Review and Current Research of Vitamin as

Functional Ingredients

Wahyu Dwi Saputra

Laboratory of Nutrition
Department of Science of Food Function and Health
Division of Bioscience and Biotechnology for Future Bioindustries
 2
Lecture contents

1) Introduction to the Regulation of Health Foods in

Japan

2) Review of the General Vitamin Functions

3) Review of the Current Research on Vitamin K and

its structurally related compound function
 Topic 1

Introduction to the Regulation of Health Foods in Japan

 4
Introduction
“Health food” is a generic term for food products, whether foods, drinks or
dietary supplements, that carry nutrient function and health claims or that are
designed to be more effective than common foods in helping people maintain
and promote their health, or at least are expected to have such an effect.
 5
The Japanese Regulation on Health Food
The concept of functional foods first evolved in Japan in the mid-1980s.
Japanese food scientists who were members of research teams on functional
foods sponsored by the Ministry of Education, Science and Culture, proposed
that, while the primary role of food was to provide adequate nutrients to meet
metabolic requirements and the secondary role was to provide gustatory and
olfactory satisfaction, there existed the tertiary function of promoting a state
of well-being and better health and reducing the risk of disease.

“Foods with Health Claims” are health foods that conform to

the safety and efficacy standards set by the Ministry of
Health. They consist of two categories: “Foods with Nutrient
Function Claims” and “Foods for Specified Health Uses”
(Article 5, Enforcement Regulations, Ministerial Ordinance, of Food Sanitation Act, 2001.).
 6
1. Foods for Specified Health Uses
“Foods for Specified Health Uses” are those that contain
dietary ingredients that have beneficial effects on the
physiological functions of the human body to maintain
and promote health and to improve specific health-
related conditions.
(Article 12, Enforcement Regulations, Ministerial Ordinance, of Health Promotion Act, 2003.
Article 5, Enforcement Regulations of Food Sanitation Act, 2001.)

The manufacturer who applies to the government for

approval as “Food for Specified Health Uses” for its product
must tabulate both published literatures and
unpublished or internal office reports on the
effectiveness of the product and/or its ingredients, and
provide a summary of each literature or report. The table
must include :
 in vitro metabolic and biochemical studies,
 in vivo studies
 human randomized controlled trials (RCT)
 Furthermore, the identification of the single, specific
active ingredient of the product is required, and the
exact mechanism of action should be clarified
 7
2. Foods with Nutrient Function Claims
“Foods with Nutrient Function Claims” are intended to
supplement or complement the daily diet, in cases
where the dietary intake is insufficient due to increased
age and unhealthy dietary practices or where consumers
feel that their diet requires supplementing.
• Vitamin A
The minimum level of each vitamin or • Vitamin D
mineral contained in the daily consumption
• Vitamin E
level of “Foods with Nutrient Function
Claims” as suggested by the manufacturer • Vitamin B1
should be one-third of the RDA for the • Vitamin B2
Japanese. The maximum level is the • Niacin
same as the maximum amount of
• Vitamin B6
nutrient items in “over-the-counter”
drugs and quasidrugs. • Folic Acid
• Vitamin B12
“Foods with Nutrient Function Claims” do not need • Biotin
to be approved by the Ministry of Health before
• Pantothenic acid
they are marketed. The manufacturers do not
need to register their products with the Ministry • Vitamin C
of Health before producing or selling them. • Calcium, Iron, Zinc, Copper,
 Topic 2

Review of the General Vitamin Functions

 9
Basics of Vitamins

Vitamins are organic compounds that are required, in small

1
quantities, for normal metabolism.

In general, humans can not synthesize sufficient quantities of

2 vitamins; thus, vitamin must come from other sources – through the
diet or in pill form.

There are two categories of vitamins: water-soluble and fat-

soluble. Water-soluble vitamins include vitamin C and B vitamins.
Vitamin A, D, E, and K comprise the fat-soluble vitamins. Water
3 soluble vitamins tend to have more oxygen and nitrogen in their
structure. The fat-soluble vitamins have significant hydrocarbon
portions in their structures.The majority of water soluble vitamin
either act as coenzymes or are important in the synthesis of
coenzymes. Fat-soluble vitamins serve a variety of biochemical
functions.
 10
Nutrient Function Claims for “Foods with Nutrient Function Claims”

Nutrient Approved Statements for Nutrient Function Attention and Warnings

Claims
Vitamin B1 Vitamin B1 is a nutrient that helps to produce
energy from carbohydrates and to keep skin
and mucosa healthy.
Vitamin B2 Vitamin B2 is a nutrient that helps to keep skin
and mucosa healthy.
Niacin Niacin is a nutrient that helps to keep skin and
mucosa healthy
Vitamin B6 Vitamin B6 is a nutrient that helps to produce
energy from protein and to keep skin and
mucosa healthy
Folic Acid Folic acid is a nutrient that helps red blood cell Although this product is a nutrient that
formation. Folic acid is a nutrient that contributes to the normal growth of the fetus,
contributes to the normal growth of the fetus. better development of the fetus will not ensue
from taking large amounts.
Vitamin B12 Vitamin B12 is a nutrient that helps red blood
cell formation.
Biotin Biotin is a nutrient that helps to keep skin and
mucosa healthy.
Pantothenic acid Pantothenic acid is a nutrient that helps to keep
skin and mucosa healthy.

Vitamin C Vitamin C is a nutrient that not only helps to

keep skin and mucosa healthy but also has an
antioxidative activity.

Nutrient Function Claims for “Foods with Nutrient Function Claims”
Nutrient Approved Statements for Nutrient
Function Claims
Vitamin A Vitamin A is a nutrient that helps in the
maintenance of nocturnal visual
acuity.
Vitamin A is a nutrient that helps to
keep skin and mucosa healthy.
Vitamin D Vitamin D is a nutrient that increases
the absorption of calcium in the
intestine and helps the formation of
bone.
Vitamin E Vitamin E is a nutrient that helps to
protect lipids in the body from
oxidation by antioxidative activity and
to keep cells healthy.
11
Attention and Warnings
Taking large amounts of this product
will not cure disease or promote
health. The indicated daily intake
should be followed. Women who are
in the first trimester of pregnancy or
desire to become pregnant should
not take this product excessively.
Taking large amounts of this product
will not cure disease or promote
health. The indicated daily intake
should be followed.
 Topic 3

Review of the Current Research on Vitamin K and its

structurally related compound function
 13
Introduction
MK-4 distribution in animal organs

Phylloquinone
(Vitamin K1)

Menaquinone-4
(Vitamin K2 subtype)

Source : Shirakawa et al. (2014). Conversion of Menaquinone-4 in Animal Organs and its Functions.

Conversion of vit. K derivatives to MK-4

Source : Nakagawa et al. (2010) Identification of UBIAD1 as a novel human menaquinone-4

biosynthetic enzyme.
 14
Japan’s Aging Population
Japan population pyramid in 2016 Japan life expectancy in 1990 &
2015

Nomura, S. et al. (2017)

National medical expenditure

Major leading death cause in Japan

Kato, R. et al. (2009)

• The elderly population dominates the
current national demographic.
• The gap between life and health
expectancy created spike in medical
expenditure.
• Alzheimer’s disease made top five leading
Nomura, S. et al. (2017) death cause for last 15 years.
 15
Global Alzheimer’s Disease
Global Cost and Prevalence of
Dementia
2500 80

Number of People (millions)

70
2000
60
$ US (Billions)

1500 50
40
1000 30
20
500
10
0 0
2015 2020 2025 2030
alzheimersresearchuk.org
cost of care prevalence
Source : Prince, M et al (2015). World Alzheimer’s Report 2015, The Global Impact
of Dementia: An analysis of prevalence, incidence, cost and trends. Alzheimer’s
Disease International.
• Alzheimer’s disease majorly contribute to the
dementia progression.
• The global burden of Alzheimer’s disease is
steadily increasing.
• Neuroinflammation has been extensively
studied as one potential cause for Alzheimer’s
disease.

Intstitute.progress.im
 16
The microglial inflammation
Brain Cell Types
IL-1β, TNF-α,
20% Neuron Cell IL-6
LPS, TNF-α
neurotoxic
function
M1: proinflammatory
microglia
80% Glia Cell :
- Astrocytes
- Oligodendrocytes Resting
- Ependymal microglia IL-10, IL-4,
- Microglia TGF-β
IL-4
M2: anti-inflammatory
microglia
neuroprotective
function

Microglia is brain-resident macrophage-like cell which is responsible for innate immunity in

central nervous system. Under persistent danger stimuli, such as by lipopolysaccharide (LPS),
microglia undergoes inflammatory reaction which in turn could induce neurotoxic condition.

Source : Subramaniam and Federrof (2017). Shematic of microglial activation and polarization and their functions.
 17
The NF-κB as a target for inflammatory modulation
LPS IL-1β, TNF-α,
IL-6

neurotoxic
Pro-inflammatory effect
microglia
LPS
Resting microglia
TLR4
NEMO
IL-1β, TNF-α,
TAK1 IKKα IKKβ
IL-6
phosporylation

p50 p65
phosporylation

IκBα p50 p65 target genes

transcription
degradation
κB site
NF-κB is one of the well-characterized molecular transactivation system which is activated during
inflammation. During infection such as by LPS, NF-κB is activated via its nuclear translocation
and binding to its recognition site to transactivate pro-inflammatory cytokines.
 18
Vitamin K and inflammation

1 Vitamin K suppresses Lipopolysaccharide-Induced inflammation in the rat.

(Ohsaki, et al., 2006)

Vitamin K suppresses the lipopolysaccharide-induced expression of inflammatory

2 cytokines in cultured macrophage-like cells via the inhibition of the activation of
nuclear factor κB through the repression of IKKα/β phosphorylation.
(Ohsaki, et al., 2010)

MK-4
Sup. Fig. 1. The injection of LPS (i.p.) induced
inflammatory expressions in mice cortex. n = 6.
* p < 0.05 vs control.

Lipopolysaccharide
DMEM
medium
MG6
Mouse microglial-derived Cells
 19
MK-4 pretreatment effect on the inflammatory cytokines
Incubation
MG6 LPS qRT-PCR/WB
MK-4
cell culture 24 h (1 ng/mL) 3h

(a) (b) (c)

0 min 10 min 30 min 60 min 0 min 10 min 30 min 60 min

(d) (e) • MK-4 pretreatment significantly
Con MK-4 Con MK-4 Con MK-4 Con MK-4
Con MK-4 Con MK-4 Con MK-4 Con MK-4 down-regulated the pro-
inflammatory cytokines mRNA
expressions.
• MK-4 inhibited the
phosphorylation of NF-κB p65,
but no change was observed in
IKKα/β phosphorylation.
• Further clarification was worth
considering.
Fig. 1.1. (a-c) Pro-inflammatory mRNA expressions in MK-4 pretreatment. (d-e) The phosphorylated expressions of IKKα/β and p65 in MK-4
pretreatment. n = 3. * p < 0.05 vs LPS. ** p < 0.01 vs LPS. (reproduced with the permission from Aoyama et al.)
 20
MK-4 effect on the NF-κB p65 sub-unit translocation (1)
Incubation
MG6 LPS Fluorescence
MK-4
cell culture 24 h (10 ng/mL) 30 min Microscopy

• Under normal condition, NF-

κB p65 sub-unit was mainly
control

located in cytoplasmic area.

• LPS induced NF-κB p65
translocation indicated by
intense staining in nuclear
parts
• MK-4 reduced NF-κB p65
stain intensity in nuclear are,
LPS

suggesting inhibition against

its nuclear translocation.

LPS
MK-4+LPS

p65
p50
p50
p65

κB site
p65 nuclei merge
Fig. 1.2. Representative images of fluorescence microscopy. Cells were fixed and labeled with anti-p65 (red) antibodies. Nuclei were stained with
Hoechst 33258 (blue). Scale bars, 20 μm.
 21
MK-4 effect on the NF-κB p65 sub-unit translocation (2)
Incubation
MG6 LPS Cytoplasmic-nuclear
MK-4
cell culture 24 h (10 ng/mL) 30 min western blot
(a) (b)

p65 cytoplasmic p65 nuclear

#
1.2 9
1 * 7.5
* 6
0.8

fold
fold

0.6
4.5 *
#
3
0.4
1.5 *
0.2
0
0 control LPS LPS + MK4
control LPS LPS + MK4 MK4 MK4

• Cytoplasmic-nuclear fractionation of NF-κB p65 sub-unit confirmed previous microscopy result.

Fig. 1.3. NF-κB p65 sub-unit in cytoplasmic and nuclear fraction after MK-4 administration. n = 3. # p < 0.05 vs control. * p < 0.05 vs LPS.
 22
Discussion (1)
LPS

Pro-inflammatory
microglia
LPS

TLR4
NEMO IL-1β, TNF-α,
TAK1 IKKα IKKβ IL-6
• MK-4 pretreatment
managed LPS-induced phosporylation
p50 p65
inflammation via
phosporylation
inhibition on NF-κB p65
sub-unit p50 p65 target genes
phosphorylation and its transcription
nuclear translocation.
MK-4
κB site
 23
Geranylgeraniol (GGOH) and inflammation
Dietary supplementation with geranylgeraniol suppresses lipopolysaccharide-
1 induced inflammation via inhibition of nuclear factor-κB activation in rats.
(Giriwono, et al., 2013)

GGOH Menaquinone-4

Lipopolysaccharide

DMEM
medium
MG6
Mouse microglial-derived Cells
 24
GGOH time on the inflammatory cytokines expressions
Incubation
MG6 LPS qRT-PCR
GGOH
cell culture 24 h (10 ng/mL) 3h
(a) (b) (c)
120 Il-1β Il-6
1000 # 800 #
cell viability (%)

90

mRNA (fold)

mRNA (fold)
800
600
60 600 *
400
30 400 *
200 200
#
0
0 0.1 1 10 100 0 0
LPS LPS
(10 ng/mL) - + + + + - + + + +
(10 ng/mL)
GGOH concentration (µM)
GGOH (µM) - 0 0.1 1 10 GGOH (µM) - 0 0.1 1 10

(d) Tnf-α (e) Cox-2

80 # 100
# • GGOH did not affect MG6
viabilities up to 10 µM.
mRNA (fold)

80
mRNA (fold)

60

40
60 * • GGOH significantly down-
* 40 regulated the pro-
20 * 20 inflammatory cytokines
0 0 mRNA expressions.
LPS LPS
(10 ng/mL) - + + + + - + + + +
(10 ng/mL)

GGOH (µM) - 0 0.1 1 10 GGOH (µM) - 0 0.1 1 10

Fig. 2.1. (a) MG6 cell viabilities in different GGOH concentrationss. (b-e) The pro-inflammatory cytokine mRNA expressions in GGOH
administration. n = 3. # p < 0.05 vs control. * p < 0.05 vs LPS.
 25
LPS time on the NF-κB-related protein
MG6 LPS
(10 ng/mL) Western blot
cell culture

P-TAK1 LPS

P-IKKαβ NEMO
P TAK1
P-p65 IKKα IKKβ
P
α-tubulin
p50 p65
P
0 10 60 120
LPS time (min)

P-TAK1 P-IKKα/β P-p65

* *
5 10 25 *
4 8 * 20
*
fold

fold

fold
3 6 15
2 4 10 *
1 2 5
0 0 nd * 0
0 30 60 120 0 30 60 120 0 30 60 120

LPS time (min) LPS time (min) LPS time (min)

Fig. 2.3. LPS administration induce inflammatory proteins expressions in time-dependent manners. n = 3. * p < 0.05 vs 0 min.
 26
GGOH on the NF-κB-related protein expressions (1)
P-TAK1 TAK1
P-IKKαβ IKKβ

α-tubulin α-tubulin
control LPS GGOH 1 µM control LPS
GGOH 10 µM GGOH 1 µM GGOH 10 µM
+LPS +LPS +LPS +LPS

P-TAK1 TAK1 P-TAK1/TAK1

1.2 1.05 1.8
1
* 1 1.5

fold
fold
fold

0.8 0.95 1.2

0.6 0.9 0.9 *
0.4 0.85 0.6
0.2 0.8 0.3
0 0.75 -2.22044604925031E-16
LPS LPS LPS
- + + + - + + + - + + +
(10 ng/mL) (10 ng/mL) (10 ng/mL)

GGOH (µM) - 0 1 10 GGOH (µM) - 0 1 10 GGOH (µM) - 0 1 10

P-IKKα/β IKKβ P-IKKαβ/IKKβ

# #
12 1.2 15
10 1 * 12

fold
fold
fold

8 0.8 9
6 0.6
6
4 * 0.4 *
2 0.2 3
0 LPS
0 LPS
0
LPS - + + + - + + +
(10 ng/mL) - + + + (10 ng/mL) (10 ng/mL)

GGOH (µM) - 0 1 10 GGOH (µM) - 0 1 10 GGOH (µM) - 0 1 10

Fig. 2.4. GGOH administration significantly down-regulated the NF-κB-related protein expressions.
n = 3. # p < 0.05 vs control. * p < 0.05 vs LPS without GGOH.
 27
GGOH on the NF-κB-related protein expressions (2)
P-p65 p65

P-IκBα IκBα

α-tubulin α-tubulin

control LPS GGOH 1 µM control LPS GGOH 1 µM

GGOH 10 µM GGOH 10 µM
+LPS +LPS +LPS +LPS

P-p65 p65 P-p65/p65

# # #
5 2 3

fold
2.5
4 *
fold

1.6
fold
2
3 1.2 *
2 * 0.8
1.5
1
1 0.4 0.5
LPS 0 0 0
- + + + LPS LPS
(10 ng/mL) - + + + (10 ng/mL) - + + +
(10 ng/mL)
GGOH (µM) - 0 1 10 GGOH (µM) - 0 1 10
GGOH (µM) - 0 1 10

P-IκBα IκBα P-IκBα/IκBα

# #
10 1.2
* 25
* *
fold

8 1 20
fold

fold
6 0.8 # 15
4
0.6
10
*
0.4
2 * 0.2 5 *
LPS
0 LPS
0 0
- + + + - + + + LPS
(10 ng/mL) (10 ng/mL) (10 ng/mL) - + + +

GGOH (µM) - 0 1 10 GGOH (µM) - 0 1 10 GGOH (µM) - 0 1 10

Fig. 2.5. GGOH administration significantly down-regulated the NF-κB-related protein expressions.
n = 3. # p < 0.05 vs control. * p < 0.05 vs LPS without GGOH.
 28
GGOH on the p65 NF-κB subunit distribution (1)
Incubation
MG6 LPS Fluorescence
GGOH
cell culture 24 h (10 ng/mL) 30 min Microscopy
(10 µM)
control
LPS
GGOH+LPS p65 Nuclei merge

Fig. 2.6. GGOH administration inhibited the p65 nuclear translocation. Scale bars, 20 μm.
 29
GGOH on the p65 nuclear translocation (2)
Incubation
MG6 GGOH LPS Fractionation
cell culture (10 µM) 24 h (10 ng/mL) and Western
Cytoplasmic Nuclear

p65 p65

tubulin lamin

control LPS GGOH+LPS control LPS GGOH+LPS

p65 cytoplasmic p65 nuclear

1.5 10 #

1.2 8

0.9 6
fold

fold

0.6 4
*
0.3 2

0 0
control LPS GGOH+LPS control LPS GGOH+LPS

• Similar to MK-4, GGOH inhibited the NF-κB p65 sub-unit nuclear translocation either
in fluorescence microscopy analysis or cytoplasmic-nuclear protein distribution.

Fig. 2.7. GGOH administration inhibited the p65 nuclear translocation. n = 3. # p < 0.05 vs control. * p < 0.05 vs LPS.
 30
Isoprenoid on the inflammatory cytokines

Incubation
MG6 Isoprenoid LPS qRT-PCR
cell culture 24 h (10 ng/mL) 3h

Geranylgeraniol Il-1β
(GGOH) 1.2
1

mRNA (fold)
0.8
*
Farnesol 0.6 *
(FOH)
0.4
0.2
0
Phytol LPS GGOH FOH POH
(POH)

Il-6 Cox-2
Tnf-α
1.2 1.2 1.2
1 * 1
mRNA (fold)

mRNA (fold)
1
* *
mRNA (fold)

0.8 0.8
*
0.8 * * *
0.6
0.6 0.6 *
0.4 0.4 0.4
0.2 0.2
* 0.2
0 0 0
LPS GGOH FOH POH LPS GGOH FOH POH LPS GGOH FOH POH

Fig. 2.8. Isoprenoid administration significantly down-regulated the pro-inflammatory cytokine genes expressions.
n = 3. p < 0.05 * vs LPS treated.
 31
Discussion (2)

LPS
TLR4

Ub P

IκBα P
K63
TRAF6 IRAK1 p50 p65 IκBα
Ub
Ub Ubiquitin
TAB2/3
K63 Proteasome
TAB1 TAK1 Ub
P
NEMO
NF-κB target genes
IKKα/β transcription
P
p50 p65
GGOH
κB site

• GGOH inhibited LPS-induced inflammation via modulation of IKKα/β events which

inhibited further down-steam signalling.
 32
Microglial induced neurotoxicity
LPS, TNF-α, IFN-γ
IL-1β, TNF-α,
IL-6

neurotoxic function
proinflammatory microglia

MK-4 exhibited protective effects against microglia-mediated SHSY5Y

neuroblastoma cell death (Yu, et al., 2016).

Conditioned medium

GGOH

MG6 Lipopolysaccharide
HT22
Mouse microglial-derived Cells
Hippocampal neuron cells
 33
GGOH on the pro-inflammatory cytokines induced by LPS_CM

Collect and transfer conditioned medium IL-1β, TNF-α, IL-6

MG6 cells New MG6 cells

LPS
GGOH Pro-inflammatory normal microglia Pro-inflammatory
(100 ng/mL)
pretreatment microglia microglia
3h
24h LPS_CM

Direct LPS

GGOH (-) GGOH (-) GGOH (-)

Il-1β Il-6 GGOH (+) GGOH (+)
c
GGOH (+) Tnf-α
1.4 1.6 3 c
1.2 a 1.4 c 2.5
mRNA (fold)

1.2 c
mRNA (fold)

mRNA (fold)
1
1 a a 2
0.8 b b
0.8 1.5
0.6
0.6
b a
0.4
0.4
1 b
0.2 0.2 0.5
0 0 0
Direct LPS LPS_CM Direct LPS LPS_CM Direct LPS LPS_CM

• LPS_CM from MG6 cells induced further inflammation in new batch MG6 cells.
• GGOH pretreatment on LPS induced MG6 cells reduced the inflammatory potent of
LPS_CM on new batch of MG6 cells.
Fig. 3.1. The subsequent inflammation in new MG6 cells induced by conditioned medium from other LPS-induced MG6 cells.
n = 3. * p < 0.05, different letters indicated significant different.
 34
GGOH on the HT22 viability by LPS conditioned medium
(a) 120
100
cell viability (%)

80 • GGOH did not induce any cell toxicity in HT22 cell until 10 µM.
60
#
40 #
• GGOH pretreatment on LPS induced MG6 cells reduced the
20 toxicity in LPS_CM induced HT22 cells.
0
0 0.1 1 10 50 100
GGOH concentration (µM) (c) Collect and transfer conditioned medium
MG6 cells HT22 cells
(b) Collect and transfer conditioned medium

GGOH LPS
MG6 cells HT22 cells pretreatment (1 µg/mL)
24h 6h HT22 cell
Direct viability
LPS LPS (24 h)
HT22 cell 120
viability

HT22 viability (%)

(24 h) 100
120
*
HT22 viability (%)

100 80

* *
80 * * 60 #
60
40
40
LPS 20
20 LPS_CM
0 0
0 10 100 1000 LPS_CM
(1 µg/mL) - + + +
LPS concentration (ng/mL)
GGOH (µM) - 0 1 10
Fig. 3.2. The indirect protection of HT22 cells by GGOH from LPS conditioned medium. n = 4. # p < 0.05 vs control. * p < 0.05 vs LPS_CM
 35
GGOH on the HT22 PI staining by LPS conditioned medium
Collect and transfer conditioned medium

MG6 cells HT22 cells

GGOH LPS
pretreatment (1 µg/mL)
PI staining
24h 6h

Control LPS_CM GGOH 1 µM+LPS_CM GGOH 10 µM+LPS_CM

PI
Phase

• GGOH pretreatment on LPS induced MG6 cells reduced the PI intensity tendencies on
HT22 cells.
Fig. 3.3. Representative images of PI positive cells in the HT22 cells incubated with conditioned medium from LPS-induced MG6 cells.
Scale bars 100 µm.
 36
Discussion (3)
LPS, TNF-α, IFN-γ
IL-1β, TNF-α,
IL-6

neurotoxic function
proinflammatory microglia

GGOH ERK1/2 Apoptosis

P

Inflammation ROS p38 Nrf2

P

Apoptosis
• GGOH indirectly protected Nrf2 target genes
JNK transcription
HT22 cell viability from the sMAF Nrf2
microglial-mediated P
ARE
neurotoxicity by inhibiting
inflammation in MG6 cells.
 37
Current Investigation
Experimental Group :
• Cont. Standard Diet (VK1 0.75 mg/kg diet = AIN93G)
• LPS Standard Diet + LPS i.p. (0.35 mg/kg)
• VK and LPS Vit. VK1 supplementation (75 mg/kg) + LPS i.p. (0.35 mg/kg)
• MK4 and LPS MK4 supplementation (75 mg/kg) + LPS i.p. (0.35 mg/kg)
1 week 2 week
sacrifice
acclimatization diet administration

behaviour test
C57BL/6J mice daily LPS i.p.
male, n=8 11-12 weeks day
Item Cont. VK1 MK-4 01 5
Corn starch 52.9486 52.9486 52.9486
Casein 20 20 20
• Behaviour test Open field test,
Sucrose 10 10 10 Passive avoidance
Soybean Oil 7 7 7
Cellulose 5 5 5 • Hippocampus & Cortex
Mineral Mix (AIN-93M) 3.5 3.5 3.5 Inflammatory markers, BDNF
Vitamin Mix 1 1 1 related-pathway, Vit K conc.
L-cysteine 0.3 0.3 0.3
Choline bitartate 0.25 0.25 0.25 • Liver and Blood Inflammatory
Tert-butyl hydroquinone 0.0014 0.0014 0.0014 biochemical markers
VK-1 0.0075
MK-4 0.0075
 38
General Conclusion

In order to maintain and improve people’s health and to

prevent chronic non-communicable diseases, a key
element is a balanced diet of staple foods (e.g.,
steamed rice), main dishes (fish, meat, chicken, eggs, tofu
etc.), and side dishes (vegetables etc.). Healthy
individuals should obtain all necessary energy,
nutrients and non-nutritious components from regular
meals. They should never take quasidrugs containing
vitamins and minerals and “health foods,” including “Foods
with Health Claims,” in place of the daily diet and regular
meals.