KR100791244B1 - Contact lens preservatives containing xanthium strumarium l. extracts as effective constituent - Google Patents

Abstract

A contact lens preservative containing xanthium strumarium L. extracts having an antibacterial function as a main active ingredient is provided to prepare a natural contact lens preservative by a stable and efficient method. A contact lens preservative contains xanthium strumarium L. extracts that are prepared by carrying out hot water extraction and purification in part. Then, the xanthium strumarium L. extracts are diluted in a dilute solution to a concentration of 1.0mg/ml-5.0mg/ml. Preferably, methanol is used as the dilute solution. The contact lens preservative containing xanthium strumarium L. extracts demonstrates excellent antibacterial activity, having an antibacterial activity band Rf value of 0.87, when measured by TLC using a methylene chloride and methanol developing solvent at the volume ratio of 94:6.

Description

CONTACT LENS PRESERVATIVES CONTAINING XANTHIUM STRUMARIUM L. EXTRACTS AS EFFECTIVE CONSTITUENT}

1: Docomari picture.

FIG. 2: A report showing the results of separating and purifying two types of antimicrobial substances having molecular weights of 386 and 230 from a dog.

3 to 125: Pictures related to the experiment of the present invention.

The present invention relates to a contact lens preservative containing the extract as the active ingredient.

Contact lenses have been devised for the purpose of vision correction and treatment, and many researches and developments have been made with the development of the material. The number of wearers has been expanded due to excellent vision correction and cosmetic benefits. Increasing the number of wearers of these contact lenses is also increasing (Erie, et al., 1993). In particular, the side effects caused by neglecting lens cleaning and care of contact lens wearers are severe from minor conjunctiva and cornea damage. It can even cause corneal ulcers (Lee, et al., 1994).

Reports have shown an increased incidence of keratitis in soft contact lens wearers, including chronic lens deposits due to prolonged wear of the lens, chronic exposure of the lens to strong adhesion bacteria, and lens deposits. Persistent irritation, prolonged presence of debris in the back of the lens, and lens cleansing and neglect have increased bacterial adhesion to the lens and caused damage to the corneal epithelium and promoted bacterial infection (Seol, et al., 1989).

Neglecting and poor cleaning of the lens provide an opportunity for bacteria to adhere and multiply in the lens, causing corneal epithelial damage and infection (Bruce and Brennan, 1990). In order to prevent various side effects caused by such bacteria, contact lenses must be washed and disinfected after getting rid of them, and care products for removing impurities on the surface of contact lenses include washing liquid, rinsing liquid, disinfecting liquid, preservative, and protein remover. .

Each time a contact lens is removed from the eye, it must be cleaned, rinsed, disinfected and stored, and the protein must be removed every few days (Kenneth and Bobby, 1996). .

There are two methods of managing contact lenses: daily washing and washing with multi-purpose solutions. However, in the modern society that prefers convenience, many contact lens wearers prefer washing with multi-purpose solutions. Soft contact lens multipurpose solutions (MPS) contain all the components that act as cleaning, rinsing, disinfecting, and preservatives, making it easier to clean, rinse, disinfect, and preserve them all at once. Increasing (Efron, et al., 1991).

Currently, many contact lens manufacturers and care products companies produce multipurpose solutions, and they have instructions for use by each manufacturer, but are classified as quasi-drugs and sold in ophthalmology, opticians, and pharmacies. Or even in the instructions it can be used like eye drops.

However, multipurpose solutions contain synthetic substances such as H ₂ O ₂ , sorbic acid, and dymed, which may cause side effects to corneas and conjunctival cells in the event of misuse or prolonged use (Tripathi and Tripathi, 1989). In addition, the use of synthetic preservatives can cause a number of complications, the most serious of which can lead to corneal clouding and corneal perforation, leading to blindness, and visual acuity of 50% to 20/50 or less in patients with infectious keratitis or corneal ulcer. Decreased or blinded (Wilhelmus, 1987; Palmer and Hyndiuk, 1993).

Conditions for contact lens cleaning and preservation are: first, antimicrobial activity in a broad range, second, rapid and durable in wearing, third, free from allergic reactions, irritation and toxicity, and fourth, in a stable state without interaction with other compounds in it. And the fifth compound must be well dissolved (Tsuda, et al., 1998). As such, contact lens cleaning and preservatives must have a high concentration of each compound in order to reduce the chances of bacterial infection as well as a wide range of antibacterial activity, but this can cause eye irritation or chemical eye trauma. It is more important than anything else.

Contact lens care solutions contain synthetic preservatives to protect the stability of the solution and to prevent the growth of bacteria. U.S. Pharmacopoeia (USP) and the Federal Food Drug Administration (FDA) require that preservatives should not induce toxic effects on tissues in contact with them (Tripathy, et al., 1992). Synthetic preservatives are known to be toxic to the eyes after prolonged use in patients with corneal and conjunctival dryness (Vajdic, et al., 1999). As such, research and development of antimicrobial natural preservatives that can supplement the toxicity and disadvantages of synthetic preservatives that cause side effects without causing toxicity to the eyes are urgently needed.

On the other hand, Xanthium strumarium L. belonging to Compositae has thick hairs all over, its stem stands straight and is about 1.5 m tall. Flowers bloom from August to September at the tip of stem with yellowish-brown head flowers. The fruit is similar to jujube seed, and has thick hairs in the shape of pericarp. It is an annual herb that grows mainly in fields or roadsides, and is widely distributed in Northeast Asia and Europe including Korea. The study on the larvae is based on the Chinese antler ( Xanthium sibiricum Patr. Ex Widd), which has excellent effects on fever, sweating, pain, pain, breeze, ulceration, skin disease, neuralgia and malignant tumors. It is known that there is a significant inhibitory effect on Staphylococcus aureus as well as Typhoid bacteria and dysentery. Chang-i-leaf has various effects such as bloating, analgesic, antipyretic, insecticide, etc. The components contained in Chang-za are xanthinin, carotenoid, alkaloid, saponine with γ-lactone structure. ), Xanthostrumarin, xanthostrunarin and oleic acid are known. As a result, Docomari with these ingredients has been established as a traditional folk remedy and widely used for the treatment of skin diseases such as eczema, as well as having various pharmacological effects, and thus antimicrobial and anticancer substances are expected. Recently, research on natural bioactive substances having new functions from herbal medicines, edible plants and various cultivated useful microorganisms (VBNC) has been actively conducted, including the East and West, and researches on the components having antibacterial and anticancer effects among natural herbal medicines. Actively done.

In particular, components such as ginnalin extracted from maple, asterisic acid extracted from sulphurous lake, beberin extracted from yellowish white or rhubarb, and hesperidin contained in citrus skin This drug has many antibacterial effects, such as herbal medicines and spices that have anti-proliferative effect on tooth decay, Streptococcus mutans, anticancer effect of Chopi extract, and anticancer effect of phenolic compounds secreted from turmeric. Among the components of medicinal plants, antibacterial and anticancer effects of various components such as flavonoids, alkaloids, glycosides, and polyphenols are known. In addition, a number of studies on antimicrobial anticancer and various physiological function effects have been conducted by separating components contained in foods including green tea (You, YS et al. (1993), Kor. J. Appl.Microbiol.Biotechnol. , 21, 187-191 .; Kim, SH et al. (1993), Kor. J. Appl.Microbiol.Biotechnol., 21, 628-634; Nagabhushan, M. and SV Bhide (1992), J. Am. Coll. Nutr., 11, 192-198; Dkai, Y. et al. (1993), J. Ferment. Bioeng., 76, 367-370; Mizuno, M. et al. (1989), Agric. Biol. Chem., 53, 959-964; Senji, S., M. et al. (1989), Agric. Biol. Chem., 53, 2307-2311).

The present inventors, as part of the search and application of new substances with a variety of biologically active functions, such as new antibacterial and anti-cancer effects from the Korean domestic dog, as well as the fact that the components extracted from each part of the dog has a wide range of antibacterial effects against bacteria and fungi The results of the separation and purification of two antimicrobial substances having molecular weights of 386 and 230 have been reported (Kim, HS et al. (1997), Kor. J. Appl. Microbiol. Biotechnol., 25, 183-188.7). Subsequent studies have shown that these antimicrobial agents have potent growth inhibitory effects on rectal cancer cell lines. However, antimicrobial effects on various cancer cell lines, antimutagenic effects, and other biologically active functions are expected to be identified. Previous studies have raised problems with the stability of isolated antimicrobials.

Therefore, the present inventors established a new method for the stable and efficient purification of all components present in the Korean domestic dog, investigating and confirming the antimicrobial and physiological effects such as mutagenicity and antimutagenicity, and searching for excellent antibacterial and anticancer substances. New natural contact lens preservatives (preservatives) have been developed to complete the present invention.

Accordingly, an object of the present invention is to provide a natural contact lens preservative (preservative) containing Xanthium strumarium L. (Korean name: Docomari, herbal name: Changja ) extract as an active ingredient, which has been found to have antimicrobial activity in various strains. There is a purpose.

Another object of the present invention is to provide a natural contact lens preservative prepared in a stable and efficient manner by partially purifying an active substance having antimicrobial and anticancer activity from Korean dogs.

Another object of the present invention is to examine the antimicrobial activity against eye disease bacteria and also to provide a novel natural contact lens preservative through eye mucosal irritation experiments using rabbits.

In order to achieve the above object, in the present invention, the antimicrobial activity against eye disease bacteria was examined using Xanthium strumarium L. extract, which was found to have antimicrobial activity, and contact lens through eye irritation experiments using rabbits. Wants to offer new natural preservatives.

In the present invention, the antimicrobial activity against ocular disease bacteria was investigated using the extract of Docomari, which was found to have antimicrobial activity in various strains, and to provide a new natural preservative through eye mucosal irritation test using rabbits.

In the following description of the present invention, detailed descriptions of related well-known structures and functions will be omitted in order not to obscure the subject matter of the present invention. Hereinafter, the preferred embodiment of the present invention will be described.

As a study of macaws, in 1953 Shrivastava claimed that macaws were valuable as a source of vegetable oil, and Moldenhawer (1957) reported as a new plant containing 38-41% oil. In addition, Dima et al. (1956) reported that dog oil seed oil is a glyceride of palmitic, stearic, oleic and linoleic acid and can be used for edible and industrial purposes, and Bhargava et al. (1961) reported that 90.8% of fatty acids in dog oil are unsaturated. Fatty acids have been reported to contain palmitic, stearic and behenic acids.

In addition, the total lipid content of chloroform-methanol extract of the seedlings was 43%, and the main component was triglyceride, the highest content of which was 1, 3-diglyceride, 1, 2-diglyceride, saturated Small amounts of fatty acids and sterols are known to be present. In addition, fatty acid composition of the seed oil of Dawley Marijuana is myristic, palmitic, palmitoleic, stearic, oleic and linoleic acid, and its contents are trace, 5.03%, trace, 2.08%, 16.98%, 75.91%, respectively. Fatty acids were reported as trace, 4.36%, trace, 2.34%, 17.14% and 76.16%, respectively (Table 1).

(Table 1) Dokomari fatty acid ingredient table

Fatty acids Retention time (min) Xanthium strumarium total seed oils (%) Myristic 3.3 trace (trace) Paimitic 5.7 5.03 (4.36) Palmitoleic 6.7 trace (trace) Stearic 10.3 2.08 (2.34) Oleic 11.9 16.98 (17.14) Linoleic 15.3 75.91 (76.16) Linolenic 18.7 -(-) Arachidic 20.7 -(-) Lauric (I.S.) 1.9

Systematic studies on the pharmacological effects of the purified components separated from Korean dogs are insufficient, and there are studies on dogs reported by the present inventors.

The inventors of the present invention have a wide range of antimicrobial effects against bacteria and fungi as a component extracted from each site of the dog as a part for the search and application of new substances with various physiologically active functions such as new antibacterial and anticancer effects from Korean dogs (Table 2). In addition to the fact that two types of antimicrobial substances having a molecular weight of 386 and 230 has been purified separately (Fig. 2).

Table 2 Antimicrobial Effects of Dogtail

Inhibitory zone (Φ, ㎜)

Strains＼Sample XE-N (10 μg) Gram-positive Mycobacterium phlei KCTC 1932 12.5 Staphylococcus aureeus KCTC 1927 10.5 Streptococcus equii 13.0 Streptococcus zooepidermicus 7.5 Bacilllus megaterium 8.0 Bacilllus subtilis 7.5 Bacilllus thermoglucosis - Bacilllus licheniformis IFO 12197 7.5 Gram-negative Escherichia coli K-12 10.5 Escherichia coli KCTC 1923 - Pseudomonas aeroginosa KCTC 1930 9.0 Pseudomonas fluorescens KCTC 1645 13.0 Enterobacter aerogenes KCTC 2190 10.5 Salmonella typhi 7.5 Serratia marcescens 7.5 Proteus vulgaris 9.0 Yeasts Cryptococcus neoformans 7.5 Hansenulla anomala B-7 - Fungus Aspergillus fumigatus IFO 5840 -

In addition, as these antimicrobial substances showed a strong growth inhibitory effect on rectal cancer cell lines, it was reported that identification of components having anti-cancer effects, anti-mutagenic effects, and various physiologically active functions against various cancer cell lines was expected. In addition, fractional extraction of Dokmari hot-water extract was performed using ether and ethyl acetate (ethylacetate), and each extract was divided into six fractions of neutral, acidic and basic. From the six fractions, five signals with excellent antimicrobial activity were separated. Among them, the signal with the highest antimicrobial activity was found to be S1, an ether neutral extract.

The docomari extract S1 was estimated to have a chemical formula of C1 ₅ H ₂ O ₃ by EIMS and NMR spectra data. The IR spectrum of the S1 compound showed a strong absorption band at 1760cm- ¹ , suggesting the lactone structure, a pentagonal ring compound. And it was confirmed that no polar group such as NH or OH group exists. The 1H NMR spectrum of S1 showed two doublets [δ 6.25 (d, J = 3.3Hz, 1H) and 5.52 (d, J = 3.3Hz, 1H)] which can be estimated as terminal olefin protons and their coupling constants Was estimated by geminal proton.

In addition, it was found that methyl signal (s, 3H) of acetyl group and one terminal methyl signal [δ 1.13 (9s, 3H)] existed at 2.15 ppm. 15 carbon signals and 20 protons, including 2 carbonyl carbons (δ 208.1, and 170.2), 2 quartnery carbons, 4 methine carbons, 5 methlene carbons, and 2 methyl carbons from 13C-NMR and DEPT spectra Could confirm. In COSY spectrum, the H-H correlation spectrum, a partial structure of ethylene unit attached to the terminal olefin group and also the acetyl group was obtained, and the presence of methine protons connected to the terminal methyl group was confirmed. Finally, the chemical structure estimated by HMQC coincided with 1H NMR and 13C NMR spectra. Thus, the structure of the compound was confirmed as tomentosin.

It will be described below with reference to a preferred embodiment of the present invention. However, these examples do not limit the scope of the present invention.

<Example>

Korean dokomari used as experimental material of the present invention is known to have an antimicrobial effect against Typhoid bacteria, dysentery bacteria, Staphylococcus aureus and the like. The antimicrobial activity against eye disease bacteria was tested using the extract of Docomari with various effects.

Experimental Example 1

The contamination patterns of soft contact lenses with time and microbial contamination after 4 hours of wearing soft contact lenses were investigated.

1. Contamination Patterns of Soft Contact Lenses over Time

One). Experimental material

The test strains used in the present invention were Staphylococcus aureus KCCM 40050, Pseudomonas aeruginosa KCCM 11803, and Enterobacter aerogenes KCCM 11783. In addition, soft contact lenses use 70% moisture content, 8.6mm base curve (BC), 14.2mm diameter, and 0.17mm (-3.00D) center thickness (one day disposable soft contact lenses, BAUSCH & LOMB, USA). It was.

2). Experiment preparation

S. aureus, P. aeruginosa , and E. aerogenes were inoculated on trypticase soy agar (pancreatic digest of casein 1.5%, papaic digest of casein 0.5%, sodium chloride 0.5%, agar 1.5%) to prepare the inoculated strains. After 2 days of incubation at 37 ° C, single colonies were diluted with phosphate-buffered saline (PBS, pH 7.4) using the McFarland standard (OD550nm) so that the total number of bacteria was 1.5 × 10 ⁸ colony-forming units (CFU) / ml. = 0.125). At this time, the components of PBS used in the culture is NaCl 0.8%, KCl 0.02%, Na2HPO4 1.44%, KH2PO4 0.024%.

In order to contaminate the microorganisms in the soft contact lens, 30 ml of S. aureus, P. aeruginosa, and E. aerogenes suspension containing 1.5 × 10 ⁸ CFU / ml were made , and 5 ml of each of the six tubes was placed side by side. . Then, the lenses were soaked in 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, and 24 hours under 37 ° C and 150rpm culture conditions.

In order to investigate the microbial contamination pattern of soft contact lens, after contacting each of 6 contact lenses in suspension contaminated with each bacteria, the lens was immersed in PBS again and centrifuged for 1 minute at 1000 × g. After removing the bacteria, scanning electron microscopy was performed.

For scanning electron microscopy, contact lenses were pre-fixed in 2.5% glutaraldehyde solution (0.1M cacodylate buffer, pH 7.4, 4 ° C) for 2 hours, washed three times with 0.1M phosphate buffer for 3 minutes, and then washed with 1% osmotic acid. After fixing for 2 hours at room temperature with (0.1 M cacodylate buffer, pH 7.4), dehydrated with ethyl alcohol, fixed to aluminum pin and coated on gold ion particles, observed by DSM 940A scanning electron microscope (Hitachi, Japan). It was.

2. Microbial Contamination after Wearing Soft Contact Lenses for 4 Hours

One). Test subject

Twenty-one university students without ophthalmological diseases except refractive error were selected on ophthalmic examination with or without contact lenses. The age ranged from 20 to 30 years, with 8 males and 13 females. There were 5 soft contact lenses and 16 non-wearers.

2). Contact lens microbial culture conditions and media composition

Twenty-one male and female college students without eye disease were given soft contact lenses and worn for 4 hours, and then each lens microorganism was cultured and analyzed. In order to minimize the contamination from the hands that may occur when wearing the lens, wash the hands with 80% ethanol, and then dry them with autoclave dried Kimwipes (215mm × 210mm, Yuhan-Kimberly, Gyeonggi-do, Korea) Was removed. Hands were washed in the same manner when the lens was removed from the eye. To investigate the implantation of bacteria against each lens, each lens was treated with LB agar (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5%, pH 7.0 ± 0.2) and YM agar (0.3% bacto yeast extract, 0.3% bacto malt extract, 0.5% bacto peptone, 1% bacto dextrose, 1.5% agar, pH 6.2 ± 0.2) plate and incubated for 3 days at 37 ℃. Inoculation method was inoculated by directly buried in the inner part of the contact lens in contact with the eye, and only colonies growing in the contact area of the lens was counted as effective colonies. In this case, the soft contact lens used was a 58% water content, a base curve (BC) 8.5mm, and a diameter of 14.2mm (one day disposable soft contact lens, ACUVUE (Johnson & Johnson)).

Experimental Example 2

The antimicrobial effects of multipurpose solutions, preservatives, and wash solutions of contact lenses were investigated.

1. Experimental Materials

The total multipurpose solution, preservation liquid and washing liquid used are 8 kinds of products of 5 manufacturers, and they are A product (B company), B product (A company), C product (A company), D product (B company) and E product ( C company), F product (S company), G product (B company) and H product (S company). These products were purchased from a commercial optician and used for this experiment. Among these products, A, C, and D products are multipurpose solutions, while B and E products are disinfection preservatives, and F, G, and H products are used as cleaning solutions.

As the test strains Bacillus subtilis KCCM 12148, Pseudomonas aeruginosa KCCM 11803, Pseudomonas fluorescense KCCM 40223, Pseudomonas stutzeri KCCM 12540, Alcaligenes faecalis subsp. faecalis KCCM 21180, Escherichia coli KCCM 11591, Staphylococcus aureus subsp. aureus KCCM 40050, Enterobacter aerogenes KCCM 11783, Escherichia coli ATCC 25922, Proteus vulgaris KCTC 2512, Korea Culture Center of microorganisms (KCCM), American Type Culture Collection, Rockwill, MD, USA and Korea Research Institute of Bioscience and Biotechnology Genetic banks (KCTC, Korean Collection for Type Cultures) were used for sale. All strains except P. vulgaris were used LB broth (10g tryptone, 5g yeast extract, 10g sodium chloride, pH 7.0 ± 0.2), and P. vulgaris was YM broth (3g yeast extract, 3g malt extract, 5g peptone, 10 g dextrose, pH 6.2 ± 0.2). In order to investigate the antimicrobial activity of lens wash solution by the agar diffusion method, P. vulgaris of YM agar (3g yeast extract, 3g malt extract, 5g peptone, 10g dextrose, 15g agar, pH 6.2 ± 0.2) was used to inoculate 100 μl of pre-culture solution, and the remaining test strains were inoculated with 100 μl of pre-culture solution in LB agar (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, 15 g agar, pH 7.0 ± 0.2). Plates were prepared. After dispensing 20 μl of each sample into a paper disc (Ø; 6mm, Whatman, Japan), the paper disc was completely dried, and then placed on a plate prepared in advance, followed by incubation at 30 ° C. and 37 ° C. for 2 days to observe the antibacterial activity.

We examined the degree of eye irritation of synthetic preservatives using multipurpose solutions and preservatives of contact lenses.

The rabbits used in the experiments of the present invention were handled according to the Guide for the Care and Use of Laboratory animals [National academy of science, 1996].

2. Experimental Animal

Group 1 (8 birds) to be administered for 4 weeks after adapting to the laboratory environment with sufficient supply of feed [Purina Korea, Seoul] and water to mature mature New Zealand White Rabbit (Yonam University, Cheonan). , Divided into 2 groups (2) to be administered for 2 weeks, a total of 10 were used. The laboratory environment was kept at an appropriate temperature (20-25 ° C) and humidity (30-35%), with 11-12 ventilation per hour and 12 hours of fluorescent lighting per day. The feed freely supplied solid feeds from Purina Korea, and the water supplied enough tap water. Experimental animals were housed in each cage to distinguish individuals. The animals were marked with oily magic inside the forearms.

All experimental animals were examined on the eyelids, conjunctiva, cornea and lens using a slit lamp immediately after arrival and one day prior to instillation of the test substance, and using a noncontact specular microscope (Konan, Japan). The density of the corneal endothelial cells, the coefficient of variation of the cell area representing polymegathism of the corneal endothelial cells, and the hexagonality, which is the inverse of the polymorphism, and safety using an ophthalmoscope. After the test, only experimental animals without abnormalities were selected and used.

3. Experimental substance

A total of three multipurpose solutions and preservatives on the market were purchased to test the degree of eye irritation with synthetic preservatives. Product A used in this experiment (company B) is a multipurpose solution, and product B (company A) and product C (company C) are used as preservatives. The main components are listed in Table 1a. It was used for 4 weeks after opening in the same way as used for lens cleaning and preservation, and was used in this experiment by following the recommendation of each manufacturer except for two (4) each group. In addition, among the three types of contact lens care solution products, B and C products must be used by neutralization treatment, and the neutralization treatment method was performed by referring to the contents written in the manual.

Table 1a: Major Components Table of Multi-Purpose Solution and Preservative Products Used in the Experiment.

Trial product Main ingredient A product (B company) Polyhexamethylene biguanide hydrochloric acid, Boric acid, Sodium chloride, Sodium phosphate, Poloxamine B product (A company) Hydrogen peroxide 3% C product (C company) Microfiltered hydrogen peroxide 3%, Sodium chloride 0.85%, Phosphonic acid, Buffered with phosphates

4. Separation of experimental group

Commercially available A, B, and C products, a total of three contact lens multipurpose solutions and preservatives were used for the ocular irritation test, and the experimental animals were divided into 4 week group and 2 week group. In case of the C product, two rats (four rats) in each group were administered to the eyes of rabbits without neutralization treatment in order to investigate the problems caused by direct administration due to user carelessness or lack of explanation of the seller, not manufacturer's recommendation. In the 4-week administration group, all of A, B, and C products were used in this experiment.

5. Administration of test substance

Observation of the eye irritation index after the 0.1ml single dose used in the Draize test is similar to the situation in which contact lenses are used because it is not suitable for the stimulation test of the contact lens multipurpose solution and the preservative solution used at least once / day. A drop (about 0.04 ml) was administered to the lower conjunctival sac. A total of three lens multipurpose solutions and preservatives were administered 4 times / day and about 0.04 ml / time. The washed subjects were treated with 50 ml of saline solution (Sino-Pharmaceuticals, Korea) 20-30 seconds after the administration of each test substance. Washing was performed.

6. Observation of eye irritation index

Draize eye test (Draize, 1944) is applied once in a 0.1 ml solution for liquids and 0.1 ml or 0.1 g conjunctival sac or cornea for solid or semi-solid particulates, and 1, 2, 3, 4, Stimulation was determined by scoring irritation in the cornea, iris and conjunctiva on day 7 and should be observed for at least 13 days if any irritation was observed. The degree of stimulus was given to the cornea by 80%, but only corneal haze was observed. However, the method of evaluating toxicity by one contact with chemicals is suitable as a test for people handling cosmetics or industrial chemicals, but the stability of eye drops or contact lens preservatives that need to be in contact with the eyes several times a day. It is not suitable for evaluation.

In this experiment, in order to observe the corneal stimulation index of the contact lens multipurpose solution and the preservative solution used several times a day, it is thought that the observation of the corneal epithelium is essential.In addition, the corneal epithelial item was added to the corneal stimulation index used in the Draize test. Corneal haze, iris and conjunctiva were adjusted by the ratio (Table 3, 4, 5).

Table 3 Scale of weighted scores for grading the severity of ocular lesion 1 (Cornea)

A. Corneal epithelium-Fluorescein stain  No stain ... ..... 0 Superficial puntate erosion ... ... 1 Small epithelial defect (longest diameter <1mm) .............. 2 Large epithelial defect (longest diameter) > 1mm) ........................... 3  B. Corneal stromal opacity (The most dense area is taken for reading)  No opacity ... 0 Scattered or diffused area (details of iris clearly visible) .................. 1 Easily discernible transluscent areas (detail of iris slightly obscured) .. 2 Opalescent area no details of iris visible, size of pupil barely discernibl .... 3 Opaque, iris invisible ................... ................. 4  C. Area of cornea involved  One quarter (or less) but not zero ......................... ... 1 Greater than one quarter-less than one half .................................. 2 Greater than one half-less than three quarters ............................... 3 Greater than three quarters up to whole cornea ... .............................. 4

Table 4 Scale of weighted scores for grading the severity of ocular lesion 2 (Iris)

A. Values  Folds above normal, congestion, swelling, circumcorneal injection (any one or all of these or combination of any there of), iris still reacting to light .................... ........................................ ..... 1 No reaction to light, hemorrhage, gross destruction (any one or all of these) ........................... ........................................ . 2

Table 5 Scale of weighted scores for grading the severity of ocular lesion 3 (Conjunctiva)

A. Redness (refer to bulbar conjuntiva & limbus)  Normal ... ............. 0 Vessels definitely injected above normal ..................... .......... 1 More diffuse deeper crimson red (involved vessels not esaily discernible) ........ 2 Diffused beefy red ............... 3 B. Chemosis  Not swollen ... .............. 0 Any swelling above normal (include nictating membrane) .......................... .1 Obvious swelling with partial eversion of lids .................................. 2 Swelling with lids about half closed 3 Swelling with lids about half closed to completely closed .............. 4 C. Discharge  Normal ... ................ 0 Any amount different from normal (does not include small amount observed in inner canthus of normal animals) .............. .............................. 1 Discharge with moistening of the lids and hairs just adjacent to the lids ...... 2 Discharge with moistening of the lids and considerable area around the eye ...... 3

Two rabbits were used for each solution to prepare the non-wash and wash groups, and the test was performed using the equipment used for the ophthalmic examination.

Stimulation of the corneal epithelium was a fluorescein paper (Haag-Streit, Switzerland) with a fluorescein solution using physiological saline on the superior conjunctival conjunctiva, and the eyelids flicker 3-4 times to stain the eye surface and slit lamps. Observation was made using the blue light of the microscope. After the primary corneal, iris, and conjunctival examinations, the pupils were shaken to observe the lens and fundus.

7. Mirror Microscopy for Corneal Endothelial Cell Observation

When light enters the cornea from the slit lamp, most of it is transmitted forward, but 0.02% of light is reflected between the endothelium and the anterior water into the cornea. This light is collected to obtain an image of the corneal endothelium. This method is a mirror microscopy, and nowadays, a non-contact mirror microscope which is not in contact with the eye due to the development of technology is widely used. In this experiment, a non-contact specular microscope (Konan, Japan) was used to show the cell area coefficient of variation and polymorphism indicating the density of corneal endothelial cells, polymegathism of corneal endothelial cells. The hexagonality of the inverse hexagonal cells was recorded and repeated three or more times based on the best images.

8. Histopathological Observation of the Eye

One). Morphological observation by optical microscope

The eyeball was removed, an incision was made in the sclera 2 to 3 mm away from the limbus, and a 360 ° incision along the limbus was made to obtain a cornea. Sections for optical microscopy of the corneal tissue were fixed in 10% neutral formalin solution, embedded in paraffin in the usual manner, and then cut into 4 μm thicknesses. Hematoxylin & Eosin (H & E) staining was performed on an optical microscope. .

2). Morphological observation by transmission electron microscope

The cornea was cut into 1 ~ 2 mm ² and fixed in 2.5% glutaraldehyde (pH 7.2) for 2 hours. After fixing for 2 hours at room temperature with 1% osmium tetroxide, dehydrated with a series of ethanol, embedded in an epon mixture, and double electron staining with uranyl acetate and lead citrate through a conventional procedure for transmission electron microscopic observation. Observed with a -7100 transmission electron microscope.

Experimental Example 3

The antimicrobial activity of Docomari extract against general bacteria and ocular disease bacteria was tested. Since the Docomari extract is more stable when present in a mixture than that which is separated and purified as a single substance, the sample purified by hydrothermal extraction was used in this experiment. Used to.

1. Experimental Materials

Xanthium strumarium L. used in this experiment was used as a Korean herbal medicine and its seeds were purchased from a construction company located in Yangnyeong-si, Jung-gu, Daegu.

2. Preparation of Sample

The preparation of the extract of Doco Mari was roughly pulverized 400g of the fruit, put in 10L of water, boiled and concentrated to the required amount while repeating the hot water extraction three times to obtain the final 2,000ml. The hot water extract was concentrated with an evaporator (EYELA Co. Japan), and these concentrated samples were dissolved in an ether extraction solvent, an organic solvent, filtered through a filter paper, and then the xanthostrumarin glycoside, an insoluble yellow substance known as a resin component and a toxic component, was removed. It was used as an antimicrobial test sample and the final 2.04 g was obtained. Also, the extract of the lizard used in this experiment was dissolved in methanol (MeOH) to prepare a sample.

3. Test strain and medium used

In order to examine the antimicrobial effect of the extract, the test strain used in this experiment was Al. faecalis subsp. faecalis KCCM 21180, Al. faecalis subsp. faecalis KCCM 40078, E. coli ATCC 25922, E. coli KCCM 11591, P. vulgais KCTC 2512, P. aeruginosa KCCM 11803, P. fluorescens KCCM 40223, S. aureus subsp. aureus KCCM 40050, E. aerogenes KCCM 11783, B. subtilis KCCM 12148 were selected and used as strains for eye disease-inducing bacteria among these test strains, P. aeruginosa KCCM 11803, P. fluorescens KCCM 40223, S. aureus KCCM 40050. The growth medium of the test strain was LB agar (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5%, pH 7.0 ± 0.2) and YM agar (0.3% bacto yeast extract, 0.3% bacto malt extract, 0.5 % bacto peptone, 1% bacto dextrose, 1.5% agar, pH 6.2 ± 0.2), and P. vulgais KCTC 2512 was used as YM agar, and the other test strains were LB agar.

4. Antibacterial activity measurement

The antimicrobial activity of ether extracts from Docomari on the general bacteria and eye diseases was measured by the agar diffusion method. That is, the test strain cultured in a petri dish was taken with a medium of 0.5 mm square and inoculated onto a PDA plate medium, and then incubated for 24 hours, and then, methanol (MeOH) and docomari ether extract 1000, which are control substances, were placed around the test strain. ㎍ / 20µl concentration, 500µg / 20µl concentration, 300µg / 20µl concentration, 1000µg / 20µl chitosan dissolved in 0.1N HCl as a comparative sample, 500µg / 10µl dokomari ether extract Samples prepared by mixing the concentration of 500 µg / 10 µl of chitosan were dispensed into paper discs (Ø; 6 mm, Whatman Co.), respectively. Each paper disc was dried and placed on each test strain, and then incubated at 30 ° C. for 48 hours. The antimicrobial activity was determined as the presence of clear zone around the paper disc and the size of inhibitory zone (clear zone). saw.

5. Isolation of Antibacterial Substances by TLC

The antimicrobial material was separated by thin-layer chromatography (TLC). In other words, spotted docomari ether extract (50µg / µl) 1 on TLC (Silica gel 60F254, Merck) plate, developed by the synergistic method at room temperature, dried, and then irradiated at 254nm UV wavelength for analysis. Methylene chloride and methanol were mixed at a volume ratio of 94: 6 as the developing solvent.

Experimental Example 4

We investigated the antimicrobial activity of the extract of Docomari on the isolates from patients with eye diseases. The antimicrobial activity of Docomari extracts against general bacteria and eye disease bacteria was examined by the same materials and methods.

1. Preparation of Sample

The preparation of the extract of Doco Mari was roughly pulverized 400g of fruit, put in 10L of water, boiled and concentrated to the required amount, and repeated hot water extraction three times. The hot water extract was concentrated with an evaporator (EYELA Co. Japan) and fractionally extracted with ether, an organic solvent. Further, the ether ether extract was dissolved in methanol (MeOH), and then prepared to be 1 mg / 20 μl.

2. Test strain and medium used

Pseudomonas aeruginosa KCTC 1637, Pseudomonas aeruginosa KCTC 2004, Pseudomonas aeruginosa KCTC 2513, Pseudomonas aeruginosa KCTC 2641 were used as test strains. In addition, among the 15 eye sample samples obtained by directly visiting the ophthalmic clinic and separating directly from the lacrimal fluid of the eye disease patient, 4 strains and Patient No. Nine strains were used as test strains of this experiment.

The reason why P. aeruginosa was selected as a test strain was the most commonly reported cause of side effects of eye, and the cause and side effects of various eye diseases have been studied. Because it is being reported.

In the growth medium of the test strain, P. aeruginosa strain was LB agar (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5%, pH 7.0 ± 0.2), Patient No. 4 strains and Patient No. For 9 strains, Nutrient agar (beef extract 3.0g, peptone 5.0g, agar 15.0g, distilled water 1.0L) was used. In addition, the concentration of the ether ether extract was prepared to be 1000 ㎍ / 20 ㎕, and then the amount of 20 μl, 40 μl, 60 μl, 80 μl, 100 μl, 120 μl on a paper disc (f; 6 mm, Whatman, Japan). And 140 µl each were dispensed. After the paper disc was completely dried, the plate was placed on a plate prepared in advance, and then cultured at 30 ° C. and 37 ° C. for 2 days, and the antimicrobial activity of docomari ether extract against eye disease-inducing bacteria was observed.

We examined the degree of eye irritation of the natural preservatives using the extract of D. koji. The degree of eye irritation was evaluated using the same materials and methods as those of the synthetic preservative using the multipurpose solution and the preservative solution of contact lenses.

3. Experimental Animal

Group 1 (8 birds) to be administered for 4 weeks after adapting to the laboratory environment with sufficient supply of feed [Purina Korea, Seoul] and water to mature mature New Zealand White Rabbit (Yonam University, Cheonan). Two groups (8 animals) to be administered for 2 weeks were divided and a total of 16 animals were used.

4. Experimental substance

600 g of the larvae were extracted with hot water (30 L), concentrated to the final 200 ml, and dialyzed with Dialysis tubing (cellulose membrane, D-9527, Sigma) using water to prepare a sample. Concentrations of 350 / ml, 1mg / ml, and 5mg / ml were used as samples, and their concentrations were obtained through MIC (minimum inhibition concentration) test.

The concentration of 350 ㎍ / ml of the extract of the larvae was obtained from the MIC test through liquid culture using Bacillus subtilis KCCM 12148 as a test strain. subtilis KCCM 12148 is the minimum concentration that forms a distinct clear zone. In addition, the concentration of 5 mg / ml of the docomari extract is the highest concentration used in the MIC test through liquid culture.

5. Separation of experimental group

Three concentrations of 350 μg / ml, 1 mg / ml, and 5 mg / ml were used for ocular mucosal irritation in rabbits. Rabbit eyes were divided into 4 weeks and 2 weeks, and 350mg / ml, 1mg / ml, and 5mg / ml concentrations of Docomari extract were added to both 2 and 4 weeks.

6. Administration of test substance

One drop (about 0.04 ml) of docomari extract (350 μg / ml, 1 mg / ml, 5 mg / ml) was administered to the lower conjunctival sac. Each test substance was administered about 0.04ml / times for 4 times / day, and in the case of 5mg / ml concentration of Docomari extract, the group that administered about 0.04ml / times for 2 times / day was added. Individuals who were washed were washed with 50 ml of physiological saline solution (Sino-Pharma, Korea) 20-30 seconds after administration.

One). Microbial Contamination Patterns of Soft Contact Lenses over Time

end. Staphylococcus aureus Soft contact lens contamination by

After scanning electron microscopy, one soft contact lens was immersed in 5 ml of Staphylococcus aureus suspension containing 1.5 × 10 ⁸ CFU / ml and examined for contamination of soft contact lenses over time. Although the surface of the contact lens was clean as shown in FIG. 5, one or two S. aureus colonies were adhered to the surface of the soft contact lens after 1 hour of immersion of the lens (FIG. 3). In addition, two S. aureus colony counts increased by 10 times compared to the S. aureus colony counts after 1 hour, and three or four S. aureus colonies were clustered and formed on the surface of the lens after 2 hours. Was observed (FIG. 4). The lens surface after 4 hours, about twice as compared to the lens surface after two hours there was observed a little change (Fig. 5), in the lens surface after 6 hours when compared with the lens surface after 2 hours S. aureus It was observed that the colony coefficient was increased, and amorphous materials were adhered to the lens surface, and the size was much increased compared to the lens surface after 2 hours (FIG. 6). After 12 hours of immersion of the lens, six to seven S. aureus colonies were clustered together to form a grape-like shape and more amorphous materials were formed (FIG. 7). After 24 hours of lens immersion, 9 or 10 S. aureus colonies clustered on the surface of the lens, forming larger clusters of grapes than the lens surface after 12 hours. Larger and more produced can be observed (FIG. 8).

I. Pseudomonas aeruginosa Soft contact lens contamination by

Scanning electron microscopy of lens contamination over time revealed no Pseudomonas aeruginosa on the surface of the soft contact lens after 1 hour of immersion of the lens, and almost also on the surface of the lens after 2 hours, 4 hours, and 6 hours. There was no. A single P. aeruginosa colony was observed on the lens surface after 12 hours of immersion of the lens (Fig. 9). On the lens surface after 24 hours, P. aeruginosa was about 10 times as large as the lens surface after 12 hours. It could be seen that it was produced, and also that many amorphous materials were adhered to the lens surface (FIG. 10).

All. Enterobacter aerogenes Soft contact lens contamination by

After contaminating the soft contact lens in the same way as S. aureus and P. aeruginosa , the surface of the lens was examined by scanning electron microscopy, and E. aerogenes was found on the surface of the soft contact lens 2 hours after the lens was immersed (Fig. 11) The adhesion of E. aerogenes was observed on the lens surface after 6 hours, but no significant change was observed when compared with the surface of the soft contact lens after 2 hours (Fig. 12). 24 hours after the lens was immersed in the lens surface it can be observed that a large amount of amorphous material adhered to the lens surface (Fig. 3).

2). Microbial Contamination after 4 Hours of Soft Contact Lens Wear

In order to examine the contamination of the lens when the soft contact lens was worn for 4 hours, each lens was inoculated onto LB agar and YM agar plates, incubated at 37 ° C. for 3 days, and then grown in the contact area of the lens. Since only counts as an effective colony, the microbial contamination of soft contact lenses was examined. As a result, a large number of bacteria and fungi were observed, and the count of colonies also varied. Bacteria were classified into single species, two species, and three species by judging the color and colony of bacteria, and the degree of bacterial adhesion after 4 hours of lens wearing showed various differences depending on the individual. The maximum number of single colonies is 39 colonies, the second type is 35 colonies, the third type is 37 colonies with the highest number, and the lowest number is single colony with 2 colonies, and the second species with 2 colonies, 3 The species appeared to be 18 colonies each (Table 6).

The degree of lens adhesion of fungi differed significantly from person to person as well as bacteria. The highest value was 18 colonies and the lowest was 1 colony, respectively.

As a result of comparing and analyzing the colony coefficients of bacteria and fungi attached to contaminated lenses in each individual, it was found that 57% (12 persons) had superior or slightly superior fungi to fungi, and almost similar. 29% (6 persons) and three colonies had lower colony counts than fungi (14%).

(Table 6) Comparative analysis of colony counts of bacteria and fungi adhered to individual contaminated lenses.

No Bacteria Fungus One 25 (Single types) One 2 Two types 2 3 Two types One 4 13 (Two types) One 5 2 (Single types) One 6 12 (Single types) 6 7 4 (Single types) 4 8 9 (Two types) 5 9 Two types 2 10 Two types One 11 39 (Single types) 16 12 20 (Single types) 13 13 18 (Three types) 9 14 9 (Two types) 3 15 Two types 18 16 Two types 6 17 35 (Two types) 8 18 28 (Two types) 15 19 9 (Two types) 11 20 37 (Three types) 13 21 28 (Two types) 11

3). Antibacterial Activity of Contact Lens Solution for General Bacteria and Eye Disease

Antimicrobial activity of 6 commercially available products was measured to determine whether the commercially available contact lens solution had antibacterial effects against general bacteria and eye disease bacteria. As a result, six commercial products (A, C, D, F, G, H) did not show antimicrobial activity against all the strains, but only two products (B, E) used as preservatives were used. In the case of dispensing on the paper disc without neutralization treatment, a clear clear zone was formed to show antibacterial activity (Table 7), showing the strongest antibacterial activity against E. coli KCCM 11591 (FIG. 14). Inhibitor zone of B product was 34mm for E. coli KCCM 11591 strain, and E product formed 29mm inhibitory zone, respectively (Table 9). However, when two products used as preservatives were neutralized by the method suggested in the manual, they did not show antimicrobial activity against all the public strains. Considering that these products must be neutralized. The antimicrobial activity of these may be little.

Table 7 Results of Antimicrobial Activity for Commercial Contact Lens Care Solution Products

Inhibitory zone (Ø, mm)

                    Test articles Strains B E P. vulgaris KCTC 2512 E. aerogenes KCCM 11783 S. aureus KCCM 4005050 P .. aeruginosa KCCM 11803 P. fluorescens KCCM 40223 P. stutzeri KCCM 12540 Al. faecalis KCCM 21180 E. coli ATCC 25922 E. Coli KCCM 11591 B. subtilis KCCM 12148 13.5 18.0 17.0 14.0 16.0 29.5 23.5 19.5 34.0 16.0 15.5 18.0 14.5 13.5 15.5 27.5 22.5 18.5 29.0 15.5

4). Eye irritation test of synthetic preservatives using multipurpose solution and preservative solution of contact lens

end. Epithelial and endothelium observation in rabbits administered A product (4 times / day, non-washed group)

A product is a multi-purpose contact lens solution that can be washed, rinsed, disinfected, and stored. It is recommended that the solution not be applied directly to the eyes, but if used directly on the eyes, one drop of multipurpose solution may get into the eyes.

A test product was applied to the rabbit 4 times a day and the condition of the rabbit was observed. The results of slit lamp microscopy and mirror microscopy were summarized in Table 8, and the shapes of each were listed in FIGS. 15 to 19. . Corneal epithelial damage occurred locally at 1 week after instillation of the test substance A (Fig. 15), and damage and irritation were accompanied by corneal and conjunctiva at 2 weeks after instillation (Figs. 15 and 17). Overall corneal epithelial injury and significant conjunctival irritation were observed (FIGS. 18 and 19). Mirror microscopic examination of corneal endothelial cells in rabbits showed an increase in endothelial cells, but this was due to the deviation between the tests, which could be analyzed statistically as the population size increased and at least did not cause significant damage to the endothelial cells. have.

(Table 8) Microscopic examination result table in which topical A product, which is a test substance, was observed in rabbits four times a day and observed the state of rabbits

                                            Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 2695 0 0 Treatment one week One 0 2 2673 0 0 Treatment two weeks One 0 2 2932 0 1 + 1 + 0 Treatment four weeks One 0 4 3012 0 2 + 1 + 0

Corneal epithelial examination by light microscopy showed that corneal epithelium, parenchyma, Descemet's membrane and endothelial cells were relatively well preserved (FIG. 20).

Transmission electron microscopy of the corneal cornea after treatment with product A revealed slight nuclear condensation and mitochondrial edema at a magnification of 10,000, but the junctional complexes including tight junctions were well preserved and the cell morphology was relatively well maintained. Figure 21).

I. Epithelial and Endothelial Observation in Rabbits Administered A Product (4 times / day, Washing Group)

A product was applied to the rabbit 4 times a day and washed, and then the progress was observed. The results of slit lamp microscopy and mirror microscopy were summarized in Table 9, and the shapes of each were listed in FIGS. 22 to 25. Corneal epithelial damage occurred locally at 1 week after the administration of A, a test substance (Fig. 22), and the damage and irritation of the cornea and conjunctiva were accompanied by weaker than non-washed subjects from 2 weeks after the eyedrops. 23) At 4 weeks of instillation, corneal epithelial damage was seen in two quadrants, and weak conjunctival irritation was observed (FIGS. 24 and 25).

In the microscopic examination of corneal endothelial cells, the endothelial cells did not change significantly.

(Table 9) Table A of the inspection result of microscopic observation after injecting A product into the rabbit four times a day and washing.

                                               Group: washed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 2824 0 0 Treatment one week One 0 One 2739 0 0 Treatment two weeks One 0 One 2816 0 1 + 0 + 0 Treatment four weeks One 0 2 2732 0 1 + 0 + 0

The results of optical microscopy showed that the endothelial cells were not visible at the cutting plane because the embeding was oblique at 40 magnification, but corneal epithelium and parenchyma were relatively well preserved. The preserved state was shown (FIGS. 26 and 27).

All. Epithelial and Endothelial Observation in Rabbits Treated with Neutralized B Product (4 times / day, non-washed group)

It is recommended that product B be neutralized as a preservative, and the neutralized solution was used as a test substance in this experiment after a certain period of time using a neutralizing tablet as specified in the manufacturer's instructions.

B was used as a test substance in the rabbit 4 times a day and observed the state, and the results of slit lamp microscopy and mirror microscopy are summarized in Table 10, each type is listed in Figures 28 to 32. No damage was observed on corneal epithelium at 1 week after instillation of the test substance B (Figs. 28 and 29), and from 2 weeks of instillation, slight corneal damage and conjunctival irritation were observed (Fig. 30). In general, weak corneal epithelial damage was observed, and conjunctival stimulation was weakly observed (FIGS. 31 and 32).

(Table 10) Microscopic examination result table after injecting B product, a test substance, into the rabbit four times a day

                                            Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 2747 0 0 Treatment one week 0 0 0 2724 0 0 Treatment two weeks One 0 2 2801 0 1 + 0 + 0 Treatment four weeks One 0 4 2701 0 1 + 0 + 0

Mirror microscopic examination of corneal endothelial cells showed no significant changes in endothelial cells.

In the examination by optical microscope, corneal epithelium, parenchyma, Descemet's membrane and endothelial cells were relatively well preserved (FIGS. 33 and 34).

Transmission electron microscopy showed epithelial cells of varying sizes at 2,000 magnifications, which appear to vary with the angle of the cut plane. At 12,000 magnification, well-preserved tight junctions and desmosomes were observed and the cell morphology was relatively well maintained (FIGS. 35 and 36).

la. Epithelial and Endothelial Observation in Rabbits Treated with Neutralized B Product (4 times / day, Washing Group)

After applying B product as a test substance to the rabbit 4 times a day and washing, the progress was observed. The results of slit lamp microscopy and mirror microscopy are summarized in Table 11, and the shapes of each of them are shown in FIGS. 37 to 40. Enumerated. Corneal epithelial damage was not observed at 1 week after instillation of the test substance B (Fig. 37). There was corneal epithelial injury in two quadrants, but no conjunctival stimulation was observed (FIG. 39, 40).

(Table 11) Microscopic examination result table after eyedropping B product as a test substance 4 times a day and washing

                                            Group: washed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 2865 0 0 Treatment one week 0 0 0 2659 0 0 Treatment two weeks One 0 One 2949 0 0 Treatment four weeks One 0 2 2808 0 0

In the microscopic examination of corneal endothelial cells, the endothelial cells did not change significantly.

As a result of examination under an optical microscope, corneal epithelium and parenchyma, Descemet's membrane and endothelial cells were relatively well preserved at both the magnification 40 and the magnification 100 (Figs. 41 and 42).

hemp. Epithelial and Endothelial Observation in Rabbits Treated with Neutralized C Product (4 times / day, non-washed group)

The C product can be disinfected and preserved as a contact lens preservative, and it is recommended to be used by neutralization treatment, and it was used as a test substance of this experiment by neutralization treatment as indicated in the manual. After a certain period of time using a platinum catalyst disk, it is recommended to rinse the lens and wear it on the eyes, and not to use it directly on the eyes.

C product, a neutralized test substance, was applied to the rabbit four times a day to observe the condition. The results of the slit lamp microscope test and the results of the mirror test microscope are summarized in Table 12, and the respective forms are listed in FIGS. 43 to 48. Local eye corneal epithelial damage was observed at 1 week after the administration of the neutralized C product, which was a test substance, and accompanied by mild conjunctival irritation (FIGS. 43 and 44). In the second week, the corneal epithelium was slightly damaged (Fig. 45, 46), similar to the first week (Fig. 45, 46). Observed (FIG. 47, 48). In the microscopic examination of corneal endothelial cells in rabbits, the endothelial cells appeared to be reduced, but no statistical methods were used to obtain meaningful conclusions.

Table 12 shows the results of the microscopic examination of the neutralized experimental substance C, which was applied to rabbits four times a day.

Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 2849 0 0 Treatment one week One 0 One 2732 0 1 + 0 + 0 Treatment two weeks One 0 One 2645 0 1 + 0 + 0 Treatment four weeks One 0 3 2624 0 1 + 0 + 0

Corneal epithelium, parenchyma, Descemet's membrane and endothelial cells were relatively well preserved by optical microscopy (FIGS. 49 and 50).

Transmission electron microscopy showed that the tight junctions and desmosomes were well preserved at magnification of 5,000, and the cell morphology was relatively well maintained. At a magnification of 10,000, the form of intracellular mitochondria was found to be relatively clear (FIGS. 51 and 52).

bar. Epithelial and Endothelial Observation in Rabbits Treated with Neutralized C Products (4 times / day, Washing Group)

After neutralizing the C product using platinum catalyst disk, the neutralized C product was applied to the rabbit four times a day and washed, and then the condition was observed.The result of slit lamp microscopy and mirror microscopy Table 15 summarizes the results, and each form is listed in FIGS. 53 to 58. Local topical corneal epithelial injury and mild conjunctival irritation occurred 1 week after instillation of the test substance, C, into rabbits (FIGS. 53 and 54). After instillation of C-products in rabbits, the cornea and conjunctiva of the cornea and conjunctiva were similar to the first week (Figs. 55 and 56). Weak conjunctival stimulation was observed (FIG. 57, 58).

Table 15 Results of eye irritant test of article C after neutralization four times per day

                                               Group: washed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 2832 0 0 Treatment one week One 0 One 2747 0 1 + 0 + 0 Treatment two weeks One 0 One 2688 0 1 + 0 + 0 Treatment four weeks One 0 3 2732 0 1 + 0 + 0

In the microscopic examination of corneal endothelial cells in rabbits, the endothelial cells did not change significantly.

As a result of optical microscopic examination, corneal epithelium, parenchyma, descemet and endothelial cells in rabbits were relatively well preserved at 40 and 100 magnification (Figs. 59 and 60).

four. Epithelial and Endothelial Observation in Rabbits Treated with Unneutralized C Products (4 times / day, non-washed group)

C products can be disinfected and preserved as a preservative, and after a certain period of time using a platinum catalyst disc, the lens is rinsed with physiological saline and recommended to be worn on the eyes. In addition, it is recommended not to use the solution directly on the eyes, but if the lens is worn directly on the eyes due to the wrong explanation of the seller or the carelessness of the user, about 1 drop of preservative may get into the eyes. In this experiment, assuming such a case, the test material that was not neutralized was directly administered into the rabbit.

The C product, a test substance, was applied to the rabbit four times a day and the condition was observed. As a result, in one animal, severe corneal damage was observed on the first week of administration and on the third day of administration, and further administration of the test substance was stopped and observation was continued for two to four weeks.

The results of slit lamp microscopy and spectroscopic microscopy are summarized in Table 16. Each type is listed in FIGS. 61 to 66. Corneal epithelial damage was observed from 1 week after instillation of untreated neutralized C product into rabbits, and moderate conjunctival irritation was observed (Fig. 61, 62). Conjunctival stimulation was accompanied (FIG. 63, 64), and severe corneal epithelial injury and moderate corneal opacity were seen at 4 weeks, and severe conjunctival stimulation was observed (FIG. 65, 66). Mirror microscopic examination of corneal endothelial cells in rabbits was not possible from 1 week of administration.

Table 16 Results of eye irritant test of article C before neutralization four times per day

                                               Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 3236 0 0 Treatment one week One 0 2 uncheckable 0 1 + 1 + 1 Treatment two weeks One One 4 uncheckable 0 2 + 2 + 2 Treatment four weeks 2 2 4 uncheckable 0 2 + 2 + 3

In the light microscopic examination, corneal edema in the rabbit, loss of epithelium and endothelium, parenchyma, and detachment of the descemet's membrane were observed, indicating that there was severe damage (FIGS. 67 and 68).

Ah. Epithelial and Endothelial Observation in Rabbits Treated with Unneutralized C Products (4 times / day, Washing Group)

The unneutralized C product was applied to the rabbit four times a day, washed, and then observed. The result showed that the cornea and conjunctiva in the rabbit were severe in the washed group, as in the non-washed group after one week. Injuries were accompanied by discontinuation of further administration of the test material and continued observation until 2 and 4 weeks.

 The results of the slit lamp microscope test and the results of the mirror microscope test are summarized in Table 17, and the shapes of each are listed in FIGS. 69 to 74. Corneal epithelial damage occurred in two quadrants at 1 week after instillation of untreated neutralized C product into rabbits, and conjunctival irritation was observed (FIGS. 69 and 70). At 2 weeks, the cornea and conjunctiva were accompanied by general damage and irritation (Figs. 71 and 72). Observed (Fig. 73, 74).

Table 17: Results of eye irritant test of article C before neutralization four times per day Group: washed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 2958 0 0 Treatment one week One 0 2 2890 0 1 + 0 + 1 Treatment two weeks One 0 4 3095 0 1 + 1 + 1 Treatment four weeks One One 4 2958 0 1 + 2 + 2

In the microscopic examination of corneal endothelial cells in rabbits, the endothelial cells did not change significantly.

The cornea in rabbits was examined by optical microscopy, and as a result of the loss of epithelial and endothelial cells at 2 weeks of observation, the descemet's membrane was detached (Figs. 75 and 76). Was lost (FIG. 77, FIG. 78).

5). Antimicrobial Activity of the Extracts from Kodo-Tak against General Bacteria and Eye Disease

end. Antimicrobial Activity of the Extracts from Kodo-Tak against General Bacteria and Eye Disease

The antimicrobial effect of Xanthium strumarium out the ether extract of the bacteria and disease germs can not size a (mm) of the paper disc and generates or without inhibitory zone (clear zone) of the clear zone around the results, B. subtilis KCCM 12148 against the strain It showed strong antimicrobial activity, S. aureus KCCM 40050, Al. faecalis KCCM 21180, P. aeruginosa KCCM 11803, Al. faecalis KCCM 40078 and P. fluorescens KCCM 40223 strains showed antimicrobial activity, respectively.

Antimicrobial activity of B. subtilis KCCM 12148 strain was 13.0 mm in clear zone of 1000μg / 20μl of Docomari extract and 11.0mm of clear zone of 500μg / 20μl of Docomari extract. Further, the eye disease causing bacteria, S. aureus KCCM 40050, P. aeruginosa KCCM 11803, P. fluorescens KCCM eotneunde exhibit antimicrobial activity about 40 223 strains of Xanthium strumarium extract 1000㎍ / 20㎕ concentration for S. aureus KCCM 40050 clear The zone was 11.5 mm, clear zone with 8.0 μg of Docomari extract 500 μg / 20 μl was 8.0 mm, and clear zone with 1000 μg / 20 μl of Docoe extract for P. aeruginosa KCCM 11803 strain was 9.0 mm, P. fluorescens Clear zones of 1000 μg / 20 μl concentration of Docomari extract against KCCM 40223 strain exhibited antimicrobial activity of 7.0 mm, respectively.

I. Confirmation of antimicrobial substance by TLC

Eight different bands were identified as a result of spreading the ether extract on the TLC plate. Each band was recovered, dissolved in methanol, and concentrated under reduced pressure to confirm the antimicrobial activity. As a result, Rf values (development rate values in the range of 0 to 1) showed antimicrobial activity only in bands of 0.87 and 0.74, and Rf value of 0.87 The band showed strong antimicrobial activity and the band of Rf value of 0.74 showed antimicrobial activity, but the antimicrobial activity was relatively low (FIG. 79).

All. In solid medium B. subtilis  MIC test for KCCM 12148 strain

MIC test results of docomari ether extract against B. subtilis KCCM 12148 strain in LB agar (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5%, pH 7.0 ± 0.2), 100 μg / 20 extract At concentrations below ㎕, only traces of the clear zone were formed (FIG. 80). However, clear zones began to form at 200 μg / 20 μl concentration of Docomari extract, and clear inhibitory zones were observed at concentrations of 500 μg / 20 μl or more (FIG. 81).

As a result of the MIC test of the B. subtilis KCCM 12148 strain in the solid medium, inhibitory zone was formed when the concentration of the extract was 200 μg / 20 μl.

la. In liquid medium B. subtilis  MIC test for KCCM 12148 strain

MIC test results of Docomari ether extract against B. subtilis KCCM 12148 strain in LB agar (1% tryptone, 0.5% yeast extract, 1% sodium chloride, 1.5%, pH 7.0 ± 0.2) at 350 μg / ml Growth was 0 and inhibition was 100%. At 100 ㎍ / ml concentration, growth was 1.50 and inhibition was 17%. At 200 ㎍ / ml concentration, growth was 1.40, inhibition was 23%, and growth was 0.90 and inhibition 50%, respectively. Table 18.

Table 18 Results of MIC test on B. subtilis KCCM 12148 at LB broth

Concentration (/ ml) Growth (OD660nm) Inhibition (%) Control 1.81 10 1.79 One 20 1.76 2 30 1.75 3 50 1.54 15 80 1.52 15 100 1.50 17 200 1.40 23 300 0.90 50 350 0 100 400 0 100 450 0 100 500 0 100 800 0 100 1,000 0 100

As a result of the MIC test in the above liquid medium, it was found that when the concentration of the docomari extract was 350 / ml, no further bacterial growth was achieved.

6). Antimicrobial Activity of the Extracts of Kodo (Taxella japonica) Extract on Eye Disease Bacteria

P. aeruginosa KCTC 1637, P. aeruginosa KCTC 2004, P. aeruginosa KCTC 2513, and P. aeruginosa KCTC 2641 strains were used to examine the antimicrobial effect of Docomari ether extract against ocular disease bacteria. In addition, the patient No. 4 strains and Patient No. Nine strains were used as test strains of this experiment.

As a result, 1000 mg / 20 μl of Docomari ether extract formed a 13 mm diameter clear zone for P. aeruginosa KCTC 2004 and P. aeruginosa KCTC 2641 strains. 4 strains, 7.0 mm, Patient No. In 9 strains, a clear clear zone having a diameter of 10.0 mm was formed (FIG. 82).

In the case of dispensing the paper disc by varying the amount of the docomari ether extract 1000㎍ / 20μl, it was found that a larger and clear clear zone is formed as the amount of dokomari ether extract increases (Fig. 83, Table 19).

Table 19: Antibacterial activity of Xanthium strumarium L. extract

                                         Inhibitory zone (f, mm)

                1000/20 Strain 20 40 60 80 100 120 140 Patient No. 9 10.0 15.5 17.0 18.5 19.5 20.0 20.5

These results suggest that Docomari ether extract is the most commonly reported cause of eye side effects, and also to P. aeruginosa , an eye disease-inducing bacterium that has been reported to cause side effects of contact lens wear. It has been shown to have strong antibacterial activity against strains isolated from patients with eye diseases, while also having antibacterial activity.

7). Ocular Membrane Stimulation Test of Natural Preservatives Using Extracts

end. Epithelial and Endothelial Observation in Rabbits Administered with Rabbit Extract (350 / ml) (4 times / day, non-washed group)

To investigate the stability of the extract, the degree of eye irritation in rabbits was measured by injecting docomari extracts into rabbits.

That is, 350 mg / ml of Docomari extract was applied to rabbits four times a day, and the state was observed. The results of slit lamp microscopy and mirror microscopy were summarized in Table 20. Enumerated. Corneal epithelial damage was not observed from 1 week to 4 weeks after instillation of 350 μg / ml of Docomari extract, a test substance (Figs. 84 to 86), and mirror microscopic examination of corneal endothelial cells showed different changes of endothelial cells. I couldn't see it.

Table 20 Results of eye irritant test of 350 μg / ml Xanthium strumarium L.

         four times per day

                                            Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 3184 0 0 Treatment one week 0 0 0 3003 0 0 Treatment two weeks 0 0 0 2849 0 0 Treatment four weeks 0 0 0 3012 0 0

Corneal epithelium, parenchyma, Descemet's membrane and endothelial cells were relatively well preserved after the injection of 350 g / ml of docomari extract into the rabbit 4 times a day, 2 weeks, and 4 weeks. 86, 87).

I. Epithelial and Endothelial Observation in Rabbits Treated with Docomari Extract (350 ㎍ / ml) (4 times / day, Washing Group)

After rinsing 350 μg / ml concentration of Dokomari extract into the rabbit four times a day and washing, the condition was observed. The results of slit lamp microscopy and mirror microscopy were summarized in Table 21. Listed up to 90. Corneal epithelial damage was not observed from 1 to 4 weeks after instillation of docomari extract, a test substance in rabbits (Figs. 88 to 90), and mirror microscopic examination of corneal endothelial cells showed different changes in endothelial cells. There was no.

Table 21. Results of eye irritant test of 350 μg / ml Xanthium strumarium L.

four times per day

                                        Group: washed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 3322 0 0 Treatment one week 0 0 0 3344 0 0 Treatment two weeks 0 0 0 3355 0 0 Treatment four weeks 0 0 0 3472 0 0

Moreover, as a result of rabbit eye examination by optical microscope, corneal epithelium, parenchyma, descemet and endothelial cells of rabbit eye were relatively well preserved (Figs. 91 and 92).

All. Epithelial and Endothelial Observation in Rabbits Treated with Docomari Extract (1mg / ml) (4 times / day, non-washed group)

1 mg / ml Dokomari extract was applied to rabbits four times a day, and the condition was observed. The results of slit lamp microscopy and mirror microscopy were summarized in Table 22, and the forms of each were listed in FIGS. 93 to 95. . No damage was observed in corneal epithelium from 1 to 4 weeks after instillation of docomari extract, a test substance in rabbits (Figs. 93-95), and mild conjunctival irritation was observed at 4 weeks.

Table 22. Results of eye irritant test of 1 mg / ml Xanthium strumarium L.

        four times per day

                                       Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 3076 0 0 Treatment one week 0 0 0 2659 0 0 Treatment two weeks 0 0 0 2906 0 0 Treatment four weeks 0 0 0 3086 0 1 + 0 + 1

Mirror microscopic examination of corneal endothelial cells in rabbits showed no significant changes in endothelial cells. Moreover, corneal epithelium, parenchyma, Descemet's membrane and endothelial cells in rabbits were relatively well preserved by optical microscopy (Figs. 96 and 97).

After spotting Docomari extract (1mg / ml) four times a day in rabbits, the rabbits were examined by transmission electron microscopy, which showed well-arranged wing cells and relatively well preserved tight junctions and desmosomes at 8,000 magnification. It was found that the morphology of the cells was relatively well maintained (FIG. 98).

la. Epithelial and Endothelial Observation in Rabbits Treated with Docomari Extract (1mg / ml) (4 times / day, washing group)

Docomari extract 1mg / ml concentration was applied to the rabbit 4 times a day and washed, and then the state was observed. The results of slit lamp microscopy and mirror microscopy are summarized in Table 23. Each type is shown in Figs. Enumerated.

Table 23. Results of eye irritant test of 1 mg / ml Xanthium strumarium L.

           four times per day

                                            Group: washed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 3067 0 0 Treatment one week 0 0 0 2976 0 0 Treatment two weeks 0 0 0 2732 0 1 + 0 + 0 Treatment four weeks 0 0 0 2976 0 0

No damage was observed in corneal epithelium from 1 week to 4 weeks after topical docomari extract in rabbits (Figs. 99-101), but there was a slight conjunctival irrigation at 2 weeks, but it disappeared.

Mirror microscopic examination of corneal endothelial cells in rabbits showed no significant changes in endothelial cells.

In the examination by optical microscope, corneal epithelium, parenchyma, Descemet's membrane and endothelial cells in rabbits were preserved relatively well (Figs. 102 and 103).

hemp. Epithelial and Endothelial Observation in Rabbits Treated with Docomari Extract (5mg / ml) (2 times / day, non-washed group)

Docomari extract 5mg / ml concentration twice a day in the rabbit eye was observed to observe the state, the results of slit lamp microscopy and mirror microscopy are summarized in Table 24, each form is listed in Figure 104 ~ 106. No damage was observed on corneal epithelium until 2 weeks after the administration of docomari extract, which was a test substance, but mild corneal epithelial damage was observed at 3 weeks after instillation and mild corneal epithelial damage at 4 weeks after instillation.

Table 24. Results of eye irritant test of 5 mg / ml Xanthium strumarium L.

           two times per day

                                        Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 2739 0 0 Treatment one week 0 0 0 2994 0 0 Treatment two weeks 0 0 0 3236 0 1 + 0 + 0 Treatment four weeks One 0 One 2898 0 1 + 0 + 0

Mirror microscopic examination of corneal endothelial cells in rabbits showed no significant changes in endothelial cells.

Moreover, corneal epithelium, parenchyma, Descemet's membrane and endothelial cells in rabbits were relatively well preserved by optical microscopy (FIGS. 107 and 108).

bar. Epithelial and Endothelial Observation in Rabbits Treated with Docomari Extract (5mg / ml) (Twice / day, Washing Group)

Docomari extract 5mg / ml concentration in the rabbit eye drops twice a day and washed and observed the state, the results of slit lamp microscopy and mirror microscopy results are summarized in Table 25, each form is shown in Figure 109 ~ 111 Enumerated. No damage was observed in corneal epithelium from 1 week to 3 weeks after instillation of the testosterone extract, but mild corneal epithelial damage and conjunctival irritation were observed at 3 weeks after instillation. FIG. 111), the mirror microscopic examination of corneal endothelial cells showed no significant change of endothelial cells.

Table 25 Results of eye irritant test of 5 mg / ml Xanthium strumarium L. two times per day

                                     Group: washed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 3154 0 0 Treatment one week 0 0 0 2849 0 0 Treatment two weeks 0 0 0 2610 0 1 + 0 + 0 Treatment four weeks One 0 One 2688 0 1 + 0 + 0

Moreover, corneal epithelial, parenchymal, descemetal and endothelial cells were relatively well preserved in rabbit corneal examination by optical microscope (FIGS. 112 and 113).

four. Epithelial and Endothelial Observation in Rabbits Treated with Docomari Extract (5mg / ml) (4 times / day, non-washed group)

The concentration of 5 mg / ml docomari extract was added to the rabbit four times a day and the condition was observed. The results of the slit lamp examination and the spectroscopic microscope examination are summarized in Table 26, and each form is listed in FIGS. 114 to 116. Corneal epithelial damage was not observed from 1 to 2 weeks after instillation of Docomari extract, a test substance, and mild corneal and conjunctival irritation were observed at 4 weeks after instillation (Fig. 114 to 116). Mirror microscopy showed no significant changes in endothelial cells.

Table 26.Results of eye irritant test of 5mg / ml Xanthium strumarium L. four times per day

                                    Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 3436 0 0 Treatment one week 0 0 0 3278 0 0 Treatment two weeks One 0 One 3355 0 1 + 0 + 0 Treatment four weeks One 0 3 3257 0 1 + 0 + 0

Corneal epithelial examination by optical microscope showed that corneal epithelium, parenchyma, Descemet's membrane and endothelial cells were relatively well preserved (Fig. 117, Fig. 118).

After spraying docomari extract (5mg / ml) into the rabbit four times a day, the cornea in the rabbit was examined by transmission electron microscopy, and the microvilli of the well-arranged endothelial cells and relatively well preserved tight at 7,000 magnification. Junctions and desmosomes could be seen and the cell morphology was relatively well maintained (FIGS. 119, 120).

Ah. Epithelial and Endothelial Observation in Rabbits Treated with Docomari Extract (5mg / ml) (4 times / day, washing group)

Docomari extract 5mg / ml concentration in the rabbit eye drops four times a day and washed and observed the state, the results of slit lamp microscopy and mirror microscopy results are summarized in Table 27, each form is shown in Figure 121 ~ 123 Enumerated. Corneal epithelial damage was not observed from 1 to 2 weeks after topical docomari extract in rabbits, mild conjunctival irritation at 3 weeks, and localized corneal epithelial damage in 1 quadrant at 4 weeks. Weak conjunctival stimulation was observed (FIGS. 124, 125).

Table 27: Results of eye irritant test of 5 mg / ml Xanthium strumarium L.

         four times per day

                                     Group: nonwashed group

Ocular region Corneal epithelium Corneal stroma Involved area Corneal endothelium (cell / mm2) Iris Conjunctiva Pretreatment 0 0 0 3436 0 0 Treatment one week 0 0 0 3278 0 0 Treatment two weeks One 0 One 3355 0 1 + 0 + 0 Treatment four weeks One 0 3 3257 0 1 + 0 + 0

In the microscopic examination of corneal endothelial cells in rabbits, the endothelial cells did not change significantly.

Furthermore. In the corneal examination of rabbits by light microscopy, corneal epithelium and parenchyma, Descemet's membrane and endothelial cells in rabbits were relatively well preserved (Fig. 109, Fig. 110).

In the present invention, the antimicrobial activity of the contact lens multipurpose solution and the preservative solution together with the docomari extract and the ocular mucosa stimulation test were simultaneously performed. The antimicrobial activity of the extract of the extract for the test strain was much better than that of the contact lens multipurpose solution and the preservative solution. Appeared.

In addition, as a result of eye irritation test, the contact lens multipurpose solution and the preservative solution had some corneal damage and conjunctival stimulation. Appeared.

In addition, it can be seen that the extract of the present invention, Dokmari Mari, can perform a sufficient role as a natural preservative to replace the existing synthetic preservative in contact lenses.

The present invention described above is not limited to the above-described embodiments, and various substitutions, modifications, and changes can be made without departing from the technical spirit of the present invention, and any person having ordinary skill in the art Various modifications and imitations may be made by the description of the present specification, but it is obvious that the present invention is not outside the scope of the present invention.

As described above, the present invention allows the preservative before and after use of the contact lens by using the preservative solution containing the natural extract of the extract, and by the antimicrobial activity of the extract, the antimicrobial activity for the contact lens and the conventional multi-purpose solution and the preservative solution, which are used more than the antimicrobial activity. There is an effect that can provide a natural contact lens preservative that is excellent.

In addition, as a result of eye irritation test, the contact lens multipurpose solution and the preservative solution had some corneal damage and conjunctival stimulation. It has been shown that there is no effect to provide an excellent natural contact lens preservative.

In addition, the present invention is a very useful invention that the effect that the extract of Docomari can play a sufficient role as a natural preservative to replace the existing synthetic preservative in the contact lens.

Claims (5)

A contact lens preservative containing an extract of Docomari as an active ingredient. The method according to claim 1; The contact extract is a contact lens preservative containing the extract extract as a active ingredient, characterized in that partially purified after hot water extraction. The method according to claim 1 or 2; A contact lens preservative containing the extract of Docomari as an active ingredient, characterized in that the dilution concentration of the Docomari extract by diluent is 1.0mg / ㎖ ~ 5.0mg / ㎖. The method according to claim 3; A contact lens preservative containing dicot extract of Docomari hot water extract as an active ingredient, characterized in that methanol. The method according to claim 1 or 2; A contact lens preservative containing an extract of Docomari as an active ingredient, characterized in that the Rf value of the antimicrobial activity band of the Docomari extract determined by TLC method using a methylene chloride and methanol developing solvent in a volume ratio of 94: 6 is 0.87.

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