KR102126635B1 - Pharmaceutical composition comprising luteolin-7-o-diglucuronide, apigenin-7-o-diglucuronid isolated from perilla frutescens var. acuta leaf extract for improving ocular fatigue - Google Patents

Abstract

Regarding the pharmaceutical composition for the improvement of ophthalmic symptoms of VDT (Visual Display Terminal) syndrome of a luteolin-7-O-diglucuronide or apigenin-7-O-diglucuronide compound contained in Chazgi extract, the Chazgi leaf freeze-dried product of the present invention And it was confirmed that the active ingredients of the spray dried luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide compounds are contained equally. Increased NO and cGMP content related to eye relaxation in ciliary muscle cells (rCSMCs) isolated from rat eyes, [Ca ²⁺ ] i Ion content was reduced. In addition, cGMP content was increased through animal experiments. These compounds can be used as a composition useful for preventing and improving eye fatigue by increasing cGMP content in ciliary muscle cells isolated from rat eyes.

Description

Pharmaceutical composition for improving symptoms of ophthalmic symptoms of VDT (Visual Display Terminal) syndrome comprising luteolin-7-O-diglucuronide or apigenin-7-O-diglucuronide isolated from Chazgi leaf extract {PHARMACEUTICAL COMPOSITION COMPRISING LUTEOLIN-7- O-DIGLUCURONIDE, APIGENIN-7-O-DIGLUCURONID ISOLATED FROM PERILLA FRUTESCENS VAR. ACUTA LEAF EXTRACT FOR IMPROVING OCULAR FATIGUE}

The present invention relates to a pharmaceutical composition for improving ophthalmic symptoms of VDT (Visual Display Terminal) syndrome comprising an active ingredient luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide isolated from Chazgi leaf extract.

Chazgi ( Perilla) frutescens (L.) Britton var. acuta (Thunb.) Kudo) is a medicinal plant belonging to the pizza plant family, dicotyledon family, and honeybee family, and is distributed in Korea and China.

Stems are straight, square, purple, and fragrant on poles, leaves are coarse, broad ovate, pointed at the end, rounded or somewhat wedge-shaped, serrated on edges, hairy on both sides, especially veins Long hairs on the top and long lobes. Flowers bloom in light purple in August-September, and run as gunshot wounds on the ends of stems, branches, and upper leaves. The calyx is divided into two, the upper one is 3 rows again, and the lower one is 2 rows, and there are hairs inside and outside the tube. The corolla is a short normal form, the lower part is slightly longer than the upper part, the stamen is 2 gangungye, and the fruit is round in the subdivision and is contained in the calyx. This species has a purple color on the whole, and the branch is round and has a net pattern. Leaves and stems are used for medicinal purposes, and young leaves and seeds are edible.

Young leaves are difficult to distinguish from perilla leaves, and sugar made from perylaldehyde in teaspoon is 2,000 times stronger than normal sugar, so it is used for tobacco, intestine, and toothpaste. The leaves that are not purple but green are called clean leaves (for. viridis). Jang Yeop-Yeop has a white flower and has a stronger scent than Chajgi.

On the other hand, the eye recognizing the object is composed of membrane-like tissues that play an important role for visual functions such as the reception of light, present in the innermost layer of the eye, and the retina is composed of 10 layers, for example from the outside. Retinal pigment epithelial layer, neuroepithelial layer, outer boundary layer, surgical granule layer, outer reticular layer, inner granule layer, inner reticular layer, ganglion cell layer, nerve fiber layer, and inner boundary layer formed in the following order.

Light irradiated to the retina from the outside world is transmitted from the inner boundary membrane side to the layer of the retina and is received by visual cells (mesenchymal cells and abstract cells) as photoreceptor cells present in the neuroepithelial layer. In visual cells, light is converted into nerve signals, signals are processed by various neurons present in the retina, and information is finally transmitted through the optic nerve from the ganglion cells present on the surface of the retina to the cerebral center.

In the highly developed mechanical civilization, eyes are easily fatigued due to various environmental pollution, excessive viewing of television, and excessive use of personal computers and electronic entertainment equipment, and visual acuity is reduced due to decreased ability to adapt during night driving or night work. have. In order to prevent such a decrease in vision, various medicines, natural foods, and the like have been enjoyed, and among them, vision improvers based on anthocyanides extracted from wild blueberries are widely used at home and abroad.

In the present invention, the equivalence of luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide contained in freeze-dried and spray-dried powders of Chagigi extract for the purpose of utilizing industrialization of traditional plant resources in Korea was confirmed. It is intended to provide a pharmaceutical composition for improving the symptoms of ophthalmic symptoms of VDT (Visual Display Terminal) syndrome comprising an active ingredient isolated from the extract of the leaf.

Patent Publication No. 10-2013-121324 filed and published by the present inventors in connection with Chazgi discloses a high-value-added functional health food composition that acts to relieve hangover using Chazgi leaf extract as an active ingredient. Korean Patent Publication No. 10-2008-10030 relates to a health functional food containing a mixture of herbal extracts (Rubus coreanus), golden (Scutellaria baicalensis) and clove (Syzygium aromaticum), the use of contact lenses, eye fatigue , VDT syndrome, dry eye syndrome, normal aging phenomenon, relates to a health functional food composition having a function of maintaining eye health by preventing oxidative damage to eyes caused by menopause. Korean Patent Publication No. 10-2002-62866 has an excellent effect on improving eyesight, so it has 85 w/w% of anthocanoside oligomers, which are used as raw materials for vision-improving drugs, health supplements, and food. Disclosed are a method for producing the above-mentioned extract and a composition containing the same. In Korean Patent Publication No. 10-1997-6124, wild blueberry extract dry powder, one or two or more types of herbal extracts dry powder selected from shorter, goji, tea stalk, ingot, heartworm, pendulum, dongguja, creation or nutrition It has been disclosed a health nutrition composition having a vision improving effect by mixing as a main component.

The present invention is to provide a pharmaceutical composition for improving eye fatigue and a health functional food composition containing a compound separated from Chazgi leaf extract, a natural resource in Korea, as an active ingredient.

In order to solve the above problems, the present invention provides a pharmaceutical composition for improving ophthalmic symptoms of VDT (Visual Display Terminal) syndrome comprising a compound isolated from Chazgi extract as an active ingredient. The compound isolated from the Chazgi leaf extract is characterized in that it is at least one flavonoid glycoside compound selected from the group consisting of luteolin-7-O-diglucuronide or apigenin-7-O-diglucuronide, and the Chazgi leaf extract is water, 1 to 5 carbon atoms. It contains an extract soluble in any one of the alcohol or a mixed solvent thereof.

The extract provides a method for preparing a flavonoid glycoside compound composed of a process of identifying a structure by NMR and MS by separating the fraction prepared by performing diaion HP-20 resin to produce a fraction of chagigi leaves by preparative HPLC.

The VDT (Visual Display Terminal) syndrome of the pharmaceutical composition for improving ophthalmic symptoms, respectively, according to a conventional method, tablets, capsules, pills, granules, liquid, powder, flakes, pate, syrup, etc. Visual Display Terminal) Syndrome is used as a pharmaceutical composition for improving symptoms.

The pharmaceutical composition for improving the symptoms of ophthalmic symptoms of VDT (Visual Display Terminal) syndrome containing the compound isolated from the Chazgi leaf extract of the present invention, particularly luteolin-7-O-diglucuronide or apigenin-7-O-diglucuronide as an active ingredient NO, cGMP related to relaxation of ciliary myocyte smooth muscle cells isolated from human eyes and [Ca ²⁺ ] i Through reduction of content and animal experiments, it was confirmed that it was effective in improving eye fatigue by increasing the shape muscle smooth muscle relaxation and related factors, cGMP. From this, luteolin-7-O-diglucuronide or apigenin-7-O-diglucuronide compound extracted from Chagigigi is a domestic natural resource, useful pharmaceutical composition and health functional food that has an eye symptom improvement effect of VDT (Visual Display Terminal) syndrome It can be used as a composition.

1 is a diagram showing a compound contained in the extract of chazgi leaf through HPLC analysis. (1) represents luteolin-7-O-diglucuronide, (2) apigenin-7-O-diglucuronide, and (3) rosmarinic acid.
2 is a diagram showing the inhibitory effect of ROS production of Chazgi leaf extract using C ₂ C ₁₂ cells. A: MTT assay (cytotoxicity) B: ROS measurement
Figure 3 is a diagram showing the relaxing effect of Chagigigi leaf extract from ciliary muscle isolated from rabbits.
FIG. 4 is a graph showing cGMP and cAMP content changes of Chazgi leaf extract in aortic smooth muscle (hASMCs). A: cGMP content for 15, 30 and 60 minutes B: cAMP content for 15, 30 and 60 minutes
5 is a diagram showing the inhibitory effect of PDE5A and PDE3A activity of Chazgi leaf extract on aortic smooth muscle (hASMCs). A: PDE5A activity B: PDE3A activity
6 is a graph showing the change in the concentration of [Ca ²⁺ ] i of Chazgi leaf extract in aortic smooth muscle (hASMCs).
7 is a diagram showing the effect of inhibiting the [Ca ²⁺ ] i content induced by ET-1 of Chazgi leaf extract in aortic smooth muscle (hASMCs).
Figure 8 shows a schematic diagram of the separation of luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide compounds isolated from Chazgi leaf extract.
Figure 9 shows the results of luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide components isolated from Chazgi leaf extract. (A-1) Compound structure of luteolin-7-O-diglucuronide, (A-2) HPLC analysis of luteolin-7-O-diglucuronide. (A-3) UV spectrum of luteolin-7-O-diglucuronide, (B-1) compound structure of apigenin-7-O-diglucuronide. (B-2) HPLC analysis of apigenin-7-O-diglucuronide. (B-3) UV spectrum of apigenin-7-O-diglucuronide.
Figure 10 shows the equivalence of luteolin-7-O-diglucuronide and apigenin-7-diglucuronide compounds according to the freeze-drying and spray-drying methods of Chazgi leaf hot water extract. FD; freeze-dried, lyophilized, SD; spray-dried
11 is a diagram showing the effect of cytotoxicity and nitric oxide (NO) production on the myocardial cells isolated from the eyes of the SD rats of Chazgi leaf extract. Cytotoxicity (A) and NO content (B) of lyophilisates of Chazgi extract
12 is a diagram showing the amount of cGMP and cAMP content changes for the myocardial cells isolated from the eyes of the SD rats of Chazgi leaf extract. The amount of cGMP (A) and cAMP (B) of the lyophilisate of Chazgi leaf extract.
FIG. 13 is a graph showing the change in concentration of [Ca ²⁺ ] i for myocardial cells isolated from the eyes of SD rats of Chazgi leaf extract. ( A) The effect of lyophilized extract of Chazgi extract on the content of [Ca ^{2 +} ] i of ciliary muscle cells isolated from SD rats, (B) The effect of lyophilized extract of Chazgi extract from ciliated muscle cells isolated from SD rats [Ca ^{2 +} ] i content change
FIG. 14 is a graph showing changes in cGMP content in rat eyes after oral administration of 100, 200 mg/kg of Chazgi leaf extract for 3 days after irradiating light to the rat eyes to induce fatigue.
15 shows the cGMP content change amount for the cGMP content change for ciliary muscle cells isolated from the eye of the SD rat for the compound isolated from the Chazgi leaf extract.

The pharmaceutical composition for improving ophthalmic symptoms of VDT (Visual Display Terminal) syndrome of the present invention contains a compound separated from Chazgi leaf extract as an active ingredient. In the following examples of the present invention, as a compound separated from Chazgi leaf extract, luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide compound will be described as an example. In order to optimize industrialization, differences in luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide contents of Chazgi extract according to the drying method were confirmed.

It was confirmed that the plinoid compound improved the fatigue of the eye by relaxing the contracted ciliary muscle by increasing the cGMP content, which is the relaxation factor of the ciliary muscle of the eye.

In the present invention, the flavonoid glycoside compound is at least one selected from the group consisting of luteolin-7-O-diglucuronide represented by Formula 1 below, and apenin-7-O-diglucuronide represented by Formula 2 below.

Example 1.Aortic smooth muscle cells ( in vitro ) And rabbit eyes Shape ( ex vivo ), In the control point (human application test) Eye fatigue effect of Chazgi extract

1. Chazgi leaf hot water extraction manufacturing

3 kg of chazgi leaves (dry leaf) was extracted with 10 times distilled water and hot water was extracted at 100° C. for 3 hours. The extracted water extract was concentrated under reduced pressure and freeze-dried to obtain 650 g of hot water lyophilized tea leaves.

2. Analysis of the components of Chazgi extract using HPLC

The HPLC device used for the analysis of the components of the tea leaves extract was a YL 9100 HPLC system, and a Triart C18 plus (250 x 4.6 mm, 5 um, YNC co. Ltd) was used as the column. The mobile phase is methanol (mobile phase A) and distilled water for HPLC (mobile phase B, 0.1% formic acid), and the ratio of methanol is 30% (0-10 minutes) to 30-50% (10-30 minutes), 60% (35 -40 minutes), 60-70% (40-45 minutes), 70-100% (45-53 minutes), 100% (53-56 minutes) and finally 30% (56-60 minutes), The flow rate was analyzed at 325 nm using a 1 mL/min, UV/VIS (9120) detector.

Figure 1 shows the compound contained in the extract of chachigi leaves through HPLC analysis. As a result of analyzing the hot water extract of Chazgi leaves using HPLC, it was confirmed that the major substances, rosmarinic acid, luteolin-7-O-diglucuronide, and apigenin-7-O-diglucuronide compounds were contained. In the figure, (1) represents luteolin-7-O-diglucuronide, (2) apigenin-7-O-diglucuronide, and (3) rosmarinic acid.

3. C ₂ C ₁₂ ROS measurement using cells

C ₂ C ₁₂ cells were dispensed into a 5x10 ⁵ cells/mL 48 well plate, treated with hydrogen peroxide (H ₂ O ₂ , 200 μM) for 2 hours to induce oxidative stress, and then chazgi extract (50, 100, 200) μg/mL) was reacted for 24 hours. Cytotoxicity was measured by MTT method. To perform ROS measurement, the medium was removed, washed twice with PBS, and treated with 1% Triton X-100 (PBS) to lyse cells for 10 minutes at 37°C. DCF-DA (10 μM) was reacted in the dark at room temperature for 30 minutes. After the reaction, cells were washed twice with cold PBS, and measured at excitation 485 nm and emission 530 nm using a fluorescence spectrometer.

Figure 2 shows the inhibitory effect of ROS production of Chazgi leaf extract using C ₂ C ₁₂ cells. A shows the result of MTT assay (cytotoxicity) and B shows the result of ROS measurement. It was confirmed that Chazgi leaf extract (50, 100 and 200 μg/mL) inhibits concentration-dependent inhibition of ROS production, which is an oxidative stress substance induced by H ₂ O ₂ without toxicity of C ₂ C ₁₂ cells.

4. Rabbit eye shape muscle relaxation rate measurement

Rabbits (2.4-2.7 kg) were purchased as experimental animals and adapted to the environment and used in this test. The zoletyl-rumpoon mixture (1:2) was subjected to general anesthesia by intramuscular injection and the eyeballs were removed from the experimental animals. The extracted eye was cut in half from the equatorial part of the eye by incising the steel membrane, and after removing the lens, the ciliary muscle was carefully separated from the steel membrane. The separated ciliary muscle was cut into samples of 3 mm wide by 6 mm long to obtain specimens.

The ciliary muscle section was passed through a mixed gas of 95% oxygen and 5% carbon dioxide, and Krebs-Henseleit (CaCl ₂ 1.5mM, NaCl 118 mM, KCl 4.7 nM, MgSO ₄ 1.1 mM, KH ₂ PO ₄ 1.2 mM, NaHCO ₃ 25 mM, glucose 10 mM; pH 7.4) was added to the solution to perform the experiment. The contraction of ciliary muscle was suspended by applying a load of 1 g using a tension transducer. The segment of ciliary muscle was stabilized for 90 minutes. After stabilization, it was shrunk by adding carbachol (100 μM/mL). After the last stimulation, Chazgi leaf extract (100, 200 μg/mL) was added to ciliary muscle to check the relaxation rate.

Figure 3 shows the relaxing effect of Chazgi leaf extract in ciliary muscle isolated from rabbits. It was confirmed whether Chazgi leaf extract affects relaxation of ciliary muscle isolated from rabbits. That is, after contracting the ciliary muscle of the rabbit using carbachol (100 μM/ml), the relaxation rate was confirmed by adding 100 and 200 μg/mL of the Chagigi extract. The group to which distilled water (control) was added did not affect the contracted ciliary muscle, whereas 200 μg/mL of Chagigi extract significantly relaxed the ciliary muscle contracted by carbachol.

5. Relaxation mechanism using aortic smooth muscle cells

5.1. cGMP and cAMP content measurement

Primary human aortic smooth muscle cells (hASMCs) were purchased from ATCC (American Type Culture Collection, PCS-100-012, Manassas, VA, USA). Vascular cell basal medium with vascular smooth muscle cell growth kit added 5% Cultured in CO ₂ incubator. hASMC was dispensed into 5x10 ⁵ cells/well in ⁶ wells and stabilized for 24 hours. After stabilization, 3-isobuytyl-1-methylxanthine (IBMX, 1 mM) was pretreated for 10 minutes, and then Chazgi leaf extract (50, 100, 200 μg/mL) was reacted for 15, 30, and 60 minutes. The cGMP and cAMP contents of Chazgi extract were measured using an ELISA kit.

Figure 4 shows the cGMP and cAMP content changes of Chagigigi leaf extract in aortic smooth muscle cells. A shows cGMP content results for 15, 30 and 60 minutes, and B shows cAMP content results for 15, 30 and 60 minutes. That is, the extract of Chazgi leaf (50, 100 and 200 μg/mL) increased the concentration of cGMP related to relaxation of the ciliary muscle in a concentration-dependent manner, and also cGMP in response to the reaction time (15-60 minutes) of Chazgi leaf extract. It was confirmed to increase the content. On the other hand, Chazgi leaf extract did not affect the cAMP content change.

5.2. Phosphodiesterase (PDE) inhibition

PDE5A and PDE3A activity was measured according to the method of the kit (BPS Bioscience, San Diego, CA). That is, Reaction mixture (PDE5A 10 ng/ml, PDE3A 20 ng/ml, FAM-Cyclic-3',5'-GMP, FAM-Cyclic-3',5'-AMP 200 nM) was added in 50 uL increments, respectively The leaf extract (50, 100, 200 μg/mL) was added. The reaction mixture was reacted at room temperature for 1 hour. Diluted binding agent (100 uL) was added and the reaction mixture was reacted for 1 hour again. Fluorescence polarization of each sample was measured at excitation 480 nm and emission 528 nm.

FIG. 5 shows the inhibition of PDE5A and PDE3A activity of Chazgi leaf extract in aortic smooth muscle cells. A shows the result of PDE5A activity, B shows the result of PDE3A activity. Chazgi leaf extract (50, 100, 200 μg/mL) inhibited PDE5A activity in a concentration-dependent manner, but did not affect PDE3A activity inhibition. That is, the PDE5A activity at the concentrations of Chagigi extract 50, 100, and 200 μg/mL was 51.23±0.29, 42.42±0.13, and 36.58±0.37%, respectively. Therefore, it was found that Chazgi extract affects the relaxation of smooth muscle by cGMP mechanism.

5.3 [Ca ²⁺ ] i Content measurement

As a calcium ion-sensitive fluorescent material, acetoxymethyl-ester form fura-2/AM (fura-2/AM; Molecular probes, Eugene, OR) was used as a calcium ion marker. In the light-blocked state, the cells were treated with 5 μM fura-2/AM, 0.001% F127 in HEPES buffer and reacted for 60 minutes at room temperature. The water buffer washed several times with HEPES buffer is allowed to flow for 5 minutes to stabilize. Chazgi hot water extracts (50, 100 and 200 μg/mL) were treated in order for 100 seconds by concentration. After stabilizing the cells for 5 minutes in the same manner, the Chazgi leaf hot water extract was treated at a concentration of 100 seconds.

In addition, cells were treated with endothelin-1 (ET-1; 10 nM) for stimulation for 100 seconds, and then Chazgi hydrothermal extract (50, 100 and 200 μg/mL) was treated in order for each concentration for 100 seconds. At this time, all buffers and chemicals were processed by gravity perfusion. The light from the lamp was selectively exposed to cells at a wavelength of 340 nm and 380 nm through a computer control wheel. Pictures were taken at 340 nm and 380 nm every 2 seconds, and the emitter fluorescence light that passed through the 515 nm long-pass filter passed the Cooled CCD camera to obtain a 340 nm/380 nm ratio value by a digital fluorescence analyzer.

Figure 6 shows the change in the concentration of [Ca ^{2 +} ] i of Chazgi leaf extract in aortic smooth muscle cells. In other words, the extract of aortic smooth muscle cells (rCSMCs) against the extract of tea leaves, that is, the extract of leaves of leaves (50, 100 and 200 μg/mL) is concentration-dependent and the concentration content of [Ca ²⁺ ] i related to contraction of ciliary muscle is concentration-dependent. Reduced.

7 shows the effect of inhibiting [Ca2+] i content induced by ET-1 in aortic smooth muscle cells of Chazgi leaf extract. After adding ET-1 (10 uM) to hASMCs cells to increase the content of [Ca2+] i , and then adding 50, 100 and 200 ug/ml of Chazgi leaf extract to measure the concentration of [Ca ^{2 +} ] i , Chazgi leaf extract significantly reduced the [Ca ^{2 +} ] i concentration.

Example 2. Isolated from SD rat Shape Through cells Chajgi Eye fatigue effects of luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide contained in extracts

1. Preparation of hot water extract from Chagisgi leaf and separation of planoid glycoside compounds

1-1. Chazgi leaf water extract preparation

3 kg of Chagigi dried leaves was extracted with distilled water at 100° C. for 3 hours. The extract extract was concentrated under reduced pressure, and then freeze-dried and spray-dried to obtain Chazgi leaf hot water lyophilisate (650 g, 21.6%) and spray dried product (669 g, 22.4%).

1-2. Separation of active ingredients using Diaion HP-20 resin

To separate the active ingredient from the Chazgi leaf extract, 2L of the Chazgi leaf hot water extract was added to Diaion HP-20 resin, and water and methanol were sequentially eluted at a ratio of 30:70, 50:50, 70:30, 0:100. Did. Finally, 2 L of acetone was eluted to obtain 5 fractions. Compound 1 (150 mg, purity 96.7%) and compound 2 (50 mg, purity 96.4%) were obtained using preparative Waters HPLC for the first small fraction (30:70) of the 5 fractions.

Figure 8 shows a schematic diagram of the separation of luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide compounds isolated from Chazgi leaf extract.

1-3. Structural identification using NMR

The structures obtained in Example 1-2, compound 1 and compound 2 were identified using NMR and MS as structures of luteolin-7-O-diglucuronide and apenin-7-O-diglucuronide, respectively. Figure 9 shows the results of luteolin-7-O-diglucuronide, apigenin-7-O-diglucuronide components isolated from Chazgi leaf extract. (A-1) Compound structure of luteolin-7-O-diglucuronide, (A-2) HPLC analysis of luteolin-7-O-diglucuronide. (A-3) UV spectrum of luteolin-7-O-diglucuronide, (B-1) compound structure of apigenin-7-O-diglucuronide. (B-2) HPLC analysis of apigenin-7-O-diglucuronide. (B-3) UV spectrum of apigenin-7-O-diglucuronide.

The analysis structures of NMR and MS of luteolin-7-O-diglucuronide and apenin-7-O-diglucuronide identified respectively are as follows.

1) Analytical structure of luteolin-7-O-diglucuronide

Compound 1 , buff powder, ESI-MS: 639 (M+H) ⁺ . ¹ HNMR (500MHz,pyridine-d5)δ4.25~4.74(1H,H-1GluA2),4.38~4.76(1H,d,J=7.2Hz,H-1GluA1),4.92(1H,d,J=9.5Hz ,H-5``),5.57(1H,d,J=8Hz,H-1'''),6.05 (1H, d, J = 7Hz, H-1''), 6.82 (1H, s, H -3), 7.13 (1H, d, J = 2Hz, H-6), 7.17 (1H, d, J = 2Hz, H-8), 7.21 (1H, d, J = 8.5 Hz, H-5') , 7.43 (1H, dd, J = 2.0, 8.5 Hz, H-6'), 7.85 (1H, d, J = 2 Hz, H-2').

¹³ CNMR(125MHz,pyridine-d5)δ72.43(C-4''),73.16(C-4'''),76.02(C-2'''),76.80(C-5''),77.40 (C-3''),77.62(C-3'''),78.06(C-5'''),83.89(C-2''),95.61(C-8),100.06(C-1''),100.64(C-6),103.77(C-3),106.57(C-1'''),106.75(C-10),114.49(C-2'),116.57(C-5'),119.47(C-6'),122.46(C-1'),147.47(C-3'),151.61(C-4'),157.55(C-5),162.46(C-9),163.47(C- 7), 165.00 (C-2), 171.79 (C-6'''), 172.37 (C-6'''), 182.61 (C-4).

2) Analysis structure of apenin-7-O-diglucuronide

Compound 2 , buff powder, ESI-MS: 623 (M+H) ⁺ . ¹ HNMR(500MHz,pyridine-d5)δ4.25~4.65(1H,H-1GluA2),4.38~4.77(1H,d,J=7.2Hz,H-1GluA1),4.95(1H,d,J=7Hz, H-5``),5.58(1H,d,J=8Hz,H-1'''), 6.08 (1H, d, J = 7Hz, H-1''), 6.81 (1H, s, H- 3), 7.13 (1H, d, J = 2Hz, H-6), 7.16 (1H, d, J = 2Hz, H-8), 7.20 (1H, d, J = 8.5 Hz, H-5'), 7.29 (1H, dd, J = 2.0, 8.5 Hz, H-6'), 7.80 (1H, d, J = 8.5 Hz,

¹³ CNMR(125MHz,pyridine-d5):72.46(C-4''),73.16(C-4'''),76.02(C-2'''),76.83(C-3''),77.43( C-5``),77.63(C-3'''),78.06(C-5'''),83.95(C-2''),95.60(C-8),100.06(C-1''),100.78(C-6),103.69(C-3),106.57(C-10),106.79(C-1'''),116.57(C-3'),116.57(C-5'),121.82(C-1'),128.74(C-2'),128.74(C-6'),157.56(C-5),162.46(C-9),162.52(C-4'),163.58(C-7),164.63(C-2),171.78(C-6'''),172.36(C-6'''),182.64(C-4).

2. Comparison of Compound Equivalence According to Hot Water Extraction and Drying Method

The equilibrium of the lyophilized and spray-dried compounds of Chazgi leaf hot water extract was analyzed by HPLC. The HPLC apparatus used was a Waters series HPLC system (Waters corporation 34 Maple street Milford, MA), and a column was used Triart C18 plus (250 x 4.6 mm, 5 um, YNC co. Ltd). The mobile phase is methanol (mobile phase A) and distilled water for HPLC (mobile phase B, 0.1% formic acid), and the ratio of methanol is 30% (0-10 minutes) to 30-50% (10-30 minutes), 60% (35 -40 minutes), 60-70% (40-45 minutes), 70-100% (45-53 minutes), 100% (53-56 minutes) and finally 30% (56-60 minutes), The flow rate was analyzed at 254 nm using a 1 mL/min, photodiode array (2998) detector.

Figure 10 shows the equivalence of luteolin-7-O-diglucuronide and apigenin-7-diglucuronide compounds according to the freeze-drying and spray-drying methods of Chazgi leaf hot water extract. FD; freeze-dried, lyophilized, SD; It indicates spray-dried and spray-dried.

After washing 3 kg of the chiller with distilled water, 30 L of distilled water was added, and the mixture was heated and extracted at 100° C. for 3 hours with an electric water heater. The extracted solution was filtered through a 400 mesh filter cloth, and then concentrated under reduced pressure using a vacuum rotary concentrator. Half of the concentrated hot water extract was freeze dried using a freeze dryer, and the other hot water extract was spray dried using a spray dryer. The luteolin-7-O-diglucuronide contents of lyophilized and spray dried extracts of Chagigi leaf were 40.14 mg/g and 41.11 mg/g, respectively, and apigenin-7-O-diglucuronide contents were 13.04 mg/g, 14.01 mg/, respectively. Equivalence was confirmed by g.

3. Separation of ciliary muscle and cell culture from rat eyes

The ciliary muscle was isolated from the eyes of 3-4 week old Sprague-Dawley rats for experiments with ciliary muscle cells of Chazgi leaf extract containing Luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide compounds as main components. That is, the separated eye was cut in half, and the corneal portion was placed in a 15 mL centrifuge tube to which papain solution was added, and reacted at 37°C for 90 minutes. The cell suspension was transferred to a new 15 mL centrifuge tube and centrifuged at room temperature (300 xg, 5 min). After removing the supernatant, the cells were immediately cultured in DMEM/F-12 (Invitrogen-Gibco, Grand Island, NY, USA) medium.

4. Measurement of morphogenetic cell viability and NO isolated from rats of Chazgi leaf extract

The ciliary muscle cells (rCSMCs) isolated from the rat eye were dispensed at 1 x 10 ⁴ cells/well in a 96 well plate using a medium containing 10% FBS and cultured for 3 days. After 3 days of incubation, luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide compounds were added at a concentration of 50, 100 and 200 μg/mL Chazgi leaf extract containing as a main component, and added to 24 at 37°C CO ₂ incubator It was reacted for an hour. After the reaction, 100 μL of each WST-1 solution was added and reacted at 37° C., and measured using a microplate reader at a wavelength of 450 nm using the method suggested by the manufacturer.

On the other hand, NO measurement was performed by dispensing ciliary muscle cells (rCSMCs) isolated from the rat eye at 5 x 10 ⁴ cells/well in a 96 well plate using a medium containing 10% FBS and incubating for 3 days. After 3 days of incubation, luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide compounds were added at a concentration of 50, 100 and 200 μg/mL Chazgi leaf extract containing as a main component, and added to 24 at 37°C CO ₂ incubator It was reacted for an hour. The NO measurement for the supernatant was measured using the Griess reaction.

FIG. 11 shows the cytotoxicity and NO content of ciliary muscle cells (rCSMCs) isolated from the eyes of SD rats for Chazgi leaf extract. The cytotoxicity (A) and NO content (B) of the lyophilisate of Chazgi extract. Chazgi leaf extract 50, 100 and 200 μg/mL increased the NO content in a concentration-dependent manner without cytotoxicity.

5. Chazgi leaves Of extract From rats Separated Shape Through cells cAMP And cGMP Content measurement

The myocardial cells (rCSMCs) isolated from the rat eye were dispensed into 5x10 ⁵ cells/well in ⁶ wells and stabilized for one day. After stabilization, 3-isobuytyl-1-methylxanthine (1 mM) was pretreated for 10 minutes, and then Chazgi leaf extract was reacted for 15 minutes. The cGMP and cAMP contents of Chazgi extract were measured using an ELISA kit.

FIG. 12 shows cGMP and cAMP content changes of ciliary muscle cells (rCSMCs) isolated from rat eyes for Chazgi leaf extract. The amount of cGMP (A) and cAMP (B) of the lyophilisate of Chazgi leaf extract. The amount of cGMP (A) and cAMP (B) of the lyophilisate of Chazgi leaf extract. Chazgi leaf extract (50, 100 and 200 μg/mL) increased concentration-dependently the cGMP content related to relaxation of ciliary muscle in a concentration-dependent manner, whereas Chazgi leaf extract did not affect cAMP content change.

6. [Ca] through ciliary muscle cells isolated from rats of Chazgi leaf extract ²⁺ ] i Content measurement

As a calcium ion-sensitive fluorescent material, acetoxymethyl-ester form fura-2/AM (fura-2/AM; Molecular probes, Eugene, OR) was used as a calcium ion marker. In the light-blocked state, the cells were treated with 5 μM fura-2/AM, 0.001% F127 in HEPES buffer and reacted at room temperature for 60 minutes. The water buffer washed several times with HEPES buffer is allowed to flow for 5 minutes to stabilize. Chazgi hydrothermal extract (50, 100 and 200 μg/mL) was treated in order for 100 seconds by concentration. After stabilizing the cells for 5 minutes in the same manner, Chazgi hydrothermal extract was treated for one second at a concentration.

At this time, all buffers and chemicals were processed by gravity perfusion. The light from the lamp was selectively exposed to cells at a wavelength of 340 nm and 380 nm through a computer control wheel. Pictures were taken at 340 nm and 380 nm every 2 seconds, and the emitter fluorescence light that passed through the 515 nm long-pass filter passed the Cooled CCD camera to obtain a 340 nm/380 nm ratio value by a digital fluorescence analyzer.

Figure 13 shows the change in ca2+ concentration of ciliary muscle cells (rCSMCs) isolated from rat eyes for Chazgi leaf extract. ( A) The effect of lyophilized extract of Chazgi extract on the content of [Ca ^{2 +} ] i of ciliary muscle cells isolated from SD rats, (B) The effect of lyophilized extract of Chazgi extract from ciliated muscle cells isolated from SD rats [Ca ^{2 +} ] i It is the amount of content change. Chazgi leaf extract (50, 100 and 200 μg/mL) concentration-dependently reduced [Ca ²⁺ ] i concentration content related to ciliary muscle contraction.

7. Measurement of cGMP content through animal test of Chazgi leaf extract

The experimental animals were male SD rats (Samtaco), 5-6 weeks old, weighing 180-200 g. During the entire experimental period, solid feed and water were supplied freely, and were raised under conditions of temperature 23±3℃, humidity 50±20%, and darkness for 12 hours. The experimental animals were adapted for 1 week in the breeding room and used for the experiment. All experiments were conducted in accordance with the guidelines of the IACU guidelines and the guidelines for the management and use of laboratory animals in the Natural Resources Research Center of the Jeonnam Biotechnology Industry Promotion Agency.

In the animal test group, the normal group that did not receive light on the last day after ingesting only distilled water for 3 days, the control group that irradiated light for 15 minutes on the last day after ingesting only distilled water for 3 days, and the tea extract group for 3 days After ingestion (100, 200 mg/kg), they were classified into four groups irradiated with light for 15 minutes on the last day, and 5 animals were assigned to each group. After sacrificing the animals, the eyes were removed from the SD rats and immediately washed with PBS. Homogenized the washed eyes, centrifuged, and the supernatant was taken and its content was measured using a cGMP ELISA kit.

14 is a result of measuring changes in cGMP content in rat eyes after oral administration of 100, 200 mg/kg of Chazgi leaf extract for 3 days after inducing fatigue by irradiating light to the rat eyes. Chazgi leaf extract (100 and 200 mg/kg) significantly increased the cGMP content compared to the control group irradiated with light only in the group ingesting the concentration of 200 mg/kg, and thus it was confirmed that it has an effect on eye shape muscle relaxation.

8. Chazgi leaves From the extract Separated luteolin -7-O- diglucuronide , apigenin Measurement of cGMP content of -7-O-diglucuronide compound

The ciliary muscle cells (rCSMCs) isolated from the rat eye were divided into 5x10 ⁵ cells/well in ⁶ wells and stabilized for one day. After stabilization, 3-isobuytyl-1-methylxanthine (1 mM) was pretreated for 10 minutes, and then luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide compounds separated from Chazgi leaf extracts were 0.01, 0.05, 0.1 And reacted at a concentration of 1 μg/mL for 15 minutes. The cGMP content of these compounds was measured using an ELISA kit.

15 shows cGMP content change of ciliary muscle cells (rCSMCs) isolated from rat eyes for Chazgi leaf extract. The luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide compounds (0.01, 0.05, 0.1 and 1 μg/mL) isolated from the extract of Chazgi leaves are components that increase cGMP content related to relaxation of ciliary muscle. Confirmed. Therefore, it was found that the luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide components among the compounds contained in the Chazgi leaf extract affected the eye fatigue effect.

Example 3: Human application test of eye control

1. Human body test method

This study was conducted with the approval of the Medical Device Institutional Review Board (IRB License No.: DSGOH-033) of Oriental Hospital attached to D University.

Subjects were screened for a total of 35 volunteers aged 18 and over and under the age of 60 who voluntarily agreed in writing after receiving the explanation of this study. If there is no congenital or chronic disease, there are no pathological symptoms or findings as a result of medical examination, and the blood test and vital signs are within the normal range, and the equivalent spherical refractive error is ??3.00D or more. A total of 30 people were selected. In this study, Chazgi extract and placebo control group were consumed for 1 week, and close-range work (VDT) was performed for 2 hours before the last intake.

2. Controlled proximal examination

The control point is the first time with the'Push-up' method covered with one eye at a distance of 40 cm after correcting the distance refraction abnormality and then keeping a close eye on the number of short-range targets and pulling it closer to the uncovered eyes of the subject. The distance of the blurring condition was measured. After examining the right eye and the left eye, both eyes were examined, and the average value measured three times was used.

Table 1 shows the amount of change in the control point before and after intake of the Chazgi leaf extract group and the placebo group. The results of comparing the average control point change after Chajgi's hot water extract group and placebo group after performing visual short-range work for 2 hours before and after intake are as follows. In the right eye of the Chazgi hydrothermal extract group, the control roots decreased statistically significantly from 8.68±2.98 cm before ingestion to 7.83±3.08 cm after ingestion (p<0.001). In the left eye, the control point decreased significantly from 8.38±3.13 cm before ingestion to 7.67±3.21 cm after ingestion (p<0.001). In both eyes, control points decreased statistically significantly before and after ingestion from 7.96±2.97 cm before intake to 7.27±3.25 cm after ingestion (p<0.001).

In the right eye of the placebo group, there was a statistically significant control point increase from 8.90±2.60 cm before ingestion to 9.63±2.40 cm after ingestion (p<0.001). In the left eye, the control point increased significantly from 8.60±2.49 cm before ingestion to 9.43±2.42 cm after ingestion (p<0.001), and in both eyes, from 9.00±2.45 cm before ingestion to 9.67±2.48 cm after ingestion, and statistically before and after ingestion. As a result, the control point was significantly increased (p<0.001).

Changes in the control point before and after intake of the Chazgi leaf extract group and the placebo group Control Point (cm) Before After Change t-test p-value A Right eye 8.68±2.98 7.83±3.08 -0.85±1.32 t = -3.53 p = 0.00 ^* Left eye 8.38±3.13 7.67±3.21 -0.71±1.21 t = -3.20 p = 0.00 ^* Binocular 7.96±2.97 7.27±3.25 -0.69±1.46 t = -2.60 p = 0.01 ^* B Right eye 8.90±2.60 9.63±2.40 +0.73±0.79 t = 5.12 p = 0.00 ^* Left eye 8.60±2.49 9.43±2.42 +0.83±1.12 t = 4.09 p = 0.00 ^* Binocular 9.00±2.45 9.67±2.48 +0.67±0.84 t = 4.33 p = 0.00 ^*

Unit: cm, ^* : P<0.05 A: Chazgi extract group B: placebo control group

By using the luteolin-7-O-diglucuronide and apigenin-7-O-diglucuronide compounds contained in Chazgi leaf extract, a natural resource in Korea, as an active ingredient, eye fatigue containing these compounds that can be safely used without side effects even after long-term use It can be usefully used as a pharmaceutical composition and health functional food composition for improving vision with improvement of eyesight by improving and confirming the equivalence of lyophilized and spray dried compounds, reducing manufacturing production cost and increasing import substitution and farm income through industrialization You can expect.

Claims (7)

Pharmaceutical composition for improving ophthalmic symptoms of VDT (Visual Display Terminal) syndrome comprising luteolin-7-O-diglucuronide or apigenin-7-O-diglucuronide compound obtained from Chazgi leaf extract as an active ingredient According to claim 1, wherein the compound is chazgi leaf extract is separated and purified through freeze-drying or spray-drying VDT (Visual Display Terminal) Syndrome for improving the symptoms of the pharmaceutical composition The method of claim 1, wherein the compound is isolated and purified from water extract of chagigi leaf VDT (Visual Display Terminal) syndrome pharmaceutical composition for improving eye symptoms The method of claim 3, wherein the compound is isolated and purified by adding Chajgi water extract to the Diaion HP-20 resin column, pharmaceutical composition for improving the symptoms of VDT (Visual Display Terminal) syndrome delete According to claim 1, wherein the compound is a pharmaceutical composition for improving the ophthalmic symptoms of VDT (Visual Display Terminal) syndrome having an activity to increase the cGMP content of ciliary muscle cells According to claim 1, wherein the pharmaceutical composition is a pharmaceutical composition for improving the symptoms of VDT (Visual Display Terminal) syndrome consisting of a formulation selected from tablets, capsules, liquids

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