Investigating the Bioactive Constituents of the Edible ...
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University of ConnecticutOpenCommons@UConn
Honors Scholar Theses Honors Scholar Program
Summer 5-1-2015
Investigating the Bioactive Constituents of theEdible Blue-green Alga Spirulina platensisGeorgette Appiah-PippimUniversity of Connecticut - Storrs, [email protected]
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Part of the Medicinal Chemistry and Pharmaceutics Commons
Recommended CitationAppiah-Pippim, Georgette, "Investigating the Bioactive Constituents of the Edible Blue-green Alga Spirulina platensis" (2015). HonorsScholar Theses. 408.https://opencommons.uconn.edu/srhonors_theses/408
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Investigating the Bioactive Constituents of the Edible Blue-
Green Alga Spirulina platensis
Author: Georgette Appiah-Pippim
Faculty Advisor: Marcy Balunas, Medicinal Chemistry, Pharmaceutical
Sciences
Honors Advisor: Kristen Kimball, Physiology and Neurobiology
University of Connecticut Honors Program
Date Submitted:
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Abstract
More people die annually from cardiovascular disease than from any other cause. Two
risk factors for cardiovascular disease are hyperlipidemia and inflammation. Current drugs that
are prescribed to regulate lipid levels often have adverse effects such as liver dysfunction. Blue-
green algae (BGA), also known as cyanobacteria, have been consumed for years for their health
benefits because they are believed to increase energy and prevent disease. One genus of edible
blue-green algae is Spirulina plantesis (SP, Spirulina). Various studies have shown that Spirulina
may have anti-cancer and anti-inflammation and anti-bacterial properties. In a previous study,
Spirulina platensis lowered triglyceride and cholesterol levels in hyperlipidemic rats. The rats
were fed a Spirulina supplemented diet and their lipid levels were measured by their fecal output
and intestinal absorption. However, the study did not involve isolation of the compounds
responsible for this biological activity. The goal of this project is to isolate the specific
compound(s) responsible for the anti-inflammatory and hypolipidemic effects of Spirulina
platensis.
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Introduction
Cardiovascular disease is the number one cause of death in men and women in the United
States (Ford 2012). Cardiovascular disease is defined as changes in the physiology and structure
of the heart or blood vessels that impairs function (AHA 2015). This research project focused on
two risk factors for cardiovascular disease: hyperlipidemia and inflammation. Inflammation is an
immune system response to harmful stimuli. Hyperlipidemia is an increase in triglycerides in the
blood plasma (Gotto 1977). Hypercholersterolemia is a subset of hyperlipidemia defined as an
increase in total cholesterol levels in the blood (Gotto 1977).
Hypercholesteromlemia can be familial or can be triggered by lifestyle and diet (Haines
2013). The two processes involved in cholesterol regulation are cholesterol synthesis and
cholesterol absorption from the diet. Cholesterol synthesis begins in the mevalonate pathway
(Figure 1). The rate-limiting enzyme in cholesterol biosynthesis is 3-hydroxy-3-methyl-glutaryl-
CoA Reductase (HMG-CoA Reductase or HMGR), the enzyme that converts HMG-CoA into
mevalonate (Haines 2013). Mevalonate is converted to squalene and then to lanosterol. There are
two pathways that convert lanesterol to cholesterol: the Kandutch-Russell pathway and the Bloch
pathway. The Kandutch-Russell pathway converts lanosterol to lathosterol and 7-
dehydrocholesterol before converting it to cholesterol while desmosterol is the intermediate in
the Bloch pathway (Wu 2014) (Figure 2). The intermediates of cholesterol synthesis are used as
biomarkers to determine the rate of in vivo cholesterol synthesis.
Figure 1. Enzymes and products of
Figure 2. Biosynthesis of cholesterol through the Bloch and Kandutsch
2014).
Acetate HMG
Kan
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Enzymes and products of the mevalonate pathway (Haines 2013).
Biosynthesis of cholesterol through the Bloch and Kandutsch-Russell Pathway (
GCoA Mevalonate Lanosterol
Desmosterol Cholesterol
Lathosterol
7-
dehydrocholesterol
BlochPathway
andutsch-Russell Pathway
Russell Pathway (Wu
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Dietary cholesterol absorption begins in the small intestine. Cholesterol particles form
micelles and pass from the lumen of the intestine into the blood (Elshourbagy 2014). Cholesterol
is insoluble in the plasma and is transported as lipoproteins. The types of lipoproteins in
circulation are high-density lipoproteins (HDL), intermediate density lipoproteins (IDL) low-
density lipoproteins (LDL) and very low-density lipoproteins (VDL) (Elshourbagy 2014). The
level of density of the lipoproteins represents the amount of protein they carry, with HDL
composed of the highest level of protein (Elshourbagy 2014). HDL and LDL are the most
commonly monitored lipoproteins. HDL and LDL are colloquially referred to as “good
cholesterol” and “bad cholesterol” respectively. HDL facilitates reverse cholesterol transport, the
process in which excess cholesterol from cells is returned to the liver to be degraded
(Elshourbagy 2014). Dyslipidemia is a decrease in the amount of HDL. LDL transports
cholesterol from the liver to peripheral tissues. A buildup of LDL in the plasma leads to
inflammation and atherosclerosis (Elshourbagy 2014).
LDL buildup in blood vessels puts stress on the vessel walls (Salisbury 2014). This stress
damages the vessel walls inducing the inflammatory response. Macrophages and other immune
cells recruited as part of the inflammatory response secrete of pro-inflammatory cytokines such
as tumor necrosis factor alpha (TNFα) and interleukin-1 beta (IL-1β,IL-1B) (Salisbury2014).
These cytokines further progress the inflammatory state. Animal studies have shown that HDL
decreases inflammation by removing LDL from vessel walls (Elshourbagy 2014).
The lipid hypothesis states that high LDL levels correlate positively with cardiovascular
disease risk and high HDL levels correlate negatively (Tobert 2003). In 1984, the National
Institute of Health began to urge physicians to monitor the diets and nutrition of their patients
with hypercholesterolemia in order to control their LDL levels. The first drugs employed to treat
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hypercholesterolemia inhibited enzymes at the end stages of the mevalonate pathway. However,
most of these drugs had adverse side effects such as the development of cataracts and toxicity.
As a result, research shifted to the rate-limiting enzyme in cholesterol synthesis, HMGR. The
first HMGR inhibitor was called compactin. It was isolated from a bacterial broth culture by a
Japanese scientist in the 1970’s. The most common class of HMGR inhibitors is the statin
family. Statins bind competitively to the enzyme’s active site. The first clinically approved statin,
lovastatin, was isolated from a fermented Aspergillus terreus broth (Tobert 2003). Eight
derivatives of lovastatin and compactin have been synthesized (Figure 5). Other treatments for
hypercholesterolemia include fibrates, bile acid sequestrates, and nicotinic acid.
Current treatment of hyperlipidemia focuses on monitoring specific lipid levels and
reducing all cardiovascular disease risk factors. Data derived from clinical trials and
observational studies have led to the development of three health questions recommended by the
American Heart Association to determine therapy options. Rather than basing therapies solely on
LDL levels, treatment intensity is decided based on age, comorbidity, in addition to LDL levels
(Pines 2014). The most common drug therappies for hypercholesterolemia are statins in
combination with diet modification. Side effects of statin use include muscle and liver damage,
impairment of cognitive function, and increased blood sugar (Dimmit 2015).
Lipid metabolism and inflammation are interconnected. Circulating cholesterol and lipids
are known to stimulate the inflammatory response (van Diepen 2013). Pro-inflammatory
cytokines also have a direct effect on lipid metabolism. As a result of this connection, lipid-
lowering drugs can also have anti-inflammatory effects. Statins, niacin, and fibrates decrease
inflammation by interacting directly with inflammatory pathways (van Diepen 2013). Non-
steroidal anti-inflammatory drugs (NSAIDs) are a major class of drugs used to treat pain and
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inflammation. Aspirin is an NSAID that decreases inflammation by reducing the synthesis of
prostaglandins. High-dose aspirin treatment also reduce lipid and cholesterol levels (van Diepen
2013). However, a study found that patients with cardiovascular disease who use the NSAIDs
diclofenac and rofecoxib are five times as likely to suffer a myocardial infarction than patients
with cardiovascular disease that don’t use those NSAIDs. (Schjerning 2014).
One mechanism of action to reduce inflammation is to target pro-inflammatory cytokines.
In low doses, a drug called methotrexate, serves as an anti-inflammatory drug by block the pro-
inflammatory cytokine interlukin-1beta from binding to its receptor (van Diepen 2013).
Methotrexate has been shown to increase HDL levels in patients with rheumatoid arthritis (van
Diepen 2013). Possible side effects from methotrexate use include gastrointestinal bleeding and
edema. Tocilizumab, infliximab, and canakunimab also target pro-inflammatory cytokines.
These drugs decrease the levels of the pro-inflammatory cytokines tumor necrosis factor alpha,
interleukin 6 and interleukin-1beta respectively (van Diepen 2013). Specifically, these drugs
disrupt cytokine signaling by degrading the cytokine receptors. Anti-cytokine drugs can have
adverse gastrointestinal effects and interact with medicines such as birth control and blood
thinners.
As a result of the side effect of common drug therapies, research on natural products with
hypolipidemic properties is necessary. Historically, natural products have been the source of
many drugs and drug therapies. Between the years 1981 and 2010, 34% of drugs based on small
molecules came from natural product sources (Harvey 2015). Researchers have been analyzing
techniques that have utilized natural products for years, such as traditional Chinese medicine, in
an effort to find natural products that match their targets.
Figure 5. Structures of lovastatin and compactin drug derivatives
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Structures of lovastatin and compactin drug derivatives (Tobert 2003)..
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Natural product extracts usually contain multiple compounds, which can be obstacle or an
advantage. An array of compounds could mean that a single natural product could work on
multiple targets. On the other hand, inactive compounds present in larger amounts may block the
activity of less abundant active compounds. Complications with isolation and synthesizing of
natural products have caused a decline in natural product drug discovery over the past two
decades (Harvey 2015).
Table 1 shows 23 natural compounds and extracts that decreased or inhibited HMGR and
had additional hypolipidemic effects. The compounds and extracts are divided into in vivo and in
vitro studies. Compounds are further divided by the method of action: transcriptional or
enzymatic regulation.
One of the particularly interesting compounds is naringenin, a flavonoid from grapefruit,
exerts hypolipidemic effects in vitro. Rat hepatocytes treated with 200µM of naringenin had a
60% reduction in triglyceride production. Naringenin also increased LDLR expression and
decreased HMGR and HMG-CoA synthase (HMGCS) expression (Goldwasser 2010). HMGCS
is an enzyme involved in cholesterol biosynthesis. This compound has great medicinal potential
because it has shown anti-inflammatory and hypolipidemic effects in vivo and in vitro.
Additionally, the effect of naringenin on fatty acid oxidation and cholesterol biosynthesis
indicates that it may play an important role in the regulation of lipid metabolism (Goldwasser
2010).
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Table 1. Natural Products with hypolipidemic properties.
Co
mp
ou
nd
/Extr
act
So
urc
e M
eth
od
of
Acti
on
Do
sage
Ref
eren
ce
In
vit
ro
Am
ylo
id b
eta
Am
ylo
id p
recu
rsor
pro
tein
In
hib
its
HM
GR
n
/a
Gri
mm
20
07
Ag
lyco
ne i
sofl
avo
nes
K
ore
an s
oyb
ean p
aste
In
hib
its
HM
GR
3
0 µ
g/1
50
µl
Su
ng 2
012
Inh
ibit
ory
pep
tide
GF
PD
GG
G
lycin
e s
oja
B
inds
com
pet
itiv
ely t
o H
MG
R
IC50 1
.5µ
M
Pak
200
8
Lip
id e
xtr
act
N
ost
oc c
om
mu
ne
Inh
ibit
s S
RE
BP
-2 d
ecre
asin
g H
MG
R m
RN
A.
100
mg
/L
Ras
mu
ssen
20
08
M2
/Co
lum
bam
ine
Rh
izom
a c
opti
dis
P
hosp
ho
ryla
tes
AM
PK
, A
MP
K i
nh
ibit
s H
MG
R i
n t
he
liv
er
15µ
M
Cao
201
3
Ro
xy
losi
de
Ro
sa d
am
asc
ena
In
hib
its
HM
GR
0
.12
mg
/mL
K
wo
n 2
01
0
In
viv
o
Alg
ae p
ow
der
S
chiz
ochytr
ium
D
ecre
ased
in
test
inal
ch
ole
ster
ol
abso
rpti
on
an
d H
MG
R
mR
NA
10g
/kg
bw
W
u 2
00
3
Ber
beri
ne
Cop
tis
ch
inen
sis
Dec
reas
ed h
epati
c c
hole
ster
ol
upre
gu
late
d L
DL
R
200
mg
/kg
bw
Ch
ang
201
2
Ber
gam
ot
po
lyp
hen
ols
C
itru
s b
erg
am
ia
Sel
ecti
ve
inh
ibit
ion
of
HM
GR
2
0 m
g/d
ay
Mo
llac
e 20
11
β-a
myri
n a
ceta
te a
nd
β-a
my
rin
pal
mit
ate
Wri
gh
tia t
om
en
tosa
lea
ves
In
hib
its
HM
GR
by b
indin
g t
o a
ctiv
e si
te
10m
g/k
g b
w
Maury
a 20
12
Dia
llyld
isu
lfid
e a
nal
ogs
All
ium
sat
ivu
m
Dec
reas
e H
MG
R m
RN
A
20m
g/k
g b
w
Rai
200
9
Eth
ano
lic
extr
act
S
ym
plo
cos
racem
osa
Rox
b
bar
k
Inh
ibit
ed l
iver
HM
GR
4
00
mg
/kg
bw
Du
rkar
20
14
Gin
ger
ol
Zin
gib
er o
ffic
inal
e In
creas
ed L
DL
R m
RN
A,
dec
rease
d H
MG
R m
RN
A
400
mg
/kg
bw
Nam
mi
201
0
Met
han
oli
c ex
tract
C
ela
stru
s pa
nic
ula
tus
seed
In
hib
its
HM
GR
6
5 m
g/k
g
Pat
il 2
010
Met
han
oli
c ex
tract
A
co
nit
um
hete
roph
yll
um
In
hib
its
HM
GR
4
00
mg
/kg
Su
bas
h 2
01
2
Mo
no
terp
ene
ger
anio
l L
emo
ng
rass
oil
D
ecre
ases
tra
nsl
atio
nal
eff
icacy
of
HM
GR
1
00
mg
/kg
bw
Jay
achan
dra
n
201
5
Nari
ngenin
G
rapef
ruit
In
duce
s L
DL
R t
ran
scri
pti
on
, in
hib
its
SR
EB
P d
epen
den
t
HM
GR
200
µM
G
old
wass
er
20
10
So
pho
rico
side
Sop
ho
ra j
ap
on
ica
Do
wn
reg
ula
tes
SR
EB
P
10µ
M
Wu 2
01
4
Ste
rol
extr
acts
S
chiz
ochytr
ium
sp
Up
reg
ula
tes
LD
LR
dow
n r
eg
ula
tes
HM
GR
0
.30
g/k
g
Ch
en 2
013
Red
koji
extr
act
Red
ko
ji
Inh
ibit
s H
MG
R
n/a
W
u 2
00
3
Ru
tin
P
lan
t-deri
ved
die
tary
fla
van
oid
D
ecre
ased
HM
GR
mR
NA
2
00
µM
C
hen
g 2
01
1
RV
S g
lyco
pro
tein
R
hus
vern
icif
lua
Sto
kes
Inh
ibit
s H
MG
R
100
mg
/kg
Su
n 2
00
6
Tan
nic
aci
d
Pla
nts
D
ecre
ased
HM
GR
mR
NA
lev
els
0.0
2%
w/w
D
o 2
011
Figure 6. Structure of naringenin
Figure 7. Stuctures of active bergamot flavonoids (Mollace 2011
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Structure of naringenin (Goldwasser 2010).
ergamot flavonoids (Mollace 2011).
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Another citrus plant with bioactivity is bergamot (Figure 7). Bergamot is known for its
high content and diversity of flavonoids. In a clinical study, polyphenol fractions of bergamot
juice were freeze-dried and turned into 500mg tablets. These tablets were given to an
experimental group of patients with hypercholesterolemia once a day. The control group of
hypercholesterolemic patients was given a daily 500mg placebo pill. After 30 days, patients who
took the bergamot pills had a 20% reduction in total cholesterol and LDL cholesterol (Mollace
2011). HPLC analysis of the bergamot fraction revealed five flavonoids that made up 26-28% of
the active fraction (Mollace 2011)
Spirulina platensis (also referred to as SP and Spirulina) is a prokaryotic blue green alga
also known as cyanobacteria. These organisms are photosynthetic but do not have plant cell
walls, nuclear membranes, or internal organelles, resembling bacterial cells (Singh 2007).
Spirulina was first isolated from a freshwater river in 1827 (Ciferri 1983). Indigenous
populations in Africa and Mexico are known to have cultivated Spirulina from lakes and
consumed the dried alga (Ciferri 1983). Spirulina has many bioactive components such as
chlorophylls, carotenoids, amino acids, minerals, phycocyanin and secondary metabolites (Singh
2007) (Figure 8a). Spirulina is known as a superfood because of the abundance of nutrients it
contains. The amounts of these various components depend on the environment in which the
Spirulina is grown (Singh 2007). Cyanobacteria are known to have a diverse range of biological
activity such as anti-malarial, anti-inflammatory, and anti-viral activity (Figure 8b).
Figure 8. A) Composition of Spirulina platensis.
(Singh 2007).
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Spirulina platensis. B) known biological activity of biological activity of cyanobacteria
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In a previous studies done by Dr. Ji-Young Lee, the anti-inflammatory and hypolipidemic
properties of two edible genera of blue-green algae have been shown (Yang 2011). An extract of
the blue green algae Nostoc commune var. sphaeroides Kützing decreased the mRNA levels of
pro-inflammatory cytokines, HMGR and the LDL receptor (LDLR) in RAW macrophages and
HepG2 cells. In vivo studies of edible blue green algae with the C57BL/6J mice model of
hyperlipidemia have had similar results. Mice fed a high fat diet supplemented with Nostoc and
Spirulina for 4 weeks showed a change in the composition of intestinal bacteria and a decrease in
plasma triglyceride and cholesterol levels (Rasmussen 2009). These hypolipidemic effects are
attributed to an increased intestinal absorption of lipids and an increased fecal output (Ku 2013).
The long-term effects of Spirulina and Nostoc were studied in vivo by supplementing the diets of
mice with the BGA for six months. The mice from the experimental group showed no adverse
affects in comparison to the control group.
Although these studies have demonstrated the anti-inflammatory and hypolipidemic
properties of blue-green algae, the compounds responsible the bioactivity are unknown. The goal
of my project was to isolate the compound(s) responsible for the effects of Spirulina platensis.
Compound structures will be determined and these will be tested on biological assays.
Experimental
Fifty grams of dried Spirulina was obtained from Earthrise Nutritionals in Irvine, CA.
The dried algae was weighed and incubated overnight in 300mL of a 2:1 ratio of
dichloromethane (DCM) to methanol. After incubation, the alga and solvent were poured into a
funnel with coarse filter paper to separate the solvent from the alga. The alga was incubated 8-10
times until an exhaustive extraction was performed, resulting in 6.43 g of crude extract. The
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crude extract was rehydrated with hexanes, combined with silica (Pittsburg, PA) and passed
through a flash column. Solvents (500mL each) were passed through the column with added air
pressure according to the following ratio: A: 100% hexanes, B: 10% ethyl acetate
(EtOAc)/hexanes, C: 20% EtOAc/hexanes, D: 40% EtOAc/hexanes, E: 60% EtOAc/hexanes, F:
80% EtOAc/hexanes, G: 100% EtOAc, H: 25% MeOH/EtOAc, I: 100% MeOH, J: 100% DCM
and 100% MeOH.
GAPSP1 fractions A-J were analyzed using liquid chromatography mass spectroscopy
(LCMS) with Agilent ESI single quadrupole mass spectrometer (Santa Clara, CA) paired to
Agilent high performance liquid chromatography (HPLC) system with a G1311 quaternary
pump, G1322 degasser, and a G1315 diode array detector using an Eclipse XDB-C18 (4.6 × 150
mm, 5 µm) RP-HPLC column. Fractions that had shown activity in the previous study were also
tested on the Agilent HPLC system.
The bioactivity of SP was tested for HMG-CoA Reductase (HMGR) expression by
treating a HepG2 cell with the GAPSP1 fractions dissolved in dimethyl sulfoxide (DMSO) at a
concentration of 50 µg/mL of media. The HepG2 cells were incubated for 24 hours. Interleukin-
1β (IL-1b) expression was tested using RAW 264.7 mice macrophage bioassay. The fractions
were applied to at a concentration of 1 µg/mL. Before fractions were added, the macrophages
were treated with 100 ng/mL of lipopolysaccharide (LPS) to stimulate inflammation.
After analysis of the LCMS chromatograms, GAPSP1C was selected for further
fractionation by reverse phase solid phase extraction (SPE) on a DSC-18 500mg column using
60 mL of the following solvent systems: 1: 25% MeOH/ wawter (H2O) 2: 50% MeOH/H2O 3:
75% MeOH/H2O 4: 100% MeOH 5: 100% EtOAc.
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GAPSP1C fractions 1-5 were analyzed using LCMS. GAPSP1C4 was analyzed on HPLC
using 75% ACN and 70% ACN. GAPSP1C4A, GAPSP1C4B, GAPSP1C4W, GAPSP1C4A.E
were collected from GAPSP1C4 using HPLC at 60% ACN over 30 minutes (Figure 5).
GAPSP1C4B was 60% pure and 0.84 mg of the compound was isolated (Figure 6). GAPSP1C
and GAPSP1C4B proton NMR spectra were recorded on a Brüker Avance 500 MHz
spectrometer (500.13 MHz 1 H, 125.65 MHz 13C).
Results
In our previous study, the D fraction (40% EtOAc/hexanes) eluted during the normal
phase flash column chromatography of the initial crude SP extract, reduced IL-1B and HMGR
expression (Figure 9). Further HPLC isolation of the D fraction yielded compounds,
CSKSP1D1+2. CSKSP1D1+2 was isolated at 40% ACN and eluted at 8.5-9.5 minutes. The
isolated compounds also had a repressive effect on HMGR and IL-1B (Figure 10).
A similar peak to those found in the original compound was found in the C fraction.
GAPSP1C (Figure 15) eluted at 20% EtOAc/hexanes during normal phase flash column
chromatography of the crude SP extract. SPE of GAPSP1C yielded five fractions. Compound 1
and 2 were most prevalent in GAPSP1C4 but were also present in fractions GAPSP1C3, and
GAPSP1C5. HPLC isolation at 60% ACN for 30 minutes yielded GAPSP1C4A eluted at 17
minutes, GAPSP1C4B eluted at 26 minutes, GAPSP1C4A.E, and GAPSP1C4W (Figure 16).
GAPSP1C4B is 60% pure and contains a similar peak to that found in CSKSP1D1+2 (Figure
17). GAPSP1 fractions A, B, H, I, J repressed IL-1b expression in RAW 264.7 macrophages
(Figure 14). The fractions did not show significant activity against HMGR expression.
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Figure 9. Bioactivity of CSKSP fractions in A) HepG2 cells and B) RAW 264.7 macrophages
(Ku 2014).
HMGR
Contr
ol
SP A
SP B
SP C
SP D
SP E
SP F
SP G
SP H
SP I
0.0
0.5
1.0
1.5
2.0
*
*****
**
Rela
tive E
xp
ressio
n
IL-1ββββ
Contr
ol
SP A
SP B
SP C
SP D
SP E
SP F
SP G
SP H
SP I
0.0
0.5
1.0
1.5
2.0
** ****
****** ******
*
Re
lati
ve
Ex
pre
ss
ion
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Figure 10. Bioactivity of CSKSPD fraction and CSKSPD1+2 in A) HepG2 cells and B) RAW
264.7 macrophages (Ku 2014).
Control SP-D 1+2 All Else0.0
0.5
1.0
1.5
***
***
***
IL-1ββββ
Re
lati
ve
Ex
pre
ss
ion
HMGR LDLR0.0
0.5
1.0
1.5
2.0
2.5Control
SP-D
1+2
All Else
a
c c
b
a
bc c
Re
lati
ve
Ex
pre
ss
ion
19
Figure 11. A) HPLC chromatogram and B) mass trace of CSKSPD1+2.
Current Chromatogram(s)
min2 4 6 8 10 12 14
mAU
-200
-100
0
100
200
300
400
500
600
DAD1 A, Sig=254,2 Ref=off (140729_MJB 2014-07-29 13-44-01\001-0101.D) DAD1 B, Sig=209,2 Ref=off (140729_MJB 2014-07-29 13-44-01\001-0101.D)
DAD1 C, Sig=299,2 Ref=off (140729_MJB 2014-07-29 13-44-01\001-0101.D)
Current Chromatogram(s)
min5 10 15 20 25 30
0
5000000
10000000
15000000
20000000
25000000
30000000
35000000
40000000
MSD1 TIC, MS File (C:\CHEM32\1\DATA\140729_MJB 2014-07-29 13-44-01\001-0101.D) ES-API, Pos, Scan, Frag: 70, "Pos_Sc MSD2 TIC, MS File (C:\CHEM32\1\DATA\140729_MJB 2014-07-29 13-44-01\001-0101.D) ES-API, Neg, Scan, Frag: 70, "Neg_Sc
Figure 12. Proton NMR spectrum of CSKSPD1+2
20
spectrum of CSKSPD1+2.
21
Figure 13. GAPSP1 fraction tree.
Figure 14. IL-1b expression in RAW 264.7 macrophages after treatment with GAPSP1
fractions.
GAPSP1C4A(
0.83mg(
GAPSP1C4B(
0.48mg(
GAPSP1C4A.E(
1.69mg(
GAPSP1C4W(
4.2mg(
HPLC(
60%ACN,1ml/min,30(min(
GAPSP1C1(0.97mg(
GAPSP1C2(6.06mg(
GAPSP1C3(1.82mg(
GAPSP1C4(13.76mg(
GAPSP1C5(21.69mg(
SPE(
SP(6.43g(
A(540.22mg(
B(185.29mg(
C(74.86mg(
D(813.74mg(
E(98.88mg(
F(46.32mg(
G(47.68mg(
H(2496.29mg(
I(809.74mg(
J(123.39mg(
Flash(column(
22
Figure 15. A) HPLC chromatogram and B) mass trace of GAPSP1C.
min5 10 15 20 25 30
mAU
-1400
-1200
-1000
-800
-600
-400
-200
0
DAD1 A, Sig=254,2 Ref=off (140718_SMG 2014-07-18 16-48-21\009-0901.D) DAD1 B, Sig=209,2 Ref=off (140718_SMG 2014-07-18 16-48-21\009-0901.D) DAD1 C, Sig=299,2 Ref=off (140718_SMG 2014-07-18 16-48-21\009-0901.D)
min5 10 15 20 25 30
0
5000000
10000000
15000000
20000000
25000000
MSD1 TIC, MS File (C:\CHEM32\1\DATA\140718_SMG 2014-07-18 16-48-21\009-0901.D) ES-API, Pos, Scan, Frag: 70, "Pos_Sc
MSD2 TIC, MS File (C:\CHEM32\1\DATA\140718_SMG 2014-07-18 16-48-21\009-0901.D) ES-API, Neg, Scan, Frag: 70, "Neg_Sc
Instrument 1 3/30/2015 10:23:26 AM ASHLEY Page
23
Figure 16. HPLC chromatogram of GAPSP1C4 at 60%ACN over 30 minutes. GAPSP1C4A,
GAPSP1C4B, GAPSP1C4A.E, GAPSP1C4W are highlighted.
Current Chromatogram(s)
min10 20 30 40 50
mAU
-15
-10
-5
0
5
10
15
20
25
DAD1 A, Sig=254,2 Ref=off (GEORGINA\GAPSP1C4000006.D) DAD1 B, Sig=209,2 Ref=off (GEORGINA\GAPSP1C4000006.D) DAD1 C, Sig=299,2 Ref=off (GEORGINA\GAPSP1C4000006.D)
24
Figure 17. A) HPLC chromatogram and B) mass trace of GAPSP1C4B.
Current Chromatogram(s)
min5 10 15 20 25 30
0
10000000
20000000
30000000
40000000
50000000
60000000
70000000
MSD1 TIC, MS File (C:\CHEM32\1\DATA\150107_SMG 2015-01-07 16-47-13\002-0201.D) ES-API, Pos, Scan, Frag: 70, "Pos_Sc MSD2 TIC, MS File (C:\CHEM32\1\DATA\150107_SMG 2015-01-07 16-47-13\002-0201.D) ES-API, Neg, Scan, Frag: 70, "Neg_Sc
Instrument 1 3/30/2015 10:24:57 AM ASHLEY Page 1
Current Chromatogram(s)
min5 10 15 20 25 30
mAU
-800
-600
-400
-200
0
DAD1 A, Sig=254,2 Ref=off (150107_SMG 2015-01-07 16-47-13\002-0201.D) DAD1 B, Sig=209,2 Ref=off (150107_SMG 2015-01-07 16-47-13\002-0201.D)
DAD1 C, Sig=299,2 Ref=off (150107_SMG 2015-01-07 16-47-13\002-0201.D)
Figure 18. Proton NMR of GAPSP1C4
25
Proton NMR of GAPSP1C4
26
Discussion
Re-isolation of CSKSPD1+2 proved to be challenging because there was only a very
small amount of the compounds isolated. This limited the number of analyses that could be
performed. Also, large scale isolation of this compound was difficult because the active
compound was a very small portion of the crude Spirulina extract. Further analysis will be done
to determine if both compound 1 and 2 are active and if they act synergistically.
The original mass that was sought after in the GAPSP1 fractions was 369.1. This was the
mass of the most prevalent peak in the initial SP D fraction. Conversely, the isolated bioactive
compound, CSKSPD1+2, does not contain a peak with that mass. Future studies will pursue the
masses present in CSKSPD1+2.
GAPSP1C fraction was not active in the HMGR or IL-1B bioassays. This may be a result
of more abundant nuisance compounds overshadowing the anti-inflammatory or HMGR-
repressing compounds that are present in smaller amounts. GAPSP1C4B will be tested in the
HMGR and IL-1B bioassays to analyze the bioactivity. However, because only 0.48 mg were
isolated, more of the compound will be necessary for structure determination.
CSKSPD1+2 activity against HMGR and IL-1B mRNA demonstrates that the
compounds are active transcriptionally. Future studies should analyze if the compounds also
inhibit the enzymatic activity of HMGR and/or prevent IL-1B from binding to its receptor.
Conclusion and Future Directions
The goal of this study was to isolate the compounds responsible for the hypolipidemic
and anti-inflammatory effects of the edible blue-green alga, Spirulina platensis. Initial
27
fractionation of a crude Spirulina extract resulted in a bioactive D fraction which was further
fractionated into the bioactive compounds CSKSPD1+2. A second fractionation of a crude
Spirulina extract yielded a fraction, GAPSP1C that contained similar peaks to the original active
D fraction. The peak of interest was isolated in GAPSP1C4B, but not enough of the compound
was isolated to determine the chemical structure. Future studies will involve re-isolation of the
compound and comparison of the bioactivity of GAPSP1C4B to CSKSPD1+2. Additionally, the
synergistic activity of these two compounds and the other bioactive fractions will be analyzed.
28
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