KR100748352B1 - Liver-protective and improvable health food containing bamboo sap - Google Patents

Abstract

A health food containing bamboo sap is provided to be effective in prevention and improvement of liver disease caused by extraneous substance or virus infection by inhibiting liver toxicity through antioxidative action on N-acetyl-p-benzoquinoneimine as liver-toxic substances increased by cytochrome P450 enzymes. The health food contains bamboo sap or its freeze dried powder and additionally food acceptable excipients. Bamboo sap is obtained by cutting a bamboo grown for a predetermined period at 2 to 3 node portions around May or June, sterilizing the cut portion and connecting to a sterilized tube. The collecting time is about 12hr and the amount of the bamboo sap collected is about 1 to 2L per bamboo. The bamboo sap is freeze-dried, ground and stored at -20deg.C.

Description

Health food for protecting and improving liver containing bamboo sap {LIVER-PROTECTIVE AND IMPROVABLE HEALTH FOOD CONTAINING BAMBOO SAP}

1 is a graph showing the amount of glutathione reduced by APAP, a liver damage substance,

2 is a graph showing the amount of glutathione reduced by CCl ₄ , a liver damage substance,

Figure 3 is a photograph showing the liver tissue of the mouse taken with an optical microscope,

Figure 4 is a graph showing the results of the antioxidant test for reactive harmful oxygen species in hepatocytes.

The present invention relates to a health food for liver protection and improvement, including bamboo sap, and more particularly, by containing a sap or lyophilisate of bamboo sap together with a pharmaceutically acceptable carrier to prevent external substances or viral infections. It relates to a health food for liver protection and improvement, including bamboo sap for preventing liver damage and improving liver function.

Recently, the mortality rate of liver cancer is the world's number one in the health level of Koreans, and the third mortality rate in chronic liver disease was also the third most recently.

In particular, the cause of the highest mortality rate among liver disease in Korea is viral hepatitis, but death from cirrhosis is reported to be 5-10 times higher in the West than viral hepatitis.

In general, the liver is a large buffering organ and does not show up well in the early stages of the disease but is found only to worsen. Liver cirrhosis is the last stage that commonly occurs when chronic liver disease progresses. Chemicals, viral hepatitis, metabolic diseases such as biliary tract diseases, autoimmune diseases, etc., but the cause is often unknown.

In cirrhosis, overall liver function decreases such as hepatic blood flow decrease, hepatic blood flow decrease, metabolic enzyme function decrease, blood protein quality, quantitative change and bile flow change.

In addition, the liver is known to be closely related to mental activity by the blood storage and circulation, blood volume control and defense detoxification in the human body, and our body is always exposed to pollutants and toxic substances according to industrialization Is constantly suffering from detoxification.

Moreover, the serious damage is liver damage caused by mental stress. In case of mental rest, damaged hepatocytes are repaired, but in an urgent modern society, the mental rest cannot be afforded, and the human body is protected by mental stress, heavy drinking, and smoking. Detoxification can cause abnormalities in the immune system, causing other diseases.

Acetaminophen (Acetaminophen, APAP) is a material well known as analgesics, brings damage of time treatment at a high concentration (Drug Metab. Rev. 24 (1992 ) 367-407.), Carbon tetrachloride (CCl ₄₎ also between the micro- It has been reported to produce cytotoxicity by generating trichloromethyl pro radicals by cytochrome P450 in vitro (Toxicology 112, 131-140).

Acetaminophen or carbon tetrachloride is mostly released from the body by binding to gluconide or sulfate, and some of the N-acetyl-p-benzoquinoneimine is produced by sitechrome P450 enzymes ( Proc. Natl. Acad. Sci. USA 81 (1984) 1327-1331), in combination with glutathione in the liver under normal conditions, effectively inhibits toxicity (Drug Metab. REv. 13 (1982) 539-553. 187 (1973) 185-194. Pharmacol.Exp. Ther. 187 (1973) 185-194. Pharmacol.Exp. Ther. 187 (1973) 211-217).

However, when administered at high concentrations, gluconide or sulfate is saturated, and acetylbenzoquinone amine production is increased by cytokron P450 enzymes, which rapidly reduce the amount of glutathione by binding to glutathione in the liver.

In addition, acetylbenzoquinone imine, which failed to bind glutathione, binds to intracellular macromolecules, causes lipid peroxidation of the cell membrane, and also interferes with the regulation of intracellular calcium, resulting in cell death (Pharmacol. Exp. Ther). 187 (1973) 211-217. Metab. Rev. 24 (1992) 367-407 Drug Metab. Rev. 29 (1997) 59-77.).

On the other hand, bamboo (Phyllostachys) is a Gramineae plant, which is known to have more than 1,250 species around the world, and bamboo has been cultivated for a long time in Korea. It is distributed in the area of North Gyeongsang Province, and is distributed in coastal areas of Gangwon-do and Chungcheongnam-do and Jeju Island.

Since ancient times, bamboo has been used as a folk medicine for the treatment of hypertension, sweating, stroke, etc. It is also known as an antiseptic effect, and stem, epidermis, bamboo shoots and bamboo thread have been used as a medicine for treating diseases, but recently, Like sap, sap is collected and drinking.

While many studies have been conducted on the antimicrobial activity of bamboo leaves, research on bamboo sap has only been conducted on the sampling amount, the sampling method, and the availability of the distilled water.

A sap is a liquid that flows through conduits or dead ends and is a relatively dilute solution in which inorganic salts, nitrogen compounds, carbohydrates, enzymes, and plant hormones are dissolved. The sap is composed of 99.3% water and 0.7% solids, but usually 'Water differentiation' is very different from water.

The custom of drinking plant sap as a healthy drink has a long history in the Soviet Union, China, and Japan. It is a folk medicine that is effective in diuresis, constipation, gastrointestinal diseases, high blood pressure, blood sugar control, neuralgia, and is highly interested in the components of these sap. In industrialized countries, sap maple syrup is processed in Canada and the United States to produce sugar or syrup, and birch sap is collected and sold as healthy drinks in Hokkaido, Japan.

Bamboo sap can collect more sap than cypress and birch, and it is known to be effective in dislocation, diuresis, nerve stabilization and suppression of heart disease, and elimination of waste products due to its high content of sugar and inorganic components. In addition, compared to the sap collected from other species, the content of sugar and inorganic components is high, the value of drinking water is high, and antimicrobial activity is shown to be great, so it can be used as a natural antibacterial substance.

In Korea, some studies have been conducted on the development of bamboo processing technology, that is, bamboo arts and crafts technology. Until now, however, there has been little development of technology using pharmacological efficacy or functionality of bamboo resources. The research on the activity of antimicrobial activity against is ongoing.

In addition, some research on bamboo resources in foreign countries has been conducted in China and Japan. Especially, in China, bamboo resources have been used in various ways, but mainly focus on industrialization of building materials using bamboo, and bamboo is a functional food. The technology used as a material is still in its infancy, and at present it is a state of developing and distributing bamboo beverages called 'jukgun' and 'juk flavor' coated with rice by preparing bamboo leaf extract has also been developed.

As such, gorgo sap, maple sap, birch sap, etc. have been studied variously on the pharmacological effect, but the research on the bamboo sap has not been much, especially the pharmacological effect has not been reported yet.

As a result, the inventors of the present invention continued to confirm that bamboo sap not only protects the liver function but also improves the damaged liver function, thus leading to the completion of the present invention.

The present invention provides a health food for liver protection and improvement, including bamboo sap that inhibits liver toxicity through antioxidant activity against acetylbozoquinone imine, an intrahepatic toxic substance increased by hepatic cytochrome P450 enzymes. There is this.

The present invention to achieve the above object is to provide a functional health food for liver protection and improvement, including bamboo sap or lyophilized powder of bamboo sap.

Hereinafter, with reference to the accompanying drawings will be described a preferred embodiment of the present invention;

In order to manufacture the health food of the present invention, first harvesting the sap of bamboo, looking at the sampling process, the second to third year-old bamboo, such as Byeongjongjuk and wangdae, cotton pad to cut the sap 2-3rd section of the ground Put the vinyl tube on the back part of the sap and connect it to the container for 12 hours. The sap per bamboo is about 1 ~ 2L, and it should be collected from early dawn and finished before noon.

Bamboo sap is easily deteriorated because it contains some sugars and amino acids, especially since it must be harvested from May to June. Although it was difficult to commercialize such products, it could be distributed in the sap state by sterilization and packaging.

Bamboo sap thus collected is a safe and non-toxic natural product that protects the liver through antioxidant activity that inhibits the oxidation of acetylbozoquinone imine, a hepatic toxic substance increased by hepatic cytochrome P450 enzymes. The effect of improving the damaged liver is excellent.

Example 1 Collection of Bamboo Sap and Treatment of Lyophilized Powder of Bamboo Sap

1. Bamboo sap was collected around May-June, and the bamboo sap was collected by connecting the sterilized tube to the cut part by cutting the second to third nodes of bamboo grown for a certain period of time. Bamboo sap will be collected in a sanitized container, the sap collection time is about 12 hours, the sap collection amount was about 1 to 2 liters per bamboo.

2. The bamboo sap thus collected is powdered through lyophilization and stored at -20 ° C until use, and dissolved in animal cells at a concentration of 500mg / ml before treatment to give a 0.22㎛ net size. The membrane was filtered using a membrane filter and stored at −20 ° C., and used for the experiment.

Example 2 Experimental Reagents and Animals

Experimental reagent

The following reagent was purchased and used.

Acetoaminophen (APAP), Alanine Amino Transferase (ALT) and Aspartate Amino Transferase (AST) Kit, Thiobarbituric Acid (TBA), L-ascorbic Acid, Pyrus Sulfate (Ferrous sulfate), Hydrogen peroxide (phenyl) methoxylsulfonyl fluoride and glutathione (GSH) were purchased from Sigma chemical co.

2. Experimental Animal

The experimental animals used 25 ~ 30g of laboratory mouse (ICR mouse), 8 weeks old, and the feed (purina korea) and water were freely ingested. The temperature of the kennel was 21 ~ 24 ℃ and the relative humidity was 40 ~ 80%. The light in the kennel was also adjusted to repeat day and night every 12 hours.

Example 3 Hepatoprotective Effect of Bamboo Sap against Substances That Cause Liver Toxicity

1. Infusion of bamboo sap lyophilizate and liver damage caused by chemicals

Bamboo sap lyophilized powder was dissolved in 1, 10, 50 mg / kg orally for 3 days, and control group was orally administered only with saline, and pretreated with lyophilized powder of bamboo sap. (CCL4 18 μg / kg) was intraperitoneally induced to cause artificial liver damage. After 18 hours, mice were anesthetized with ethanol and blood and livers were collected.

2. Determination of Alanine Amino Transferase and Aspartate Amino Transferase Activities in Blood

The amount of alanine amino transferase and aspartate amino transferase activity released into the blood upon liver injury was measured according to the Reitman-Frankel method.

Briefly, after 18 hours after treatment, the serum collected from the heart was reacted with the substrate solution at 37 ° C. for 30 minutes to produce pyruvate, and then the coloring solution was added. After 20 minutes, 0.4N-sodium hydroxide was added. When (NaOH) was mixed to give the color development, the absorbance was measured at 505 nm.

Table 1. Measurement of ALT and AST Activity in Blood

division Control Acetoaminophen (APAP) Carbon tetrachloride (CCl ₄ ) Bamboo Sap (mg / kg) 0 0 One 50 200 0 One 50 200 Serum ALT (U / liter) 38 ± 2 2532 ± 321 2102 ± 241 805 ± 135 186 ± 53 4210 ± 731 3861 ± 623 2253 ± 431 453 ± 139 Serum AST (U / liter) 23 ± 2 1331 ± 224 1082 ± 135 505 ± 171 86 ± 13 4210 ± 731 3861 ± 623 2253 ± 431 453 ± 139

As can be seen from Table 1, alantoaminotransferase and aspartate aminotransferase activity levels of hepatic cells released from liver cells due to liver damage caused by acetoaminophen and carbon tetrachloride were measured. Compared with the pretreatment group, activity decreased depending on the treatment concentration.

3. Lipid Peroxidation in Liver

To determine the liver damage protection effect of bamboo sap lyophilized powder (bamboo sap) on lipid peroxidation produced by acetoaminophen, 100 mg of liver was poured into 10 ml of buffer (Tris-HCL), and then crushed by a homogenizer and then peroxidated. Absorbance at 532 nm was measured at 532 nm for the thiovabitroic acid pigment, which was formed by reacting malondialdehyde (MDA), a lipid degradation product, with 2-Thiobarbituric acid (TBA) at 100 ° C for 20 minutes. .

The results are shown in Table 2 below.

Table 2. Measurement of Lipid Peroxidation in Soy Sauce

division Control Acetoaminophen (APAP) Carbon tetrachloride (CCl ₄ ) Bamboo Sap (mg / kg) 0 0 One 50 200 0 One 50 200 Hepatic Lipid Peroxidation (MDA.nmol / g) 4.2 ± 0.9 9.6 ± 1.2 8.2 ± 0.8 6.3 ± 0.4 4.8 ± 0.8 13.1 ± 1.1 12.8 ± 0.2 7.8 ± 0.7 5.6 ± 0.1

Phospholipids in cell membranes are broken down into lipid endoperoxide radicals and lipid endoperoxide radicals by the attack of free radicals, and then decomposed to form MDA. Lipid peroxidation is widely used as an indicator of oxidative stress (Johansson, I et al., 1985; Ayene, IS et al., 1992).

In vivo, lipid peroxide works with proteins, RNA, and DNA, which are components of cells, to cause mitochondrial dysfunction and biochemical conclusions, thereby attracting attention as a substance that promotes disease and aging.

Particularly, since lipid peroxide is well formed in the biofilm and polyunsaturated fatty acid, which is a major component of the biofilm, is changed in fat composition by lipid peroxide formed of free radicals, the function of the biofilm is reduced, and fluidity is reduced, thereby transporting material, which is the main function of the membrane. And reduced permeability, which impedes maintenance of homeostasis (Plaa, GL, 1976; Curtis, MT, 1984; Lacko, AG, 1984).

As can be seen in Table 2, the results of measuring the level of lipid peroxidation (LPO) in order to confirm the hepatotoxicity defense mechanism by oxidative stress (Oxidative stress) results in the aceto group pretreated with lyophilized powder of bamboo sap Compared with the aminophene-only group, the concentration-dependent decrease was observed, and 200 mg / kg pretreatment was similar to the lipid peroxidation level of the control group.

Example 4 Recovery of Glutathion (GSH) Reduced by Liver Injury

After the liver was washed with distilled water, it was dried for a while, weighed, and then the amount of glutathione was measured using a portion of the liver.In this method, 100 mg of liver was extracted into 0.02M EDTA, followed by a homogenizer. To break down the organization.

After centrifuging the aqueous solution for 30 minutes at 12,000 rpm, only 100 µl of the supernatant was taken, and 1.8 µl phosphate-EDTA buffer and 100 µl of phthalidealdehyde (O-Phthalialdehyde) were added to the 100 µl supernatant. After 15 minutes, the absorbance is measured at 412 nm with a flowometer.

The results are shown in Figures 1 and 2, Figure 1 is a graph showing the amount of glutathione reduced by APAP, a liver damage material, glutathione reacts with the toxic substances in the cell to detoxify the toxic substances and out of the body It acts to release quickly.

In addition, glutathione was consumed a lot in order to reduce liver damage caused by APAP, but the amount of glutathione consumed by APAP was reduced by the action of lyophilized powder of bamboo sap.

This is a phenomenon that occurs when the production of liver damage caused by the metabolism of APAP in the hepatocytes is reduced, the generated liver damage is detoxified, or the production of glutathione increases.

The increase in glutathione could not be confirmed in this experiment, but it could be considered that the sample was involved in the reduction of the production of liver damage or detoxification of the generated liver damage, indicating that hepatocellular damage caused by APAP was caused by bamboo sap. The results indicate that the lyophilized powder of the lyophilized powder is inhibited or reduced.

And, Figure 2 is a graph showing the amount of glutathione reduced by the liver damage CCl ₄ , glutathione reacts with the toxic substances in the cell to act to detoxify the toxic substances, and to quickly exit the body.

In addition, glutathione was consumed a lot in order to reduce liver damage caused by CCl ₄ , but the amount of glutathione consumed by CCl ₄ was decreased by the action of lyophilized powder of bamboo sap.

This may be a phenomenon that occurs when the production of liver damage caused by the metabolism of CCl ₄ in hepatocytes is reduced, the generated liver damage is detoxified, or the production of glutathione is increased.

The increase in glutathione could not be confirmed in this experiment, but it could be seen that the sample was involved in reducing the production of liver damage or detoxification of the generated liver damage, which is responsible for the hepatocellular damage caused by CCl ₄ . The results indicate that the lyophilized powder of sap is suppressed or reduced.

And, Figure 3 is a photograph showing the liver tissue of the mouse taken with an optical microscope, it can be confirmed that hepatic cells around the blood vessels damaged by hepatic necrosis and natural death of the cells by APAP, and the sample can be processed simultaneously In this case it was confirmed that this phenomenon is significantly reduced.

4. Determination of aniline hydroxylase activity

The measurement of aniline hydroxylase related to P450 2E1 was quantified by measuring the amount of hydrosilaniline (4-Hydroxy aniline) produced using aniline as a substrate by absorbance at 630 nm.

Simply stated, aniline (5-20 mM), 10 uM NADPH, regenerating system and microsome samples were mixed in 0.1 M potassium phosphate buffer (pH 7.4) and then a 37 ° C shacking water bath. incubator for 20 minutes, and then the reaction was terminated by adding 20% TCA, mixed with 5% phenol and 2.5N sodium hydroxide (NaOH), and then added with 2.5N sodium carbonate (Na ₂ CO ₃ ) for 30 minutes. The color was developed while standing at room temperature.

Absorbance was measured at 630 nm to calculate the activity from the hydrosyl aniline (4-hydroxyaniline) calibration curve.

Table 3. Aniline hydroxylase Activity

division Control Acetoaminophen (APAP) Bamboo Sap (mg / ml) 0 0 One 50 200 AH 13.56 ± 0.9 13.58 ± 1.2 10.26 ± 0.7 8.3 ± 1.5 5.7 ± 0.5

The activity of AH (aniline hydroxylase) decreased in proportion to the concentration of the sap of bamboo sap. These results indicate that the hepatocellular damage damaged by acetaminophen or carbon tetrachloride is recovered by the sap of saline enzymes (ALT). , AST) and hepatic lipid peroxidation levels, and hepatocellular histological observations. The effects of hepatoprotective effects were dependent on the concentration of bamboo sap.

Example 5 Investigation of Changes in Reactive Oxygen Species (ROS) Related to Hepatotoxicity Induction

1. Hepatocyte isolation and hepatocyte primary culture for in vitro experiments

Isolation and Culture of Hepatocytes

Hepatocytes were separated by the collagenase perfusion method according to Dickins and Peterson's method, and Han's balanced solution (HBSS) was used as a perfusion buffer solution.

100 mg of collagenase was transferred to a container containing perfusion buffer solution to release liver cells from the liver, and the hepatocyte suspension was passed through a nylon mesh having a pore size of 250 μm. Got it.

The filtrate thus obtained was transferred to a sterile tube and centrifuged three times at 50 × g for 4 minutes to obtain only liver parenchymal cells, and finally single cells were suspended in culture medium (William's E).

In addition, hepatocytes were cultured at 37 ° C., and replaced with fresh medium 3-4 hours after the start of culture to remove the cells that did not adhere to the bottom of the culture vessel. The sample was processed.

2. Determination of Reactive Oxygen Species (ROS)

Determination of reactive harmful oxygen species in hepatocytes

The amount of reactive noxious species in hepatocytes was measured using a fluorescent probe, 2 ', 7'-dichlorofluorescein diacetate (2', 7'-Dichlorofluorescein diacetate; DCF-DA).

2 ', 7'-dichlorofluorescein diacedate in the culture solution was incubated at 25 μM per well for 15 minutes and treated with bamboo sap lyophilized powder and tributyl hydrophosphate (0.5 mM) to induce intracellular ROS. Intracellular peroxides formed after the reaction were measured for fluorescence at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.

As can be seen in Figure 4, the amount of reactive ROS was decreased in proportion to the treatment concentration of lyophilized powder (bamboo sap) of bamboo sap.

Taken together, hepatocellular damage damaged by acetoaminophen and carbon tetrachloride reduces liver damage in liver by reducing antioxidant oxidative stimulation of bamboo sap lyophilized powder (bamboo sap) through liver. Reduction of serum levels of enzymes (ALT, AST) and lipid peroxidation in hepatocytes was confirmed by measuring reactive reactive oxygen species (ROS) related to inducing hepatotoxicity in hepatocytes. Investigate.

As described above, the bamboo sap or the lyophilized powder of the bamboo sap included in the health food according to the present invention not only prevents liver disease caused by foreign substances or viral infections such as acetoaminophen and carbon tetrachloride, but also a biological modulator for improving liver function. It may be useful clinically as.

Furthermore, the health food of the present invention can be formulated as a bamboo sap itself, or lyophilized powder or granules and tablets of bamboo sap, and excipients that are acceptable as food additives in lyophilized powder of bamboo sap or bamboo sap. It can be mixed and used suitably according to a conventional method.

As described above, in the detailed description of the present invention, specific embodiments have been described, but various modifications may be made without departing from the scope of the present invention.

Therefore, the substantial scope of the present invention should not be limited by the above-described embodiment, but should be defined by the same structure as the claims as well as the claims described below.

The present invention configured as described above provides an effect that can be usefully used as a beverage or functional health food to prevent and improve epilepsy caused by foreign substances or viral infections.

Claims (1)

Functional health food for liver protection and improvement, including bamboo sap or lyophilized powder of bamboo sap.

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