KR100436428B1 - Extract of the seeds of nelumbo nucifera gaertn. having liver cell protective effect and liver injury preventive or therapeutic effect, and a composition containing same - Google Patents

Abstract

The present invention is an extract of the yeonja (nelumbo nucifera Gaertn.) Extract and hepatocellular protection, liver damage prevention and treatment containing the same as an active ingredient, and for inhibiting mutation by liver cancer-causing agents The present invention relates to a composition, wherein the extract of the lotus root is safe to the human body while showing excellent liver protection, and may be usefully used for protecting liver cells and preventing or treating liver damage.

Description

Extract of lotus root meat and composition comprising the same showing hepatocellular protection and prevention or treatment of liver damage [EXTRACT OF THE SEEDS OF NELUMBO NUCIFERA GAERTN. HAVING LIVER CELL PROTECTIVE EFFECT AND LIVER INJURY PREVENTIVE OR THERAPEUTIC EFFECT, AND A COMPOSITION CONTAINING SAME}

The present invention is for extracting the medicinal herb (Mature fruit of Nelumbo nucifera Gaertn.) Extract and hepatocellular protection, liver damage prevention and treatment containing the same as an active ingredient, and for inhibiting mutation by liver cancer-causing agents The present invention relates to a composition, wherein the extract of the lotus root is safe to the human body while showing excellent liver protection, and may be usefully used for protecting liver cells and preventing or treating liver damage.

The liver is located between the digestive system and the circulatory system of the human body to perform the function of protecting the whole body from in vitro substances from outside. Once in vivo, the in vitro material is passed through the liver, so the liver is more at risk of being exposed to many toxic substances in addition to nutrients, which is more likely to be damaged. The liver is an organ with excellent regenerative capacity, and if it is damaged slightly, it will return to normal, but if the damage persists, part of the liver tissue will be completely destroyed and liver function will be reduced. . When these liver damage becomes chronic, liver fibrosis or cirrhosis of the liver, regardless of the cause progresses to liver cancer. Liver fibrosis, cirrhosis, liver cancer, etc. are currently a disease that does not have a clear treatment and treatment, the mortality rate is also a disease. Therefore, preventing and treating liver damage before it becomes chronic is very important to inhibit the progression of liver fibrosis, cirrhosis or liver cancer.

Currently used therapeutic agents for liver disease include silymarin and biphenyl dimethyl dicarboxylate (DDB). Silymarin is a new drug prepared by extracting and separating thistle from Silybum marianum seeds, and DDB has been isolated from Schizandra chinensis at the Chinese Institute of Chinese Medicine (Beijing) since 1980. It is widely used in therapy. In the case of acute liver disease, rest and a high protein diet and a sufficient supply of vitamins are mainly used, and pharmacotherapy that is burdened by the liver is preferably avoided. Therefore, the drugs such as silymarin, DDB, etc. are not used as a prophylactic agent but as a therapeutic agent, i.e., only when there is a need for treatment of hepatitis.

The liver is a silent organ, with no symptoms in the early stages of the disease, but only when the degree of liver damage is severe. Accordingly, in recent years, efforts have been made to find natural substances that can easily be prevented as well as to prevent liver damage without burdening the liver.

Carbon tetrachloride (CCl ₄ ) is a hepatotoxic sample that is widely used to study liver toxicity using liver cell culture, liver slice culture, and direct administration to mice or rats. Carbon tetrachloride is a liver cytochrome (cytochrome) is metabolized by metabolic enzymes such as P ₄₅₀ is a very reactive form a strong free radical CCl _3, and Cl free radical CCl ₃ that the complex function in the liver is the accumulated triglycerides or Oxidation of fatty acids in membrane phospholipids leads to rancidity of lipids, which combine with oxygen to form organic peroxides through lipid peroxidation. Lipid peroxidation then accumulates fat in the liver, reduces protein synthesis, breaks down glycogen and destroys cytoplasmic enzymes in blood vessels, resulting in necrosis of liver tissue (Recknagel, RO, et al., Pharmacol. Rev. 19: 145-208, 1967; Chang, IM, et al., Drug and chemical toxicology 6 (5): 443-453, 1983).

Aflatoxin B1 (AFB ₁ ) is a secondary metabolite produced by fungi, which are parasitic in grains, and is known to humans as a carcinogenic substance about 3,750 times higher than dimethylnitrosamine and mainly targets the liver. AFB ₁ is metabolized in the body by cytochrome P ₄₅₀ to form a hepatotoxic epoxide form, which reacts with DNA, RNA, proteins, etc. in the cell to form adducts, resulting in mutations, cancers, etc. (Eaton, DL et al., Annu. Rev. Pharmacol. Toxicol. 34: 135-72, 1994).

On the other hand, lotus root is the dried fruit of the perennial lotus, Nelumbo nucifera Gaertn., Belonging to the Nymphaceae, which grows in a pond in the south-central part of our country. It is used to treat diarrhea, lowering the well, stabilizing the mind, and stopping the bleeding in Korean medicine. Non-herb diarrhea, low back pain, oil well, uterine bleeding, crayfish, and nervous breakdown. In addition, lotus roots are used for porridge and hot pot cooking as a nutritional formula for examinees or weak people (Eng-hak Dictionary, Magpie Publisher, p.700, 1998; Herbology, Korean Pharmacy Society, p.852-855, 1995; Book, p. 70, 1988).

The present inventors have found that intake is easy to study cases to find a natural substance that can prevent as well as treat the liver damage is safe to human efforts result yeonjayuk used for human consumption (Changshu a fruit of Nelumbo nucifera Gaertn.) Protect liver extract is excellent The present invention was completed by confirming that it may be useful for protecting human liver cells, preventing and treating liver damage, and inhibiting mutations caused by liver cancer causing substances.

It is an object of the present invention to provide a natural extract that is easy to ingest, safe for the human body, and can prevent as well as treat liver damage.

Another object of the present invention is to provide a composition for protecting liver cells, preventing or treating liver damage, and inhibiting mutations by a liver cancer-causing agent containing the natural extract as an active ingredient.

In order to achieve the above object, the present invention provides a lower alcohol extract of soft meat (mature fruit of Nelumbo nucifera Gaertn.) Excellent in liver protection and excellent in biosafety.

In addition, the present invention provides a pharmaceutical composition for protecting liver cells, preventing and treating liver damage, and inhibiting mutations by a liver cancer-causing agent, containing the lower alcohol extract of the soft lotus meat as an active ingredient.

Hereinafter, the present invention will be described in more detail.

Firstly, the present invention provides a lower alcohol extract of lotus root (mature fruit of Nelumbo nucifera Gaertn.) With excellent liver protection and excellent biosafety.

The extract of mature fruit of Nelumbo nucifera Gaertn. Can be obtained by extracting from a soft extract with a conventional extraction method using a lower alcohol.

The lower alcohol extraction method adds 1 to 10 times, preferably 2 to 3 times lower alcohol to the dried soft meat and heats it at 60 to 100 ° C, preferably 80 to 90 ° C for 1 to 12 hours, preferably After refluxing for 2 to 3 hours, cooling and filtration, and concentrated under reduced pressure using a rotary evaporator to obtain a viscous soft alcohol lower alcohol extract, wherein the filtration process may be repeated two or more times for the remaining residue. As the lower alcohol as the extraction solvent, methanol, aqueous methanol solution, ethanol, aqueous ethanol solution, normal propanol, iso-propanol, normal butanol, or a mixture thereof may be used, and ethanol of 60 to 80% is most preferred. In the present invention, by using 80% ethanol as a preferred embodiment to obtain the soft ethanol extract.

The lower alcohol extracts extracted from lotus root meat by the above extraction method were treated with carbon tetrachloride to cause acute liver injury. Hepatoprotective effects such as decreased levels, and hepatocytes isolated from normal rats show hepatocellular protective effects such as decreased AST levels in hepatocyte culture medium when hepatic cell damage caused by carbon tetrachloride. As a result, it was confirmed that the soft ethanol extract of the present invention does not cause liver toxicity even at high concentrations in the normal group, and has an effect of preventing and inhibiting liver damage in the experimental group induced liver damage by carbon tetrachloride. In addition, the soft ethanol extract of the present invention exhibits the effect of inhibiting mutagenicity by aflatoxin B1, a liver cancer-causing substance, and inhibiting hepatocyte damage by aflatoxin B1 in hepatocytes isolated from normal rats.

The present invention also provides a pharmaceutical composition for protecting liver cells, preventing and treating liver damage, and inhibiting mutations by liver cancer-causing agents, which contain the lower alcohol extract of the soft lotus meat as an active ingredient.

The pharmaceutical composition comprising the lotus leaf extract of the present invention as an active ingredient, which has been shown to have excellent liver protection and high human safety, is useful for protecting liver cells, preventing and treating liver damage, and inhibiting mutations by liver cancer-causing agents. Can be used.

The pharmaceutical composition of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of general drug replacement therapy.

That is, the pharmaceutical composition may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used may be used. Are prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose ( Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium, stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized replacement therapeutic suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular.

Typical daily dosages of the extract of the lotus root meat of the present invention range from 1 to 2,000 mg / kg body weight, preferably from 10 to 500 mg / kg body weight, and may be administered once or in several divided doses. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex, weight of the patient and the severity of the patient, and thus the dosage should in any way be within the scope of the invention. It is not intended to limit.

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

The following examples are merely illustrative of the present invention, but the content of the present invention is not limited by the examples.

Example 1 Preparation of Soft Potato Extract

Yin-yak (Moon fruit of Nelumbo nucifera Gaertn.) Purchased from Hongin Dangdo Pharmaceutical Co., Ltd. (Iksan, Jeonbuk) was confirmed and verified by Professor Kim Yun-chul of the College of Pharmacy, Wonkwang University. 3 g of 80% ethanol was added to 600 g of dry soft meat, refluxed for 3 hours while heating to 90 ° C., cooled and filtered, and concentrated under reduced pressure using a rotary evaporator to obtain 21.5 g of viscous soft ethanol extract.

Example 2 Determination of Liver Damage Prevention Effect of Ethanol Extract of Soft Root Meat

Male mice weighing 22-25 g (Korea Experimental Animal Center; Chungbuk Negative) were acclimated to the laboratory environment for one week and were fed with sufficient feed and water. To determine the liver damage prevention effect of soft ethanol extract, ethanol extract was dissolved in physiological saline and orally administered to mice by using sonde for 3 days. Intraperitoneal injection. The number of each group and experimental animals is as follows.

Group 1 (n = 6) normal group saline administration group

Group 2 (n = 6) soft ethanol extract administration group (25 mg / kg / day, p.o.)

Group 3 (n = 6) soft ethanol extract administration group (100 mg / kg / day, p.o.)

Group 4 (n = 6) soft ethanol extract administration group (250 mg / kg / day, p.o.)

Group 5 (n = 8) Induced liver damage by carbon tetrachloride after physiological saline administration

Group 6 (n = 8) induced liver damage by carbon tetrachloride after administration of ethanol extract (25 mg / kg / day, p.o.)

Liver damage induced by carbon tetrachloride after group 7 (n = 8) soft ethanol extract administration (100 mg / kg / day, p.o.)

Group 8 (n = 8) induced liver damage by carbon tetrachloride after administration of soft ethanol extract (250 mg / kg / day, p.o.)

Groups 1 and 5 were orally administered with the corresponding amounts of saline, and the other groups were lotus root ethanol extract using Sonde. 1 hour after the last oral administration of the extract for 3 days, 1 to 4 groups were injected with a corresponding amount of corn oil (oriental flow product), and 5 to 8 were injected once with intraperitoneal carbon tetrachloride mixed with corn oil. After 24 hours, the blood was taken from the carotid artery of the mouse, left at room temperature for at least 1 hour, and then centrifuged to obtain serum, and the serum was stored at minus 20 ° C.

Serum ALT and AST levels were measured using ARKRAY's Spotchem automatic analyzer II (Spotchem SP4410, Japan), and the results are shown in Table 1 below.

Military number ALT (IU / ℓ) AST (IU / ℓ) Military number ALT (IU / ℓ) AST (IU / ℓ) One 37 ± 12 163 ± 96 5 4269 ± 528 1675 ± 334 2 33 ± 15 148 ± 87 6 4080 ± 542 1320 ± 893 3 37 ± 12 167 ± 53 7 3910 ± 1671 1442 ± 412 4 39 ± 19 174 ± 94 8 1550 ± 792 805 ± 320

From the results of Table 1, it can be confirmed that the soft ethanol extract of the present invention has an effect of inhibiting liver damage caused by carbon tetrachloride.

Example 3 Measurement of Hepatoprotective Activity of Ethanol Extract of Soft Root Meat

This experiment was carried out to confirm the hepatocyte protective effect of ethanol extract of lotus root meat. Male rats (Korea Experimental Animal Center; Chungbuk Negative) weighing 220-250 g were adapted to the laboratory environment for 1 week and were fed with sufficient feed and water.

First, hepatocytes were isolated from normal rats by the method of Sieglen et al. (Seglen, PO, Methods Cell Biol . 13: 29-83, 1976), and then 1 × 10 ⁶ cells / column in a collagen coated 24-well plate. Three hours after adding hepatocytes to the wells, the wells were washed with HBSS to remove unbound cells and incubated again for 16 hours. Culture medium was Williams media E containing ^10-8 M Insulin, 10% FBS, ^10-6 M Dexamethasone, Penicillin (100 IU / mL), Streptomycin (100 μg / mL) (William's Media E, Gibco BRL) was used. In order to induce hepatocellular damage by carbon tetrachloride, the cells were changed to a culture medium containing 5 mM carbon tetrachloride and soft ethanol extract, and cultured for 1.5 hours, and the AST levels in the culture medium were measured. In this case, carbon tetrachloride was used as a solvent, and ethanol extract was used as a solvent. In order to measure the survival rate of the hepatocytes, the culture medium containing 5 mM carbon tetrachloride and the extract was removed, washed with HBSS, and the culture solution containing 0.5% MTT was added and incubated for 2 hours. After removal of the culture medium containing MTT, DMSO was added to dissolve formazan produced by living hepatocytes, and the absorbance at 550 nm was determined. The number of each group and experimental animals is as follows.

Group 1 (n = 4) Normal-treated with ethanol (1%) and DMSO (1%) as solvent

Group 2 (n = 4) ethanol (1%) and soft ethanol extract treatment (50 μg / ml)

Group 3 (n = 4) ethanol (1%) and soft ethanol extract treatment (10 μg / ml)

Group 4 (n = 4) ethanol (1%) and soft ethanol extract treatment (5 μg / ml)

Group 5 (n = 4) treated with ethanol (1%) and soft ethanol extract (2.5 μg / ml)

Group 6 (n = 4) ethanol (1%) and soft ethanol extract treatment (1 μg / ml)

Treatment with DMSO (1%) and 5 mM carbon tetrachloride used as Group 7 (n = 4) solvent

Group 8 (n = 4) treated with 5 mM carbon tetrachloride and soft ethanol extract (50 μg / ml)

Group 9 (n = 4) 5 mM carbon tetrachloride and soft ethanol extract treatment (10 μg / ml)

Group 10 (n = 4) 5 mM carbon tetrachloride and soft ethanol extract treatment (5 μg / ml)

Group 11 (n = 4) 5 mM carbon tetrachloride and soft ethanol extract (2.5 ㎍ / ㎖)

Group 12 (n = 4) treated with 5 mM carbon tetrachloride and soft ethanol extract (1 μg / ml)

After adding the culture solution and incubating for 1.5 hours, the AST value secreted into the culture solution was measured by using ARKRAY's Spotchem automatic analyzer II (Spotchem SP4410, Japan), and the results are shown in Table 2 below.

Military number AST (IU / ℓ) Survival rate (%) Military number AST (IU / ℓ) Survival rate (%) One 43 ± 14 100 ± 5 7 266 ± 71 69 ± 4 2 34 ± 15 102 ± 3 8 84 ± 42 93 ± 7 3 37 ± 17 105 ± 17 9 59 ± 13 95 ± 5 4 35 ± 16 98 ± 5 10 81 ± 39 101 ± 5 5 36 ± 13 106 ± 9 11 125 ± 27 96 ± 6 6 39 ± 16 99 ± 5 12 198 ± 28 89 ± 4

As shown in Table 2, it can be seen that the soft ethanol extract of the present invention has a hepatocellular protective effect by carbon tetrachloride.

Example 4 Antimutagenic Effects of Ethanol Extracts of Soft Roots on Aflatoxin B1

Salmonella typhimurium TA100 strain (University of California, Berkeley) was incubated overnight in a nutrient broth, 0.1 ml of DMSO suspension of soft ethanol extract was incubated at 37 ° C for 30 minutes at 37 ° C. . 2.5 ml of top agar containing histidine / biotin was added to the minimal glucose agar medium, solidified and incubated at 37 ° C. for 48 hours to count the return mutations. The results are shown in Table 3 below.

Military number Lotus root extract (µg / plate) % Inhibition Military number Lotus root extract (µg / plate) % Inhibition One 1000 100 4 100 77 2 500 100 5 50 59 3 250 100 6 10 25

As shown in Table 3, it was confirmed that the soft ethanol extract of the present invention has an effect of inhibiting mutagenicity by aflatoxin B1.

Example 5 Hepatocellular Protective Effect of Ethanol Extracts of Soft Roots against Aflatoxin B1

Hepatocytes were isolated from F344 rats (Korean experimental animals, Chungbuk-negative), added to the collagen-coated 24 well plates to give 1 × 10 ⁶ cells / well, and after 3 hours, the wells were washed with HBSS and unbound cells. Removed and incubated again for 3 hours. Cultures were used Williams media E (Gibco BRL) containing ^10-8 M insulin, 10% FBS, ^10-6 M dexamethasone, penicillin (100 IU / ml), streptomycin (100 μg / ml). To induce hepatocyte damage by aflatoxin B1, change to a culture medium containing 1 μM aflatoxin B1 and soft-potency ethanol extract, and incubate for 48 hours, followed by MTT method (Monks et al., J. Natl. Cancer Inst . 83: 757-766 , 1991) cell viability was measured.

Military number process Survival rate Aflatoxin B1 (μM) Lotus root extract (㎍ / ㎖) One 0 0 100% 2 One 0 27.2% 3 One 100 74.3% 4 One 250 103.3% 5 One 500 131.5%

From the results in Table 4, it was confirmed that the soft ethanol extract of the present invention has the effect of inhibiting hepatotoxicity caused by aflatoxin B1.

Example 6 Acute Toxicity Test

The mice were administered with various doses of the ethanol extract composition of Example 2 of Example 2 through an oral route for the experimental group 5 consisting of 10 mice per group, and observed for 7 days, and the maximum allowable doses were measured. Indicated.

Count Dose (mg / kg) Lethal Maximum dose (mg / kg) 10 1000 0/10 - Mouse 10 2000 0/10 - 10 3000 0/10 - Control 10 6000 0/10 More than 6000 10 0 0/10

As shown in Table 5, the soft ethanol extract of the present invention has a maximum allowable dose of 6,000 mg / kg or more at the time of oral administration. The composition can be administered safely to a living body.

As described above, the ethanol extract of the lotus leaf (mature fruit of Nelumbo nucifera Gaertn.) Of the present invention exhibits excellent liver protection and is safe for the human body, so that the pharmaceutical composition containing it as an active ingredient may protect liver cells, prevent liver damage, and It can be useful for the treatment and suppression of mutation by liver cancer causing agent.

Claims (6)

An extract of lotus root extract (mature fruit of Nelumbo nucifera Gaertn.) Extracted with lower alcohol, which is viscous and exhibits mucosal protection, hepatocellular protection, prevention and treatment of liver damage, and mutagenesis induced by liver cancer-causing agents. The method of claim 1, Lotus root extract, characterized in that the lower alcohol is ethanol. A pharmaceutical composition for protecting hepatocytes, which contains a lower alcohol extract of lotus root meat as an active ingredient. A pharmaceutical composition for preventing liver damage and for inhibiting mutations by a liver cancer-causing agent, which contains a lower alcohol extract of lotus root meat as an active ingredient. The method according to claim 3 or 4, Pharmaceutical composition, characterized in that the lower alcohol is ethanol. The method according to claim 3 or 4, A pharmaceutical composition, characterized in that the dosage of the lower alcohol extract of the lotus root is 1 to 2,000 mg / kg body weight / day.