Hyung-Sik Seo ^{1 *}, Dong-Jin Lee ¹

This experimental study was designed to investigate the safety and the effectiveness of Samjeong pharmacopuncture solution (SPS) manufactured by using a the lowtemperature extract on process.

To identify the safety and the effectiveness of using SPS as eye drops, we performed applied eye irritation tests on rabbits and antibacterial tests for Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, and Candida albicans. The eye irritation test was performed according to the toxicity testing regulation of the Korea Food & Drug Administration (2009. 8. 24, KFDA 2009-116). After SPS had been applied on the left eye of the rabbits, eye irritation in the cornea, iris and conjunctiva was observed on the 1st, 2nd, 3rd, 4th & 7th day. After SPS had been dropped on bacterial species that cause keratitis, the minimum inhibition concentration and the size of the inhibition zone were measured. The anti-bacterial potency was also measured by taking the size of inhibition zone.

After SPS had been administered on the left eye of the rabbits, none of nine rabbits were found to show abnormal signs or weight changes. After SPS had been administered on the left eye of the rabbits, no eye irritation in the cornea, iris and conjunctiva was observed on the 1st, 2nd, 3rd, 4th & 7th day. No specific response was detected in MIC for bacterial species Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Aspergillus niger, Fusarium oxysporum, and Candida albicans after SPS had been applied.

This study suggests that SPS is a non-toxic and non-irritant medicine that does not cause any of eye irritation in rabbits, but it has no antibacterial effects on bacterial species that are well known to cause keratitis. These results suggest that more research is required on extracts from herbal medicines for treating keratitis.

Antibacterial effect, Eye drops, Eye irritation, Samjeong pharmacopuncture solution (SPS)

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Eye drops are a medical treatment applied to various ophthalmological diseases, so they must be safe and non-irritating as they directly affect the eyes. Especially, the fact that no blood vessels exist in the cornea makes localized eyedropping more available and effective than oral medication; besides, effective cleansing of bacteria and toxins can be mostly achieved through the proper use of eye drops [1]. However, recently there have been no standardized pharmaceutical companies manufacturing eye drops for clinical use in korean medicine. For that reason, many pharmacopunctures manufactured by the Korea Pharmacopuncture Institute, which has aseptic facilities almost equivalent to the GMP level, are considered to be the most appropriate materials for making eye drops.

In order to utilize Pharmacopuncture solutions (PSS) from the Korean Pharmacopuncture Institute as eye drops, there have been constant experimental trials on ascertaining the safety and the effectiveness of saline solution, Hwangryeonhaedoktang, Bovis Calculus (B), Fel Ursi (U), Bovis Calculus & Fel Ursi (BU) and Bovis Calculus & Fel Ursi &+ Moschus (BUM) PSS [2-8]. However, PSS manufactured through distillation and alcohol immersion haven't shown any significant antibiotic efficacy in many testings so far. Thus, PSS with antibiotic effects need to be produced by using new extraction processes. Thereupon, this experiment on eye irritation and antibiotic effects was conducted with Samjeong pharmacopuncture solution (SPS), which was manufactured using a low-temperature extracting process.

SPS used in this study was manufactured using a low-temperature extracting process and was provided by the Korea Pharmacopuncture Institute. The prescription is given in Table 1.

In this study, animal experiments were conducted under the approval of the Pusan National University Hospital Institutional Animal Care and Use Commitee (PNUH 2011-031). Nine New Zealand white rabbits (male: 5, female: 4) aged about 3∼4 months (weight: 2.0∼3.0 kg) were used in this study and snuffles, ear mites, coccidium infection states and conditions of fur and excrement were checked on arrival. They had been observed during one week of domestication at the breeding farm. Ophthalmological tests were performed on the conjunctiva, eyeball and cornea 24 hours before the experiment.

Rabbits were bred in the rabbit cage (420W×500D×310H ㎜) made of stainless and had free access to feed (Sinchon Co.) and water. The environment was maintained at a constant temperature (21±2℃) and humidity (60%).

The cell line used in this experiment was provided by Korean Collection for Type Culture (KCTC). Staphylococcus aureus (KCTC 1916), Staphylococcus epidermidis (KCTC 1917) and Pseudomonas aeruginosa (KCTC 2004) were cultured in Trypic Soy Agar. Aspergillus niger (KCTC 6906) was cultured in Malt Extract Agar. Fusarium oxysporum (KCTC 16322) was cultured in Potato Dextrose Agar, and Candida albicans (KCTC 7965) was cultured in Yeast Mold Agar.

Eye irritation tests were conducted following the toxicity testing regulation of the Korea Food & Drug Administration (2009. 8. 24, KFDA 2009-116). Both eyes of all the laboratory rabbits had been tested 24 hours before the experiment began, among which rabbits with normal corneas were selected. SPS, 0.1 ㎖ was dropped on an eye of each of the nine rabbits, and after 20 ∼30 seconds, three out of these were washed with 20 ㎖ of warm saline solution for a minute while the others were left untouched to be treated as a control group. After the application of SPS, changes in the weight and clinical symptoms on the 1st, 2nd, 3rd, 4th and 7th day were observed. The eye irritation tests were evaluated, with the maximum points set at 80 points on cornea response, 10 points on iris response and 20 points on the conjunctiva response, adding up to a total score of 110. If injury still remained on the affected eyes afterwards, the eye-dropping was applied every three days over 13 days (Tables 2,3, and 4).

"The mean index of ocular irritation (MIOI)" which had been obtained from the division of the total score of "the individual index of ocular irritation (IIOI)" by the number of rabbits, "the index of acute ocular irritation (IAOI)", which is the maximum value of "the mean index of ocular irritation (MIOI)" during observation and the day-7 IOII (indivisual ocular irritation index) were used in order to evaluate the degree of eye irritability (Table 5)

Sterile water was added to the lyophilized ampule, and the medium was coated with 1∼2 droplets of the cell line for 16∼ 24 hours under 35±1℃. This single colony was then moved to the new medium and subcultured for another 16∼24 hours under 35±1℃. A yeast fungus was cultured for 3 days and a mold was cultured for a week. Several colonies separated from the 10 ㎖ sterile saline solution were suspended, and the number of germs in the colony was counted up to 2.5∼10×10⁹ cells/㎖ through a microscope in order to be used as a source of inoculum. Mold was made to 2.5∼10×10⁹ cells/㎖ in the saline solution with 0.03% detergent and was used as the inoculum source. The prepared inoculum sources were coated with 0.4 ㎖ each on the medium and left to dry for 2∼3 minutes with a plate lid slightly open on a clean bench. Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa were cultured in Tryptic soy agar, Candida albicans was cultured in Yeast malt agar, Aspergillus niger was cultured in Malt extract agar, and Fusarium oxysporum was cultured in Potato Dextrose Agar. SPS, 50 ㎕ of sample was dropped on the germ-seeded medium with a sterilized filter disc on top of it. Equal amounts of sterile water and Vioflox (Ofloxacin) were used for negative and positive control, respectively. The antibiotic potency was examined by measuring the clear zone after culturing (2∼7days) each cell line at an appropriate temperature. To measure the minimal inhibitory concentration (MIC), we difuted an appropriate amount of the sample, and we performed the experiment in exactly the same way as above.

No abnormalities related to general conditions like weight, appearance, feed, water consumption, tremor, spasm, diarrhea, coma, drowsiness, contraction and dilatation of pupils, feces and urine, and disposal per day were found during this experiment (Table 6 and 7).

After SPS had been administered only on the left eyes, no eye irritation on the affected cornea, iris and conjunctiva of the nine rabbits from both the washed and the non-washed groups was observed compared with the right eyes (control group) (Table 8), (Fig 1).

SPS didn't show any antibacterial effects on Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium oxysporum. On the other hand, Vioflox (Ofloxacin), the control group, shows strong antibacterial effects on Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa, forming an inhibition zone of over 1 ㎝. However, no antibacterial effects were seen on Candida albicans, Aspergillus niger and Fusarium oxysporum (Fig 2).

Currently, korean medical doctors are having difficulty treating ophthalmological patients because no standardized pharmaceutical company manufacture eye drops, which can possibly lead to a scale-down in our medical treatment range. Therefore, readily available forms of eye drops desperately need to be immediately developed in korean medical ophthalmology as various kinds of eye drops are already prevalent in western medicine. Especially, aseptic forms of eye drops are needed.

PSS is an aseptic treating material that is obtained from single or compound herbs through various extract on methods, and is applied in many clinical fields, chiefly by injection, for musculoskeletal or internal problems. However, the range of korean medical ophthalmological treatments could be expanded through this new method to aseptically manufacture PSS for eye drops.

To utilize PSS as eye drops, several experiments have chiefly focused on proving the safety and the effectiveness of anti-inflammatory herbs [2-8], and "A clinical case report of Hwangryeonhaedok pharmacopuncture soluton" was reported by the Korean Medical Society [9]. PSS manufactured through distillation and alcohol immersion have been found not to trigger any eye irritation in the safety and the effectiveness experiments so far, but different and improved extraction methods are still required because no significant antibacterial effects have been detected.

In low-temperature extraction, compound herbs are decocted and then separated to undergo decompression and lowtemperature distillation. The outside temperature is maintained at 120℃, and the inside temperature is maintained at 60℃ during decompression and distillation. This method is recommended to maximize the efficacy of herbs and will replace the distillation method sooner or later [10].

Thus, by using SPS obtained from low-temperature extraction, we performed an antibacterial activity experiment on eye irritation by using six kinds of infectious keratitis-causing cell lines: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Candida albicans, Aspergillus niger and Fusarium oxysporum [11-14]. No abnormalities were found in weight and general conditions during the experiment, and no eye irritation of the cornea, iris and conjunctiva in both the washed and the non-washed groups treated with SPS was observed, so the safety of SPS is assured.

In the antibacterial activity experiment, 50 ㎕ of SPS showed no antibacterial effects on the six kinds of cell lines with infectious keratitis-causing germs, yeast fungus and mold. Even an increased amount up to 200 ㎕ showed no such effects.

SPS is a non-toxic and non-irritant medicine which does not cause any of eye irritation in rabbits, but it has no antibacterial effects on bacterial species that are well known to cause keratitis. Consequently, the fact that SPS causes no eye irritation secures its safety, but the lack of effectiveness in antibacterial activity indicates that constant research on a new extract on method is still needed.

This research was supported by a Pusan National University Hospital Research Grant, 2011.

1. Seo HS. Eye Irritation Test of Soyeom Pharmacopuncture Solution for Eye Drop. Journal of Pharmacopuncture. 2008;11(2):75-9.
2. Kang EK, Seo HS. The Experimental study of safety and efficacy in using Soyeom Pharmacopuncture solution as eye drops. Journal of Pharmacopuncture. 2009;12(1):21-33.
3. Seo HS. The Experimental Study on Anti-bacterial Potency of Hwangryunhaedoktang Herbal-acupuncture & Extract on Staphylococcus aureus & Staphylococcus epidermidis. Journal of Pharmacopuncture. 2006;9(2):99-103.
4. Seo HS. The Experimental Study of Safety and Efficacy in Using Bovis Calculus Pharmacopuncture Solution as Eye Drop. Journal of Pharmacopuncture. 2009;12(3):61-72.
5. Seo HS. Eye Irritation Test of Fel Ursi & Bovis Calculus Pharmacopuncture Solutions for Eye Drop. Journal of Pharmacopuncture. 2008;11(3):99-104.
6. Han NY, Seo HS. The Experimental Study on the Effect of Fel Ursi & Bovis Calculus Pharmacopuncture Solution in Bacterial Species Which Cause Keratitis. Journal of Pharmacopuncture. 2010;13(2):101-10.
7. Seo HS. Eye Irritation Test of Fel Ursi, Bovis Calculus & Moschus Pharmacopuncture Solutions for Eye Drop. Journal of Pharmacopuncture. 2008;11(3):105-11.
8. Seo HS. The Experimental Study of Efficacy in Using Fel Ursi, Bovis Calculus & Moschus Pharmacopuncture Solution as Eye Drop. Journal of Pharmacopuncture. 2009;12(3):41-7.
9. Lee CW, Park IB, Kim SW, Ahn CB, Song CH, Jang KJ, et al. The Clinical Study on Chronic Conjunctivitis or Xerophthalmia Treated with The Hwangryunhaedock-tang Herbal Acupuncture Therapy. Korean Journal of Acupuncture. 2003;20 (4):77-84.
10. Korean Pharmacopuncture Institute. Pharmacopuncturology. Seoul: Elsevier Korea, 2011:10.
11. Hahn YH, Hahn TW, Choi SH, Choi KY, Wee WR, Kim KS, et al. Epidemiology of Infectious Keratitis [I] A Multi-center Study. J Korean Ophthalmol Soc. 1998;39(8):1633-51.
12. Hahn YH, Lee DJ, Kim MS, Choi SH, Kim JD. Epidemiology of Fungal Keratitis in Korea: A Multi-center Study. J Korean Ophthalmol Soc. 2000;41(7):1499-1508.
13. Hahn YH, Hahn TW, Tchah HW, Choi SH, Choi KY, Kim KS, et al. Epidemiology of Infectious Keratitis II: A Multi-center Study. J Korean Ophthalmol Soc. 2001;42(2):247-65.
14. Seo HS. The Experimental Study on Antibiosis of Decoctions Made by Cassiae Semen, Celosiae Semen and Buddlejae Flos. Journal of Pharmacopuncture. 2010;13(2):75-83.