EFFECT OF ALOE GEL IN ENHANCING
DIABETIC WOUND HEALING
Djanggan Sargowo, Yudha Handaya,
Askandar Tjokroprawiro
Brawijaya Medical School
Malang
1
BACKGROUNDS
DM
2003 (197 millions)2010 ( 50%)
2025 (333 millions/6,3% population)
77% Developed Country
(Lauderdale 2006, Goycheva,et al 2006)
WHO Indonesia : 4th largest population with DM
2000(5,6 millions)2006(14 millions); 2025 (6,5% of
population)
(sidartawan 2007)
2010 ( 7 jt) no 92030 (12jt)No 6 (IDF)
(Tjokropawiro 2010)
DM (15% DFU)
amputation ( 84% DFU)
Surabaya3,8% Fountain IVDFU
(Beem et al 2007,Tjokropawiro 2011)
2
WAGNER CLASSIFICATION
Grade 0 pre-ulcerative lesion, healed
ulcers, presence of bony
deformity
Grade 1 superficial ulcer without
subcutaneous tissue
involvement
Grade 2 penetration through the
subcutaneous tissue (may
expose bone, tendon, ligament
or joint capsule)
Grade 3 osteitis, abscess, or
osteomyelitis
Grade 4 gangrene on the forefoot
Grade 5 gangrene on the entire foot
3
# Micro/Macroangiopaty 
diabetic foot ulcers
 Wound Healing
Wound healing complex mediated by :
vascular, neuronal, cellular & immune
factors,
Wound healing on DM
↓growth factor & pro-angiogenic cytokines
matrix deposition & remodelling
Angiopathy
Neuropathy
combination
(David et al 2002)
(Brownlee2005,Ralf et al 2005)
Imbalance Wound Healing Phases
WOUND HEALING DISORDERS
4
DMHyperglycemia↑ ROS 
↑ apoptosis angiogenesis dysfunction (#tube formation) 
↓ WH ↑ diabetic foot ulcers (DFU) progression amputation
(David et al 2002, Hesham et al 2006 ,Beem 2007, Tjokropawiro 2010)
DM↓ VEGF  ↓ Ca cytosolic activity + ↓eNOS ↓Homing EPC
↓ Angiogensis  # WH ulkus DM
(Costanza et al 2002, Pradhan et al 2007,Singhan et al 2007)
DFU proper management 
Herbal therapy  enhance angiogenesis
(Larijani et al 2008)
5
HIPERGLIKEMIA (DM)
HYPERGLYCEMIA
SCHIFF BASE <1 day
AMADORI 1-7 days
P
↑PKC
↑ROS
↑TNF
↑NFκB
↓NO
b↑AGE
>1 week
A
H
↑HEKSOSAMINE PATWAY (H)
A
↑AR
GFAT
↑NFκB, ↓eNO, ↑TGFβ
↑NAD(P)H OXIDASE
↑ET-1, ↑PAI-1, ↑VEGF
↑TGFβ,↑MMPs
↓CELL PROLIFERATION
OXIDATIVE
STRESS
STRESS OXIDATIVE
INSULIN SENSITIVITY
↑OH+
↑TNFα
↑SORBITOL
↓GSH
↑TNFα
Β-CELL FUNCTION
“PAHAM”:PKC,AGE,HEKSOSAMINE,AR,MITOCONDRIAL DYSFUNCTION
MIKROANGIOPATI & MAKROANGIOPATI
MACRO-MICROANGIOPATHY
NEUROPATHY
MOTORIC
WEAKNESS
ATROPHY
DEFORMITY
↑PLANTAR PRESSURE
CLAVUS
SENSORY
↓PROTECTION
↓SENSATION
TRAUMA
AUTONOM
#INFLAMMATION 2-7 DAYS
MACROPATHY
BM CAPILLARY THICKENNING
A-V SHUNT
↑BLOOD FLOW
EDEMA NEUROPATHY
↓RESPONSE TO INFECTION
#INFLAMMATION
↓NEUTROPHIL
↓MONOCYTE
AMPUTASI
AMPUTATION
MICROPATHY
ANHIDROSIS
DRY SKIN
FISSURE
↑SYMPATHY PRESSURE
OSTEOARTHROPATHY
HEMOSTASIS 1 HOUR
VASCULOPATHY
ENDOTHELIAL DYSFUNCTION
# ANGIOGENESIS
↓KERATINOCYTES
↓PROLIFERATION
#PROLIFERATIOM 2-20 DAYS
PENYEMBUHAN
ULKUS KAKI DM
#GAMGGUAN
WOUND
HEALING
ATEROSCLEROSIS
ISCHEMIA
↓NUTRITION
↓CAPILLARY BLOOD FLOW
↓FIBROBLAST
↓GF
↓CITOKINES
#REMODELLING 6WEEK-3 MONTHS
AMPUTASI
AMPUTATION
Gambaran Kegagalan Patofisiologi Ulkus diabetes dan Patologi Penyembuhan ulkus diabetes (Robert et al 2006,
Gugliucci 2007, Pradhan et al 2007, Tjokroprawiro 2010).
6
ALOE VERA
herbal high potency for diabetic wound
healing (Wendell, 2000).
Easy to find, antiinflammation (C-glukosyl
chromone), immune modulator, wound
nutrition (Wendell, 2000)
clinical Aloe gel  modulate immunity,
blood glucose stabilizer,↓ pain ; ↑ arteriolar
diameter (Somboonwong et al., 2000).
Aloe (gel): 98% water ;Acetylated
mannan>60% active compound  1 mg aloe
gel (acemanan 0,6mg) (Somboonwong et al,
2000; Kusmawati,2007 )
7
Acemannan
Β-(1-4)-linked glukomanan acetylated and
manosaasetilasi residu
Polysaccharides doses 20 µg/mL  ↑ enhance WH, stimulate
cellular proliferation,↑ migration and cytokines production
Stimulate oxygen consumption, ↑ angiogenesis & ↑ sintesis (Laura
et al., 2002).
oral ↑WH fibroblast proliferation and induce expresssion of
KGF-1 and VEGF (Ramamoorthy, 1996; Hamman, 2008).
8
Problem of the Study
herbal Aloe gel  angiogenesis
WH DM
•
•
•
•
•
•
wound healing
VEGF expression
eNOS
EPC
CEC
EPC/CEC ratio
9
Aim of the Study
PROVE :
herbal Aloe gelangiogenesisWH DM
wound healing
VEGF expression
 eNOS,
 EPC
CEC
EPC/CEC ratio
10
CONCEPTUAL THEORY
ALOE GEL
VEGF 
eNOS 
CEC 
EPC 
ANGIOGENESIS 
ERK-1/2
Cellular proliferation 
DIABETIC WOUND HEALING 
11
Hipotesis
wound
diameter
Dose of
30,60,120
(mg/day)
Aloe gel
Angiogenesis
Diabetic wound
healing
VEGF, eNOS ,EPC
,CEC ,
Rasio EPC/CEC
Expression and
amount
12
MATERIALS AND METHODS
Study Design
True experimental in vivo study designed by post
test only control grup design
Subject of study
Male strain wistar rats induced by STZ to make
Diabetic rats and wounded and treated by Aloe
vera gel orally
13
SCHEME OF STUDY PROTOCOL
(ROCKET SYSTEM MODIFICATION, TJOKROPRAWIRO, 1978)
S
T
R
A
I
N
W
I
S
T
A
R
+
A
L
O
E
DAY
14
0
0
PRA
PENELITIAN
PRE- STUDY
A0
N:15
R
A
N
D
O
M
B0
E0
WOUND
n:3
DM 0
WOUND
n:3
B14
DATA
30
WOUND + ALOE
GEL 30 MG
n:3
C14
DATA
DM 60
WOUND + ALOE
GEL 60 MG
n:3
D14
WOUND + ALOE
GEL 120 MG
n:3
E14
DM120
G
E
L
PRE- STUDY
0
A14
STUDY
14
DAY
28
A
:Vascular Endothelial Growth Factor
:endothelial Nitric Oxide Synthase
:Endothelial Progenitor Cells
:Circulating Endothelial Cells
: NORMAL RATS
B
: UNTREATED RATS
C
: DIABETIC RATS+TREATMENT
D
: DIABETIC RATS+TREATMENT
E
: DIABETIC RATS+TREATMENT
PRE-STUDY: 0-14 days bedore treatment
STUDY
: 0-14 hari after treatment
Ale gel
: glycoprotein and polysacchaarides aloe vera
DATA
DATA
MAKING OF DIABETIC RATS
Injection of STZ 60mg/kg (IP)
for 14 days (2 weeks)
Diabetes if blood glucose level >200mg/dl
D
A
N
A
T
A
L
A
I
A
S
N
I
A
S
L
D
Y
A
S
T
I
A
S
RESULT
HASIL
DATA
VEGF,eNOS,
EPC,CEC
Ø WOUND
PARAMETER
MAIN PARAMETER
VEGF
eNOS
EPC
CEC
VEGF,eNOS,
EPC,CEC
Ø WOUND
PENELITIAN
STUDY
NON
DM
C0 DM
D0
14
VEGF
eNOS
EPC
CECs
: immunohistochemistry
: immunohistochemistry
: CD34 (Flowcitometri)
: CD45(FITC)+CD146(PE)
(Flowcitometri)
Ø wound : wound dimeter
DATA ANALYSIS
Anova (simple anova)
Linear Regression
MAKING OF WOUND
ketamin (10mg/kg/iv)
Wound diameter 2,5 cm depth 0,2 cm
Wash the wound with Nacl 0,9%
SAMPLE POPULATION
p
(t-1)(r-1)≥ 15
t=±treatment
r=±repetition
14
R
E
P
O
R
T
STUDY TIMELINE
PRE
MAKING OF ALOE GEL
MAKING OF DIABETIC RATS
study
Stage I study
Effect of Aloe Gel wound
diameter
stage II study
(IMMUNOHISTOCHEMISTRY)
Effect of Aloe Gel Expression of
VEGF and eNOS
stage III study
(FLOWCYTOMETRI)
Effect of Aloe Gel amount of EPC,
CEC and EPC/CEC ratio
15
RESULTS
14th Day, measurement of :
•
• Wound diameter
Immunohistochemistry of dermal tissue
• Blood for Flowcitometry
16
Measurement of Wound Diameter
Normal Rats
p = 0.000
Diabetic rats
DM + 30 mg
DM + 60 mg
DM + 120 mg
Treatment
n
Mean  SD (p ≤ 0,05)
Normal Rats
3
1,100,14 (a
Diabetic Rats
3
1,190,12(a
Diabetic rats + 30 mg aloe
gel
Diabetic rats + 60 mg aloe
gel
Diabetic rats + 120 mg
aloe gel
3
0,320,01(b
3
0,270,02(b
3
0,410,02(b
17
Vascular Endothelial Growth Factor (VEGF) Expression
Normal Rats
Diabetic Rats
DM+30mg
DM+60mg
DM+120mg
treatment
n
Mean  SD (p ≤ 0,05)
Normal Rats
3
24,672,00 (a
Diabetic Rats
3
25,334,51(a
Diabetic rats + 30 mg aloe
gel
3
28,335,86(a
Diabetic rats + 30 mg aloe
gel
3
34,675,03(a
Diabetic rats + 30 mg aloe
gel
3
25,674,73(a
p = 0.676
18
Endothelial Nitric Oxide Synthase (eNOS) Expression
Normal Rats
p = 0.000
Diabetic rats DM + 30 mg
DM + 60 mg
DM + 120
mg
Treatment
n
Mean  SD (p ≤ 0,05)
Normal Rats
3
24,671,73 (b
Diabetic Rats
3
30,672,08(a
Diabetic rats + 30 mg
aloe gel
3
30,001,00(a
Diabetic rats + 60 mg
aloe gel
3
34,332,08(a
Diabetic rats + 120 mg
aloe gel
3
42,001,00(b
19
Amount of Endothelial Progenitor Cells (EPCs) CD 34
Non DM
hari ke-14
(kontrol -)
Normal Rats
DM hari ke-14
(kontrol +)
DM + aloe gel 30 mg
hari ke-14
Diabetic rats DM + 30 mg
n kelompok = 3
DM + aloe gel 60 mg
hari ke-14
DM + 60 mg
DM + aloe gel 120 mg
hari ke-14
DM + 120
mg
Treatment
n
Mean  SD (p ≤ 0,05)
Normal Rats
3
16,880,59 (a
Diabetic Rats
3
10,471,14(a
Diabetic rats + 30 mg
aloe gel
3
10,649,34(a
Diabetic rats + 60 mg
aloe gel
3
12,111,16(a
Diabetic rats + 120 mg
aloe gel
3
13,751,63(a
p = 0.457
20
Amount of Circulating Endothelial Cells (CECs) CD 45
Non DM
hari ke-14
(kontrol -)
Normal Rats
DM hari ke-14
(kontrol +)
DM + aloe gel 30 mg
hari ke-14
Diabetic rats DM + 30 mg
DM + aloe gel 60 mg
hari ke-14
DM + 60 mg
DM + aloe gel 120 mg
hari ke-14
DM + 120 mg
Treatment
n
Mean  SD (p ≤ 0,05)
Normal Rats
3
3,161,68(bc
Diabetic Rats
3
14,762,28(a
Diabetic rats + 30 mg
aloe gel
3
7,361,22(b
Diabetic rats + 60 mg
aloe gel
3
4,890,83(bc
Diabetic rats + 120 mg
aloe gel
3
2,200,74(c
p = 0.000
21
PATH ANALYSIS
22
DISCUSSION
23
Effect administration of Aloe gel on wound
diameter
 Wound
diameter
Dosis
(mg/day)
Aloe gel
•
•
•
↑ wound contraction
 Diabetic
wound healing
Anti inflammatory
fibroblast proliferation
collagen dan
glycosaminoglikan
synthesis
(Heggers 1993; Jia et al 2008;Kwack et al 2009 ,;Liu et al., 2010;
Fernanda et al., 2009;Jettanacheawchankit et al 2009)
24
Effect administration of Aloe gel on Expression of VEGF and
eNOS Dermal Tissue
Dosis
(mg/day)
Aloe gel
↑ Angiogenesis
 Diabetic
wound healing
 Wound
diameter
VEGF 
eNOS 
EPC mobilization
(Heggers 1993; Choi et al 2002; Tanimoto et al 2002;Duda et al 2004;
Young et al 2004;Kim et al 2007;Pradhan et al. 2007; Beem et al. 2007;
Singhan et al 2007; Chen et al 2007).
25
Effect administration of Aloe gel on amount of
EPCs and CECs circulation
 Wound
diameter
Dosis
(mg/day)
Aloe gel
↑ Angiogenesis
 Diabetic
wound healing
migration
/Homing EPC
CECs
Spinetti et al. 2008;Albiero et al., 2011;Oren et al., 2010;McClung et al
2005;Fiorina et al. 2010;Sibal et al 2009;Spinetti et al 2008;Nababan et al.,
2007;Boos et al 2006
26
EPC/CEC RATIO AFTER ADMINISTRATION OF ALOE GEL
Treatment
Mean  SD (p ≤ 0,05)
Normal rats
6,53,41 (c
Diabetic rats
0,720,1(a
Diabetic rats + 30 mg aloe
gel
Diabetic rats + 60 mg aloe
gel
Diabetic rats + 120 mg aloe
gel
2,180,16(ab
2,510,84(ab
6,351,99(c
aloe gel  herbal pro-angiogenic keep survival
EPCs during mobilization and homing into wound
 increase amount of EPCs to damage
endothelial cells from vascular wall (CECs).
27
wound
diameter
cm2
Dose
120(mg)
Aloe gel
•
Hipotesis
statistically
proved
(P<0,05)
Angiogenesis
wound healing
Ratio
EPC/CEC
VEGF, eNOS
,EPC
,CEC
Exresion (ng/ml)
and amount
EPC/CEC ratio
Novel (new)
Base of therapy
Prognostic factor
28
29
30
31
THE ROLES OF HSP 70 / HSP 72
on Wound Healing
HEALTHY WOUND
DIABETIC WOUND
DM WITH 5 DYSFUNCTION : KERATINOCYTE, PLATELET,
ENDOTHELIUM, MACROPHAGE, MYOFIBROBLAST
c-myc
-catenin
VEGF
LIMITED NEOVASCULOGENESIS
AND WOUND HEALING
LIMITED
EPC HOMING
IMPROVED BY
LOCAL SDF-1
INJECTION
IMPROVED BY HBO
THERAPY
Blood Glukose
Should be <
200
Decreased
eNOS Activation
Mekanisme Penyembuhan luka Normal dibanding Diabetes (Beem et al 2007, Tjokropawiro 2011)
Conceptual Theory
Vascular Complication of Diabetes
Ischemia/hypoxia
VEGF
VEGFR-2
ROS
PLC-γ
DAG
IP3
PKC
C a 2+
cSrc
FAK
CaM
Raf-1
MEK
eNOS
PI3K
p38MAPK
Calpain
ERK1/2
PIP3
HSP27
Akt/PKB
NO
HIF-1α, AP-1,
NFκB, Ets-1
MMPs, uPA,
PAI-1
Paxilli
n
prolifera
tion
EPC Homing,
CEC ↓
Migration
vascular
permeability
Angiogenesis
Diabetic wound healing
33
Active Compound of Aloe Gel (Hamman, 2008)
CLASS
Anthraquinones/anthrones
ACTIVE COMPOUND
Aloe-emodin, aloetic acid, anthranol, aloin A and B (barbaloin),
isobarbaloin,emodin, cinnamic acid ester
mannanasetilasi, glucomannan
Pure Mannan ,
asetilasi,glucogalactomannan,galactan,
Chromones
galactogalacturan,arabinogalactan, galactoglucoarabinomannan,
pectic,xylan, cellulose
8-C-glucosyl-(2’-O-cinnamoyl)-7-O-methylaloediol A,8-Cglucosyl-(S)aloesol, 8-C-glucosyl-7-O-methyl-(S)-aloesol, 8-C glucosyl-7-O-methylaloediol, 8-C-glucosyl-noreugenin, isoaloeresin D, isorabaichromone,
neoaloesin A
Enzymes
Alkaline phosphatase, amylase, carboxypeptidase,
catalase,cyclooxidase, cyclooxygenase, lipase, oxidase,
phosphoenolpyruvate carboxylase, superoxide dismutase
Inorganic substances
Calcium, chlorine, chromium, copper, iron, magnesium, manganese,
potassium, phosphorous, sodium, zinc
Other substances (Organic and fat)
Asam Arachidonic,asam linolenic, steroids (campestrol,cholesterol,
âsitosterol), triglicerides, triterpenoid, gibberillin, lignins,Potassium
sorbate, salicylic acid, uric acid
Asam Amino Non-essential and essential
Alanine, arginine, asam aspartic,asam glutamic,
glycine,histidine,hydroxyproline, isoleucine, leucine, lysine,
methionine, phenylalanine, proline, threonine, tyrosine, valine
Protein
Saccharides
Vitamin
Lectins, lectin-like substance
Mannose, glucose, L-rhamnose, aldopentose
B1, B2, B6, C, â-carotene, choline, folicá acid-tocopherol
34