KR20140047790A - A pharmaceutical comprising lactuca indica var. laciniata for immunity enhancement - Google Patents

Abstract

The present invention relates to a composition comprising lactuca indica var. laciniata as an active ingredient for immunity enhancement and, more specifically, to a technical feature capable of enhancing immunity by facilitating generation of nitric oxide (NO), anti-allergic activity, and antioxidant activity. Accordingly, the composition of the present invention can be provided as pharmaceutical compositions, functional health foods, cosmetics, or animal feeds which can be used for various diseases caused by lack of immune or abnormal conditions.

Description

A composition for enhancing immunity, which contains a wild chrysanthemum as an active ingredient {A pharmaceutical comprising Lactuca indica var. laciniata for immunity enhancement}

The present invention relates to a composition for enhancing immunity, containing Lactuca indica var. Laciniata as an active ingredient.

More specifically, it possesses technical characteristics that can increase the production of nitric oxide (NO) by generating synergy with IFN-γ naturally occurring in the body during the immune process to promote the production of iNOS and TNF-α. have.

It also possesses technical features that can enhance normal immune capacity by inhibiting systemic anaphylactic reactions and passive skin anaphylactic reactions and inhibiting histamine release from mast cells.

In addition, by promoting the antioxidant activity has a technical feature that can enhance the immunity by improving the lack or abnormal state of the immune state due to oxidative stress.

Immunity is a phenomenon in which the internal environment of a living body defends against an antigen that is an external factor, and can be classified into innate immunity that reacts nonspecifically to an antigen and acquired immunity having a memory function for the antigen.

Here, the antigen refers to various external factors such as pathogenic microorganisms, foods, chemicals, drugs, pollen, animal stools, and the like.

Most infections are defended by innate immunity, and macrophages in the process work mainly, and acquired immunity works by maintaining a close relationship between lymphocytes (T lymphocytes, B lymphocytes) and macrophages.

That is, free macrophages move immediately upon activation by various stimuli to perform a predetermined task at each site. Macrophages are activated by the action of interferon- [gamma] (IFN- [gamma]) produced by T lymphocytes activated by antigenic stimulation, thereby enhancing immune function.

As such, macrophages are involved in innate immunity (primary immune function) and at the same time control acquired immunity (immune immunity) by lymphocytes, and are also directly involved in performing various immune functions by amplifying the effect by immune enhancers.

When macrophages are involved in immune action, ie antimicrobial action, antiviral action and anticancer action, nitric oxide (NO) produced by macrophages acts as an important mediator of immune action.

In other words, when culturing cancer cells and macrophages together, macrophages stimulated with lipopolysaccharide (LPS) were found to inhibit the metabolism of cancer cells. Nitrate was found to be excreted.

It has also been demonstrated that macrophages are activated by recombinant interferon-γ (rIFN-γ) and LPS to generate nitrites and nitrates, which are derived from NO generated in macrophages.

The expression of nitric oxide (N0) is associated with DNA damage, which is known to promote the expression of wild-type p53, which in turn causes apoptosis of cancer cells.

In addition, NO may be induced in the cancer cell itself, which is induced by automatic expression of iNOS (inducible NO synthase, NOS-II).

Therefore, NO requires antigen recognition, but it can be said that it performs immune function through the process of inducing the non-specific death of antigen.

iNOS can be expressed when exposed to LPS and inflammation-induced cytokines, in particular inflammation-induced cytokines such as tumor necrosys factor-α (TNF-α).

TNF-α is expressed during microbial infection or tissue damage to promote immune function, which is increased by expression of interferon-γ (IFN-γ). Increased TNF-α increases NO production of macrophages induced by IFN-γ through autocrine action.

Therefore, nitric oxide (N0) generated by the activation of macrophages in the overall immune process can be seen to play a major role, the activation of macrophages is basically achieved by IFN-γ.

In addition, NO production according to the activation of macrophages is further increased by the expression of iNOS and TNF-α, an inflammation-inducing cytokine, to perform immune action.

An allergy response can be seen as an immune hypersensitivity reaction to external stimuli, which can occur in the respiratory, skin, gastrointestinal tract, or the whole body.

In particular, histamine is mainly stored in mast cells and is released when the cells are damaged by wounds or bacterial infections. The released histamine plays an important role in immune function by dilatating blood vessels and increasing their permeability, allowing white blood cells and many plasma proteins to move quickly to the site of bacteria.

However, excessive secretion of histamine due to excessive immune response may cause allergic reactions and anaphylaxis.

Specifically, compound 48/80, a potent degranulation inducer, releases about 90% of intragranular histamine from mast cells, and thus, the use of an appropriate amount of compound 48/80 allows direct and convenient study of systemic anaphylaxis. .

In addition, the surface of mast cells has a high affinity receptor (FcεRI) for IgE (immunoglobulin E), so the immunological specificity of mast cells is mediated by IgE binding to this receptor. Passive dermal anaphylaxis reactions in which IgE molecules on the surface of mast cells form crosslinks by multivalent antigens and release various chemical mediators such as histamine, serotonin, leukotriene, and prostaglandin.

Degranulated chemical mediators in this anaphylactic reaction cause anaphylaxis, allergic asthma, allergic rhinitis, allergic otitis media, and atopic dermatitis.

Therefore, the systemic anaphylactic reaction by Compound 48/80, the passive dermal anaphylactic reaction by IgE antibody, and the inhibitory action on histamine release from mast cells are important in antiallergic action.

On the other hand, as the causes of stress, ultraviolet rays, radiation, chemicals (pesticides, pesticides, medicines, nitrogen compounds), instant foods, smoking, and other environmental pollution have increased in modern times, some of the oxygen that enters the body has to be stabilized. Active oxygen, which is not reduced and has an unpaired electron, is becoming a problem.

Because oxidative stress caused by free radicals causes degeneration and destruction of lipids, proteins, DNA, enzymes, tissues, etc., free radicals cause cancer, stroke, arteriosclerosis, diabetes, joint diseases, as well as adult diseases and men. It also causes infertility.

In addition, active oxygen causes abnormalities in the sympathetic nervous system, parasympathetic nervous system, and immune system, which are the autonomic nervous system that maintains homeostasis of the human body, and as a result, infectious diseases such as atopy, type A, hepatitis B and cold, flu, and swine flu Occurs.

Specifically, when harmful substances and heavy metals accumulate in the body, free radicals, which are toxic substances, increase, and cellular stress increases, and the sympathetic nervous system and parasympathetic nervous system are highly toxic hormones. By releasing and adrenaline to further harm the cells.

This reaction not only weakens the immune system and acts as a cause of various diseases, but also fails to cope with the stimulation of pollutants or invasion of pathogens from the outside, and increases diabetes, obesity, and heart disease, which are emerging as a problem of adolescent diseases, Many studies have revealed that the weak physical strength is a weak state to endurance, endurance, mental strength.

Acting as an antioxidant can be divided into enzymatic and non-enzymatic systems. Enzyme-based materials are produced in-house and include antioxidant enzymes such as superoxide disputase (SOD), catalase, and peroxidase. have.

Non-enzymatic substances are nutrients that have to be absorbed from the outside of the body. They are in the form of low-molecular compounds and help the action of polymeric antioxidants (enzymes) in the body or remove active oxygen by themselves. Beta-carotene, vitamin A, vitamin C (ascorbic acid), vitamin E (tocopherol) and flavonoids are representative antioxidants.

The synthetic antioxidants developed so far include butylated hydroxyl anisole (BHA), butylated hydroxyl toluene (BHT) and nordihydro-guaiaretic acid (NDGA). Natural antioxidants include tocopherol, gossypol, sesamol, lecitthin, cephalin, ether extract components of red pepper, extracts from wheat germ, soybean, pepper, and tomato, and extracts of mainly florboloid pigments.

However, synthetic antioxidants are toxic in vivo and may cause allergies and tumors, and also have weaknesses such as being easily destroyed when heat is applied once due to weak temperature.

On the other hand, the Applicant utilizes Lactuca indica var. Laciniata as an active ingredient to increase nitric oxide production, to suppress anapalaxi reactions, to inhibit histamine release, and to promote antioxidant activity to enhance normal immune capacity. To provide a composition that can be made, a brief look at the following.

Lactuca indica var.laciniata is a year-old herb belonging to the compositae and contains triterpenoids, sesquiterpene lactones, β-Amyrin, Stigmasterol, and β-sitosterol. It has a unique bitterness and flavor, and has been used as a herb and ssamchae in the private sector.

Its shape is different from that of Youngia sonchifolia (Bunge) Maxim, Youngia denticulata (Houtt.) Kitamura, and Ixeris dentata, which are grown and sold in farms. The ingredients are also different.

In oriental medicine, Wanggogi is called Sanwageru, and it is clearly distinguished because it is called medicinal herb, bracts, and moths. Wang Dangus is known for its indigestion and loss of appetite in Chinese medicine.

On the other hand, when looking at the prior patent literature related to Wanggoe-extract, Korean Patent Laid-Open Publication No. 10-2008-0032944 describes an antioxidant substance separated by the root, stem and leaf of Ixeris sonchifolia Hance.

Second, the Republic of Korea Patent Publication No. 10-0553982 describes a composition for treating liver disease containing the extracts of the apple, bitter and Ixeris sonchifolia.

Third, Korean Patent Laid-Open Publication No. 10-2004-0018733 describes a composition for treating viral liver disease containing an extract of Younia sonchifolia.

 Fourth, the Republic of Korea Patent Publication No. 10-2011-0139582 discloses a composition having a hangover relief and ethanol liver detoxification action using the perilla and perilla perilla.

In summary, the composition using the king eel extract is capable of enhancing normal immune ability by increasing nitric oxide production, inhibiting the release of histamine, and promoting antioxidant activity, by only having ethanolic liver detoxification effect. It is not described.

The present invention is to provide a composition that can enhance the immune capacity by increasing the production of nitric oxide using Lactuca indica var. Laciniata as an active ingredient.

The present invention is to provide a composition that can utilize the Lactuca indica var. Laciniata as an active ingredient to inhibit the anaphylaxis reaction and to inhibit the histamine release to promote anti-allergic activity to enhance normal immune capacity .

The present invention is to provide a composition that can enhance the immune capacity by promoting the antioxidant activity by utilizing Lactuca indica var. Laciniata as an active ingredient.

The present invention is to provide a composition that can improve and treat a variety of diseases that can occur due to lack or abnormal immune capacity by enhancing the immune capacity by utilizing the Lactuca indica var. Laciniata as an active ingredient.

The present invention is to provide a general health food, health functional food, beverages, tea, sweets, cosmetics, livestock feed that can enhance immunity by using the King Ole (Lactuca indica var. Laciniata) as an active ingredient.

The present invention is to provide a composition for dietary supplement, health functional food, cosmetics and livestock feed containing Lactuca indica var. Laciniata as an active ingredient.

The present invention is to provide a composition comprising a dry powder obtained by drying and grinding the roots, stems and / or leaves of the king eel.

The present invention provides an extract of King Dried Algae obtained by extracting with high purity lower alcohol or water.

The present invention provides a variety of solvent fraction extract by extracting the King Ddung with low alcohol to obtain an alcohol extract, diluting the alcohol extract again with distilled water and repeating fractionation with an organic solvent.

The composition according to the present invention may be provided as an anticancer pharmaceutical composition, an antiallergic pharmaceutical composition, an antistress pharmaceutical composition, or an antioxidant pharmaceutical composition, each composition using a food-acceptable additive. It is manufactured and provided as a health functional food.

The health functional food using the composition according to the present invention may be provided in various forms, such as functional drinks, health supplements, tea, confectionary.

In order to use the present invention as a pharmaceutical composition can be prepared by a known method in the pharmaceutical field, powders, granules, tablets, capsules by themselves or mixed with a pharmaceutically acceptable carrier, excipient, diluent, etc. Or in the form of an injection or the like. They may also be administered orally or parenterally.

The effective dosage of the composition according to the present invention may be appropriately selected depending on the absorption of the active ingredient in the body, the rate of water activation and excretion, the age, sex and condition of the patient, the severity of the disease to be treated and the like.

The compositions according to the invention can be used in cosmetics or feed for animals, and in each case can be prepared using additives or additives that are acceptable in cosmetics or feed.

Formulations such as pills, granules, beverages, tablets, capsules and the like can be prepared using the compositions according to the invention, in which case additives may be added to prepare each formulation.

Composition containing the active extract of Lactuca indica var. Laciniata according to the present invention, by synergizing with IFN-γ naturally occurring in the body during the immune process, by promoting the production of iNOS and TNF-α, It has a significant effect that can increase the production of nitric oxide (NO).

The composition containing the Lactuca indica var. Laciniata according to the present invention as an active ingredient has a remarkable effect of enhancing the immunity of the human body by increasing the production of nitric oxide (NO).

The composition containing Lactuca indica var. Laciniata according to the present invention as an active ingredient has a remarkable effect of inhibiting systemic anaphylactic reactions and passive dermal anaphylactic reactions.

The composition containing Lactuca indica var. Laciniata according to the present invention as an active ingredient has a remarkable effect of inhibiting the release of histamine from mast cells.

The composition containing the Lactuca indica var. Laciniata according to the present invention as an active ingredient, has a remarkable effect to enhance the normal immunity by controlling the immune hypersensitivity reaction by inhibiting the anaphylactic reaction and histamine release have.

The composition containing the King Lax (Lactuca indica var. Laciniata) according to the present invention as an active ingredient has a remarkable effect of promoting antioxidant activity and antilipid peroxidation activity.

Composition containing the active extract of Lactuca indica var. Laciniata according to the present invention as an active ingredient, the remarkable effect that can enhance the immunity by improving the lack of or abnormal state due to oxidative stress by promoting antioxidant activity Holds.

Composition comprising the active ingredient (Lactuca indica var. Laciniata) according to the present invention, various diseases caused by lack of immunity or abnormalities, such as cancer, adult diseases, stress, stroke, allergic diseases, stress diseases, It can be widely used for the prevention and treatment of inflammatory diseases and cardiovascular diseases, and has a remarkable effect that can be usefully used as various functional foods.

1 is a diagram showing the results of experiments on the expression of iNOS of King Dwarf.
Figure 2 is a diagram showing the experimental results for the production of TNF-α of King Dwarf.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may properly define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the matters described in the Examples, Experimental Examples, Preparation Examples, and drawings described herein are only the most preferred examples of the present invention, and do not represent all of the technical ideas of the present invention, and therefore, these are replaced at the time of the present application. It should be understood that there may be various equivalents and variations that can be made.

Example  1. Preparation of Dried Dried Rice Flour

After removing sterilized roots, stems and / or leaves with ozone water, they are dried at low temperature using a cold air dryer or dehumidifier below 55 ℃ to process chlorophyll alive. Next, it is pulverized by a grinding | pulverization type grinding | pulverization method with little heat generation, and green algae powder is produced.

Here, general sun drying or drying above 60 ° C. is preferably avoided, because chlorophyll of plant cells is destroyed, browned, vitamin C and antioxidants are reduced, and pharmacological efficacy is reduced. This is because the value of the material is reduced.

Depending on the design conditions, after the sterilization wash, lyophilized, and then pulverized may be prepared Wang Dye powder.

Example  2. Preparation of King Dwarf Extract

1 kg of King Dried Algae powder prepared in Example 1 was extracted by cold extraction in 100% methanol for one week to obtain a methanol extract (210.5 g). 1000 ml of distilled water was added thereto, and then 1000 ml of hexane was added, and the mixture was partitioned into a water layer and a hexane layer. 1000 ml of ethyl acetate was added to the water layer again, the mixture was partitioned into an ethyl acetate layer and a water layer, and 500 ml of butanol was further added to the water layer, and finally fractionated into a butanol layer and a water layer, and each process was repeated three times.

The fractions obtained above were concentrated in vacuo to give 34.3 g, 5.7 g, 15.6 g, and 141.5 g of hexane, ethyl acetate, butanol and water fractions, respectively.

Depending on the design conditions, the roots, stems and / or leaves of the King Dd. May be dried first at 35 ° C. to remove moisture from the surface, then ground and processed into fresh juice to be used instead of powder.

Experimental Example  1. Macrophages by King's Mines NO  Investigate the impact on generation

In order to investigate the effect on the production of nitric oxide (NO) in macrophages, which are important immune responsible cells in vivo,

Experimental Example  1-1. Laboratory Animals, Reagent Preparation, and Macrophage Culture

As the experimental animals, C57BL / 6 male mice between 8-12 weeks purchased from the Korean Experimental Animal Center (Daejeon) were used.

2.5 ml injection of 4% thioglycollate (TG, Thioglycollate, purchased from Difco Laboratories (Detroit, MI, USA)) into the abdominal cavity of mice, and after 3-4 days the abdominal cavity was RPMI 1640 (Sigma Chemical Co. (St. Louis) Purchased from DMEM (Life Technologies (Grand Island, NY, USA)) containing 10% FBS (purchased from Life Technologies (Grand Island, NY, USA)) to remove red blood cells ) And aliquot to 4-well to give _2.5 × 10 ⁵ cells per well to CO ₂ Lt; / RTI > After about 3 hours, the culture medium was exchanged to remove cells that did not adhere to the culture vessel surface, and only the attached macrophages were used for the experiment.

Experimental Example  1-2. NO  Generation measurement

In the medium containing the peritoneal macrophages alone or rIFN-γ (purchased from Genzyme (Munchen, Germany)) (5U / ml), the concentration of bitter extract prepared as in Example 2 (10ug / ml, 100 ug / ml, 1000 ug / ml) and the amount of nitrite accumulated in the medium for 48 hours was measured. LPS was used as a control.

In other words, the amount of NO generated from macrophages was mixed with 100 μl of Grease's reagent and 100 μl of the nitrite, an oxide of NO accumulated in the medium, and after 10 minutes, 550 nm using an ELISA reader (manufactured by Bio-Tek Instruments Co.). Absorbance at was measured. The concentration calculation of nitrite was determined using sodium nitrite as a reference material.

Experimental Example  1-3. NO  Generation test result

LPS (10 μg / ml)
(Control group) Nitrite Concentration (μM) rIFN-γ (5U / ml) King Mino (μg / ml) - - - <4 + - - 20 ± 0.03 + - + 122.5 ± 12.27 * - 1000 - 14.5 ± 0.15 + 10 - 55.2 ± 14.25 + 100 - 65.2 ± 12.22 * + 1000 - 36.61 ± 0.04 *

The experimental results shown in Table 1 are four independent experiments, each represented by the mean value and the standard error. All data were expressed as the mean ± SD, and the significance test was performed by the student's t-test. The significance level was * p <0.05.

As shown in [Table 1], some NO production was also induced by King Albinus alone, and the NO synthesis was markedly increased especially at the concentration of 100 ug / ml when treated with rIFN-γ.

Wang-Ok-Dong-Shi has similar effects to LPS used as a control, but unlike LPS, NO means activation of macrophages without toxicity. In other words, Wang Dingus is known to have anti-cancer and anti-viral effects due to immune enhancement by immune cell activation.

Experimental Example  2. by the excavation iNOS  Investigate the impact on expression

The following experiments were carried out to investigate the effects on the production of iNOS when Rhwang-Nang-Jin was treated with rIFN-γ.

Experimental Example  2-1. iNOS  Expression measurement

Peritoneal macrophages (5x10 ⁶ cells per well) were incubated with treatment of rIFN-γ (10 U / ml) for 6 hours. Next, King Dipper (100 ug / ml) or LPS (10 ug / ml) was treated for 12 hours.

Whole cell lysates were made by boiling peritoneal macrophages in sample buffer (62.5 mM Tris-HCL, pH 6.8, 2% SDS, 20% glycerol, 10% 2-mercaptoethanol). Proteins in cell lysates were separated by 7% SDS-polyacrylamide gel electrophoresis and transferred to nitro cellulose paper. The membrane was blocked for 1 hour at room temperature with 5% skim milk in PBS (phosphate-buffered saline) -tween-20. Next, anti-iNOS antibody was treated and cultured. After washing three times in PBS-tween-20, the blots were incubated with the secondary antibody for 30 minutes. Antibody-specific proteins were visualized by chemilumine-sence detection system (Amersham Corp. Newwark, NJ)

Experimental Example  2-2. iNOS  Expression test result

1 is a diagram showing the results of experiments on the expression of iNOS of king exophorus. (Western blot analysis) 1 is a control, 2 is rIFN-γ, 3 is rIFN-γ + LPS, 4 is rIFN-γ + mongolia (100 ug / ml).

Referring to FIG. 1, it can be seen that iNOS protein expression in peritoneal macrophages is significantly increased when rIFN-γ and king cattle are treated together as compared with rIFN-γ alone.

Experimental Example  3. by the excavation TNF investigating the effects on -α production

In order to investigate the effect on the production of TNF-α when ryeul-si was treated with rIFN-γ, the following experiments were carried out.

Experimental Example  3-1. TNF -α generation measurement

Experiments were performed in which the abdominal macrophages (2.5x10 ⁵ cells per well) were treated with king mulberry alone or rIFN-γ (10 U / ml) at concentrations of 0, 1, 10 and 100 ug / ml.

The amount of TNF-α was measured by ELISA method after 24 hours incubation. Three independent experiments yielded mean ± SEM values. * p <0.05 is for macrophages alone, ** p <0.05 is for rIFN-γ alone.

Experimental Example  3-2. TNF -α generation test result

Figure 2 is a diagram showing the results of experiments on the production of TNF-α of king mutant (TNF-α: ng / ml) Referring to Figure 2, even when the treatment of the wild mutant alone TNF-α production It can be seen that the concentration-dependent increase.

In addition, when treated with rIFN- [gamma], synergistic action with the king-daughter was found to be more dependent on the production of TNF- [alpha] than the case of the single-daughter.

In summary, Experimental Examples 1 to 3, while IFN-γ and king cattle excreted naturally occurring in the body during the immune process, synergistic action, but promote the production of iNOS and TNF-α, thereby increasing the production of NO It can be seen that it can enhance the immunity of.

Experimental Example  4. King Dwarf Anti-allergy  Activity investigation

The anti-allergic effect of king cattle was examined through animal experiments. First, the inhibitory effect was investigated by oral administration of King Ddella to experimental animals that induced the systemic anaphylactic reaction and passive skin anaphylactic reaction. Next, the peritoneal mast cells of rats were isolated and examined for the effect of King D. perilla on histamine release from mast cells.

Experimental Example  4-1. Systemic Anaphylaxis  Preparation of reaction suppression experiment

As experimental animals, mice (ICR mice) purchased from the Korean Experimental Animal Center (Daejeon) were used. The air cleaning system maintained at a temperature of 22 ± 2 ° C, a humidity of 55 ± 10%, and a cleanness of 0.045㎛. It was bred in -flow room.

Reagents used in the experiment were all Sigma Chemical Co. (St. Louis, Mo., USA).

Before the experiment, the extract of King Dried Algae extract was prepared with physiological saline or T-load buffer A (10 mM HEPES, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl ₂ , 1 mM MgCl ₂ , 5.6 mM glucose, 0.1% bovine serum). Dissolved in albumin).

In the cell experiment, the drug was used by filtration with a 0.20 μm filter.

Experimental Example  4-2. Systemic by Compound 48/80 Anaphylaxis  Reaction suppression experiment

Compound 48/80 (8 mg / Kg), a non-immune stimulant against mast cells, was injected into the mouse abdominal cavity to induce a systemic anaphylactic response.

Royal lax and physiological saline were orally administered at various concentrations 1 hour before compound 48/80 administration, and mortality was examined 1 hour after compound 48/80 injection. Each group used 8 mice.

Mortality was calculated as follows.

Mortality (%) = total number of dead mice × 100 / total number of experimental mice

Experimental Example  4-3. Systemic by Compound 48/80 Anaphylaxis  Reaction suppression results

Dose of King Dill
(g / Kg BW) Compound 48/80
(8mg / Kg BW) death rate
(%) 0 (physiological saline administration) + 100 0.1 - 0 0.1 + 25 0.05 + 62 0.01 + 72.5 0.005 + 100

Referring to [Table 2], the oral administration group of physiological saline showed a mortality rate of 100%, whereas the oral administration group of king cattle was significantly reduced in mortality. Thus, Wang-Du-lung suppressed the systemic anaphylactic reaction.

Experimental Example  4-4. Manual skin Anaphylaxis  Reaction Experiment Preparation

The effect of inhibiting the typical passive dermal anaphylactic response induced by oral-administered king cattle in a specific immunological method was analyzed using an experimental animal model.

Rats (Sprague-Dawley rats) purchased from the Korean Experimental Animal Center (Daejeon) were used as the experimental animals, and the air cleaning system maintained at a temperature of 22 ± 2 ° C., a humidity of 55 ± 10%, and a cleanness of 0.045 μm. It was bred in a laminar air-flow room.

Reagents used in the experiment were all Sigma Chemical Co. (St. Louis, Mo., USA).

Before the experiment, the extract of King Dried Algae extract was prepared with physiological saline or T-load buffer A (10 mM HEPES, 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl ₂ , 1 mM MgCl ₂ , 5.6 mM glucose, 0.1% bovine serum). Dissolved in albumin).

In the cell experiment, the drug was used by filtration with a 0.20 μm filter.

Experimental Example  4-5. IgE  Passive Skin by Antibody Anaphylaxis  Inhibition experiment for the reaction

The effect of the king scallop was compared with the control group in which physiological saline was administered by quantifying the leakage of pigment by incision of local skin.

More specifically, the following will be described. An IgE antibody dependent passive dermal anaphylactic response model was generated by intradermal injection of anti-DNP IgE into the dorsal skin of mice followed by DNP-HSA injection into the tail vein.

Cleanly dehydrated mouse skin was injected intradermal with 100 ng of anti-DNP IgE each 24 hours prior. The injection site was marked with water insoluble red ink. DNP-HSA was used diluted to 4% Evans Blue in 1 mg DNP-HSA / ml dissolved in phosphate buffered saline.

Physiological saline and various concentrations of king mackerel were administered orally 1 hour before the reaction, and 40 minutes after the reaction, the animals were dyed and stained skin areas were taken to quantify the Evans blue amount. In order to analyze the amount of Evans blue, the dissected tissue was added and dissolved in 1 ml of 1.0N KOH, extracted with 9 ml of acetone and phosphoric acid mixture (5:13), and absorbance was measured at a wavelength of 620 nm.

Experimental Example  4-6. IgE  Passive Skin by Antibody Anaphylaxis  Inhibition results for the response

King Mince Dose
(g / Kg BW) Amount of dye (mg / L) Inhibition Rate (%) ± Standard Error 0 (physiological saline administration) 13.83 ± 0.45 0 ± 11.12 0.1 2.83 ± 1.50 80.45 ± 4.56 * 0.05 6.91 ± 0.29 51.08 ± 2.83 * 0.01 9.82 ± 0.66 27.45 ± 3.21

Table 3 shows the results of quantifying the leaked pigment, which is expressed as the mean value and the standard error in three independent experiments (n = 8), respectively. (* P <0.01; significant compared to control)

Referring to [Table 3], King Dried Cattle (0.01-0.1 g / kg BW) inhibited the passive skin aniphylactic response in a concentration-dependent manner, and showed a significant inhibition at 0.1 g / kg.

Experimental Example  4-7. Histamine release inhibition preparation

The free inhibition of histamine from the mast cells by Compound 48/80 was tested using the king exophore.

After rats were anesthetized with chloroform, about 30 ml of T-load buffer B (NaCl, NaHCO ₃ , KCl, NaH ₂ PO ₄ , glucose) containing 0.1% gelatin was injected into the abdominal cavity and lightly massaged the abdominal wall for 90 seconds, followed by abdominal wall centerline. Carefully dissected and the fluid containing the peritoneal cells was removed with a Pasteur pipette. The peritoneal cells were repeatedly centrifuged twice at 150 x g for 10 minutes, and then the supernatant was discarded and resuspended in 1 ml of T-load buffer B.

Mast cells in the cell suspension were separated and purified using 22.5% w / v methazamide. That is, 1 ml of suspension was gently added to 2 ml of 22.5% w / v methazamide and centrifuged carefully at 400 x g for 10 minutes to prevent mixing. At this time, only precipitated cells were taken.

Mast cells isolated as described above (2 × 10 ⁵ cells / ml) were stabilized by culturing for 20 minutes in a medium mixed with α-MEM and FBS in a 1: 1 ratio, and then cultured by treating the wangjueul by concentration. After 15 minutes, 48/80 (6 mg / L) was treated, and after 10 minutes, only the supernatant was taken and analyzed for the amount of histamine released.

Experimental Example  4-8. Rat  Histamine Free Inhibition from Peritoneal Mast Cells

In order to measure the histamine released from the abdominal mast cells, 500 μl of the sample was placed in an eppendorf tube, 450 μl of 0.1 N-HCl, 50 μl of 60% perchloric acid solution, mixed, and centrifuged (1,500 rpm, 20 min). 800 μl was placed in a test tube containing 500 μl of 5 N-NaOH solution, 3 ml of distilled water, 10 ml of n-butanol, and 1.2 g of NaCl, followed by stirring for 20 minutes, followed by centrifugation (2,000 rpm, 10 min).

8 ml of butanol layer was placed in a 50 ml test tube, and 3 ml of 0.1 N-HCl solution and 10 ml of n-heptane were added thereto, stirred for 20 minutes, and centrifuged (2,000 rpm, 10 min). To 2 ml of the aqueous layer obtained, 400 μl of 1 N-NaOH solution and 100 μl of 1% o-phthaldialdehyde solution were reacted for 3 minutes in a water bath (37 ° C.), and then 200 μl of 3 N-HCl solution was added thereto. After mixing, the fluorescence was analyzed by fluorescence absorbance (λex = 360 nm, λem = 440 nm).

The histamine release rate (%) was calculated as follows.

Inhibition rate (%) = (Histamine sheep without wild boil minus-Histamine sheep without wild boil minus) × 100 / Histamine without wild boil minus

Experimental Example  4-9. Rat  Results of Histamine Free Inhibition from Peritoneal Mast Cells

King Mince Concentration (mg / mL) Inhibition Rate (%) ± Standard Error 0 (physiological saline administration) 0 ± 2.42 0.1 59.25 ± 2.71 * 0.05 42.62 ± 2.82 * 0.01 26.08 ± 2.45 * 0.005 12.45 ± 2.82 *

The results are shown as mean and standard error in three independent experiments (n = 6), respectively (* P <0.01; significant compared to control).

Referring to [Table 4], it can be seen that the king's cattle (0.005-0.1 mg / mL) inhibited the histamine release induced by compound 48/80 from mast cells in a concentration-dependent manner.

Experimental Example 4 summarized, the result of oral administration of King Ddang to experimental animals that caused the systemic anaphylactic reaction and passive skin anaphylactic reaction suppressed the reaction in a concentration-dependent manner.

In addition, as a result of treatment with perilla mast cell, peritoneal mast cells inhibited histamine release from mast cells in a concentration-dependent manner.

Therefore, King Dwarf can enhance normal immune ability by activating anti-allergy.

Experimental Example  5. Investigation of the Antioxidant Activity of the Dried Red Pepper

In order to investigate the antioxidant activity of the King Ddang, the extract of Example 2 was used, but the anti-oxidant activity was investigated. In addition, using the powder prepared in Example 1 to produce a fraction according to Example 2 to investigate the antioxidant activity.

Experimental Example  5-1. Preparation of Antioxidant Activity of King Dried Cattle

In order to investigate the antioxidant activity, the antioxidant activity measurement method using free radical 1, 1-diphenyl-2-picrylhydrazyl (1, 1-diphenyl-2-picrylhydrazyl, DPPH) (Blois, Nature, 188: 1199, 1958; Choi, et al., Kor. J. Phamacogn, 24: 299-303, 1993).

4 ml of methanol was added to the glass test tube, and sample compounds were added at different concentrations (1.5-30 µl). Then, 1 ml of the DPPH (0.15 mM) solution was added thereto, reacted at room temperature for 30 minutes, and absorbance was measured at 517 nm. RC ₅₀ (μg / ml) indicates the concentration of the compound that reduces the value of the control group to which nothing is added by 50%.

Experimental Example  5-2. Antioxidant Activity Test Results

Antioxidant Activity of Fresh Juvenile Fractions Fraction Yield (g) Antioxidant activity (RC ₅₀ : ㎍ / mL) Hexane fraction 0.32 68 Ethyl acetate fraction 2.68 38 Butanol fraction 5.2 35 Water fraction 76.7 33 α-tocopherol (comparative group) 14

Antioxidant Activity of Extracts from Korean Dried Algae Powder Fraction Yield (g) Antioxidant activity (RC ₅₀ : ㎍ / mL) Methanol extract 207.8 36 Hexane fraction 35.8 21 Ethyl acetate fraction 6.2 3 Butanol fraction 15.7 8 Water fraction 142.5 13 α-tocopherol (comparative group) 14

As can be seen from [Table 5] and [Table 6], the ethyl acetate fraction and the butanol fraction showed strong antioxidant activity and the water fraction showed some activity, but the powder methanol extract was the strongest overall.

Experimental Example  6. King Dwarf Anti-lipid peroxidation  Activity investigation

In order to investigate the anti-lipid peroxidation activity of the King Ddang, the extract of Example 2 was used to produce anti-lipid peroxidation activity. In addition, using the powder prepared in Example 1, but produced a fraction according to Example 2 to investigate the anti-lipid peroxidation activity.

Experimental Example  6-1. King Dwarf Anti-lipid peroxidation  Active Experiment Preparation

In order to investigate antilipid peroxidation activity, mice were examined using Okawa (Ohkawa, et al., Anal. Biochem., 95: 351, 1979).

First, the microsomes were fractionated from the liver of the mice, separated and suspended in 100 ml Tris-HCl buffer (pH 7.4). The obtained microsomal fraction 0.3% and the compound of each concentration were mixed, 500 μM FeSO ₄ · 7H ₂ O was added to the mixture, and the mixture was shaken at 37 ° C. for 30 minutes, followed by 20% TCA (3M trichloroacetic acid: 2.5M ammonium chloride). = 1: 1) was added to stop the reaction.

Through the reaction, the lipid peroxide is produced according to the concentration of the compound malondialdehyde (Malondialdehyde, MDA) is reacted with thiobarbituric acid (TBA), lipid peroxidation reaction by measuring the TBA reaction product The% lipid peroxidation inhibitory activity can be calculated from Equation 1 below.

Where T is the test group in which the peroxidation reaction was added with the compound, C is the test group in which the peroxidation reaction occurred without the compound, and B was the value obtained by measuring absorbance at 530 nm, respectively, in the control group without the peroxidation reaction. to be.

Experimental Example  6-2. King Dwarf Anti-lipid peroxidation  Active Experiment Results

Antilipid Peroxidation Activity of Fresh Juvenile Extracts Fraction Yield (g) Lipid peroxidation inhibitory activity (RC ₅₀ : ㎍ / mL) Hexane fraction 0.32 > 100 Ethyl acetate fraction 2.68 40 Butanol fraction 5.2 75 Water fraction 76.7 > 100 α-tocopherol 5

Antilipid Peroxidation Activity of the Powder Extracts Fraction Yield (g) Lipid peroxidation inhibitory activity (RC ₅₀ : ㎍ / mL) Methanol extract 207.8 > 100 Hexane fraction 35.8 > 100 Ethyl acetate fraction 6.2 20 Butanol fraction 15.7 30 Water fraction 142.5 > 100 α-tocopherol (comparative group) 5

As can be seen from Tables 7 and 8, the ethyl acetate fraction showed strong antilipid peroxidation activity, but also a little activity in the butanol fraction.

Claims (10)

Composition for immune enhancement, containing King Lax (Lactuca indica var. Laciniata) as an active ingredient. The method according to claim 1,
The royal extract is a composition, characterized in that the powder or extract. The method according to claim 1,
The composition is characterized in that the immune enhancement by increasing the production of nitric oxide (NO). The method according to claim 1,
The composition is characterized in that the immune enhancement by promoting anti-allergic activity. The method according to claim 1,
The composition is characterized in that the immune enhancement by promoting antioxidant activity. 3. The method of claim 2,
The extract is a composition, characterized in that extracted with water or high purity lower alcohol. 3. The method of claim 2,
The extract is characterized in that the extract is extracted with high purity lower alcohol extract, high purity lower alcohol and then further fractionated with hexane, ethyl acetate, butanol or water, respectively. Health functional food for immune boosting containing Lactuca indica var. Laciniata as an active ingredient. Cosmetic for strengthening immunity which contains Lactuca indica var. Laciniata as an active ingredient. Livestock feed for immune enhancement containing Lactuca indica var. Laciniata as an active ingredient.

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