This document summarizes research on the wound healing effects of a glycoprotein fraction isolated from Aloe vera. It describes methods used to isolate the glycoprotein fraction (G1G1M1D12) and evaluate its effects on keratinocyte proliferation, migration, and epidermis formation in raft culture. Results showed G1G1M1D12 enhanced keratinocyte multiplication and migration. It also improved epidermis formation and accelerated wound healing in mice. The conclusion is that G1G1M1D12 possesses wound healing properties by stimulating keratinocyte proliferation and migration.

1. 8/3/2012 1
The wound-healing effect of a glycoprotein fraction isolated from
Aloe vera
Deepa kumari karn
B pharm 7th sem
Seminar VI

2. CONTENT
• Introduction
• Materials and methods
• Results
• Discussion
• Conclusion
• References
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3. Introduction
• Aloe vera belonging to family Liliaceae
• Known as healing plant or Silent healer
• The effect of active component from Aloe vera
upon cell proliferation and activity was
isolated and studied
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4. • Numerous research showed that Aloe vera
have various therapeutic activities such as
immunomodulator, anti-inflammatory, wound
healing
• Raft culture system was used to permit long
term culture at air-liquid interface
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5. Materials and methods
• All reagents were of analytical grade
purchased from Sigma-Aldrich
Isolation of glycoprotein fraction
G1G1M1D12
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6. 8/3/2012 6

7. Thymidine uptake assay
• Use to evaluate the effect on cell proliferation
by measuring the thymidine uptake in
presence of aloe gel
• The amount of thymidine uptake was
measured by ascintillation counter
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8. 8/3/2012 8
SCC seeded on to
a 96 well tissue
culture plated
5000 cells per well
Aloe fraction +
serum free
medium applied
Thymidine added,
washed by PBS
and dried
Solubilized in
NaOH
Neutralized with
Hcl

9. Preparation of keratinocyte
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10. Effect of G1G1M1D12 on kerationcyte
migration
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Normal human keratinocytes were cultured to confluence
in a 6 well cultured plate and culture medium
Wound created
Well were washed 4 times with PBS to remove cellular
deloris
Aloe fraction was added to culture and the control
received PBS
Wound restoration was photography at 16, 25 and 40h
after injury

11. Effect of G1G1M1D12 on epidermis formation
from keratinocytes in raft culture
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Aloe
fractio
n was
applied
to cell
with
serum
free
media
at 0,
0.05,
0.5 and
50µg
per mL
every 2
days
Part of
epiderma
l tissue
formed
by 3
weeks of
culture
was
taken
Fixed
in
Carnoy
solutio
n
Washe
d with
60%
and
80%
ethanol
Put in
paraffi
n
blocks
for
morpho
logical
compar
ison

12. Effect of G1G1M1D12 on wound healing in
hairless mice
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10 male hairless
mice were used
under phenobarbital
sodium anaesthesia
Experimental
animal receive
G1G1M1D12 with
gentamicin
Control receive
vehicle only
Ointment was
changed daily
Wound area was
photographed and the
size of the wound was
estimated using an
image analyzer

13. RESULTS
Isolation and characterization of proliferation
stimulating glycoprotein fraction
G1G1M1D12
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Fig: Effect of various fractions of Aloe vara on thymidine uptake by squameous
carcinoma 13 cells

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Fig: High performance liquid chromatography of acid hydrolysate obtained from
carbohydrate moiety of G1G1M1D12

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Fig: Amino acid composition of glycoprotein fraction G1G1M1D12 estimated using
an amino acid analyser

16. Effect of G1G1M1D12 on migration of
keratinocytes
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17. Effect of G1G1M1D12 on the architecture of
epidermis in raft culture
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18. 8/3/2012 18
a) In absence of
G1G1M1D12 fraction
b) 0.05 µg per ml
G1G1M1D12 fraction
c) 0.5 µg per ml
G1G1M1D12 fraction
d) 50 µg per ml
G1G1M1D12 fraction
Fig: Effect of glycoprotein fraction G1G1M1D12 on the formation of epidermis
from keratinocytes in raft culture

19. Effect of G1G1M1D12 on wound healing
hairless mice
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Fig: Effect of G1G1M1D12 on wound healing hairless mice

20. a) Data represent relative percentage of wound
size
b) Wound area on day 10 after treatment 1)
control and 2) experimental
c) Morphology of wound area of control at day
10
d) Morphology of wound area of experimental at
day 10
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21. DISCUSSION
• Aloe has long been known for its various
pharmacological effects
• According to Yagi et al aloe components
aloesin, mannose-6 phosphate, glycoprotein
and aloe-emodin have reported to stimulate
cell proliferation
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22. • According to Maehiro et al various factors are
known to affect cell migration such as EGF,
interleukin(IL)-ß, nitric oxide, short chain fatty
acid
• In this experiment few cells survived in the
absence of serum and aloe fraction
G1G1M1D12
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23. • In presence of G1G1M1D12, keratinocytes,
survived and multiplied even in the serum free
media
• It indicate that the aloe fraction G1G1M1D12
stimulates cell proliferation
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24. • The aloe fraction G1G1M1D12 enhanced DNA
synthesis as well as EGF receptor expression
which suggest that EGF receptors transmitted
the cell proliferation aignal from the
G1G1M1D12
• EGF receptor level were increased by
G1G1M1D12 in the artificial tissue raised in
the raft culture
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25. • Keratinocyte didn’t invade into the matrix
• Keratinocyte in the basal layer of the raft
culture expressed fibronectin receptor in a
dose dependent manner
• G1G1M1D12 affect cell proliferation rather
than differentation
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26. CONCLUSION
• There have discrepant observations on wound
healing effect of aloe fraction
• This experiment clearly demostrated that the
glycoprotein fraction G1G1M1D12 enhances
keratinocyte multiplication and migration,
expression of proliferation related vactor and
epidermis formation
• Thereby leading to wound healing
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27. REFERENCES
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 Chol WS, Son WB, Son SY, Park IY, Lee KS and Chung HM (2001),
Wound healing effect of glycoprotein fraction isolated from Aloe vera.
British Journal of Dermatology 145:535-545.
 Yagi A,Egusa T, Arase M et al (1997), Isolation and characterization of the
glycoprotein fraction with a proliferation promoting activity on human and
hamster cells in vitro from aloe gel. Planta Med 63:18-21
 Maehiro K, Walamabe S, Hirose M et al (1997), Effects of epidermal
growth factor and insulin on migration and proliferation of primary
cultured rabbit gastric epithelial cells. J Gastroenterol 32:572-578