
68
]. In a cohort
of 32 women in the second and third trimester of pregnancy, oral Chlorella supplementation (6 g/day)
for 12–18 weeks decreased markers of anemia as compared to the control group [
68
], suggesting that
Chlorella supplementation significantly reduces the risk of pregnancy-associated anemia.
Nutrients 2020,12, 2524 7 of 22
Table 4. Mineral content of 13 commercially available Chlorella products.
Minerals (Per
100 g Dry Weight) A B C D E F G H I J K L M
Sodium (mg) 5–75 65 40 80–220
10–45
5–30 80 65 47
Iron (mg)
10–130
160
121
62
350–1600
100–200 50–100
110
113
Calcium (mg) 650
513 850 500–1500
100–300 433
Potassium (mg) 970
1075 350
200–500
500–1500
1020
Magnesium (mg) 350
250
23–420 298
Zinc (mg) 2 1
Copper (mg) 1 1
Phosphorus (mg) 1600 1320
Manganese (mg) 5
Selenium (Se) is an essential trace mineral that serves as a fundamental nutrient to human
health. It is a component of selenoproteins, such as thioredoxin reductase and glutathione peroxidases,
and protects
against intercellular oxidative damage [
69
71
]. Therefore, low levels of Se show various
pharmaceutical activities, including antitumor and antiaging eects; however, high levels of Se induce
the generation of reactive oxygen species. Generally, the organic forms of Se are more bioavailable and
less toxic than the inorganic forms of Se. Selenite (SeO
32
) and selenite (SeO
42
) are the predominant
forms of Se in freshwater. Microalgae act as a major transporter of Se from water to filter-feeders
and other organisms. Although most plant species accumulate less than 25
µ
g Se/g dry weight [
72
],
some microalgae species can accumulate Se at high concentrations (100
µ
g Se/g dry weight) [
73
].
Se is
essential for many algae and functions to protects them from oxidative damage. Sun et al. [
74
]
indicated that C. vulgaris can accumulate Se at high concentrations (857
µ
g/g dry weight) when grown
under Se concentrations of 0–200 mg/L in a growth medium and that relatively low Se concentrations
(
75 mg selenite/L
medium) positively promotes C. vulgaris growth and acts as an antioxidant by
inhibiting lipid peroxidation and intracellular reactive oxygen species. The maximum accumulation
of organic Se was found at 316
µ
g/g dry weight under relatively low Se (75 mg selenite/L medium)
conditions [
75
], indicating that C. vulgaris is an ecient Se accumulator and that Se-enriched Chlorella
cells might be useful for human supplementation.
2.3. Pigments
Carotenoids are secondary metabolites in the most abundant naturally occurring pigments that
participate in various biological processes in plants, including photosynthesis, photomorphogenesis,
photoprotection, and development [
76
]. They also serve as colorants and critical components
of the human diet, such as antioxidants and provitamin A [
76
]. More than 400 carotenoids
have been identified in living organisms [
77
], and
β
-carotene, astaxanthin, lutein, zeaxanthin,
and lycopene are widely known as the major carotenoids among them. The green microalgae
Dunaliella salina produces high concentrations of
β
-carotene of up to 14% of algal dry weight [
78
].
Haematococcus pluvialis increases
astaxanthin concentration up to 4–5% of algal dry weight [
79
] under
stressful conditions.
Chlorella products
contain lower contents of total carotenoids (approximately
1.3%) [
80
], compared with the above-mentioned green algae. C. vulgaris reportedly produces lutein as
the primary carotenoid [
81
,
82
]. However, C. zofingiensis reportedly accumulates significant amounts of
astaxanthin, and it might be a suitable organism for the mass production of astaxanthin [83].
3. Pharmacological Activities of Chlorella Products
Because Chlorella cells contain various nutrients and biologically active compounds, the eects
of Chlorella supplementation on preventing the development of various diseases has been studied in
rats and mice, including disease-specific model animals. These animal studies have been useful for
elucidating the specific health eects of Chlorella supplementation. Moreover, the eects of Chlorella
supplementation on mitigating a variety of diseases in humans have been investigated. These studies
have used either C. vulgaris or C. pyrenoidosa because these species are commercially available as
Chlorella products.
Nutrients 2020,12, 2524 8 of 22
3.1. Antihypertensive Eects
Hypertension increases the risk of cardiovascular disease [
84
]. Antihypertensive compounds in
foods have been identified using a stroke-prone spontaneously hypertensive (SHRSP) rat model,
which is
genetically predisposed to hypertension and cerebral stroke [
85
]. Sansawa et al. [
86
]
investigated the eects of dried Chlorella powder (C. regularis) on blood pressure, cerebral stroke lesions,
and the life span of SHRSP rats. In 12-week-old SHRSP rats fed Chlorella (5%, 10%, and 20%) for
13 weeks
, elevated blood pressure significantly decreased in the 10% and 20% Chlorella groups compared
with the untreated controls. Serum total cholesterol levels were significantly lower in all Chlorella groups,
and their average life span was more than that of the controls. To characterize the antihypertensive
compounds in Chlorella,Chlorella powder was fractionated into hot-water-soluble, lipid-soluble,
and residual
fractions. Blood pressure was significantly lower in rats fed the lipid or residual fraction
but not in those fed the hot-water-soluble fraction. The lipid fraction contained substantial amounts
of carotenoids, which are potent antioxidants, and phospholipids, which mediate aorta collagen and
elastin metabolism. The residual fraction contained a high level of arginine,
which increases
the
production of endothelium-derived relaxing factor. These beneficial eects of Chlorella powder on
SHRSP rats might result from synergism between its numerous bioactive compounds.
To evaluate whether daily Chlorella supplementation can reduce blood pressure in subjects
with mild to moderate hypertension, a pilot study was conducted in 24 participants administered
C. pyrenoidosa (10 g of Chlorella tablets and 100 mL Chlorella extract) [
87
]. After two months of
Chlorella supplementation, the average heart rate and sitting systolic and diastolic blood pressures
only slightly changed. On the other hand, for some subjects with mild to moderate hypertension,
Chlorella supplementation reduced or maintained their sitting diastolic blood pressure.
Arterial stiness is a well-established risk factor of cardiovascular disease [88]. Previous studies
have reported that antioxidants [
89
], potassium [
90
], and n-3 unsaturated fatty acids [
91
] decrease
arterial stiness. Nitric oxide (NO), derived from arginine in the vascular endothelium, is an important
modulator of arterial stiness [
92
]. Chlorella products contain antioxidants, vitamins, potassium,
arginine, and n-3 unsaturated fatty acids. To evaluate the eects of Chlorella supplementation on arterial
stiness, a single-blinded, placebo-controlled crossover study was conducted in 14 young participants
who were administered C. pyrenoidosa (6 g/day) or placebo for four weeks, with a 12-week washout
period between trials, in a randomized order [
93
]. No dierences were observed in blood pressure or
heart rate before and after supplementation in both the placebo and Chlorella groups. Brachial-ankle
pulse wave velocity, a measure of arterial stiness, decreased in the Chlorella group but not in the placebo
group [
93
]. A similar trial in 32 middle-aged and older subjects reports that the brachial-ankle pulse
wave velocity decreased after Chlorella supplementation but not after placebo supplementation [
94
].
These changes in brachial-ankle pulse wave velocity with Chlorella supplementation correlated with
the plasma NOx level. These results suggest that Chlorella supplementation decreases arterial stiness
in both younger and older subjects.
The ecacy of Chlorella supplementation in reducing cardiovascular risk factors was assessed in a
meta-analysis of 19 randomized controlled trials including 797 subjects [
95
]. This study concluded that
Chlorella supplementation improves total cholesterol levels, low-density lipoprotein cholesterol levels,
systolic blood pressure, diastolic blood pressure, and fasting blood glucose levels but not triglyceride
levels, high-density lipoprotein cholesterol levels, and body mass index.
3.2. Antihypercholesterolemic and Antihyperlipemic Eects
Elevated total cholesterol and triglycerides and abnormal metabolism of lipoproteins and
apolipoproteins are responsible for an increased risk of cardiovascular disease [
96
98
]. The indigestible
components of foods, such as dietary fiber, decrease serum cholesterol by inhibiting the intestinal
absorption of neutral steroids [
99
]. Chlorella supplementation reportedly decreases serum cholesterol
levels in model animals [
100
]. To identify the bioactive compounds responsible for this eect,
the indigestible
fraction of C. regularis powder was isolated and characterized, revealing a content of
Nutrients 2020,12, 2524 9 of 22
43% crude protein, 37.3% dietary fiber, 6.9% carbohydrate, 5.4% moisture, 4.3% crude fat,
and 2.7%
ash [
101
]. Rats fed a diet with 5.3% of this indigestible fraction demonstrated lower serum and
liver cholesterol levels and higher fecal neutral steroid levels as compared with those fed a diet of
12.7% Chlorella powder. Both Chlorella powder and the indigestible fraction exhibited a high bile-acid
binding capacity
in vitro
. Furthermore, the indigestible fraction increased the hepatic mRNA levels
of cholesterol 7
α
-hydroxylase, which is the rate limiting enzyme for cholesterol catabolism and
bile-acid synthesis [
102
]. These results indicate that the indigestible fraction of Chlorella possesses
hypercholesteromic activity, which improves cholesterol catabolism via the upregulation of hepatic
cholesterol 7α-hydroxylase expression.
Chlorella supplementation is also reported to decrease serum cholesterol levels in hyperlipemia
and mild hypercholesterolemic patients in a small, open-label trial [
103
]. To evaluate the preventive
role of Chlorella in maintaining serum cholesterol levels against excess dietary cholesterol intake,
a double-blind
, randomized, placebo-control study was conducted in 63 mildly hypercholesterolemic
subjects treated with either C. vulgaris (5 g/day) or placebo for four weeks [
104
]. A similar trial
investigated cholesterol levels in 34 participants administered 510 mg of dietary cholesterol from three
eggs concomitantly with either Chlorella (C. vulgaris) (5 g/day) or a matched placebo for
4 weeks [105]
.
Participants on the three-egg diet alone exhibited significant elevation in serum total cholesterol,
low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol levels. The administration
of Chlorella in addition to the three-egg diet significantly suppressed these increases in total cholesterol
and low-density lipoprotein cholesterol levels and significantly increased serum lutein and
α
-carotene
levels [
105
]. In mildly hypercholesterolemic subjects, Chlorella administration resulted in marked
changes in total cholesterol, triglycerides, lutein/zeaxanthin, and
α
-carotene levels as well as a significant
decrease in very low-density lipoprotein cholesterol, apolipoprotein B, non-high-density lipoprotein,
and high-density lipoprotein/triglyceride levels [
104
]. These results suggest that Chlorella might inhibit
the intestinal absorption of dietary and endogenous lipids. In addition, the observed changes in
serum lipids may be associated with changes in serum carotenoids. These results suggest that daily
consumption of Chlorella provides potential health benefits by reducing the levels of serum lipid risk
factors, such as triglycerides and total cholesterol, in mild hypercholesterolemic subjects.
3.3. Antidiabetic Eect
Type 2 diabetes, accounting for 90–95% of all diabetes cases, is a severe health problem aecting
over 380 million people worldwide [
106
]. Elevated blood glucose levels, insulin resistance, and low
insulin sensitivity are the main characteristics of patients with type 2 diabetes [
107
], resulting in serious
conditions, including arteriosclerosis, renal damage, and retinopathy [
108
]. In a streptozotocin-induced
animal model of diabetes, several studies have been conducted to elucidate the mechanisms underlying
the antidiabetic activity of Chlorella supplementation [
109
111
]. Shibata et al. [
109
] evaluated the eects
of Chlorella supplementation on antioxidant status and cataracts by feeding a diet containing 7.3% (w/w)
Chlorella powder (C.regularis) to 11-week old rats with streptozotocin-induced diabetes. After 11 weeks
of supplementation, serum lipid peroxide levels (an index of oxidative status) and blood glycated
hemoglobin were lower in Chlorella-supplemented rats than in control rats; however, the serum glucose
level did not dier between groups. Chlorella supplementation delayed the development of lens
opacities. These results indicate that Chlorella supplementation might be beneficial for preventing
diabetes complications such as cataracts, possibly due to the activity of its antioxidant compounds.
Cherng and Shih reported potential hypoglycemic eects of Chlorella supplementation in
streptozotocin-induced diabetic mice [
110
]. Oral administration of Chlorella 60 min before glucose
administration (0.5 g/kg body weight) resulted in a transient hypoglycemic eect at 90 min after
glucose administration without an increase in insulin secretion. Chlorella supplementation increased
2-deoxyglucose uptake in the liver and soleus muscles of streptozotocin-treated mice and was likely
the cause of the observed hypoglycemic eects [111].
Nutrients 2020,12, 2524 10 of 22
The prophylactic eect of Chlorella (C. vulgaris) supplementation on diabetes was studied by
Vecina et al. [112]
, who investigated body weight, lipid profile, blood glucose, and insulin signaling in
liver, skeletal muscle, and adipose tissue in high-fat diet-induced obese mice. Chlorella supplementation
improves glycemic control in obesity and diabetes because it decreases insulin resistance caused by
increased expression of glucose transporter 4 via the activation of protein kinase B phosphorylation
in skeletal muscle. Chlorella supplementation combined with aerobic exercise training showed more
pronounced eects on the improvement of glycemic control via increased activation of muscle
phosphorylation signaling in type 2-diabetic rats [113].
A randomized, double-blind, placebo-controlled human study was conducted in 28
borderline-diabetic participants treated with either Chlorella (8 g/day) or placebo for 12 weeks [
114
].
The expression
levels of 252 genes, including six associated with type 2 diabetes, diered between
the two groups. Notably, the mRNA expression level of resistin, an insulin resistance inducer,
was significantly
lower in the Chlorella group than in the placebo group and correlated with the
expression levels of hemoglobin A
1c
, tumor necrosis factor-a, and interleukin-6 [
114
], all of which are
involved in glucose metabolism and/or inflammation.
3.4. Hepatoprotective Eect
Li et al. [
115
] demonstrated that C. vulgaris extract has a potent hepatoprotective eect on carbon
tetrachloride-induced acute hepatic injury in mice. Chlorella extract of 50, 100, or 200 mg/kg of diet,
was administered to mice every other day for four weeks, and carbon tetrachloride was administrated
intraperitoneally 3 h after the final Chlorella supplement. Carbon tetrachloride treatment increased serum
alanine and aspartate aminotransferases levels, lipid peroxidation, and cytochrome P450 expression
and decrease in reduced glutathione and cellular antioxidant defense enzyme levels; all of these
changes were significantly lower in the Chlorella (100 and 200 mg/kg diet) groups.
Although hepatocyte
necrosis was mildly diminished in the 50 mg/kg Chlorella-treated group, it was absent in the 100 and
200 mg/kg Chlorella-treated groups. These results indicate that Chlorella extract has a protective eect
on carbon tetrachloride-induced acute hepatic injury in mice, presumably due to the inhibition of
carbon tetrachloride-induced cytochrome P450 activation and the activation of antioxidant enzymes
and free radical scavengers.
Non-alcoholic fatty liver disease (NAFLD) is a group of metabolic disorders that involving
abnormal fat accumulation of more than 5–10% in hepatocytes [
116
]. It aects 10–35% of the
world population [
117
]. NAFLD includes steatosis, non-alcoholic steatohepatitis, fibrosis, cirrhosis,
and hepatocellular
carcinoma [
118
]. Most NAFLD patients have at least one characteristic metabolic
syndrome, including insulin resistance, hypertension, dyslipidemia, diabetes, and obesity [
119
].
Seventy NAFLD patients were randomly administered C. vulgaris (1.2 g/day) or placebo for eight
weeks [
120
]. The mean body weight and serum concentrations of liver enzymes were significantly lower
in the Chlorella group than in the placebo group, and the serum insulin concentration was significantly
higher in the Chlorella group than in the placebo group. Therefore, Chlorella supplementation may
have beneficial eects on reducing weight and serum glucose levels and improving inflammatory
biomarkers as well as liver function in NAFLD patients [120,121].
To evaluate the safety and ecacy of Chlorella (C. pyrenoidosa) in patients chronically infected with
hepatitis C virus genotype 1, patients received daily oral supplement of Chlorella (both of Chlorella
extract and tablets) for 12 weeks [
122
]. The majority (approximately 85%) of the patients exhibited a
significant decrease in alanine aminotransferase levels from Week 0 to Week 12. Patients with decreased
alanine aminotransferase level showed a tendency toward decreased hepatitis C virus load.
3.5. Detoxification Eect
Dioxins are a group of polychlorinated dibenzo-p-dioxin and dibenzofuran-related compounds
that are industrial contaminants and ubiquitous environmental pollutants [
123
]. These compounds are
easily absorbed in the mammalian gastrointestinal tract [
124
] and then stored in the liver,
adipose tissue
,
Nutrients 2020,12, 2524 11 of 22
and breast milk due to their lipophilic properties [
125
]. An incident involving the consumption of
cooking oil contaminated with dioxins had tragic eects [
126
]. To investigate the eects of Chlorella
supplementation on fecal excretion of dioxins, rats were administered dioxin-contaminated rice oil [
127
].
The rats were fed 4 g of a 10% (w/w)Chlorella (C. vulgaris) diet or a control diet (without Chlorella)
once during the five-day experimental period, and the amounts of fecal dioxins were measured.
The fecal
dioxin levels were significantly greater in the Chlorella group than in the control group.
In addition
,Chlorella supplementation significantly inhibited the gastrointestinal absorption of dioxins
(approximately 2–53% decrease). These results indicate that Chlorella supplementation might be useful
in promoting dioxin excretion.
Heterocyclic amines have been established as carcinogenic chemicals that form when amino
acids, sugars, and creatine in muscle meats (beef, pork, fish, and poultry) react with one another
during cooking at high temperatures [128]. To evaluate the eect of Chlorella supplementation on the
detoxification of carcinogenic heterocyclic amines, a randomized, double-blind, placebo-controlled
crossover study with Chlorella supplementation (100 mg/day) for two weeks was conducted [
129
].
Chlorella supplementation decreased urinary excretion of the predominant metabolite of carcinogenic
heterocyclic amines [
129
], suggesting that Chlorella either inhibits the intestinal absorption of heterocyclic
amines or inactivates carcinogenic compounds.
Methylmercury is a neurotoxic metal compound that is converted from inorganic mercury by
microorganisms in aquatic environments and is then accumulated in fish and shellfish through marine
food chains [
130
]. Therefore, the major route of human exposure to methylmercury is the consumption of
seafood [
130
]. In many countries, pregnant women are cautioned against consuming large fish,
such as
tuna, to prevent fetal exposure [
131
]. As Chlorella consumption is reported to increase the excretion of
methylmercury and lower tissue mercury levels in methylmercury-treated mice [
132
],
an open-label
clinical trial was performed to estimate the eects of Parachlorella beijerinckii supplementation (9
g/day) for three months on mercury concentrations in the hair and blood of healthy subjects [
133
].
Chlorella supplementation
reduced mercury levels in both the hair and blood [
133
]. Fecal excretion is
the major route of methylmercury elimination (90%) in humans [
134
]. Most of the methylmercury in
the liver is secreted as a glutathione complex via the bile duct, with a small portion excreted in the
feces [
135
]. The dietary fiber in Chlorella cells increases the amount of feces excreted by humans [
136
].
Dietary fiber has been shown to absorb some methylmercury
in vitro
[
132
]. These observations suggest
that the observed lowering of hair and blood mercury levels in Chlorella-treated participants may result
from the promotion of fecal methylmercury excretion via accelerated bile secretion, the binding of
methylmercury to dietary fiber in the intestinal tract, and increased feces production.
3.6. Immunomodulatory Eects
Allergic disease is a prevalent aberrant immune responsive against innocuous environmental
proteins (antigens) [
137
]. Allergen-specific CD4
+
T cells involved in the initiation of allergic reactivity
can develop into either type 1 or type 2 helper T cells [
138
]. CD4
+
T cells stimulated in the presence of
interleukin-12 and
γ
-interferon can develop into type 1 helper T cells [
138
], while interleukin-4 promotes
the development of type 2 helper T cells and inhibits the generation of type 1 helper T cells [
139
].
Since type 1
and 2 helper T cells regulate each other, interleukin-12 functions not only to induce the
type 1 helper T-cell response but also to regulate the type 2 helper T-cell response [
140
]. Interleukin-12
strongly suppresses the production of IgE by preventing type 2 helper T-cell development [
141
].
Allergen-specific IgE induces the pathogenesis of allergic disorder [142].
Hasegawa et al. [
143
] described the eects of a Chlorella (C. vulgaris) hot-water extract on
antigen specific response in mice. A 2% (w/w)Chlorella hot-water extract diet or control diet
(without Chlorella extract) was given to mice for two weeks before intraperitoneal administration
of casein/complete Freund’s adjuvant (an immunostimulant). Mice that received the hot-water
extract exhibited suppressed IgE production and mRNA expression of interleukin-6 involved in the
type 2
helper T-cell response. They also exhibited increased levels of interleukin-12 and g-interferon
Nutrients 2020,12, 2524 12 of 22
mRNA, increasing the
type 1
helper T-cell response and suppressing the type 2 helper T-cell response.
These results
suggest that Chlorella hot-water extract supplementation might be useful for suppressing
allergic responses with a predominant type 2 helper T-cell response. To clarify the mechanisms
underlying the immunomodulatory activity of Chlorella hot-water extract, soluble polysaccharides
were isolated from C. pyrenoidosa hot-water extract and characterized [
144
]. GC-MS analysis indicated
that the major monosaccharide components of the soluble polysaccharides are rhamnose (31.8%),
glucose (20.4%)
, galactose (10.3%), mannose (5.2%), and xylose (1.3%). These soluble polysaccharides
were intraperitoneally administrated (100 mg/kg of body weight) to 6–8-week-old mice.
After 24 h
,
lipopolysaccharide as an antigen was administrated to mice, and their serum was collected 1.5 h
later [
144
]. The soluble polysaccharides induced interleukin-1b secretion in macrophages via the toll-like
receptor protein kinase signaling pathway. Interleukin-1
β
is one of the most important mediators of
inflammation and host responses to infection [
145
]. These results suggest that Chlorella hot-water-soluble
polysaccharides could be used as an agent source to stimulate anti-microorganism activity.
Halperin et al. [
146
] evaluated the eect of C. pyrenoidosa supplementation (200 or 400 mg) on the
immune response to influenza vaccination. After 28 days of Chlorella supplementation, the antibody
response to the influenza vaccine was not elevated in the overall study population but was increased
in participants aged 50–55 years.
Salivary secretory immunoglobulin A (SIgA) plays a crucial role in mucosal immune function
and is the first line of defense against pathogenic microbial invasion in human [
147
]. To evaluate
whether Chlorella supplementation increases salivary SIgA secretion in humans, a blind, randomized,
crossover study
was conducted in participants administered Chlorella (C. pyrenoidosa) (6 g/day) or
placebo for four weeks [
148
]. Although no dierence was observed in salivary SIgA levels before and
after placebo ingestion, salivary SIgA levels were significantly elevated after Chlorella ingestion than at
baseline. The SIgA secretion rate increased significantly after Chlorella supplementation. These results
suggest that four-week Chlorella supplementation increases salivary SIgA secretion and improves
mucosal immune function in humans.
Natural killer cells are the predominant innate lymphocyte subsets that mediate antitumor and
antiviral responses [
149
]. To evaluate the eect of Chlorella supplementation on natural killer cell
activity and early inflammatory response in humans, a randomized, double-blinded, placebo-controlled
trial was conducted in healthy adults ingested with Chlorella (C. vulgaris) (5 g/day) or placebo [
150
].
After eight weeks of supplementation, serum interferon-
γ
and interleukin-1
β
levels were significantly
elevated and that of interleukin-12 tended to increase in the Chlorella group. Natural killer cell activities
were significantly elevated in the Chlorella group. These results suggest a beneficial immunostimulatory
eect of short-term Chlorella supplementation that increases natural killer cell activity and produces
interferon-γ, interleukin-12, and interleukin-1β.
3.7. Antioxidant Eects
C. vulgaris hot-water extract [
151
] and acetone extract [
152
] are reported to have antitumor activity.
AChlorella aqueous extract containing substantial amounts of antioxidants also exhibit antiproliferative
activity in human hepatoma cells [
153
]. Lipophilic pigments, including carotenoids antheraxanthin,
zeaxanthin, and lutein, extracted from Chlorella cells were observed to significantly inhibit the growth
of human colon cancer cells [
154
]. These results suggest that the antitumor activity of Chlorella
might be the synergistic eect of multiple bioactive compounds.
Romos et al. [155]
reported that
Chlorella supplementation can modulate immunomyelopoietic activity and disengage tumor-induced
suppression of various cytokines and related cell activities in tumor-bearing mice. Interestingly,
a 63.1-kD antitumor glycoprotein was isolated from the culture supernatant of
C. vulgaris
strain
CK22 [
156
,
157
], and its chemical and antitumor properties were characterized [
158
],
suggesting possible
contribution of this glycoprotein toward the observed antitumor activity.
Alzheimer’s disease is a severe neurodegenerative condition aecting humans [
159
].
The erythrocytes
of Alzheimer’s disease patients are known to be in an excessively oxidized
Nutrients 2020,12, 2524 13 of 22
state [
160
].
α
-Tocopherol and carotenoids such as lutein are important lipophilic antioxidants in human
erythrocytes [
161
]. Erythrocyte lutein levels were found to be significantly lower in Alzheimer’s
disease patients than in normal subjects [
162
]. Oral intake of lutein capsules increases lutein levels
and prevents phospholipid hydroperoxide accumulation in human erythrocytes [
163
], suggesting
that dietary lutein has the potential to act as an important antioxidant in erythrocytes and thus may
have beneficial eects in Alzheimer’s disease patients. According to the labels on Chlorella products D
and M, the products contain substantial amounts of lutein (approximately 200 mg/100 g dry weigh).
A randomized
, double-blind, placebo-controlled human study was conducted to evaluate the eects
of Chlorella supplementation (8 g Chlorella/day/person; equivalent to 22.9 mg lutein/day/person) on
phospholipid hydroperoxide and lutein levels in erythrocytes [
164
]. After two months of Chlorella
supplementation, erythrocyte lutein levels increased 4.6-fold, but tocopherol levels did not change [
164
],
suggesting that daily Chlorella intake may be eective for improving and maintaining erythrocyte
antioxidant status and lutein levels in humans. These results suggest that Chlorella supplementation
contributes to maintaining the normal function of erythrocytes and has beneficial eects on Alzheimer’s
disease-related dementia in humans.
Major depressive disorder is a widespread mental disorder that greatly impairs the quality of
life of humans. Approximately 12% of people experience at least one episode of depression during
their lifetime [
165
]. Although various antidepressant drugs are available for treating depression,
a considerable proportion of patients are not responsive to these drugs and some experience side
eects [
166
,
167
]. Therefore, alternative antidepressant drugs with adequate ecacy and safety are
needed. The therapeutic eect of dried C. vulgaris extract administration (1.8 g/day) for six weeks
was evaluated in patients with major depressive disorder [
168
]. After treatment, the participants
exhibited improvements in physical and cognitive symptoms of depression [
168
]. As oxidative stress is
an important pathophysiological mechanism underlying major depressive disorder,
major depressive
disorder has been eectively reversed via antioxidant therapy [
169
,
170
]. These observations suggest
that the therapeutic eectiveness of Chlorella supplementation may result from the action of its
antioxidant nutrients and compounds [171].
3.8. Other Eects
Stress is well known to disturb homeostasis, impairing immunological functions.
Chlorella supplementation
reportedly stimulates the pool of hematopoietic stem cells and activates
leukocytes [
172
]. To further understand the influence of Chlorella (C. vulagaris) supplementation on
hematopoiesis, hematopoietic cell populations in the bone marrow of mice subjected to a single or
repeated stressor were measured [
173
]. Reduced numbers of hematopoietic progenitors in the bone
marrow were observed after treatment with either stressor. Both stressors induced a decrease in
mature myeloid and lymphoid populations but did not aect pluripotent hematopoietic progenitors.
Both stressors
reduced the levels of interleukin-1
α
and interleukin-6. Chlorella supplementation
prevented the changes produced by both stressors in all of the parameters tested, suggesting that
Chlorella supplementation is an eective tool for the prophylaxis of myelosuppression caused by single
or repeated stressors.
Stressors are processed in the brain through the activation of several types of neurons.
Immediate early genes such as c-fos are extensively used to map brain areas involved in
stress responses [
174
].
Using c-fos
expression, Oueiroz et al. [
175
] evaluated the eect of
acute pretreatment with Chlorella (C. vulgaris) on the peripheral and central responses to forced
swimming stress in rats.
Chlorella supplementation
produced a significant reduction in stress-related
hypothalamic–pituitary–adrenal axis activation due to decreased corticotrophin releasing factor gene
expression in the hypothalamic paraventricular nucleus and a lower adrenocorticotropic hormone
response. Hyperglycemia induced by the stressor was similarly reduced. These results suggest that
Chlorella supplementation might reduce the impact of stressors.
Nutrients 2020,12, 2524 14 of 22
A hot-water extract of Chlorella (C. pyrenoidosa) increased the lifespan of superoxide dismutase-1
mutant adults of Drosophila melanogaster in a dose-dependent manner (200–800
µ
g/mL) [
176
].
An active
compound was purified and identified as phenethylamine, an aromatic amine, which exhibited no
superoxide dismutase-like activity. Treatment with this compound extended the lifespan of the
mutant flies at very low concentration (60 ng/g diet) [
176
]. Similarly, supplementation of C. sorokiniana
(
4 mg/mL
) reportedly increased the lifespan of D. melanogaster by 10% increase as compared to a
control diet, likely due to the increased mRNA expression of antioxidative enzymes (Cu/Zn-superoxide
dismutase and catalase) [177].
However, for the beneficial eects described above, no human study has been conducted.
4. Conclusions
Commercially available Chlorella products contain a variety of nutrients essential for humans,
as well as a large amount of good quality protein, dietary fibers, and polyunsaturated fatty acids,
including
α
-linolenic and linoleic acids. In particular, Chlorella products contain vitamins D
2
and B
12
,
which are absent from plant-derived food sources, and larger amounts of folate and iron than other
plant-derived foods. Mounting scientific evidence of the health benefits of daily Chlorella consumption
has been presented in animal and human studies. The pharmacological activities reported in Chlorella
studies include immunomodulation, antioxidative activity, and eects against diabetes, hypertension,
and hyperlipidemia. The beneficial eects of Chlorella might involve synergism between multiple
nutrient and antioxidant compounds. Overall, the information regarding bioactive compounds in
Chlorella is limited. Thus, new bioactive compounds responsible for its pharmacological activities may
be identified in future studies.
Author Contributions:
T.B., E.O., M.F., and F.W. conceived of and designed the study. T.B. and F.W. wrote the
original draft. E.O. and M.F. reviewed and edited the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.
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