Therapeutic Effect of the Preparation of Lithospermi Radix and Gardeniae Fructus Extracts on the Burn and Wound healing

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Therapeutic Effect of the Preparation of
Lithospermi Radix and Gardeniae Fructus
Extracts on the Burn and Wound healing

• Dong-Hoon Min
• College of pharmacy, Woosuk University

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Introduction
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Burn 1) First degree burn Damage to the outer
layer of skin(epidermis), causing pain, redness
and swelling 2) Second degree burn Damage to
both outer skin and underlying tissue
layers (epidermis and dermis), causing pain,
redness, swelling and blistering 3) Third
degree burn Damage extends deeper into
tissues (epidermis, dermis and hypodermis),
causing extensive tissue destruction. The skin
may feel numb.
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Lithospermi Radix
- Root of Lithospermum Erythrorhizon Sieb. -
Naphthoquinone pigment shikonin,
acetylshikonin, isobutylshikonin, etc. -
Treatment of burn, excision wound, eczema,
blister as antifebrile, antiphlogistic
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Figure 1. Molecular structure of shikonin R H
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Gardeniae Fructus
- Fruits of Gardeniae Jasminoides Ellis. -
iridoid glycoside geniposide, gardenoside,
genipin, etc - Accelerate choler secretion
and anti-inflammatory, analgesic effect
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Figure 2. Molecular structure of geniposide
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Scope of studies
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- Therapeutic effect of skin disease
Burn and Wound healing 1)
Hydrogel preparations containing Lithospermi
radix(LR) and Gardeniae fructus extracts 2)
Studies on skin permeation, anti-inflammatory
effects, reduction rate of burn wound size
and therapeutic periods
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Experiment
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• Materials
• - Lithospermi radix
• - Gardeniae fructus
• - Shikonin (Wako. Chem. Co. Japan)
• - Geniposide (Sigma. Chem. Co. USA)
• - ?-Carrageenan ? (Sigma. Chem. Co. USA)

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• 2. Apparatus
• High performance liquid chromatograph
• (M920, Youngin
  Co., Korea)
• Skin permeation tester
• (FCDV-15, Lab
  Fine, Korea)
• - Viscometer (Brook Lab, Inc.)
• - Digital pletysmometer
• (LE7500, Panlab
  s.l., Spain)
• Handscopy
• (USB microscope M2, Scalar Co.,
  Japan)

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• 3. Animal and Bacteria
• 1) Animals
• Hairless mouse (Male, 25 5g, Charles River Lab.,
  USA)
• Rat (S.D, Male, 200 20g, Damul Science Co.,
  Korea)
• 2) Bacteria
• - Gram positive
• S. aureus (ATTC 29213), B. Subtilis (ATTC
  6633),
• S. pneumoniae (ATTC 49619), S. epidermis (ATTC
  12228)
• - Gram negative
• P. aeruginosa (ATTC 9027), K. pneumoniae (ATTC
  10039),
• E. coli (ATTC 35150), S. paratyphi (ATTC
  13428)

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4. Gel preparations of containing LR and GF
extracts
Add LREP and water with stirring
Add GFEP and water with stirring
Add Propylene glycol, Labrasol, Ethanol and
Nano-ATP water with stirring
Add Carbopol 940 with stirring (50?)
30 min
triethanolamine dropping
pH 7.0
Make gellation
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• 5. Determination of shikonin and
• acetylshikonin,
  geniposide
• - Shikonin Acetylshikonin
• Method High performance liquid chromatograph
  
• Column µ-Bondapak C18 3.9300 ?
• Mobile phase acetonitrile water acetic
  acid
• triethanolamine (70300
  .30.3 v/v)
• Detector UV detector 548 ?
• Geniposide
• Method High performance liquid chromatograph
• Column µ-Bondapak C18 3.9300 ?
• Mobile phase acetonitrile water (1585
  v/v)
• Detector UV detector 240 ?

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• 6. Skin Permeation Test
• Animal hairless mouse
• Apparatus Franz diffusion cell
• Skin areas 1.77cm2
• Receptor phase
• Propylene glycol pH 7.4 phosphate buffer
• - Shikonin, acetylshikonin and geniposide was
• determined by HPLC

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7. Determination of Residual Amount in
Epidermic Endodermic Tissue
Material Epidermic Endodermic tissue
Freezing Refrizerator (-66?, 24hr)
Homogenizing Tris buffer (pH 7.4) Solvent
t-butyl ethyl ether Assay HPLC Determination

• 8. Determination of Viscosity
• Brook-Field Viscometer
• - 50?, 37?, 20?

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9. Effect of anti-inflammatory - Inhibition
of Swelling Test Edema Inducer 1
?-Carrageenan(Type IV) Soln. - Observed edema
after 6, 12, 24, 48hrs - Inhibition rate
1-Vd/Vc100        
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• 10. Antibacterial effect
• Bacteria Gram() and Gram(-) bacteria 8
  species
• Experiment
• 1) Minimum inhibitory concentration
• Agar serial dilution method
• 2) Inhibition rate of growth
• Broth serial dilution method
• (600nm UV transmission)

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• 11. Effect of Burn Healing
• Animal Mouse
• - 2nd degree burn
• - Apply to LR, GLC, GLN gel

• 12. Effect of Wound Healing
• Animal Mouse
• Excision wound
• - Apply to LR, GLC, GLN gel

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Results
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Table ?. Formulas of Each Hydrogel Preparations
Preparation GF Gel LR Gel GLC Gel GLN Gel
GFEP 1.0 - 1.0 1.0
LREP - 1.0 1.0 1.0
Carbopol 940 1.0 1.0 1.0 1.0
Propylen glycol 20.0 20.0 20.0 20.0
Labrasol 10.0 10.0 10.0 10.0
Ethanol 10.0 10.0 10.0 10.0
Triethanol amine 1.5 1.5 1.5 1.5
Distil. Water 56.5 56.5 55.5 50.0
Nano-ATP - - - 5.5
Total 100.0 100.0 100.0 100.0
GFEP Gardeniae  fructus extract powder LREP
Lithospermi  radix extract powder
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Table ?. Comparision of Contents(mg) of
Geniposide, Shikonin and
Acetylshikonin in Gel Preparations.
  Geniposide Shikonin Acetylshikonin
GF Gel 291.3 - -
LR Gel - 2.9 15.8
GLC Gel 293.2 3.0 15.7
GLN Gel 295.7 3.2 16.3
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Table ?. Viscosity of each hydrogels under
various temperatures by spindle
number RV 5 of Brookfield viscometer (Factor
400)
Formulations Viscosity(cps) Viscosity(cps) Viscosity(cps)
Formulations 50? 37? 20?
GF gel 16666 20200 21700
LR gel 17540 20000 22400
GLC gel 13160 14400 17850
GLN gel 13540 15666 19933
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Figure 3. Amount of permeated shikonin in GF and
LR hydrogels for 8 hours through
excised hairless mouse skin Key -- LR
Gel -?- GLC Gel -?- GLN Gel
Significantly different from LR Cream (Plt0.05)
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Figure 4. Amount of permeated acetylshikonin in
GF and LR hydrogels for 8 hours
through excised hairless mouse skin Key
-- LR Gel -?- GLC Gel -?- GLN
Gel Significantly different from LR
Cream (Plt0.05)
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Figure 5. Amount of permeated geniposide in GF
and LR hydrogels for 8 hours
through excised hairless mouse skin Key
-?- GF Gel -?- GLC Gel -?- GLN
Gel
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Table ?. Permeation Parameters of Various Gels
Through Excised Hairless Mouse
Skin
sample sample Parameters Parameters Parameters
sample sample Cumulative amount for 8 hr (?/?) Js TL
GF Gel Geniposide 45.771.30 5.720.160 0.780.16
GF Gel Shikonin - - -
GF Gel Acetylshikonin - - -
LR Gel Geniposide - - -
LR Gel Shikonin 8.440.15 1.060.023 0.750.13
LR Gel Acetylshikonin 21.260.79 2.660.097 0.910.34
GLC Gel Geniposide 55.901.07 6.890.130 1.030.18
GLC Gel Shikonin 8.770.16 1.110.020 0.770.23
GLC Gel Acetylshikonin 23.550.66 2.910.081 0.930.26
GLN Gel Geniposide 57.201.50 7.150.190 1.010.21
GLN Gel Shikonin 9.380.18 1.170.022 0.690.19
GLN Gel Acetylshikonin 25.150.57 3.140.070 0.810.15

Each data represents the mean SE from 5
experiments.
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Figure 6. Comparision of Residual Geniposide,
Shikonin and Acetylshikonin on
Epidermic Tissue after 8 hours
under Various Preparations.
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Table ?. Inhibitory effects of formulations on
swelling of rat hind-paw induced
by carrageenan(1)
Formulation Time(?) Time(?) Time(?) Time(?) Time(?)
Formulation 0 hr 6 hr 12 hr 24 hr 48 hr
Control 1.290.03 2.040.01 1.870.01 1.800.03 1.700.03
GLC 1.280.01 2.050.02 1.830.02 1.700.01 1.540.03
GLN 1.310.01 2.080.02 1.800.02 1.650.02 1.480.03
Each data represents the meanSD from 5
experiments Significantly different
from control group (Plt0.05)
Significantly different from control group
(Plt0.01)
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Figure 7. Rate of edema in carrageenan(1)
induced foot edema in rats under
formulations Key -?- Control       
-?- GLC Gel        -?- GLN Gel
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Figure 8. Inhibitory effects of formulations on
swelling of rat hind-paw induced by
carrageenan(1)    Key GLC   
       ? GLN     Significantly different
from GLC gel (Plt0.01)
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Table ?. Antibacterial Activity of GLC and GLN
Microorganism(ATCC No.) Minimum inhibitory concentration(?/?) Minimum inhibitory concentration(?/?)
Microorganism(ATCC No.) GLC GLN
Staphylococcus aureus (29213) 100 50
Bacillus subtilis (6633) 100 50
Streptococcus pneumoniae (49619) 100 50
Staphylococcus epidermis (12228) 100 50
Pseudomonas aeruginosa (9027) gt500 gt250
Klebsiella pneumoniae (10039) gt500 gt250
Escherichia coli (35150) gt500 gt250
Salmonella paratyphi (13428) 100 50
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Figure 9. Inhibition Rate of Bacterial Growth of
GLC and GLN gel     Key GLC gel      
     ? GLN gel     Significantly different
from GLC gel (Plt0.05)     Each bar represents
the mean SE from 5 experiments.
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Figure 10. Comparison of reduction rate of GF and
LR hydrogels on thermal burn
model Key -?- Control       -- LR
Gel        -?- GLC Gel -?- GLN Gel
Significantly different from GLC Gel
(Plt0.05)
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Figure 11. Comparision of Therapeutic period of
GF and LR hydrogels              to burn
healing
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A
B
Figure 14. Photomicrographs of the process of
burn healing (A,B) A Thermal burn model
B Thermal burn model of hydrogels treated
group after 4 hours
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C
D
Figure 15. Photomicrographs of the process of
burn healing (C,D) C A scrab peels away
D Heal a burn
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Figure 12. Comparison of reduction rate of GF and
LR hydrogels on excision wound
model Key -?- Control       -- LR
Gel        -?- GLC Gel -?- GLN Gel
Significantly different from GLC Gel
(Plt0.05)
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Figure 13. Comparision of Therapeutic period of
GF and LR hydrogels              to wound
healing
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A
B
Figure 16. Photomicrographs of the process of
wound healing (A,B) A Excision wound
model B Excision wound model of hydrogels
treated group after 4 hours
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C
D
Figure 17. Photomicrographs of the process of
wound healing (C,D) C A scrab peels away
D Heal a wound
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Conclusion
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• Determination of shikonin, acetylshikonin
  geniposide
• were seperated by HPLC.
• Skin permeation rates of GLN gel were more
  increased
• than other gel preparation.
• Anti-inflammatory activities of GLN gel were
  more effective
• than that of GLC gel.
• Absorption ratio of shikonin, acetylshikonin and
  geniposide
• were more than residual amount significantly.

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• Anti-bacterial activities of GLN gel were more
  effective
• than that of GLC gel.
• The reduction rates of GLN gel treated group
  were increased
• rapidly than that of other gel groups in burn
  wound skin.
• Therapeutic period of GLN gel treated group were
  shorter
• than that of other gel groups in burn wound
  skin.
• GLN gel would be a suitable preparation to
  increase
• the therapeutic effects for the burn and wound
  heal-
• ing, anti-bacterial and anti-inflammatory
  effects.