Transcript 슬라이드 1
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 Introduction ■ 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. Lithospermi Radix - Root of Lithospermum Erythrorhizon Sieb. - Naphthoquinone pigment : shikonin, acetylshikonin, isobutylshikonin, etc. - Treatment of burn, excision wound, eczema, blister as antifebrile, antiphlogistic Figure 1. Molecular structure of shikonin * R: =H Gardeniae Fructus - Fruits of Gardeniae Jasminoides Ellis. - iridoid glycoside : geniposide, gardenoside, genipin, etc - Accelerate choler secretion and anti-inflammatory, analgesic effect Figure 2. Molecular structure of geniposide Scope of studies - 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 Experiment 1.Materials - Lithospermi radix - Gardeniae fructus - Shikonin (Wako. Chem. Co. Japan) - Geniposide (Sigma. Chem. Co. USA) - λ-Carrageenan Ⅳ (Sigma. Chem. Co. USA) 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) 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) 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 5. Determination of shikonin and acetylshikonin, geniposide - Shikonin & Acetylshikonin Method ; High performance liquid chromatograph Column ; μ-Bondapak C18 3.9×300 ㎜ Mobile phase ; acetonitrile : water : acetic acid : triethanolamine (70:30:0.3:0.3 v/v%) Detector ; UV detector 548 ㎚ - Geniposide Method ; High performance liquid chromatograph Column ; μ-Bondapak C18 3.9×300 ㎜ Mobile phase ; acetonitrile : water (15:85 v/v%) Detector ; UV detector 240 ㎚ 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 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℃ 9. Effect of anti-inflammatory - Inhibition of Swelling Test Edema Inducer : 1% λ-Carrageenan(Type IV) Soln. - Observed edema after 6, 12, 24, 48hrs - Inhibition rate = [1-Vd/Vc]*100 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) 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 Results Table Ⅰ. 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 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 1.5 1.5 1.5 1.5 Distil. Water 56.5 56.5 55.5 50.0 Nano-ATP® - - - 5.5 100.0 100.0 100.0 100.0 Carbopol 940 Triethanol amine Total GFEP* : Gardeniae fructus extract powder LREP** : Lithospermi radix extract powder Table Ⅱ. 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 Table Ⅲ. Viscosity of each hydrogels under various temperatures by spindle number RV 5 of Brookfield viscometer (Factor : 400) 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 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 (P<0.05) 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 (P<0.05) 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 Table Ⅳ. Permeation Parameters of Various Gels Through Excised Hairless Mouse Skin Parameters sample GF Gel LR Gel GLC Gel GLN Gel Cumulative amount for 8 hr (㎍/㎠) Js TL Geniposide 45.77±1.30 5.72±0.160 0.78±0.16 Shikonin - - - Acetylshikonin - - - Geniposide - - - Shikonin 8.44±0.15 1.06±0.023 0.75±0.13 Acetylshikonin 21.26±0.79 2.66±0.097 0.91±0.34 Geniposide 55.90±1.07 6.89±0.130 1.03±0.18 Shikonin 8.77±0.16 1.11±0.020 0.77±0.23 Acetylshikonin 23.55±0.66 2.91±0.081 0.93±0.26 Geniposide 57.20±1.50 7.15±0.190 1.01±0.21 Shikonin 9.38±0.18 1.17±0.022 0.69±0.19 Acetylshikonin 25.15±0.57 3.14±0.070 0.81±0.15 Each data represents the mean ±SE from 5 experiments. 12 Amount localized(g/cm2) 10 GF Gel LR Gel GLC Gel GLN Gel 8 6 4 2 0 Shikonin Acetylshikonin Geniposide Figure 6. Comparision of Residual Geniposide, Shikonin and Acetylshikonin on Epidermic Tissue after 8 hours under Various Preparations. Table Ⅴ. Inhibitory effects of formulations on swelling of rat hind-paw induced by carrageenan(1%) Time(㎖) Formulation 0 hr 6 hr 12 hr 24 hr 48 hr Control 1.29±0.03 2.04±0.01 1.87±0.01 1.80±0.03 1.70±0.03 GLC 1.28±0.01 2.05±0.02 1.83±0.02 1.70±0.01* 1.54±0.03* GLN 1.31±0.01 2.08±0.02 1.80±0.02* 1.65±0.02** 1.48±0.03** Each data represents the mean±SD from 5 experiments * ; Significantly different from control group (P<0.05) ** ; Significantly different from control group (P<0.01) Figure 7. Rate of edema in carrageenan(1%) induced foot edema in rats under formulations Key : -◇- ; Control -○- ; GLC Gel -●- ; GLN Gel 70 * Edema Inhibition Rate(%) 60 50 40 30 20 10 0 12 24 48 Time(h) Figure 8. Inhibitory effects of formulations on swelling of rat hind-paw induced by carrageenan(1%) Key ; ■ : GLC □ : GLN * ; Significantly different from GLC gel (P<0.01) Table Ⅵ. Antibacterial Activity of GLC and GLN Microorganism(ATCC No.) Minimum inhibitory concentration(㎍/㎖) 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) >500 >250 Klebsiella pneumoniae (10039) >500 >250 Escherichia coli (35150) >500 >250 100 50 Salmonella paratyphi (13428) 50 * Inhibition Rate (%) 40 * 30 20 10 0 S. aureus S. epidermidis Microorganism Figure 9. Inhibition Rate of Bacterial Growth of GLC and GLN gel Key ; ■ : GLC gel □ : GLN gel *Significantly different from GLC gel (P<0.05) Each bar represents the mean ±SE from 5 experiments. 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 (P<0.05) * * Figure 11. Comparision of Therapeutic period of GF and LR hydrogels to burn healing 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 C D Figure 15. Photomicrographs of the process of burn healing (C,D) C : A scrab peels away D : Heal a burn 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 (P<0.05) * * Figure 13. Comparision of Therapeutic period of GF and LR hydrogels to wound healing 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 C D Figure 17. Photomicrographs of the process of wound healing (C,D) C : A scrab peels away D : Heal a wound Conclusion • 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. • 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 healing, anti-bacterial and anti-inflammatory effects.