KR20060016627A - Health food for the prevention or treatment of osteoporosis containing rat bean extract as an active ingredient - Google Patents

Abstract

The present invention relates to a health food containing a rat eye extract having an effect on osteoporosis or treatment, specifically, a mouse eye extract having an effect on preventing or treating osteoporosis by promoting expression of genes involved in bone formation in osteoblasts. It relates to a health food containing. The health food containing the rat eye extract of the present invention is not only effective in preventing or treating osteoporosis, but also contains various nutrients such as various amino acids, vitamins, and calcium, and thus may be usefully used in osteoporosis patients and the general public.

Osteoporosis, Rat Beans, Health Food

Description

Health food comprising extracts of Rhynchosia Volubilis as active ingredients for prevention or treatment of osteoporosis}             

Figure 1 shows the process of separating and purifying the rat eye extract of the present invention from the rat bean,

FIG. 2 shows that BMP1a (Bone morphogenic protein 1a), BMP2a (Bone morphogenic protein 2a), and BMP3a (Bone morphogenic protein 3a) genes, which are involved in bone formation, are promoted as a result of treating rat eye extract of the present invention. An electrophoretic photograph showing a graph and a graph analyzing the same,

Figure 3 shows that as a result of treating the rat eye extract of the present invention to osteoblasts, the expression of OTN (osteonectin), OTC (osteocalcin), COL1 (type I collagen), TG2 (transglutaminase 2) genes involved in bone formation is promoted It is an electrophoretic photograph showing and a graph analyzing it,

Figure 4 is a graph showing the results of toxicity testing at the cellular level of the rat eye extract of the present invention.

The present invention relates to a health food containing rat eye extract having an effect on the prevention or treatment of osteoporosis, specifically, a mouse eye bean having an effect on the prevention or treatment of osteoporosis by promoting the expression of genes involved in bone formation in osteoblasts. It relates to a health food containing the extract.

Osteoporosis is a condition in which bone mineralization is reduced due to the reduction of bone tissue, and thus the bone marrow cavity is widened. Bone mass is influenced by several factors, including genetics, nutrition, hormone changes, differences in exercise and lifestyle, and the causes of osteoporosis include old age, lack of exercise, low weight, smoking, low calcium diet, menopause, Ovarian ablation and the like are known. On the other hand, although there are individual differences, blacks have lower bone resorption levels than whites, resulting in higher bone mass, usually the highest in 14-18 years of age, and decreasing by about 1% per year. Especially in women, bone reduction continues after 30 years of age, and when menopause reaches bone growth rapidly due to hormonal changes. In other words, estrogen concentration rapidly decreases at the end of menopause, when a large amount of B-lymphocytes are generated, and B-cell precursors (pre-B cells) accumulate in bone marrow, which causes IL-6 As the amount increases, the activity of the osteoclasts increases, resulting in a decrease in bone mass.

As described above, osteoporosis is unavoidable for elderly people, especially postmenopausal women, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing. It is also known that there is an estimated $ 130 billion market around the world for bone disease treatment and is expected to grow further. have.

Materials currently used for the treatment of osteoporosis include estrogen, androgen anabolic steroids, calcium preparations, phosphates, fluoride preparations, ipriflavones, and vitamin D3. In 1995, Merck, USA, used aminobisphosphonate, and in 1997, Lilly Co., USA, used raloxifene, which acts as a selective estrogen receptor modulator (SERM), to osteoporosis. It was developed as a new drug.

       Estrogen, which is most widely used as a therapeutic agent for osteoporosis, has a problem of increasing the incidence of endometrial cancer and breast cancer and provoking thrombosis, cholelithiasis, hypertension, edema, breast pain, etc., in long term use. In addition, the study of post-menopausal women who co-administered estrogen and progesterone showed that there is an urgent need to develop new osteoporosis therapeutics that can be safely used for a long period of time from data indicating an increased incidence of breast cancer, stroke, and pulmonary embolism. In terms of stability, it is considered necessary to develop a natural medicine for treating osteoporosis.

Rat eye bean is also known as Seomoktae (鼠目 太), a perennial perilla of legumes, and the stem and leaves are brown. In July, yellow flowers bloom in the oval pods and harvest when black and round fruits with a diameter of 5-7 mm are formed. According to ‘Boncho River Mok’, it is better to use it as a medicine while it is black and light. According to the Chinese medical classification, kidney-related grains can be eaten when you have kidney disease. Even middle of three seconds, lowering the qi to suppress all the wind (風 熱), and let the vein is blocked. Releases all poisons, including the poison of mineral medicine, and improves blood circulation. In addition, the decoction of this water is very cold because the body is very fever, chest, stuffy, painful symptoms, and all the drugs to the effect of detoxifying. The tofu made with this is cold because of its nature. If you eat fried to warm the body, soaked in alcohol and eat it is effective in wind (風 症). When boiled or steamed beans are mixed with salt or shrimp, the medicine becomes very cold. This will help you get rid of thirst.

       The common components of rat bean are protein 42.01%, fat 19.41%, carbohydrate 21.12%, fiber 42.29%, ash 5.2%, moisture 6.63%, other amino acids such as tretoran, lysine and abundant nutrients such as vitamin B1, B2 and calcium It is an excellent food containing.

Therefore, the present inventors have selected a substance that is effective in preventing or treating osteoporosis from natural products and researched to develop it as a health food. As a result, the rat eye extract of the present invention promotes the expression of genes involved in bone formation in osteoblasts and is cytotoxic. The present invention was completed by confirming that it can be usefully used as a health food for preventing or treating osteoporosis.

An object of the present invention is to provide a health food for preventing or treating osteoporosis.

In order to achieve the above object, the present invention provides a health food for preventing or treating osteoporosis containing rat eye extract.

Hereinafter, the present invention will be described in detail.

The present invention provides a health food for the prevention or treatment of osteoporosis containing rat eye extract as an active ingredient. Rat eye extract of the present invention shows the transcriptional promoting activity of the genes involved in bone formation.

Osteoblasts are cells derived from undifferentiated mesenchymal cells. During the differentiation of these cells, progenitor cells acquire specific phenotypes under the control of individual regulatory factors. BMP (Bone morphogenic protein) is a protein that plays a key role in the differentiation of undifferentiated mesenchymal cells into osteoblasts.

In order to determine the effect of the rat eye extract of the present invention on the transcription of the BMP genes, the germ cell extracts by germination days were treated to osteoblasts pretreated with H ₂ O ₂ , and the expression of the BMP gene was observed after a certain time. As measured by RT-RCR, it can be seen that the rat eye extract of the present invention promotes the expression of the BMP gene inhibited by H ₂ O ₂ (see FIG. 2 ).

In addition, Type I collagen (COL1, type I collagen), Osteocalcin (OTC, osteocalcin), Osteonectin (OTN, osteonectin) and Transglutaminase 2 (TG2, transglutaminase2) constitute bone tissue As a protein to be treated, rat germ extract according to the germination days of the present invention was treated to osteoblasts pretreated with H ₂ O ₂ , and after a period of time, the osteotintin, osthiocalcin, type I collagen and transglutaminase genes were As a result of measuring the expression by RT-RCR, it was confirmed that the rat bean extract of the present invention also promoted the expression of the genes inhibited by H ₂ O ₂ (see FIG. 3 ).

Therefore, the health food containing the rat eye extract of the present invention as an active ingredient can be usefully used for the prevention or treatment of osteoporosis by promoting the expression of the osteoporosis-related genes.

When the rat bean extract of the present invention is used as a food or beverage additive, the rat bean extract may be added as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In the case of long-term intake for health and hygiene or health control, the active ingredient can be taken for a long time because there is no problem in terms of safety. There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Preparation of Rat Eye Extract 1

1 g of distilled water was added to 200 g of Jeongseon Mountain Root Bean and extracted at 100 ° C. for 2 hours. Thereafter, the extract was filtered and the filtrate was concentrated under reduced pressure at 60 ° C and lyophilized for 72 hours (see Figure 1 ).

Preparation Example 1 Manufacturing Method of Food and Beverage

The present inventors prepared a food or beverage composition containing the rat bean extract as an active ingredient as follows.

<1-1> Preparation of nutritional supplement foods (tablets)

Rat Bean Extract 33.3%

Chlorella 8.4%

Vitamin C 1.3%

Crystalline cellulose 3.0%

Maltitol 30.0%

Lactose 20.0%

Starch 3.0%

Magnesium Stearate 1.0%

The nutritional supplement food (tablet) was prepared using the conventional method with the said composition and content.

<1-2> Preparation of nutritional supplement foods (capsules)

Rat Bean Extract 60.0%

Red Ginseng Extract 20.0%

Cinnamon Powder 18.0%

Vitamin C 1.5%

Sodium benzoate 0.5%

The nutritional supplement food (capsule) was prepared using conventional methods with the composition and content.

<1-3> Preparation of Drink 1

522 mg of honey

Chioctosanamide 5 mg

Nicotinamide 10 mg

Riboflavin Sodium Hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Orthoic acid 50 mg

Mouse Eye Bean Extract 0.48 ～ 1.28 mg

200 ml of water

The beverage was prepared by the conventional method with the said composition and content.

<1-4> Preparation of beverage 2

Rat Bean Extract 0.05%

Tofu extract (more than 2% solids) 56.47%

Licorice extract (more than 2% solids) 14.00%

Dermis Extract (2% solids) 11.00%

Liquid fructose 10.00%

5.00% honey

Citric acid 0.20%

Apple Juice 3.00%

Taurine 0.10%

Vitamin C 0.05%

Peach flavor 0.07%

Sodium benzoate 0.06%

The beverage was prepared by the conventional method with the said composition and content.

<1-5> Preparation of the ring

Rat Bean Extract 4.00%

Turmeric powder 5.00%

Cheongung Powder 7.00%

Angelica powder 7.00%

Rate powder 20.00%

Cinnamon Powder 10.00%

Rat Bean Powder 16.00%

Sulfur Sulfur Concentrate 10.00%

Honey 20.00%

Starch 1.00%

The ring was prepared using the conventional method with the said composition and content.

<1-6> production of chewing gum

Gum base 20%

Sugar 76.36 ～ 76.76%

Rat Bean Extract 0.24 ~ 0.64%

1% fruit flavor

Water 2%

Chewing gum was prepared using conventional methods in the above composition and content.

<1-7> Preparation of Candy

Sugar 50-60%

Starch syrup 39.26 ～ 49.66%

Rat Bean Extract 0.24 ~ 0.64%

Orange flavor 0.1%

Candy was prepared using conventional methods with the above composition and content.

<1-8> Preparation of Biscuits

Force Class 1 88 kg

Gravity First Class 76.4 ㎏

16.5 kg per white

2.5 kg of salt

2.7 kg of glucose

Palm shortening 40.5 kg

5.3 kg of ammo

Medium kg 0.6 kg

0.55 kg sodium bisulfite

Rice flour 5.0 kg

Vitamin B1 0.003 kg

0.003 kg of vitamin B2

Milk Flavor 0.16 ㎏

71.1 kg of water

Whole milk powder 4 kg

Substitute powder 1 kg

0.1 kg of calcium phosphate

Spray salt 1 kg

25 kg of spray oil

Rat Bean Extract 0.1 ~ 0.5 ㎏

Biscuits were prepared using conventional methods with the above compositions and contents.

<1-9> Preparation of Ice Cream

Milkfat 10.0%

Non-fat solids 10.8%

Sugar 12.0%

Starch syrup 3.0%

Emulsifying stabilizer (span) 0.5%

Spices (Strawberries) 0.15%

Water 63.31-62.91%

Rat Bean Extract 0.24 ~ 0.64%

Ice cream was prepared using conventional methods with the above composition and content.

<1-10> Preparation of Sausage

Pork 63.6%

Chicken 27.5%

Starch 3.5%

Soy Protein 1.7%

Saline 1.62%

Glucose 0.5%

Other additives (glycerine) 0.94 ～ 1.34%

Rat Bean Extract 0.24 ~ 0.64%

Sausages were prepared using conventional methods with the above compositions and contents.

<1-11> Preparation of Chocolate

Sugar 34.36-34.76%

Cocoa Butter 34%

Cocoa Mass 15%

Cocoa Powder 15%

Lecithin 0.5%

0.5% vanilla

Rat Bean Extract 0.24 ~ 0.64%

Chocolate was prepared using conventional methods with the above composition and content.

Experimental Example 1 Preparation of Osteoporosis Model and Treatment of Rat Bean Extract

In order to determine the effect of improving bone formation, Hos-TE85 cells, which are osteoblasts, were cultured and treated with 250 uM of H ₂ O ₂ for 30 minutes, and then germinated for 0, 1, 2 or 3 days. The cells were treated for 2 days such that the final concentration of the cell culture solution was 100 ug / ml.

Experimental Example 2 Measurement of the Effect of Rat Eye Bean Extract on the Expression of Genes Involved in Bone Formation.

<2-1> RNA Isolation

48 hours after the rat eye extract of <Example 1> whole cell RNA was extracted from the cultured Hos-TE85 cells using the Trizol solution method. First, 1 ml of Trizol solution (Gibco BRL, Co., USA) was added to the cells recovered by centrifugation, homogenized with an 18-21 G syringe, and centrifuged at 12,000 rpm at 4 ° C to recover the supernatant. . This was allowed to stand at room temperature for 5 minutes, and then 200 μl of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added and mixed, followed by centrifugation at 12,000 rpm for 10 minutes. After recovering the clear supernatant, the same amount of isopropanol was mixed and then placed at -20 ° C for 30 minutes to precipitate RNA pellets. Precipitated RNA was dissolved in 100 μl of DEPC treated water, and then quantified using an absorbance spectrophotometer (spectrophotometer, Hewlett Packard, Co., U.S.A.).

<2-1> cDNA Synthesis and RT-PCR

For cDNA synthesis, 1 μl of RNA extracted from each sample (5-10 μg of RNA template) was added to 1 μl of 0.5 μg / μl of a random primer, which was immediately immersed in ice after heating at 70 ° C. for 5 minutes. Then 6 μl of MMLV 5X reaction buffer, 1 μl of 10 mM dNTP, 0.5 μl of 20 U RNase inhibitor were added and DEPC-distilled water was added to a total volume of 30 μl. After the reaction mixture was left at room temperature for 2 minutes, 200 U of MMLV reverse transcriptase was added thereto, and reacted for 50 minutes at 42 ° C. for 15 minutes at 70 ° C. to synthesize cDNA.

Subsequently, PCR products were performed using the products of the reverse transcription reaction as templates. That is, 2.5 μl of 10 × buffer, 0.5 μl of 10 mM dNTP, 1 μl of upstream primer, 1 μl of downstream primer, 0.3 μl of Taq polymerase, 1.0 μl of reverse transcription reaction solution and adjusted to DEPC treated distilled water so that the total volume is 25 μl. , PCR reaction was performed once every 10 minutes at 95 ° C, 45 seconds at 95 ° C, 45 seconds at 60 ° C, 60 seconds at 72 ° C, and once at 10 ° C at 72 ° C. In this case, the housekeeping gene GAPDH (Glyceraldehyde-3-dehydrogenase), a positive control group, used primers of SEQ ID NO: 1 and SEQ ID NO: 2 , and BMP1a (bone matrix protein 1a), primers of SEQ ID NO: 3 and SEQ ID NO: 4 , and BMP2a. for the case of the case of (bone matrix protein 2a) are the primers of SEQ ID NO: 5 and SEQ ID NO: 6, BMP3a (bone matrix protein 3a ) are the primers of SEQ ID NO: 7 and SEQ ID NO: 8, OTN (Osteonectin) is SEQ ID NO: Primers of SEQ ID NO: 9 and SEQ ID NO: 10 , primers of SEQ ID NO: 11 and SEQ ID NO: 12 for OTC (Osteocalcin), primers of SEQ ID NO: 13 and SEQ ID NO: 14 for COL1 (Collagen type 1), and TG2 (Transglutaminase). In the case of type 2), primers of SEQ ID NO: 15 and SEQ ID NO: 16 were used (see Table 1 ).

<2-3> Electrophoresis and Quantitative Analysis of PCR Products

       10 μl of each PCR product was electrophoresed at 100 V for 40 min using 0.5 × TAE buffer in 1.5% agarose gel. In order to visually identify the separated PCR product, the gel was stained with 500 ml of ethidium bromide (Et-Br) solution for 20 minutes and quantitatively analyzed using GEL DOC (BIO RAD Co., U.S.A.).

As a result, the BMP1a gene showed an increase in expression of 19% compared with the control group treated with H ₂ O ₂ only 14% when treated with germinated soybean extract germinated for 1 day. When BMP2a gene was treated with ungerminated rat bean extract, expression was increased by 7% and 33% compared with H ₂ O ₂ . The BMP3a gene was increased by 6% compared to the control group and 5% of the H ₂ O ₂ treatment (see Fig. 2 ; the control on the horizontal axis of the graphs treated with both H ₂ O ₂ and mouse eye extracts on osteoblasts). is not a group, HP is a group treated only with H ₂ O _2, non-GRV is the group treated with jwinunyi bean extract did not germinate after H ₂ O ₂ treatment, 1day GRV, 2 day GRV and 3 day GRV is 1 day, 2 days and 3 days after germination of H ₂ O ₂ treated rat eye extract extract group).

The COL1 gene was restored to the control level by increasing the 5% expression level when treated with germinated soybean extract, which had been germinated for 1 day, than H ₂ O ₂ . OTN is a gene expression level of 5% than that after treatment _2%, H 2 O 2 compared with the control group man When handles jwinunyi bean extract was germinated one days was increased. TG2 gene increased 5% expression level when treated with germinated soybean extract for 1 day than H ₂ O ₂ alone, but did not return to the control level (see FIG. 3 ; In the H ₂ O ₂ and the rat bean extract was not treated group, HP is the group treated with H ₂ O ₂ only, non-GRV is the group treated with ungerminated rat bean extract after H ₂ O ₂ treatment, 1 day GRV, 2 day GRV, and 3 day GRV were groups treated with rat bean extract germinated for 1, 2 and 3 days after H ₂ O ₂ treatment).

Therefore, it can be seen that the rat eye extract of the present invention promotes the expression of genes essential for bone formation.

Experimental Example 2 Cytotoxicity Test of Rat Eye Extract

For cytotoxicity test using rat bean extract, first, Hos-TE85 (female, osteoblast) cells cultured in T-75 flask were washed 2-3 times with 1x PBS and attached using 0.025% Trypsin-EDTA. After dropping the cells, cells were recovered by centrifugation (Heraeus, Labofuge 400R) for 5 minutes at 1500 rpm. 10 ml of RPMI 1640 medium (10% FBS) was added thereto, mixed evenly, and the cells were measured using a Hemacytometer, and 100 μl of the plate was dispensed into a 96 well plate to be 1 × 10 ⁵ cells per well. When cells were grown to about 50% of the well area at 37 ° C. and 5% CO ₂ , the cells were exchanged with fresh RPMI 1640 medium, and the rat bean extract was added to each well for each concentration. Shielding the light, the cell proliferation assay solution before culture termination 1 hours (CellTiter 96 Cell Proliferation Assay ^ⓡ AQueous. Promega Co. USA) after the processing to be 20 per well ㎕ ELISA absorbance of each of the color-well reader (Bio-Tek instrument Measured at 490 nm using INC.) And cell viability in terms of percentages compared to the absorbance of the control group.

As a result, the rat eye extract of the present invention showed little change in viability of Hos-TE85 cells until the final concentration of the cell culture solution was treated to 1 mg / ml, and the cell viability was not added until 10 mg / ml was added. It can be seen that the cytotoxicity is greatly reduced (see FIG. 4 ). Therefore, the rat eye extract of the present invention has been found to be safe for human use because of low cytotoxicity.

Experimental Example 3 Oral Acute Toxicity in Rats

In order to determine the toxicity of the rat eye extract of the present invention, acute toxicity test was performed using 6-week-old SPF rats. Rat eye extract was suspended in 0.5% methylcellulose solution and administered orally at a dose of 5 g / kg / 15 ml. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were examined, and hematological and blood biochemical tests were performed. The necropsy was performed to visually examine the abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. Specifically, the rat eye extract of the present invention did not show a toxicity change in rats up to 5 g / kg and was determined to be a safe substance having a minimum lethal dose (LD ₅₀ ) of 5 g / kg or more.

The present invention relates to a health food for the prevention or treatment of osteoporosis containing rat eye extract as an active ingredient. The health food containing the rat eye extract of the present invention is not only effective in preventing or treating osteoporosis, but also contains various nutrients such as various amino acids, vitamins, and calcium, and thus can be usefully used in osteoporosis patients and the general public.

<110> Helixpharms, Inc. <120> Composition comprising Rhynchosia Volubilis extracts for          prevention and treatment of osteoporosis <130> 4p-07-12 <160> 16 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer1 <400> 1 ggtgaaggtc ggagtcaacg g 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer2 <400> 2 ggtcatgagt ccttccacga t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer3 <400> 3 gttacgacta tgtggaggtc c 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer4 <400> 4 gctgctctca ctgtgcccgt c 21 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer5 <400> 5 ctagcgacac ccacaaccct c 21 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer6 <400> 6 gaaacgcctt aagtccagct gt 22 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer7 <400> 7 ttatctgcaa gcgcaagact cta 23 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer8 <400> 8 gattgaacct cggaattgcg cc 22 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer9 <400> 9 gatgaggaca acaaccttct gac 23 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer10 <400> 10 ttagatcaca agatccttgt cgat 24 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer11 <400> 11 ctagaccggg ccgtagaagc g 21 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer12 <400> 12 atgagagccc tcacactcct c 21 <210> 13 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer13 <400> 13 agggctccaa cgagatcgag atccg 25 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer14 <400> 14 tacaggaagc agacagggcc aacgtcg 27 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer15 <400> 15 atcccatcac catcttccag 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer16 <400> 16 atgacattcc ggaagccctt 20  

Claims (5)

Health food for the prevention or treatment of osteoporosis containing rat bean extract as an active ingredient. The health food according to claim 1, wherein the health food is selected from the group consisting of nutritional supplements, beverages, pills, chewing gum, candy, biscuits, ice cream, sausages and chocolates. The method according to claim 1, wherein the rat eye extract is a health food, characterized in that by adding water to the mouse eye extract at 100 ℃ extracted and concentrated to freeze-dried. Food additives containing the rat eye extract of claim 1 as an active ingredient. Beverage composition according to claim 1 of the rat eye extract.

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