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, georgette.appiah-pippim@uconn.edu

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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

1

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:

2

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.

3

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

4

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

5

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

6

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

7

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

8

Structures of lovastatin and compactin drug derivatives (Tobert 2003)..

9

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).

10

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

11

Structure of naringenin (Goldwasser 2010).

ergamot flavonoids (Mollace 2011).

12

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).

13

Spirulina platensis. B) known biological activity of biological activity of cyanobacteria

14

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

15

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.

16

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.

17

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

18

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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