KR101317551B1 - Composition with liver protective effect and preventing and/or treating liver disease comprising extract of leaves of perilla frutescens - Google Patents

Abstract

The present invention relates to a composition for protecting liver and preventing or treating liver disease containing a functional perilla leaf extract.

Description

Composition with liver protective effect and preventing and / or treating liver disease comprising extract of leaves of perilla frutescens}

The present invention relates to a composition for protecting liver and preventing or treating liver disease containing a functional perilla leaf extract.

Perilla is an annual herb of Lamiaceae, and its origin is the eastern Asian plateau including the Korean peninsula. Perilla is a crop grown to squeeze oil. It is sesame leaves that harvest and edible leaves during growth. It was recorded for the first time in the agricultural budget (1273), and its cultivation history was longer than that of sesame seeds, and in the old books there were forest, forest, plant, water, It was called Yuma and Jima.

Perilla leaves can be eaten as a side dish such as herbs, pickles, sesame leaf kimchi, or as a spice in radish or tang. In the past, perilla has been cultivated mainly for the purpose of harvesting seed oil, but the recent growth of meat consumption, the development of the food culture, and the rapid growth of the ssam vegetable consumption market caused by the well-being craze have allowed the development of varieties for perilla and its production all year round. Therefore, it is expected that industrial value will be great if it reveals the medical function of perilla leaves and applies them to food and pharmaceuticals in various ways.

Perilla is our nation's traditional food. Perilla oil, perilla and wild perilla leaves have kept our health, and the perilla leaf, which gives our unique flavor, is the representative food that our nation enjoys and enjoys. Although it can be done, it is recognized as a low-cost income crop because of the consumption form that is consumed only by raw food and the storage of surplus at the harvest time.

Perilla aldehyde, rosemary acid, anthocyanin, caffeic acid, and the like have been reported as a substance indicating the perilla leaves. Since these ingredients have high water solubility, high intake rates can be expected when ingested.

Meanwhile, the mortality rate of liver disease and liver cancer in Korea accounts for 10% of all deaths. According to the 2005 death cause statistics published by the National Statistical Office, liver disease is ranked 6th among domestic causes of death.

However, hepatic medicines prescribed in hospitals or on the market are usually those that have preventive and therapeutic effects on acute liver injury in animals or clinical trials, most of which are synthetic chemicals that reduce damage to liver cells rather than fundamentally treat the disease. It only serves as an adjuvant treatment in the sense of doing so.

There is a need for the development of a technology to find liver protection and liver function improvement drugs from natural materials other than synthetic chemicals.

In response to these needs, various techniques for treating liver cancer and liver disease have been developed using extracts of conventional plants, for example, mushroom extracts, garlic extracts and bean sprout extracts. The prior art relates to the manufacture of medicines or functional foods with an active ingredient as a food extract exhibiting a hepatoprotective effect, and these food extracts ultimately exhibit a hepatoprotective action by significantly increasing serum ALT and AST levels.

The present invention has been made in view of the above necessity, and an object of the present invention is to provide a cultivation method for increasing the content of caffeic acid having liver protection and liver disease prevention activity.

Another object of the present invention is to provide a food composition and a pharmaceutical composition having liver protection and liver disease prevention activity.

In order to achieve the above object, the present invention provides a pharmaceutical composition for liver protection and liver disease prevention and treatment comprising perilla leaf extract.

In one embodiment of the present invention, the perilla leaves are preferably cultivated using sugar water, the concentration of the sugar water is more preferably in the range of 4% to 8% concentration, and most preferably 6% concentration It is not limited.

In another aspect, the present invention provides a functional food composition for protecting the liver comprising perilla leaf extract.

In another aspect, the present invention provides a method for producing perilla leaves with increased caffeic acid content using sugar water.

The present invention also provides a pharmaceutical composition for liver protection and liver disease prevention and treatment comprising caffeic acid as an active ingredient.

The present invention also provides a functional food composition for liver protection comprising caffeic acid as an active ingredient.

When using the compound or extract of the present invention as a medicine, a compound prepared by mixing the compound or extract of the present invention with a suitable additive is usually used.

As the additives, excipients, binders, lubricants, disintegrants, coloring agents, copulation agents, emulsifiers, surfactants, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, stabilizers, absorption accelerators, which are generally used in medicine These etc. can be mentioned, You can use combining these suitably as needed.

Examples of the excipient include lactose, white sugar, glucose, corn starch, mannitol, sorbitol, starch, α-starch, dextrin, crystalline cellulose, hard anhydrous silicic acid, aluminum silicate, calcium silicate, magnesium aluminate silicate and calcium hydrogen phosphate. There is,

Examples of the binder include polyvinyl alcohol, methyl cellulose, ethyl cellulose, gum arabic, tragacanth, gelatin, shellac, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose sodium, polyvinylpyrrolidone, macrogol and the like. Can be

Examples of the lubricant include magnesium stearate, calcium stearate, sodium stearyl fumarate, talc, polyethylene glycol, colloidal silica, and the like.

Examples of the disintegrating agent include crystalline cellulose, agar, gelatin, calcium carbonate, sodium hydrogen carbonate, calcium citrate, dextrin, pectin, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, sodium carcamelloose and carboxymethyl Starch, carboxymethyl starch sodium, and the like.

Examples of the colorant include those which are permitted to be added to medicines such as iron trioxide, yellow iron trioxide, carmine, caramel, β-carotene, titanium oxide, talc, sodium riboflavin sodium and yellow aluminum lake.

Cocoa powder, peppermint brain, aromatic acid, peppermint oil, cedar, cinnamon powder, etc. are mentioned as said copulation agent,

Examples of the emulsifiers or surfactants include stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, glycerin monostearate, sucrose fatty acid esters, glycerin fatty acid esters, and the like.

Examples of the dissolution aid include polyethylene glycol, propylene glycol, benzyl benzoate, ethanol, cholesterol, triethanolamine, sodium carbonate, sodium citrate, polysorbate 80, nicotinic acid amide, and the like.

Examples of the suspending agent include hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose, in addition to the surfactant.

Examples of the tonicity agent include glucose, sodium chloride, mannitol, sorbitol, and the like.

Examples of the buffer include buffers such as phosphate, acetate, carbonate and citrate,

Examples of the preservative include methyl paraben, propyl paraben, chlorobutanol, benzyl alcohol, phenethyl alcohol, dihydroacetic acid, sorbic acid, and the like.

Examples of the antioxidant include sulfite, ascorbic acid and α-tocopherol.

As said stabilizer, what is generally used for medicine is mentioned.

As said absorption promoter, what is generally used for medicine is mentioned.

In addition, the preparations include, for example, oral preparations such as tablets, powders, granules, capsules, syrups, troches, inhalants; Examples include suppositories, ointments, eye ointments, tapes, eye drops, eye drops, ear drops, pape, lotions such as lotions, or injections.

The oral formulation is formulated by combining the additives as appropriate. Moreover, you may coat these surfaces as needed.

The external preparations are formulated by appropriate combination of excipients, binders, copulation agents, emulsifiers, surfactants, dissolution aids, suspending agents, isotonic agents, preservatives, antioxidants, stabilizers or absorption accelerators, among others.

The injectables are formulated in appropriate combination of the above additives, in particular emulsifiers, surfactants, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, stabilizers or absorption promoters.

When the compound or extract according to the present invention is used as a medicament, its amount varies depending on symptoms and age, but usually in the case of oral preparations. 0.1 mg to 10 g (preferably 1 mg to 2 g), 0.01 mg to 10 g (preferably 0.1 mg to 2 g) for external preparations, 0.01 mg to 10 g (preferably for injections) 0.1 mg to 2 g) is used once daily or divided into 2 to 4 times.

The present invention provides a health functional food for the prevention and improvement of liver disease containing the caffeic acid or perilla leaf extract of the present invention as an active ingredient having the effect of preventing and improving liver disease. Examples of the food to which the caffeic acid or the extract of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of preventing the liver disease. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids.

The health functional beverage composition of the present invention is not particularly limited to the other ingredients other than the above-mentioned compounds as essential ingredients in the indicated ratios and may contain various flavors or natural carbohydrates as additional ingredients such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the caffeic acid or extract of the present invention may be used in various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors such as flavoring agents, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, Alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like.

The compounds of the present invention may contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the caffeic acid or extract of the present invention.

Hereinafter, the present invention will be described.

The present invention provides a step of cultivating perilla leaves using sugar water.

In addition, the extraction step of the present invention

Performing reflux cooling with distilled water on the dried perilla leaves;

Centrifuging the extract obtained from the reflux cooling extraction step to obtain a supernatant;

The supernatant filtrate is filtered under reduced pressure and lyophilized to provide a step to obtain perilla leaf extract powder.

According to a preferred embodiment of the present invention, the reflux cooling extraction step is repeated twice the extraction process for 3 hours to 4 hours at a temperature of 100 ℃ to 110 ℃ with distilled water of 2.8 liter to 3.2 liter volume of dried perilla leaves 100g This can be done by.

According to another preferred embodiment of the present invention, the centrifugation step may be performed for 25 to 30 minutes at a temperature of 7,500rpm at a temperature of 4 ℃.

Hereinafter, the present invention will be described in detail.

Caffeic acid contained in perilla leaves is known to have an anti-cancer effect because the absorption rate of the small intestine is more than 95% when consumed, and the caffeic acid has a strong antioxidant activity. In addition, the present invention has been found to have an effect of increasing the caffeic acid having a hepatoprotective effect from perilla leaves cultivated using sugar water, on the basis of this provides a method for producing an effective and simple extract from functional perilla leaves I would like to.

According to the present invention, extracts can be prepared from the perilla leaves grown using sugar water in a simple and inexpensive manner, and the food and pharmaceutical compositions comprising the prepared extracts are widely used for liver protection and prevention and treatment of liver diseases. It can be utilized.

Figure 1 shows the antioxidant capacity of perilla leaves when grown with sugar water obtained by the method according to the present invention. It can be seen that the antioxidant activity is high when 6% sugar water is used.
Figure 2 shows the content of caffeic acid, a functional substance of perilla leaf extract when grown with sugar water. It can be seen that the antioxidant activity is high when 6% sugar water is used.
Figure 3 shows the inhibition of oxidative stress of Perilla leaf extract when oxidative stress is induced in HepG2 cells, liver cells. It can be seen that the antioxidant activity is high when 6% sugar water is used. As a control, normal perilla leaves not grown in sugar water were used.
Figure 4 shows the content of glutathione, an antioxidant substance in cells when treated with perilla leaf extract in HepG2 cells, liver cells. It can be seen that the content of glutathione is high when 6% sugar water is used.
Figure 5 shows the inhibition of lipid peroxidation when oxidative stress is induced in HepG2 cells, liver cells.
Figure 6 shows the content of glutathione, an intracellular antioxidant, when orally administered perilla leaf extract in rats.
Figure 7 shows the ability to inhibit lipid peroxidation caused by oxidative stress when oral administration of perilla leaf extract in rats.
Figure 8 shows the inhibition of liver tissue damage due to oxidative stress when oral administration of perilla leaf extract in rats.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are described for the purpose of illustrating the present invention, the scope of the present invention is not to be construed as limited by the following examples.

Example  1. Perilla leaves with sugar water ( perilla frutescens Cultivate

The Miryang Agricultural Technology Center is sowing in September 2009 and harvesting 4%, 6%, and 8% sugar water after irrigation to harvest perilla leaves.

Example  2. Functional Perilla Leaves perilla frutescens Extraction of hepatoprotective substances from

Dried perilla leaves frutescens ) 100g was obtained by reflux cooling for 3 hours using 3 L of distilled water and then centrifuged at 7,500 rpm, 30 min, 4 ℃ to obtain a supernatant. To remove insoluble matter that may remain in the centrifuged supernatant, replicate the supernatant again with whatman No. 42 < / RTI > The filtered supernatant was distilled under reduced pressure and then lyophilized to extract powdered perilla leaves (plain perilla leaf PLE-I, 4% perilla perilla perilla PLE-II, 6% perilla perilla perilla PLE-III, 8% perilla perilla perilla PLE-IV).

Experimental Example  Comparison of Antioxidant Activity between Perilla Leaf Extract and Functional Perilla Leaf Extract

Total polyphenol content was quantified using Folin-Denis method. That is, add 0.25 mL of folin-ciocalteu reagent to 100 μL of the extract, dilute 0.4 mL of distilled water, add 1.25 mL of 20% Na ₂ CO ₃ saturated solution, mix well, and leave at room temperature for 40 minutes. Absorbance was measured at 725 nm. After preparing different concentrations of gallic acid as a standard, a standard curve was prepared and calculated.

The total flavonoid content was measured at an absorbance value of 430 nm after 100 μL of 2% AlCl ₃ 6H ₂ O solution was added to 100 μL of each extract and left for 5 minutes. Quercetin dihydrate was used as a standard.

2.5 mL of 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) dissolved in 40 mM HCl as a FRAP reagent was added 2.5 mL of 20 mM FeCl ₃ .6H ₂ O and 25 mL of 0.3 M acetate buffer at pH 3.6. It was. Then, 100 μL of perilla perilla leaf extract was mixed in 3 mL of FRAP reagent, and after 5 minutes at 37 ° C., absorbance was measured at 593 nm and calculated using FeSO ₄ · 7H ₂ O as a standard curve.

Radical scavenging property measured the scavenging ability of the free radical DPPH. 100 μL of each sample was added to 100 μL of 200 μM DPPH reagent dissolved in ethanol, mixed well, and stored for 30 minutes in a dark state. The change of absorbance with time was measured at 515 nm, and ascorbic acid was used as a control. SC ₅₀ (scavenging activity 50%) of each sample was the concentration of the sample required to reduce the concentration of DPPH by 50%.

Radical scavenging property also measured scavenging ability for ABTS. ABTS reagent was mixed with 7 mM ABTS and 2.45 mM potassium persulfate and left in the dark for one day before use. 20 μL of the concentration-specific sample was added to 180 μL of ABTS reagent and stored for 6 minutes in the dark. Then, the change of absorbance was measured at 734 nm. SC ₅₀ (scavenging activity 50%) of each sample was the concentration of the sample required to reduce the concentration of ABTS 50%.

Table 1 shows the results of evaluation of antioxidant activity for each group. Functional perilla leaf extract cultivated with 6% sugar water showed higher antioxidant activity than general perilla leaf extract and functional perilla leaf extract using 4% and 8% sugar water.

Experimental Example  2. Determination of Hepatoprotective Marker Contents of Perilla Leaf Extract and Functional Perilla Leaf Extract

The content of caffeic acid, known as a hepatoprotective marker, from perilla leaf extract and functional perilla leaf extract was measured by HPLC. Table 2 shows the HPLC analysis conditions for measuring caffeic acid from the extract.

Kinds Varian Prostar column Waters XTerra RP18 Column, 5 μm (4.6 × 150 mm) Mobile phase A: 0.05% trifluoroacetic acid B: MeOH
Gradient: A: B = 100: 0 → 0: 100 Residence time 9 mins Flow rate 1 mL / min Detector 340 nm

As can be seen in Figure 2, it was confirmed that the higher content of caffeine in the functional perilla leaf extract grown with sugar water than the common perilla leaf extract, the highest caffeic acid content in the functional perilla leaf extract grown with 6% sugar water Confirmed.

Experimental Example  2. Perilla Leaf Extract and Functional Perilla Leaf Extract MTT assay Through Liver protection  Active comparison

After inducing oxidative damage with t -BHP ( tert -butyl hydroperoxide) in HepG2 cells, a hepatocyte-derived cell, perilla leaf extract with 6% sugar water and wild perilla extract, which is expected to prevent oxidative damage Hepatoprotective effect of the extract was confirmed through MTT analysis (cell viability, cell viability) (see Figure 3). Perilla leaf extract and functional perilla leaf extract were confirmed to have a liver protective effect. However, it can be seen that the hepatoprotective activity of the functional perilla leaf extract cultivated using 6% sugar water was higher than the general perilla leaf extract.

Experimental Example  3. Lipid Peroxidation Inhibitory Activity of Perilla Leaf Extract and Functional Perilla Leaf Extract GSH  Through content measurement Liver protection  Active comparison

After inducing oxidative damage with t -BHP ( tert -butyl hydroperoxide) in HepG2 cells, a hepatocyte-derived cell, perilla leaf extract with 6% sugar water and wild perilla extract, which is expected to prevent oxidative damage Lipid peroxidation inhibitory activity of the extracts was measured. It was confirmed that lipid peroxidation of the control group was increased due to oxidative damage by t- BHP, but lipid peroxidation was inhibited when the perilla leaf extract and the functional perilla leaf extract were treated. However, it was confirmed that the lipid peroxidation was suppressed higher than the general perilla leaf extract when the functional perilla leaf extract was treated.

GSH contents related to intracellular hepatoprotective activity were measured when HepG2 cells, a hepatocyte-derived cell, were treated with perilla leaf extract and functional perilla leaf extract. Compared with the control group, the GSH content was increased compared to the control group when the general perilla leaf and the functional perilla leaf extract were treated, and when the functional perilla leaf extract was treated, the GSH content was increased by a significant level difference. (See FIG. 4).

Experimental Example  4. t - BHP Of Perilla Leaf Extract and Functional Perilla Leaf Extract in Hepatic Impairment Liver protection  Active comparison

After the extract and the deulkkaetip t untreated control group, the group treated only with t -BHP, deulkkaetip extract 1000 mg / kg not treated with -BHP according to the present invention as an experimental animal by setting a group of six rats was added to the diet t The group treated with -BHP, 1000 mg / kg functional perilla leaf extract according to the present invention was added to the diet and then divided into t- BHP-treated groups to set four groups. t- BHP was used for the purpose of damaging the liver of rats. After controlling diet for 5 days, 0.5 mmol / kg of t- BHP was treated in the group except the untreated control group, and then dissected after 18 hours, and ALT, AST and LDH, which are indicators of liver damage, were measured. Lipid peroxidation and GSH were measured. In addition, hematoxylin & eosin (H & E) staning was used to determine the extent of liver tissue damage.

Table 3 shows the evaluation results for each group. Referring to Table 2 below, AST, ALT, LDH in the group treated with 0.5 mmol / kg of t- BHP increased compared to the group not treated, but decreased in the group treated with perilla leaf extract 1000 mg / kg. It can be seen that in the group treated with 1000 mg / kg functional perilla leaf extract according to the present invention can be seen to decrease to a level similar to the control. In addition, referring to Table 3 below, lipid peroxidation of liver tissue was increased in the group treated with 0.5 mmol / kg t-BHP, but the group treated with perilla leaf extract and functional perilla leaf extract according to the present invention. It can be seen that the decrease. GSH content of liver tissue was decreased in the t-BHP treated group, but GSH was increased in the group treated with perilla leaf extract and the functional perilla leaf extract according to the present invention. In addition, comparing the wild perilla leaf extract and the functional perilla leaf extract according to the present invention, the AST, ALT, and LDH of the blood were confirmed to have high hepatoprotective activity in the functional perilla leaf extract, and lipid peroxidation and GSH content of liver tissue were also functional perilla leaves according to the present invention. When the extract was treated, it showed significantly higher hepatoprotective activity than common perilla leaves.

Therefore, it can be seen that the functional perilla leaf extract according to the present invention has better liver protection effect than normal perilla leaf extract against liver damage caused by t-BHP treatment.

Claims (10)

delete delete delete delete delete delete A method for producing perilla perilla leaves with increased caffeic acid content, the step of irrigating sugar water after planting perilla.  8. The method of claim 7, wherein the concentration of sugar water is in the range of 4% to 8% concentration. delete delete

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