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1 21 DOI : /KPI Received : Accepted : Key Words: Soyeom Pharmacopuncture solutionon, Keratitis, Eye-Irritation, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans. The Experimental study of safety and efficacy in using Soyeom Pharmacopuncture solution as eyedrops. Eun-Kyo Kang Hyung-sik Seo Dept. of Ophthalmology, Otorhinolaryn gology & Dermatology, Collego of Medicine, Sangi University ABSTRACT Objective : This experimental study was performed to investigate the safety of Soyeom Pharmacopunture solution manufactured by extraction of alcohol and water. To identify the use of it as eyedrops, the eye irritation test of rabbits and antibacterial test of Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, and Candida albicans was performed. Methods : 1. The eye irritation test of this material was performed according to the Regulation of Korea Food & Drug Administration( , KFDA 25-6). After Soyeom pharmacopuncture solution was administered in the left eye of the rabbits, eye irritation of the cornea, iris and conjunctiva was observed at 1, 2, 3, 4 & 7day. 2. After administering Soyeom Pharmacopuncture solution on bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans) which cause Keratitis, MIC(Minimum Inhibition Concentration) and the size of inhibition zone were measured. Anti-bacterial potency was also measured using the size of inhibition zone. Results : 1. After Soyeom pharmacopuncture solution was administered in the left eye of the rabbits, it was found that none of nine rabbits have abnormal signs and weight changes. 2. After Soyeom pharmacopuncture solution was medicated in the left eye of the rabbits, no eye irritation of the cornea, iris and conjunctiva was observed at 1, 2, 3, 4 & 7day. 3. There was no response to MIC on bacterial species (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans) after Soyeom pharmacopuncture solution was medicated. Conclusions : The present study suggests that Soyeom pharmacopuncture solution is a non-toxic and non-irritant medicine, which does not cause eye irritation in rabbits, but dosen't have anti-bacterial effects on bacterial species which cause Keratitis. These study result recommends that more research on other herbal medicines of eye drop for Keratitis are required. I 1/6 Corresponding author : Hyeongsik Seo. Department of Ophthalmology, Otolaryngology and Dermatology, Oriental Medicine Hospital, Sangji University, 283, Woosan-dong, Wonju-si, Kangwon-do, , South Korea. Tel: aran99@sangji.ac.kr

2 corneal opacity corneal scar corneal ulcer Staphylococcus, Pseudomonas aeroginosa, Fusarium II 1 1 Table 1 2 3~4 2.~3. 9 snuffle, ear mite, coccidium W 5D 31H Co. 3 KCTC Staphylococcus aureus KCTC 1916, Staphylococcus epidermidis KCTC 1917, Pseudomonas aeruginosa KCTC 24 Trypic Soy Agar Aspergillus niger KCTC 696 Malt Extract Agar Fusarium oxysporum KCTC Potato Dextrose Agar Candida albicans KCTC 7965 Yeast Mold Agar

3 g 3 2 5g 2,5 1 3 Controller impellar Buchner Funnel 5 9% alcohol alcohol 9% *9 1 magnetic stirrer 3 decompressor 9% alcohol alcohol :65, vacuum: % alcohol 9% alcohol alcohol 8% 3 9% alcohol 2 +alcohol 8 8% alcohol 7 7% alcohol 8% alcohol alcohol 7% 3 8% alcohol 3 +alcohol 7 8 7% alcohol 3 caramel alcohol g 1g % w/v saline , 2, 3, 4 7 Table 2-4

4 The Individual Index of Ocular Irritation I.I.O.I Mean Index of Ocular Irritation M.I.O.I M.I.O.I. The Index of Acute Ocular Irritation I.A.O.I. Table 5 4 Inhibition zone Minimum Inhibition Concentration, MIC ~48 2 Tryptic Soy broth 37 12rpm OD Optical Density / 25.2% Tween / 2 1 filter disc cylinder cup 3 / 5 cylinder cup 15 sealing 37 1~ / 1~3 Levofloxacin 5 Cravit III 1 1, 2, 3, 4 7 Table Staphylococcus aureus KCTC Fig. 1 2 Staphylococcus epidermidis KCTC Fig. 2 3 Pseudomonas aeruginosa KCTC 24 5 Fig. 3 4 Aspergillus niger KCTC Fig. 4 5 Fusarium oxysporum KCTC Fig. 5 6 Candida albicans KCTC Fig. 6

5 25 IV 65~9% Staphylococcus aureus Pseudomonas aeruginosa Staphylococcus spp. Staphylococus aureus Staphylococcu epidermidis Staphylococcus epidermidis Pseudomonas aeruginosa Pseudomonas 2 2~3 Aspergillus Fusarium Candida satellite lesions microabscess immune ring endothelial plaque Candida S. aureus S. pneumoniae Aspergillus 1998 Aspergillus, Fusarium Candida 2 Fusarium spp. Aspergillus spp.,

6 Candida spp. 48 Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa Aspergillus niger, Fusarium oxysporum, Candida albicans Cravit 1, 2, 3, 4 7 Table 6 1, 2, 3, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa 3 Cravit Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans 6 5 6

7 27 V VI Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans , 2, 3, Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, Candida albicans Dong Ho Yoon, Sang Uk Lee, Ouk Choi. Ophthalmology 7th. Seoul: Ilchokak. 26: 3, 111, Seok Seon Roh. 3. ( ). 27: 16, 164, 165, 245, Young Ho Hahn, Sang Joon Lee, Tae Won Hahn et al. Epidemiology of infectious Keratitis[I] A Multi-center Study. J Korean Ophthalmol Soc. 1998; 39(8): Young Ho Hahn, Tae Won Hahn, Hung Won Tchah et al. Epidemiology of Infectious Keratitis(2) : A Multi center Study. J Korean Ophthalmol Soc. 21; 42(2): : 477, 479, 488, ,. 3. :. 1996: 71, 15, 159, Min Ahn, Young Taek Jung, Hong Joo Han. A Clinical Study on Infectious l Ulcer. J Korean Ophthalmol Soc. 1996; 37(9): Jin A Lee, Hean A Kim, Won Chul Lee. Literatural study on Eye-drop. J Oriental Medical surgery, Ophthalmology & Otolaryngology. 1993; 12(2): :. 2: 127, ( ) :. 1999: Jae Ho Kim, Hong Bok Kim.. Seoul: Ilchokak. 1999: 75, 81, 82, 97, 1, Joon Young Hyon et al. Expression of IRFs and inos in the Mouse with Pseudomonas Aeruginosa Keratitis. J Korean Ophthalmol Soc. 26; 47(1): Asbell P, Stenson S. Ulcerative keratitis. Survey of 3 years' laboratory experience.

8 Arch Ophthalmol. 1982: 1: Young Ho Hahn, Young Sik Jang. Epidemiology of Staphylococcus epidermidis Keratitis. J Korean Ophthalmol Soc. 22; 43(4): Young Ho Hahn, Sang Joon Lee, Tae Won Hahn et al. Epidemiology of Pseudomonas Keratitis-A Multi-center Study-. J Korean Ophthalmol Soc. 1999; 4(9): Min Hwa Yoon, Hyun Ju Park, Ki Young Lee. A Study of antibacterial effect on Pseudomonas aeruginosa of soft contact lens multi-purpose solution(mps). J Korean Oph. Opt. Soc.. 24; 9(2): Hyoung Jun Koh, Hong Bok Kim. The Effect of Topical Antibiotics on the Experimental Pseudomonas Keratitis. J Korean Ophthalmol Soc. 1992; 33(4): Young Ho Hahn, Dong Jun Lee, Man Soo Kim, Jae Duck Kim, Si Hwan Choi. Epidemiology of Fungal Keratitis in Korea : A Multicenter Study. J Korean Ophthalmol Soc. 2; 41(7): Jack J. Kanski. Clinical Ophthalmology 5th. Seoul: Jung-Dam. 25: Yun Sang Kim, Young Soo Song, Jae Chan Kim. Fungal Keratitis Caused by Chromomycetes. J Korean Ophthalmol Soc. 23; 44(3): Yu Hwan Lee, Young Ho Hahn, Joo Hun Roh. Microbial Contamination of Topical Eye Drugs. J Korean Ophthalmol Soc. 1996; 37(5): Newell FW. Pphthalmology, 6th ed. St.Louis, CV Mosy Co. 1986: Kwang Hoon Lee, Hong Jae Chae, Kyung Chul Yoon. Analysis of Risk Factors for Treatment Failure in Fungal Keratitis. J Korean Ophthalmol Soc. 28; 49(5): Hyung Sik Seo, Young Ku Lee, Sek Seon Roh. A Literatual Study on the external treatment of eye diseases. J Oriental Medical surgery, Ophthalmology & Otolaryngology ; 1(1): Hyung Sik Seo. The Experimental Study on Anti-bacterial Potency of Soyum Herbalacupuncture on Staphylococcus aureus & Staphylococcus epidermidis. J Korean Institute of Herbal Acupuncture. 26; 9(2): ,,. In Vitro Activities of Levofloxacin and Other Commonly Used Antimicrobial Agents Aganist aerobic Bacteria ; 27(1): CRAVIT(Levofloxacin). Ewha Med J. 1996; 19(3):

9 29 Table 1. Prescription of Soyeom The Botanical Name of Herb Relative Amount(g) Taraxaci Herba 77.4 Lonicerae Flos 77.4 Rehmanniae radix 77.4 Forsythiae Fructus 77.4 Coptidis Rhizoma 47.6 Scutellariae Radix 47.6 Phellodendri Cortex 47.6 Gardeniae Fructus 47.6 Total Amount 5. Table 2. Scale of weighted scores used for grading the severity of ocular lesions (1) A. Opacity-Degree of density(area which is most dense is taken for reading) No opacity and pyosis Scattered or dense areas. Every inch of iris is clearly visible 1 Easily discernible translucent areas. Every inch of iris is slightly obscure. 2 Opalescent areas. Every inch of iris is invisible. The size of pupil is barely discernible. 3 Opaque. is not observed. 4 B. Area of corneal opacity One quarter (or less) but not zero 1 More than one quarter ~ less than one-half 2 More than one-half ~ less than three quarters 3 More than three quarters up to whole area 4 Score equals Total maximum = 8 Table 3. Scale of weighted scores used for grading the severity of ocular lesions (2) Score equals Total maximum = 8 A. Values Normal Remarkable folds, congestion, swelling, circumcorneal injection (any one of these or combination), iris still reacting to light 1 No reaction to light, hemorrhage, gross destruction (some of these or all) 2

10 Table 4. Scale of weighted scores used for grading the severity of ocular lesions (3) A. Redness (refers to ocular and palpebral conjunctiva only) Normal Vessels definitely injected above normal 1 More diffuse bright red. Each individual vessels is not observed easily. 2 Diffused beefy red 3 B. Chemosis No swelling Any swelling above normal(included nictitating membrance) 1 Obvious swelling with partial eversion of eyelids 2 Swelling with eyelids about half closed 3 Swelling with eyelids about half closed to completely closed 4 C. Discharge No discharge A little discharge(expect small amount of discharge observed in inner canthus of normal animal) 1 Discharge with moistening of the lids and hairs 2 Discharge with moistening of the lids, hairs and considerable area around the eye 3 Score equals Total maximum = 2 Table 5. Irritation index of eye Irritation Rating Evaluation value I.I.O.I M.I.O.I Day-7 I.A.O.I Noniirritant ~ 5 (after 48hrs) Minimally irritant 5 ~ 15 5(after 48hrs) Mildly irritant 15 ~ 3 5(after 4days) Moderately irritant 3 ~ 6 2(after 7days) Severely irritant 6 ~ 8 4(after 7days) Extremely irritant 8 ~ 11 3 (all of non-washing group) 1 (more than four of non-washing group) 6 (all of non-washing group) 3 (more than four of non-washing group) I.I.O.I(The Individiual Index of Ocular Irritation): total score of each animal M.I.O.I(Mean Index of Ocular Irritation): the amount of total score/tested animal No. Day-7 I.A.O.I(The Index Acute Ocular Irritation): Max among M.I.O.I on Day-7

11 31 Table 6. Eye irritation score of New zealand White male rabbits treated with Soyeom Pharmacopuncture solution Group Rabbit No. Check area Tissue score Days Total score M.I.O.I /8 1 /1 ( ) 2 /2 /8 2 /1 ( ) 2 /2 /8 3 /1 ( ) 2 /2 /8 4 /1 ( ) 2 /2 /8 5 /1 ( ) 2 /2 /8 6 /1 ( ) 2 /2 /8 7 /1 ( ) 2 /2 /8 8 /1 ( ) 2 /2 /8 9 /1 ( ) 2 /2 group : Soyeom Pharmacopuncture solution washed group group : Soyeom Pharmacopuncture solution non-washed group A: Opacity, B: Area of cornea involved A: Values A: Redness, B: Chemosis, C: Discharge Fig. 1. MIC of Soyeom Pharmacopuncture solution and cravit on Staphylococcus aureus. Soyeom Pharmacopuncture solution(3 /, 5 ) was not showed inhibition zone but cravit(levofloxacin, 5 ) was showed inhibition zone on Staphylococcus aureus.

32 12 1 29 3 Fig. 2. MIC of Soyeom Pharmacopuncture solution and cravit on Staphylococcus epidermidis.

12 Fig. 2. MIC of Soyeom Pharmacopuncture solution and cravit on Staphylococcus epidermidis. Soyeom Pharmacopuncture solution(3 /, 5 ) was not showed inhibition zone but cravit(levofloxacin, 5 ) was showed inhibition zone on Staphylococcus epidermidis. Fig. 3. MIC of Soyeom Pharmacopuncture solution and cravit on Pseudomonas aeruginosa. Soyeom Pharmacopuncture solution(3 /, 5 ) was not showed inhibition zone but cravit(levofloxacin, 5 ) was showed inhibition zone on Pseudomonas aeruginosa. Fig. 4. MIC of Soyeom Pharmacopuncture solution and cravit on Aspergillus niger. Soyeom Pharmacopuncture solution(3 /, 5 ) and cravit(levofloxacin, 5 ) were not showed inhibition zone on Aspergillus niger.

13 33 Fig. 5. MIC of Soyeom Pharmacopuncture solutionand and cravit on Fusarium oxysporum. Soyeom Pharmacopuncture solution(3 /, 5 ) and cravit(levofloxacin, 5 ) were not showed inhibition zone on Fusarium oxysporum. Fig. 6. MIC of Soyeom Pharmacopuncture solution and cravit on Candida albicans. Soyeom Pharmacopuncture solution(3 /, 5 ) and cravit(levofloxacin, 5 ) were not showed inhibition zone on Candida albicans.

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101~110 ¿õ´ã.¿ìÈ² 101 DOI : 10.3831/KPI.2010.13.2.101 Received : 10. 05. 06 Accepted : 10. 05. 27 The Experimental Study on the Effect of Fel Ursi & Bovis Calculus Pharmacopuncture Solution in Bacterial Species which cause