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Licensee OA Publishing London 2013. Creative Commons Attribution Licence (CC-BY)

F :Almelkar SI, Patwardhan AM. Bacopa monniera extract plays a signiicant role in lipofuscin

scavenging in cultured human vascular endothelial cells. OA Alternative Medicine 2013 Feb 02;1(1):5.

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Compenginterests:nonedeclared.ConflictofInterests:nonedeclared.

Allauthorscontributedtotheconcepon,

design,andprep

araonofthemanuscript,aswellas

readandapprovedthefinalmanuscript.

AllauthorsabidebytheAssociaonforMedicalEthics(AM

E)ethicalrulesofdisclosure.

Bacopa monnieraextract plays a significantroleinlipofuscin

scavenging in cultured human vascular endothelial cells

SI Almelkar1*, AM Patwardhan2

* Corresponding authorEmail: [email protected]

1Department of Cardiovascular and ThoracicSurgery, Seth G.S. Medical College and KEMHospital, Parel, Mumbai 400012, Maharash-tra, India

2Department of Cardiovascular and ThoracicSurgery, JNMC, Acharya Vinoba Bhave Rural

Hospital, Sawangi (Meghe), Wardha 442004,Maharashtra, India

AbstractIntroduction

We investigated the generation of

lipofuscin granules in human umbil-

ical vein endothelial cells that were

exposed to hydrogen peroxide (H2O

2,

100 M) for 1 h. Lipofuscin granule

scavenging was studied using the

Bacopa monniera (Brahmi) extract

(100 g) for 2 h, 4 h and 6 h expo-

sures. We used an endothelial cell

culture model to investigate the

antioxidant effects of the B.

monniera extract on lipofuscin

granules scavenging in cultured

human umbilical vein endothelial

cells.

Materials and methods

To understand the protective role

of the B. monniera extract in post-

oxidative stressed human umbilical

vein endothelial cells, an experimentwas set-up with the following

exposure regimen: pre-B. monniera

extract (100 g) for 2 h plus post-

H2O

2(100 M) for 1 h. To analyse

whether pre-oxidative stressed

human umbilical vein endothelial

cells could be protected with

exposure to post-B. monniera

extract and whether the lipofuscin

granules could be scavenged by

post-B. monniera extract treatment,

the following exposure regimen wasprovided: pre-H

2O

2(100 M) for 1 h

plus post-B. monniera extract (100

g) for 2 h.

Introduction

due to the iron-catalysed oxida-

tion/polymerisation of protein and

lipid residues3. It is believed that th-

ese changes occur not only due to

continuous oxidative stress (causing

oxidation of mitochondrial constitu-

ents and autophagocytosed materi-

al) but also because of the inherent

inability of cells to completely remo-

ve oxidatively damaged structures

(biological garbage)4. Accumulation

of lipofuscin granules is mostly obs-

erved in various neurological disor-

ders2. There are no reported studies

of B. monniera and its antioxidant

activities, which play a significant

role in lipofuscin scavenging in cult-

ured human umbilical vein endothe-

lial cells (HUVECs). Therefore, this

study is the first to show that the B.

monniera extract (BME) plays a piv-

otal role by strongly supporting thescavenging of lipo-fuscin granules in

cultured HUVECs.

Material and methodsThis work conforms to the values laid

down in the Declaration of Helsinki

(1964). The protocol of this study has

been approved by the relevant ethical

committee related to our institu-

tion in which it was performed. All

subjects gave full informed consentto participate in this study.

BME preparationThe dried herb was macerated in 95%

methanol and the crude extract of B.

monnierawas prepared and iltered

with Whatman no.1 ilter paper2. The

extract was stored at 30C5.

Harvest and expansion of HUVECs6

The isolation protocol for HUVECs

was adapted from Baudin et al. 20077.

The umbilical cord was collectedimmediately and the cord was rinsed

Ayurveda

Results

The presence of lipofuscin granule

formation was confirmed by the Zie-

hl-Neelsons staining method and

microscopic examinat-ion. Exposur-

es to H2O2 (100 M) showed the ab-

undant generation and formation of

lipofuscin granules. B. monniera ext-

ract exposures of 2 h, 4 h and 6h

(100 g), showed the abundant gen-

eration and formation of lipofuscin

granules. B. monniera extract expos-

ures of 2 h, 4 h and 6h (100 g), sho-

wed a complete absence of lipofusc-

in granules. Pre-B. monniera extract

(100 g) exposure for 2 h plus post-

H2O2 (100 M) exposure for 1 h,

resulted in extremely reduced lipof-

uscin granules. H2O2 exposure for 1

h and pre-H2O2 (100 M) exposure

for 1 h plus post-B. monniera extract

(100 g) exposure for 2 h, resultedin the moderate appearance of

lipofuscin granules.

Conclusion

This study concluded that the B. mo-

nniera extract has potential antioxi-

dant properties, which play a pivotal

role in the analysis of lipofuscin gra-

nules scavenging in cultured human

umbilical vein endothelial cells.

Medicinal plants that have been use-

ful in history are obvious choices as

sources with significant pharmacol-

ogical and biological activities1. One

such medicinal plant is Bacopa mon-

niera, commonly termed as Brahmi2.

Studies in the past have focused on

B. monnieras cognitive enhancing

effects that specifically include me-

mory, learning and concentration.

Post-mitotic cells accumulate a non-

degradable, intralysosomal substan-ce called lipofuscin which is formed

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and washed with sterile phosphate

buffered saline (PBS). One end of the

umbilical vein was clamped using

artery forceps and an enzyme cock-

tail of 0.15% collagenase type IV

(Sigma Aldrich, St. Louis, Missouri,

USA) and dispase II (Roche, Nutley,

New Jersey, USA) was illed into

the lumen cavity of the vein for

dislodging the endothelial cells (ECs).

The ECs were incubated for 20 min

at 37C. After completion of incuba-tion, the vein was lushed with M199

medium (Invitrogen, Carlsbad, Cali-

fornia, USA). The cells in suspension

were centrifuged at 1500 rpm for

10 min. Cell pellets were refreshed

twice by centrifugation and the inal

pellet was again resuspended in

an endothelial cell growth medium

(ECGM) (PromoCell GmbH, Germany)

containing 20% foetal bovine serum

(FBS) (Invitrogen, Carlsbad, Cali-

fornia, USA); isolated HUVECs were

plated in tissue culture lasks. After

12 h of incubation, the HUVECs were

fed with complete ECGM containing

20% FBS, 2 mM L-glutamine, 10 g/

mL heparin and penicillin 5 unit/mL

and incubated at 37C with 5% CO2.

Lipofuscin granules stained by Ziehl-Neelson method for control cells(without exposure), oxidative stress(H

2O

2) for 1 h8

HUVECs were cultured onto cov-

er slips until resembling a cobblestone appearance. HUVECs without

any exposure served as the control.

The HUVECs were exposed to oxida-

tive stress (H2O

2) for 1 h (100 M)

and were fixed with 4% paraform-

aldehyde and refreshed with PBS 1

(5 min 3 washes). The HUVECs

were washed with 70% isopropanol

(10 min 2 washes) and stained with

carbolfuscin for 30 min at 60oC. The

HUVECs were further rinsed in acid-

alcohol (1% HCl in 70% ethanol)until they turned light pink. They

were washed in distilled water for

5 min, counter stained (0.5% tolu-

idene blue) for 10 min and rinsed

again with distilled water for 5 min.

Isopropyl alcohol (70%) was used to

dehydrate the cells. The cover slips

were mounted with dibutyl phthalate

xylene (DPX). Stained HUVECs were

observed under a bright field micro-

scope for lipofuscin granules.

Exposure to BME for 2 h, 4 h and 6 h,pre-BME exposure for 2 h plus post-H

2O

2 for 1 h and pre-H

2O

2 exposure

for 1 h plus post-BME for 2 h tocultured HUVECs to study the eff ectof BME on lipofuscin granules8

HUVECs were cultured on cover

slips until they reached a cobble

stone morphology. HUVECs were

further processed as follows: BME

exposure for 2 h, 4 h and 6 h, pre-

BME expo-sure for 2 h plus post-H2O

2

for 1 h and pre-H2O2exposure for 1 hplus post-BME for 2 h.

All exposed HUVECs were fixed

with 4% paraformaldehyde and

refreshed with PBS 1 (5 min 3

washes). The HUVECs were washed

with 70% isopropanol (10 min 2

washes) and stained with carbolfuscin

for 30 min at 60oC. The HUVECs were

rinsed in acid-alcohol (1% HCl in

70% ethanol) until they turned light

pink. The HUVECs were washed with

distilled water for 5 min, counterstained (0.5% toluidene blue) for 10

min and rinsed again with distilled

water for 5 min. Isopropyl alcohol

(70%) was used to dehydrate the

cells. The cover slips were mounted

with DPX. Stained HUVECs were

observed under a bright field micro-

scope for lipofuscin granules.

ResultsB. monnieracrude extractA 12% yield of sticky, crude extract

was obtained from B. monniera and

further used for experimental work.

Isolation and culture of HUVECsHUVECs were isolated in clusters

of 30. HUVECs were attached to

the plate surface immediately after

seeding for 2 h. HUVECs started

expanding in clusters and reached

conluence on the 10thday (Figure 1).

On 70% conluence, the HUVECs

resembled the perfect cobblestone

morphology, and the cell density was3 105 cells/cm2.

Figure 1: HUVECs showed a cobble

stone morphology in culture.

Figure 2: HUVECs in control (with-

out treatment) showed a very low

appearance of lipofuscin granules.

Figure 3: HUVECs treated with H2O

2

(100 M) showed abundant lipofus-

cin granules.

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Lipofuscin granules in the controlcells and in cells exposed to oxidativestress (H

2O

2for 1 h)

The current investigation reveals the

appearance and occurrence of lipo-

fuscin granules in cultured HUVECs.

HUVECs without any exposure servedas controls (normal) and showed a

much lower presence of lipofuscin

granules (Figure 2).

Oxidant exposures (H2O

2 for 1 h)

to cultured HUVECs, led to lipo-

fuscin granule generation. Exposure

of H2O

2 for 1 h at a concentration

of 100 M resulted in maximum

damage to the cultured HUVECs;

the cells showed distorted cell and

nuclear membranes with copious

formations of lipofuscin granules inthe cytosol region (Figure 3).

Exposure to BME for 2 h, 4 h and 6 h,pre-BME exposure for 2 h plus post-H

2O

2for 1 h and pre-H

2O

2exposure

for 1 h plus post-BME for 2 h tocultured HUVECs to study the effectof BME on lipofuscin granules

Exposure to BME (100 g) treatmentfor 2 h (Figure 4), 4 h (Figure 5) and

6 h (Figure 6), showed the absence

of or the lack of lipofuscin granule

formation and an intact cell and

nuclear membrane. Pre-BME (100

g) exposure for 2 h plus post-H2O

2

(100 M) for 1 h, showed the pres-

ence of very reduced amounts of lipo-

fuscin granule formation with intact

nuclear membranes and very little

injury to the cell membranes (Figure

7). Pre-H2O2 (100 M) exposure for1 h plus post-BME (100 g) for 2 h,

resulted in the moderate appear-

ance of lipofuscin granule formation

with little nuclear and cell membrane

damage (Figure 8).

DiscussionCells are constantly subjected to

free-radical attack. Free radicals are

scavenged in the cells by the antioxi-

dant system present in the cell. The

unscavenged free radicals exert theircytotoxic action on cell membrane

lipids. These peroxidised membranes

are engulfed by lysosomes which

may bring about damage to the lyso-

somal enzymes. The residual body of

lysosomes that are unable to digest

cell membranes turns into lipofuscin

granules. In advanced aging, the rate

of accumulation of lipofuscin gran-

ules increases in tissues like the heart

and brain2.

B. monnierais a herb, and a varietyof studies have been carried out to

study its antioxidative properties.

Past research investigating BME

exposure in neurons, established the

lipofuscin scavenging property of B.

monniera2. We tried to investigate

the role of BME in lipofuscin scav-

enging in cultured HUVECs. HUVECs

without any exposure served as the

controls and showed very little pres-

ence of lipofuscin granules. Exposure

to 1 h of oxidative stress (H2O2) at aconcentration of 100 M resulted in

Figure 6: HUVECs treated with BME

for 6 h (100 g) showed a complete

absence of lipofuscin granules.

Figure 4:HUVECs treated with BME



Figure 7: HUVECs treated with

pre-BME (100 g) for 2 h plus post-

H2O

2 (100 M) for 1 h showed re-

duced lipofuscin granules.

Figure 5:HUVECs treated with BME



Figure 8:HUVECs treated with pre-

H2O

2(100 M) for 1 h plus post-

BME (100 g) for 2 h showed moder-

ate lipofuscin granules.

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maximum damage to the cultured

HUVECs, such as distorted cell and

nuclear membranes and abundant

formation of lipofuscin granules

in the cytosol region. This irmly

explains that dose-dependant H2O

2

exposure for 1 h brings about cellular

damage and is involved in the forma-

tion of lipofuscin granules in cultured

HUVECs.

Exposure to BME (100 g) for 2 h,

4 h and 6 h, showed the absence of

lipofuscin granule formation and

intact cell and nuclear membranes.

Pre-BME (100 g) exposure for 2 h

plus post-H2O

2 (6.25 M to 100 M)

for 1 h, showed negligible/reducedamount of lipofuscin granule forma-

tion with intact nuclear membrane

and very little injury to the cell

membrane. This illustrates that

pre-BME exposure for 2 h provides

the necessary anti-oxidative protec-

tion to cultured HUVECs while the

results from the post-H2O

2exposure

for 1 h illustrate that there was a

lack of or minimal lipofuscin granule

formation. Therefore, it is deduced

that pre-BME exposure has a protec-tive role against lipofuscin granule

formation even after post-H2O

2expo-

sures. Pre-H2O

2 (100 M) exposure

for 1 h plus post-BME (100 g) expo-

sure for 2 h, resulted in the moderate

appearance of lipofuscin granule

formation with little nuclear and cell

membrane damage. This shows that

pre-H2O

2 exposures bring about the

moderate generation of lipofuscin

granules, but after post-BME expo-

sures, the generation of lipofuscin

granules is halted due to the BME

antioxidative properties.

ConclusionThis investigation shows that BME is

an antioxidant which plays a pivotal

role in lipofuscin scavenging instressed and normal HUVECs. H2O

2

(100 M) treatment is responsible

for oxidative impair and brings about

the extreme generation or formation

of lipofuscin granules. BME expo-

sures for 2 h, 4 h and 6h (100 g),

bring about scavenging of lipofuscin

granules at different time intervals.

Pre-BME (100 g) exposure for 2 h

brings about reduced lipofuscin

formation. After post-H2O

2(100 M)

exposure for 1 h, there is no further

rise in lipofuscin. Pre-H2O2(100 M)exposure for 1 h plus pos-tBME

(100 g) exposure for 2 h, resulted

in the moderate appearance of lipo-

fuscin granules.

Abbreviations listBME, Bacopa monnieraextract; ECs,

endothelial cells; ECGM, endothe-

lial cell growth medium; FBS, foetal

bovine serum; PBS, phosphate buff-

ered saline.

AcknowledgementWe gratefully thank the Indian

Council of Medical Research (ICMR),

File No: 45/47/2010/BMS/TRM IRIS

Cell: 2010-06230, Government of

India, for providing a senior research

fellowship to our irst author. We are

also thankful to Dr NB Agrawal for

providing research funds through Dr

PK Sen, Research Society, GSMC and

KEM Hospital, Parel, Mumbai. We

thank Dr BM Kandalkar (Professor

and Head of Department, Depart-

ment of Pathology, GSMC and

KEM Hospital, Mumbai, India) for

his generosity in helping us and

providing his support with the tissue

culture laboratory. We especially

thank Mrs Seema V Sharma (Senior

Scientiic Oficer, CVTC) Mrs Rajeshri

(JSO), Mr Sunil Chaudhary, Mrs Nidhi

Taneja, Mr Janardhan and other CVTC

staff for their laboratory support.

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