KR100896700B1 - A.tuberosum Rottl. extract having an activity of activating choline acetyltransferase in brain nerve cells - Google Patents

Abstract

The present invention relates to a novel use of leek extract, more specifically leek extract having a brain neuron cholineacetyltransferas (ChAT) activation function, low molecular weight having a choline acetyltransferase activation function isolated from the leek extract The present invention relates to flavonoids, 4-hydroxy-3-methoxycinnamic acid and ferulic acid, and a purification method thereof.

The present invention isolated and purified ferulic acid, a brain neuron cholineacetyltransferase activating substance, from leek, a domestic edible biological resource that has never been studied for brain signal cells, and improved the efficiency of the purification process. Therefore, the low molecular weight active ingredient included in the leek extract can be usefully used as a new material of functional food or medicine for the treatment and prevention of brain neurological diseases.

Description

Leek extract having brain neuron cholineacetyltransferase activation function A.tuberosum Rottl. extract having an activity of activating choline acetyltransferase in brain nerve cells}

1 is a flow chart briefly illustrating a process for separating and purifying the active ingredient of the present invention from leek.

Figure 2 is a graph comparing the ChAT activity of methanol and ethanol extract of leek.

3 is a graph comparing ChAT activity according to the organic solvent fraction of leek.

Figure 4 is a graph showing the effect of ChAT activation by fractions of the active ingredient of the present invention by primary open silica gel chromatography.

5 is a graph showing the effect of ChAT activation by fractions of the active ingredient of the present invention by secondary open silica gel chromatography.

6 is a chromatogram showing purification confirmation using high pressure liquid chromatography (HPLC) of a low molecular flavonoid which is a final active material of the present invention.

FIG. 7 is a graph showing the dose dependent ChAT activity of ferulic acid isolated from leek and compared with commercial ferulic acid.

8 is a graph showing the protective effect of leek extract on TMT-induced learning and memory impaired mice.

9 is a graph showing the protective effect of leek extract on TMT-induced learning and memory impaired mice.

The present invention relates to a novel use of leek extract, more specifically, a low molecular weight having a ChAT activation function isolated from leek extract having a choline acetyltransferas (ChAT) activation function, a brain neuronal cell signaling synthetase The present invention relates to a separation of trace materials and a purification method thereof.

ChAT is known to activate the production of acetylcholine, a neuronal cell signaling material in the brain. The basal forebrain of the brain contains a large number of cholinergic neurons, which appear to be responsible for cognitive abilities. In patients with senile dementia, the amount of ChAT decreases rapidly, and the activity of ChAT decreases and the acetylcholine concentration decreases. ChAT is an enzyme that produces acetylcholine from choline and acetyl-CoA in the pre-synaptic part of the nerve and studies to administer nerve growth factor (NGF) or neurotrophin to enhance this activity. There is also progress, but it has problems with drug delivery. The human brain is protected by the blood brain-barrier, which exhibits selective permeability, making it difficult to penetrate large molecular weights (eg proteins, fats, etc.). Therefore, there is a need to solve the problem of drug delivery by searching for low molecular weight substances that can penetrate the meninges. Therefore, this study aims to efficiently isolate and purify low molecular weight trace elements that promote the activity of synthetase of neuronal neurons from edible plant resources and materialize them into raw materials of functional foods and pharmaceuticals.

To date, unlike cholinesterase inhibitors (ChEIs), there are no FDA-approved drugs reported as ChAT activators to improve patient cognition. To date, the treatment of dementia is only to relieve symptoms or slow down the progression of the disease, and the disease itself cannot be cured. Geriatric dementia is most likely to occur in elderly people over 65 years old, and according to the growing number of elderly people, senile dementia will become increasingly important in public health problems. . The anti-dementia activating components reported to date are mainly plant-derived low-molecular-weight and synthetic substances, which are frequently used as drugs for improving cognitive symptoms. As the first generation of ChEIs, tacrine, which was first approved for anti-dementia, has to be administered four times a day due to its short duration of action. The second-generation ChEIs, which are recently attracting attention, can be taken once a day with donepezil, developed in Japan, approved by the US FDA in late 1996, and sold in over 30 countries since 1997, and reduced peripheral side effects with selective inhibition. Rivastigmine is a drug developed by Novartis in the United States, approved in December 1997 in Switzerland, used in the EU and South American countries, and in preparation for approval in the United States and Canada, and introduced in September 1997 in Korea. Rivastigmine can be taken twice a day and has high specificity in the central nervous system, which significantly reduces peripheral side effects and is reported to have little liver toxicity as it is metabolized in the kidney. Metrifonate is undergoing a phase 3 clinical trial in patients with dementia and has a long duration of action as irreversible AChEIs. Another antidementia activator is ChAT activator, but as many substances as ChEIs have not been developed, there are currently no FDA-approved drugs. In Japan, ChAT activator in herbal medicine and domestic anti-dementia in Korea have not been systematically studied. Although complete treatment is not currently possible for senile dementia, efforts are being made to improve cognitive and noncognitive symptoms in patients with early-to-medium dementia.

Recently, various tonics using botanicals, botanical medicine, dietary supplement, etc. have already formed a large market, and various foods using green tea are being developed. The increase in consumer demand for them is because their effects do not appear as fast as pharmaceuticals, but they have the perception that they are effective in preventing aging diseases such as degenerative chronic diseases such as dementia through steady intake. Koreans have traditionally enjoyed tea, vegetables and other foods and medicinal products, as well as many oral and reports on their pharmacology and physiological activity. Therefore, the study of active ingredients contained in edible biological resources will be actively conducted in the future. In particular, this field research is likely to find new compounds having specific structures and functions in relatively unexplored fields compared to other biological materials. Industrial applications of extracts from food and medicinal plants containing physiological and pharmacological activities can take various forms, from pharmaceuticals to functional foods, depending on the degree of purification. As such, the low molecular weight trace elements having a ChAT activation function may be used as functional foods and adjuvant as a material for improving brain function and preventing dementia.

The present invention was achieved by confirming the high ChAT activation function in the leek extract and purifying the active material from the leek extract during the study of the availability as a functional food of the edible plant used as a food material.

Therefore, the main object of the present invention is to provide leek extract as a ChAT activator present in brain neurons.

Another object of the present invention is to provide a ChAT activated flavonoid purified from leek extract.

Still another object of the present invention is to provide a method for purifying the ChAT activated flavonoid from leek extract.

The present invention is a food plant resource that has been used as a food for a long time in searching for phytochemicals for the prevention and treatment of brain diseases in which aging diseases are rapidly increasing in edible plants and materializing them as raw materials of medicinal and functional foods. In order to secure the safety of the final material, we aimed to increase the probability of becoming a functional food material. For this purpose, we searched for a microorganism from leek, a native edible plant, to search for micromaterials that promote the activity of synthase.

In order to achieve the object of the present invention, the present invention is a brain neuron cholineacetyl containing 4-hydroxy-3-methoxycinnamic acid (4-hydroxy-3-methoxycinnamic acid, ferulic acid) as an active ingredient Provides a choline acetyltransferase (ChAT) activating composition.

[Formula 1]

In the composition of the present invention, the 4-hydroxy-3-methoxycinnamic acid (4-hydroxy-3-methoxycinnamic acid, ferulic acid) is commercially available as white crystal powder having a molecular weight of 194.19 having a CAS No. of 1135-24-6. It may be purchased as, but preferably the 4-hydroxy-3-methoxycinnamic acid (4-hydroxy-3-methoxycinnamic acid, ferulic acid) is characterized in that separated from the ethanol extract of leek.

In the composition of the present invention, the choline acetyltransferase (ChAT) is a synthase of acetylcholine, a neuronal neurotransmitter that is involved in neuropsychic functions such as learning, memory, etc. It is the most specific indicator for monitoring the functional status of cholinergic neurons. Specific loss of these cholinergic neurons is observed in Alzheimer's disease (Perry et al., 1978; Whitehouse et al., 1982). In the present invention, it was found for the first time that leek extract promotes the activity of the choline acetyltransferase.

In order to achieve the object of the present invention, the present invention provides a brain neuron choline acetyltransferase (ChAT) activating composition containing a leek extract as an active ingredient.

In the composition of the present invention, the leek extract may be extracted using water, an organic solvent or a mixed solvent thereof, which is a well-known extraction solvent for extracting conventional natural products, but preferably a lower (C1) such as methanol or ethanol of leek -5) It is characterized by an alcohol extract. Here, methanol or ethanol can also be used diluted in water at 50% -100%. The methanol or ethanol extract may be further fractionated using an organic solvent having a different polarity, and may be further purified by various chromatography.

In the composition of the present invention, the leek extract is characterized by containing 4-hydroxy-3-methoxycinnamic acid (ferulic acid), which is a low molecular flavonoids as an active ingredient.

In the composition of the present invention, preferably the leek extract or its active ingredient 4-hydroxy-3-methoxycinnamic acid (4-hydroxy-3-methoxycinnamic acid, ferulic acid) is used for the treatment and prevention of cerebral nerve disease It is characterized by. In particular, the leek extract or the active ingredient of the present invention can be effectively used for the prevention and treatment of senile dementia, such as Alzheimer's disease. In patients with senile dementia, the amount and activity of ChAT decreases rapidly, thereby lowering the concentration of acetylcholine, because the leek extract or the active ingredient of the present invention activates ChAT of brain neurons.

In addition, to achieve another object of the present invention, the present invention comprises the steps of iii) drying the leek, powdered and extracted with ethanol; Ii) sequentially fractionating the ethanol extract of the leek using hexane, chloroform and ethyl acetate; Iii) obtaining the active fraction from the chloroform fraction obtained above through a sequential first and second open silica gel column; And iii) separating the obtained active fractions using reverse pressure HPLC (high pressure liquid chromatography) to activate brain neuronal cell signaling substance synthase (ChAT) from leek extract. Provided is a method for purifying low molecular weight flavonoids, 4-hydroxy-3-methoxycinnamic acid (ferulic acid).

In the present invention, the leek extract includes all of the extracts, fractions and purified products obtained in each step of extraction, fractionation and purification, any dilution or concentrate thereof, or dried products thereof.

Leek extract which is a raw material of the present invention or an active ingredient thereof may be formulated in the form of granules, tablets, capsules or drinks by conventional methods known in the art, orally administered or parenteral administration in the form of injections. . Moreover, in order to make storage and handling easy, you may add the carrier used for normal formulation, such as dextrin and cyclodextrin, and other arbitrary preparations.

In addition, the leek extract of the present invention is appropriately administered so that the daily intake of adults 1 ~ 3000mg, 4-hydroxy-3-methoxycinnamic acid, the active ingredient of the present invention is administered so that 1 ~ 3000μg per day for adults. It is suitable. In addition, the dosage can be appropriately increased or decreased depending on age, symptoms, and the like.

The present invention was found to be highly active from the ethanol extract of leek during the search for a natural material that promotes the activity of synthesizing enzymes of neuronal cell signaling cells in 30 domestic edible plants. After drying the selected leek ( A. tuberosum Rottl.) (12kg) and grinding it finely, 1.2 kg of leek powder was shaken for 5 hours with 5 times the volume of ethanol, extracted five times, and concentrated under reduced pressure to prepare leek ethanol extract. do. Extracted crude ethanolic extract was identified as inhibitory activity by solvent partitioning according to polarity. 31 small fractions (CHCl ₃ : EtOH = 100: 0, 90:10, 80:20, 70:30, 60:40, 50 :) were subjected to 1st open silica gel column chromatography with chloroform moieties with high inhibitory activity 50, 40:60, 30:70, 20:80, 10:90, 0: 100). CHCl ₃ : EtOH = 90:10 (v / v) fractions from the fractions of 1st Open silica gel column chromatography were fractionated again using 2nd open silica gel column chromatography, and the activity was examined to determine the best fraction of CHCl. ₃ : EtOH = 96: 4 was isolated by HPLC to obtain an active material having a molecular weight of 194 m / z.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Preparation of Leek Extract and Determination of ChAT Activation Capacity Containing Brain Neurons

About 30 edible plants were tested for choline acetlytrnasferase (ChAT) activation. Among them, the methanol extract of leek (Allium tuberosum Rottl.) Showed the most significant ChAT activity effect in MC-IXC cells. In addition, the methanol and ethanol extracts of leek showed similar ChAT activity, so ethanol was selected as the extraction solvent. Figure 2 is a graph comparing the ChAT activity of methanol and ethanol extract of leek. Sample concentration was 0.25 mg / mL and ChAT activity values represent mean ± S.E (n = 3). Specifically, 1.2 kg of dried leek was dissolved in 99.5% ethanol by shaking at room temperature for 24 h. The resulting ethanol extract was filtered through Whatman filter paper. The extraction process was repeated five times. The extract was concentrated to about 210 mL in a rotary evaporator (EYELA) at 40 ° C. under reduced pressure.

In order to measure the ChAT activating ability of the leek extract (or fractions and purified products described later), 85% to 90% confluent value was obtained after growing MC-IXC cells (human brain neuroblastoma cells) producing Choline acetyltrasferase in a culture dish. When the cells were treated with Trypsin-EDTA, the cells were removed from the dish, and then cell lysis buffer (0.5% Triton X-100, 10 mM MgCl _2, 150 mM NaCl, pH 7.5) was added and homogenized. The supernatant (enzyme source) obtained after centrifugation of the homogenate was used to measure the ChAT activity. The supernatant and the reagent (50 mM K-phosphate buffer, 0.1 mM Eserine, 8 mM choline chloride, 25 M coenzyme A [acetyl-1-14C]) were mixed and incubated at 37 ° C. for 1 h. The reaction was terminated by addition of sodium tetraphenylborate. [14C] acetylcholine formed as a result of the reaction was counted using a liquid scintillation counter to determine ChAT activity. The formula for calculating ChAT activity is as follows.

Activity (%) = [(sample reaction enzyme reaction) / enzyme reaction] × 100

   Ethanol extracts of dozens of edible plant resources were searched for their activity using the ChAT activity measurement method. As a result, chives (43%), crown daisy (40%), and celery (seed) (38%) showed high activity. Showed.

        Table 1 Primary and secondary screening of ChAT activity

Scientific name Common name Primary [ChAT activity (%)] 2nd [ChAT activity (%)] .Allium tuberosum L. chinese chive 43% 15 Chrysanthemum coronarium crown daisy 40% 11.4 Apium graveolens L celery (seed) 38% 13

Example 2: Purification of ChAT-Activated Flavonoids from Leek Extract

 2-1. Sequential Organic Solvent Fractionation

To separate the ChAT active component from the leek extract, firstly, the leek ethanol extract obtained in Example 1 was fractionated using an organic solvent in the order of hexane, chloroform, ethyl acetate according to polarity. 1 is a flow chart of a fraction using an organic solvent for obtaining the active material of the present invention from leek. Since the chloroform fraction showed the most significant ChAT activity, the Chloroform fraction was separated and purified using primary and secondary silicagel column chromatography in order to obtain a ChAT active component. 3 is a graph comparing ChAT activity according to the organic solvent fraction of leek. Sample concentration was 0.25 mg / mL and ChAT activity values were expressed as mean ± S.E (n = 3). 1-3: n-hexane layers, 4-6; chloroform layers, 7-9; ethyl acetate layers.

 2-2. Primary Silica gel column chromatography

64.2 g of silica gel was packed in a glass column (2.6 × 40 cm) with chloroform. The chloroform fraction having the highest ChAT activity was evaporated at 40 ° C. and the concentrated sample (3.2 g) was dissolved in chloroform. Samples were chloroform and methanol (100: 0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10 at a flow rate of 2.5 mL / min. : 90, 0: 100). The bed volume of the solvent was 154 mL. 33 fractions were harvested and tested for choline acetyltransferase activation. Figure 4 is a graph showing the effect of ChAT activation by fractions of the active ingredient of the present invention by primary open silica-gel chromatography. The concentration of each sample was 0.25 mg / mL and the ChAT activity value was expressed as mean ± SE (n = 3). As a result, the highest fraction of 60% activity was obtained in the fifth fraction, CHCl ₃ : MeOH = 90: 10 (v / v).

 2-3. Second Silica gel column chromatography

The fifth fraction of CHCl ₃ : MeOH = 90:10 (v / v) showing the highest ChAT activation effect was evaporated at 40 ° C. to dissolve the concentrated sample (1.2 g) in chlorofom, and 23.2 g of silica gel was dissolved in CHCl. Swelled to ₃ and loaded into a 2.3 × 20 cm packed glass column. Samples were prepared by a stepwise gradient consisting of chloroform and methanol (100: 0, 96: 4, 92: 8, 90:10, 88:12, 85:15, 80:20 0: 100) at a flow rate of 2.5 mL / min. Separated. The bed volume of the solvent was 55.7 mL. 27 fractions were harvested and tested for ChAT activation effect. The concentration of each sample was 0.25 mg / mL and the ChAT activity values were expressed as mean ± SE (n = 3). 5 is a graph showing the effect of ChAT activation by fractions of the active ingredient of the present invention by secondary open silica-gel chromatography. As a result, the highest fraction was 82% in the fifth fraction CHCl ₃ : MeOH = 96: 4 (v / v).

2-4. Thin layer chromatography

Silica-gel with chloroform / methanol (88:12, v / v) solvent to separate the single substance with the fifth fraction with the highest CHAT activation effect, CHCl ₃ : MeOH = 96: 4 (v / v) It was developed by thin layer chromatography (TLC). The TLC plate was visualized under visible and ultraviolet (254 and 360 nm) to measure the Rf value. Each band from the TLC plate was scraped with ethanol and evaporated at 40 ° C., and then the ChAT activity was measured. As a result, the band with the R _f value of 0.7 to 2.2, especially 2.2 showed the highest activity, which was again evaporated at 45 ° C.

 2-5. HPLC

HPLC assay was performed to isolate the active compound from the highest activity TLC band. Using a μ-bondapakTM C ₁₈ column (3.9 × 300 mm), 0-100% methanol was scanned from 200nm to 800nm through 90 minutes linear gradient elution at 1.0 ml / min and the injection volume was 10 μl. . 6 is a chromatogram showing purification confirmation using HPLC (high pressure liquid chromatography) of a low molecular flavonoid of the final active material of the present invention. In FIG. 6, the X axis represents a delay time (min), the Y axis represents an absorbance value measured by a PDA detector on the left side, and methanol% on the right side. The delay was 25 to 35 min and the highest peak at 45 to 55% methanol concentration was detected.

2-6. Identification of final active substance

In order to identify the final active material, the highest peak of FIG. 6 was concentrated and subjected to Electron ionization (EI-MS) spectroscopy and Nuclear Magnetic Resonance (NMR) spectroscopy. For EI-MS spectroscopy, the peaks from HPLC were concentrated and dissolved in MeOH, and the spectra were recorded on a positive ion EI mass spectrometer (JMS AX505WA, JEOL, Japan). Peaks were observed in the EI-MS spectrum and the molecular weight of the active ingredient was found to be 194 m / z. ^{For 13} C-NMR spectroscopy, the peaks from HPLC are concentrated and dissolved in MeOD (methyl-d3 alcohol-dl) and run on a high resolution NMR spectrometer (Avance-600, Bruker, German) operating at 600 MHz and 25 ° C. Spectrum was recorded. ^{For 1} H-NMR spectroscopy, the peaks from HPLC are concentrated and dissolved in MeOD (methyl-d3 alcohol-dl) and run on a high resolution NMR spectrometer (Avance-600, Bruker, German) operating at 500 MHz and 25 ° C. Spectrum was recorded. ^{The 1} H / ¹³ C-NMR spectrum contains the frequency pattern of the intramolecular carbon nuclei and reflects the proximity and connectivity of adjacent atoms. ¹ H / ¹³ C-NMR chemical shifts were used to predict the chemical structure of the active ingredient. As a result of the experiment, the final active material of the present invention was identified as ferulic acid [4-hydroxy-3-methoxycinnamic acid] of the formula (1), which is a low molecular weight flavonoids having a molecular weight of about 194 m / z.

Example 3 Test of ChAT Activation Capacity of Commercial Compound (4-hydroxy-3-methoxycinnamic acid)

Ferulic acid, which is isolated from leek and ferulic acid, commercially available ferulic acid (4-hydroxy-3-methoxycinnamic acid), was used to confirm the same activating effect on ChAT as ferulic acid. The concentration-dependent treatment and the commercial compounds (4-hydroxy-3-methoxycinnamic acid, Sigma, Co, USA) were treated at the same concentration, and then the effects of ChAT activation were compared. 7 is a graph showing the dose-dependent ChAT activity of ferulic acid isolated from leek compared to commercial ferulic acid. Y values are expressed as% of control (0 mg / mL ferulic acid). FA stands for commercial ferulic acid. Ferulic acid isolated from the leek dose-dependently increased ChAT activity at concentrations of 0.05 to 4.0 mM. When comparing the ChAT activity of the isolated compound and the commercial compound, these activities were similar at 2.0 mM concentration of ferulic acid (FIG. 7). This shows that the effect of ferulic acid isolated from leek on the ChAT activity is the same as that of commercial ferulic acid.

Example 4: in vivo behavioral test of leek extract

In vivo behavioral tests were conducted on mice with ethanol extracts containing leek ethanol extract containing the final substance (Chlorohydroxy-3-methoxycinnamic acid). In other words, by using a mouse to administer a drug (trimethyltin) that inhibits memory and learning ability artificially induced memory and learning ability degradation, the effect of the active material of the present invention was administered (Y-maze test) And Passive Avoidance Test). ICR-male mice were used to measure cognitive function after one week of adaptation. Mice were divided into five groups of 12 animals per group. Table 2 below shows the composition for each experimental group. Ethanol extracts of leek (AT Rottl.) Were mixed with feed at concentrations of 400, 800 and 1200 (mg / kg BW). Mice were allowed free access to feed and water for 28 days.

[Table 2] Group composition of in vivo experiment

Normal control TMT- control A B C Basal diet + + + + + TMT-injection (2.5 mg / kg, B.W) - + + + + A.T. Rottl. (mg / kg, B.W) - - 400 800 1200

The spontaneous alternation behavior, which is regarded as a measure of immediate spatial working memory, was evaluated by the Y-maze test. No significant difference in the number of entries was found in all experimental groups. This shows that TMT treatment did not affect locomotor ability in all mice. However, mice treated with TMT alone showed significantly reduced shift behavior (about 21%) compared to the normal control. However, mice treated with leek extract showed about 14% -28% increased shift behavior compared to the TMT control (FIG. 8). 8 is a graph showing the protective effect of leek extract on TMT-induced learning and memory impaired mice. Y-axis is Alternative behavior (%).

In passive avoidance performance, TMT control mice showed significantly shorter latency times (52% reduction in step through latency) in retention trials compared to normal controls. However, when the leek extract was treated for 28 days, passive avoidance performance was significantly improved. That is, the step through latency was increased by about 33% to 43% compared to the TMT-control. The higher weight of retention time resulted in alleviation of TMT-induced memory impairment.) (FIG. 9). 9 is a graph showing the protective effect of leek extract on TMT-induced learning and memory impaired mice. The y-axis represents the step-through latency (%) in retention trials of passive evasion performance. From the experimental results, it can be seen that the leek extract of the present invention is effective in the recovery of memory and learning ability and prevention of cognitive disorders.

As described above, the present invention provides a brain neuron-containing ChAT-activated flavonoids isolated from leek extract and its purification method. ChAT-activated flavonoids of the present invention are purely separated from leek ethanol extract by a series of processes through organic solvent fractionation, silica gel column chromatography, and HPLC (high pressure liquid chromatography). The trace element thus separated was analyzed by EI-MS and ¹ H / ¹³ C NMR. The molecular weight was 194 m / z and it was found to be ferulic acid of low molecular weight flavonoids. Due to the low molecular weight, permeability is thought to be good. Therefore, it has a memory and learning-promoting effect. Therefore, it can be used as a drug for senile dementia or memory loss, and as a functional food.

Claims (5)

delete delete delete delete Iii) drying the leek to powder and extracting with ethanol; Ii) sequentially fractionating the ethanol extract of the leek using hexane, chloroform and ethyl acetate; Iii) obtaining the active fraction from the chloroform fraction obtained above through a sequential first and second open silica gel column; And, Iii) separating the obtained active fraction from the leek extract, comprising separating the active fraction at a delay time of 25 to 35 minutes (min) and methanol concentration of 45 to 55% using reverse phase HPLC (high pressure liquid chromatography). A method for purifying brain neuronal signaling substance synthetase (choline acetyltransferase (ChAT) activated 4-hydroxy-3-methoxycinnamic acid, ferulic acid).

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