KR101243115B1 - A fever reducer containing stauntonia hexaphyllafruit leaf extract - Google Patents

Abstract

PURPOSE: An antipyretic drug containing a Stauntonia Hexaphylla leaf extract is provided to ensure an antipyretic effect without cytotoxicity. CONSTITUTION: An antipyretic drug contains a Stauntonia Hexaphylla leaf extract. The extract is prepared by extracting Stauntonia Hexaphylla leaves with water, alcohol of 1-5 carbon atoms, and a mixture thereof. The extract is a fraction which is prepared by fractioning the Stauntonia Hexaphylla leaf extract with chloroform, butanol, or ethyl acetate.

Description

Antipyretic agent containing honey leaf extract {A FEVER REDUCER CONTAINING STAUNTONIA HEXAPHYLLAFRUIT LEAF EXTRACT}

The present invention relates to an antipyretic comprising a plant extract having an antipyretic effect, specifically a honey leaf extract as an active ingredient.

Antipyretic drugs (antipyretic drug) is a drug that acts to lower the body temperature in the fever state, generally called analgesic action because it also exhibits analgesic action at the same time.

The opinion currently supported mainly by the mechanism of action associated with antipyretics is that the antipyretics inhibit the biosynthesis of prostaglandin (PG) to soothe fever and enable antipyretics.

Specifically, since the prostaglandin concentration of the hypothalamic thermoregulatory center increases during fever, when the prostaglandin content of the thermoregulatory center is lowered, it is possible to suppress the exothermic effect and exert an antipyretic effect. In addition, the prostaglandins are known as mediators of pain. However, various mechanisms have been proposed in relation to the symptoms of fever.

Antipyretic agents currently prescribed include salicylic acid derivatives such as aspirin, aniline derivatives such as acetone anilide and phenacetin, and parazoron derivatives such as antipyrine, aminopyrin or sulfirin. Some anti-inflammatory drugs have antipyretic analgesic effects, such as nonsteroidal anti-inflammatory drugs such as indomethane.

As described above, although some associations may be found in relation to antipyretic analgesic action and anti-inflammatory effect, that is, anti-inflammatory effect, while there is little anti-inflammatory action, there are also powerful drugs for antipyretic action, while antipyretic action is rare but anti-inflammatory The anti-inflammatory and antipyretic and analgesic effects do not necessarily correlate directly.

Therefore, it is not only a compound derived from natural compounds, such as aniline, which causes acute poisoning, but also has a high antipyretic effect. There is a need to develop materials that do not exist.

In particular, recently, research and development on antipyretics have been actively conducted as natural products using natural raw materials to meet the needs of consumers.

KR 0234495 B KR 0329685 B KR 0484525 B KR 0922987 B

In order to meet the above requirements, an object of the present invention is to provide a composition for antipyretic with an active ingredient of a plant extract is less likely to cause problems related to side effects.

In order to achieve the above object, the present invention provides a composition for antipyretic comprising a honey leaf extract as an active ingredient. The antipyretic composition may be a pharmaceutical composition, for example, may be an antipyretic or antipyretic analgesic.

The honey leaf extract was not only excellent in the antipyretic effect, it was further confirmed that the chloroform fraction among the various solvent fractions of the hot water extract of the honey leaf has an excellent antipyretic effect compared to the other fractions in the range that the toxicity is not a problem.

The present inventors have a significant antipyretic effect of the honey leaf extract unlike other anti-inflammatory effect of the plant extract having an anti-inflammatory effect, especially the chloroform fraction of the hot water extract of the honey leaf is antipyretic compared to the fraction using other fraction solvent It was confirmed through the animal experiments that the effect is excellent, in addition, when butanol is used as the fraction solvent, the fraction yield is excellent, the property antipyretic effect is excellent and the antipyretic effect is also superior to the hexane fraction. The invention has been completed.

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

The present invention relates to a composition for antipyretic comprising honey extract, preferably honey leaf extract as an active ingredient.

The honey ( Stauntonia hexapillya) is an evergreen vine plant with the dicotyledon plant Buttercup hyacinth creeper, also known as honey beet. The honeysuckle is one male and female, the main stem extends about 5m, the leaves are shifted, palm-like double leaf consisting of 5 to 7 small leaves. Small leaves are thick, egg-shaped or oval, with flat edges. Petioles are about 6cm to 8cm long, petioles about 3cm long. Flowers blossom in May, yellowish white, hanging on inflorescences. Small flower branch of female flower becomes reddish brown in autumn, and there are many big trees and is rough. Fruits are berries, egg-shaped or oval, 5cm to 10cm long, ripened in reddish brown in October, and pulp tastes better than erosion. Seeds are oval-shaped oval black. The honey is mainly distributed in Korea, Japan, Taiwan or China. In Korea, it grows well in the valleys and forests of southern regions such as Jeollanam-do, Gyeongsangnam-do, and Chungcheongnam-do.

The honey extract may be prepared according to a conventional method for producing a plant extract, for example by extracting by adding an extracting solvent to the leaves, branches, stems, roots or bark of the honey or a pulverized product thereof, preferably the leaves of the honey It may be prepared by fractionation by adding a fractional solvent to the crude extract prepared or prepared by extraction with an extraction solvent.

The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as alcohol having 1 to 5 carbon atoms, the alcohol dilution water, ethyl acetate or acetone and a nonpolar solvent of ether, chloroform, benzene, hexane or dichloromethane, or a mixed solvent thereof. The alcohol having 1 to 5 carbon atoms may be methanol, ethanol, propanol, butanol, or isopropanol, but is not limited thereto. In addition, the alcohol dilution water may be one obtained by diluting the alcohol in water at 50% (v / v) to 99.9% (v / v).

Extraction solvent of the honey leaf extract of the present invention is preferably at least one selected from the group consisting of water, alcohol having 1 to 5 carbon atoms, alcohol dilution water and mixtures thereof, more preferably water, 1 to 4 carbon atoms It may be any one selected from the group consisting of alcohols and mixtures thereof, and even more preferably may be water. For example, the extraction process may be performed at 50 ° C. to 150 ° C., or 75 ° C. to 120 ° C., or 90 ° C. to 115 ° C., but is not limited thereto. In addition, the extraction time is not particularly limited, but may be 10 minutes to 12 hours, or 30 minutes to 8 hours, or 2 hours to 6 hours.

The honey leaf extract of the present invention may be prepared according to a conventional method for producing a plant extract, and specifically, may be a heat extraction method including a hot water extraction method, a cold extraction method, a warm extraction method, an ultrasonic extraction method, etc. Grinding extractors or fractionators may be used.

In addition, the extract extracted with the solvent is any one solvent selected from the group consisting of hexane, chloroform, methylene chloride, ethyl acetate, ethyl ether, acetone, butanol, water and mixtures thereof, preferably ethyl acetate, chloroform, Or butanol, more preferably ethyl acetate or chloroform, still more preferably ethylacetate. Two or more solvents may be used in the fractionation, and each solvent extract may be prepared by sequentially using or mixing the solvent according to the polarity of the solvent.

The thus-prepared extract or the fraction obtained by performing the fractionation process may be filtered, concentrated or dried to remove the solvent, and may be subjected to both filtration, concentration and drying. Specifically, the filtration may be performed using a filter paper or a reduced pressure filter, the concentration may be concentrated under reduced pressure using a reduced pressure concentrator, for example, a rotary evaporator, and the drying may be performed by, for example, a lyophilization method.

The antipyretic composition may be used as a medicament, or for medicinal or pharmaceutical use. In this aspect, the antipyretic composition may be a medicinal composition, and for example, may be an antipyretic or antipyretic analgesic.

When the antipyretic composition is used for medicinal or pharmaceutical purposes, the antipyretic composition may be used to suppress abnormal fever or to treat or prevent abnormal fever associated with a disease.

In this aspect, the present invention may be directed to a composition for antipyretic comprising honey leaf extract as an active ingredient. The antipyretic composition comprising the honey leaf extract as an active ingredient may be used to suppress, treat, improve or prevent abnormal fever associated with abnormal fever or disease or disease, and may be an antipyretic or antipyretic analgesic agent.

In the antipyretic composition comprising the honey leaf extract as an active ingredient, the honey leaf extract is a honey leaf hydrothermal extract, preferably a chloroform fraction, ethyl acetate fraction or butanol fraction of the honey leaf hydrothermal extract, more preferably ethyl acetate Fractions or chloroform fractions, even more preferably ethyl acetate fractions.

The abnormal exotherm means that the body temperature is abnormally high.

The antipyretic is a drug used to relieve fever or abnormal fever, and refers to a medicine used to lower heat when the body temperature becomes abnormally high. Antipyretics and antifebrin are known as antipyretics. , Apyrin or salipyrin. The antipyretic is usually called an antipyretic analgesic because it also has an analgesic alleviating effect.

The antipyretic composition comprising the honey leaf extract as an active ingredient may be directly applied to animals including humans. The animal is a biological group corresponding to a plant, and the organic material is mainly consumed as nutrients, and the digestive organ, the excretory organ, and the respiratory organ are differentiated. Preferably, the animal may be a mammal, more preferably a human.

The honey leaf extract may be used alone in the antipyretic composition, and may further include other pharmacologically acceptable carriers, excipients, diluents or subcomponents.

More specifically, when the composition comprising the honey leaf extract is used as a medicament, or for medicinal or pharmaceutical use, the honey leaf extract is mixed with a pharmaceutically acceptable carrier or excipient according to a conventional method or It can be used diluted with a diluent.

In this case, the content of the honey leaf extract in the composition may be 0.001% by weight to 99.9% by weight, 0.1% by weight to 99% by weight or 1% by weight to 50% by weight, but is not limited thereto. Depending on the method, the content of the extract can be used by appropriately adjusting to the desired content.

Examples of the pharmaceutically acceptable carrier, excipient or diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and one or more selected from the group consisting of physiological saline, but is not limited thereto, and all common carriers, excipients or diluents can be used. Two or more carriers or excipients may be used.

In addition, the antipyretic composition may be a conventional filler, extender, binder, disintegrant, anti-coagulant, lubricant, wetting agent, pH adjuster, nutrient, vitamin, electrolyte, alginic acid and salts thereof, pectic acid and salts thereof, protective colloids, Glycerin, fragrances, emulsifiers or preservatives may be further included. The components may be added independently or in combination to the honey leaf extract is the active ingredient.

In addition, the composition for antipyretic of the present invention may further include a substance recognized to have a known antipyretic effect in addition to the active ingredient.

In addition, the antipyretic agent of the present invention may further include a compound or a plant extract having a known antipyretic effect in addition to the active ingredient, and 0.1 parts by weight to 99.9 parts by weight or 0.5 parts by weight to 20 parts by weight based on 100 parts by weight of the active ingredient, respectively. It may be included in parts by weight.

When the composition is used as a medicine, it can be administered orally or parenterally. In one example, it can be administered through various routes including oral, transdermal, subcutaneous, intravenous or muscular.

In addition, the formulations of the compositions may vary depending on the method of use and may be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal .

In general, solid dosage forms for oral administration include tablets, pills, soft or hard capsules, pills, powders and granules, which may contain one or more Excipients such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Oral liquid preparations include suspending agents (SUSTESIONS), liquid solutions, emulsions (EMULSIONS) and syrups (SYRUPS) .Such as simple diluents such as water and liquid paraffin, various excipients such as wetting agents, Sweeteners, fragrances, preservatives and the like can be included.

Forms for parenteral administration include creams, looses, ONITMENTS, PLASTERS, LIQUIDS AND SOULTIONS, AEROSOLS, FRUIDEXTRACTS, (ELIXIR), INFUSIONS, SACHET, PATCH or INJECTIONS.

Furthermore, the compositions of the present invention may be formulated using suitable methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). Can be.

The dosage of the composition may be determined in consideration of the administration method, the age, sex, the severity of the patient, the condition of the patient, the degree of absorption of the active ingredient in the body, the inactivity and the drug to be used in combination. Kg body weight to 500 mg / kg body weight, 0.1 mg / kg body weight to 400 mg / kg body weight or 1 mg / kg body weight to 300 mg / kg body weight, And may be administered once or several times in divided doses. The dose is not intended to limit the scope of the invention in any way.

In addition, the present invention relates to a method for preparing a fraction of the honey leaf extract having an antipyretic effect in one aspect.

More specifically, as confirmed in one embodiment of the present invention, in the solvent fraction of the honey leaf hydrothermal extract, when the hexane is used as the fractionation solvent, the yield is too low, and the antipyretic effect is insignificant. It was evaluated as inappropriate as it may occur. On the other hand, in the case of butanol fraction, the economic efficiency was recognized with high yield, but it was confirmed that the antipyretic effect was not high. On the other hand, when ethyl acetate is used as the fractional solvent, it is not only the highest yield compared to other fractional solvents except butanol, but also excellent antipyretic effect, it is recognized that the treatment effect and economic efficiency is excellent industrially.

Therefore, the ethyl acetate fraction was judged to be excellent in industrial aspect, namely, in terms of antipyretic effect and production yield, and ethyl acetate was selected as a fraction solvent.

Specifically, the method for producing a honey fraction having the antipyretic effect is a step of preparing an ethyl acetate fraction by adding water to the honey honey ( Stauntoni ahexaphylla ) and extracting the hydrothermal extract and adding ethyl acetate to the prepared hydrothermal extract of honey. It may be a method comprising a.

The honey hot water extract manufacturing step is the honey ( Stauntoni ahexaphylla ) After adding water, for example distilled water to the leaves, 0.5 to 20 hours, preferably 1 hour to 80 ℃ to 150 ℃, preferably 90 ℃ to 130 ℃, more preferably 100 ℃ to 120 ℃ The heating may be carried out for 10 hours, more preferably for 2 hours to 8 hours.

The amount of water added based on 100 parts by weight of the honey leaf may be 100 parts by weight to 10,000 parts by weight or 500 parts by weight to 5,000 parts by weight or 1,000 parts by weight to 3,000 parts by weight.

For example, the honey hot water extract manufacturing step is the process of washing the honey leaf in flowing water; Adding 1,500 parts by weight to 2,500 parts by weight of water, more specifically distilled water, based on 100 parts by weight of the honey leaf, and heating the mixture of honey and water at 100 ° C. to 120 ° C. for 3 hours to 5 hours. It can be carried out by the method, the process of condensing the extract prepared by the method using a filter cloth, the process of concentrating the filtrate obtained by the filtration process under reduced pressure and the like, and freeze-dried the concentrated concentrate The method may further include drying the method.

The preparing of the ethyl acetate fraction may include adding ethyl acetate to the hot water extract; And it can be carried out by a method comprising the step of separating the ethyl acetate fraction.

For example, preparing the ethyl acetate fraction may include dissolving the hot water extract in distilled water; Ethyl acetate may be added to the solution and mixed with each other, and the ethyl acetate layer may be separated from the mixed solution.

The preparing of the ethyl acetate fraction may be performed by sequentially adding and mixing a solvent in the order of hexane, chloroform, ethyl acetate and butanol in a solution of hot water extract dissolved in distilled water, separating the solvent layer, It can be carried out by adding a solvent and preparing a fraction of the solvent.

In addition, the step of preparing the ethyl acetate fraction is the process of filtering the fraction prepared by the method using a filter cloth, the step of concentrating the filtrate obtained by the fraction or the filtration process through a reduced pressure concentration method, and the The concentrated concentrate may further include a step of drying by a method such as lyophilization.

In relation to the preparation of fractions of the honey leaf hydrothermal extract, the yield of the hexane fraction is 0.015%, the yield of the chloroform fraction is only 0.27%, while the yield of the ethyl acetate fraction is significantly superior to 1.05%, In addition to the excellent antipyretic effect, in the case of the honey leaf material, unlike the honey fruit, its yield is abundant and easy to store, and thus the method of producing ethyl acetate fraction of the honey leaf hydrothermal extract is economically recognized due to its high yield in fraction production efficiency. In addition, since the antipyretic effect is recognized, it is evaluated to be excellent industrially.

The honey leaf extract of the present invention is a plant-derived extract used for edible, and has no problems with side effects or safety, and as a result of MTT analysis, there is no cytotoxicity, and it is easy to produce because the yield is higher than that of honey, and antipyretic effect It is also recognized, in particular, the ethyl acetate fraction of the fraction of the honey leaf extract was confirmed that the antipyretic effect is remarkably excellent through animal experiments.

1 is a schematic diagram showing a process for preparing a honey leaf hydrothermal extract and a solvent fraction of the hydrothermal extract according to an embodiment of the present invention.
Figure 2 is a graph showing the results of measuring the cytotoxicity of the honey leaf extract by MTT assay using RAW264.7 cell line according to an embodiment of the present invention, where + is LPS (1 μg / mL) Or-means that the extract was treated,-means not treated, the SHL value of the horizontal axis represents the dose (㎍ / mL) of the honey leaf hydrothermal extract, the vertical axis is the control group not administered any sample % Of relative cytotoxicity.
Figure 3 is a graph showing the results of the antipyretic heat induced by LPS, in order to confirm the antipyretic effect of the honey leaf hydrothermal extract, using the experimental animal according to an embodiment of the present invention, the horizontal axis number is a sample Elapsed time (hour, h) after administration, the value on the vertical axis means the measured body temperature.
Figure 4 is a graph showing the results of the heat dissipation induced by the LPS, in order to confirm the antipyretic effect of the honey leaf hydrothermal extract using an experimental animal, the numerical value of the horizontal axis is a sample Elapsed time (hour, h) after administration, the value on the vertical axis is the change in body temperature measured from the body temperature measured before the administration of the sample, that is, the body temperature of the experimental animal measured before administration of the sample at the body temperature measured at that time It means minus body temperature.
5 is a graph showing the results of measuring the cytotoxicity of the fraction of the honey leaf extract by MTT assay using RAW264.7 cell line according to an embodiment of the present invention, where + is LPS (1 μg) / mL) or solvent fractions treated,-means untreated samples, each value on the horizontal axis represents the dosage (㎍ / mL) of the fraction of each fraction solvent of the honey leaf hot water extract The vertical axis represents the percentage (%) showing cytotoxicity compared to the control group which did not receive any sample.
Figure 6 is a graph showing the results of dissociating the heat generated by LPS, in order to confirm the antipyretic effect of the fraction of the honey leaf hot water extract, using the experimental animal according to an embodiment of the present invention, the numerical value of the horizontal axis Is the time elapsed after the administration of the sample (hour, h), and the value on the vertical axis is the change in body temperature from the temperature measured before the administration of the sample, that is, the experiment measured before administration of the sample at the body temperature of the experimental animal measured at that time. It means minus the body temperature of the animal.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to specific examples and comparative examples in order to help understanding of the present invention. However, the following examples are only intended to more clearly understand the present invention, and are not limited to the following examples of the present invention, the protection scope of the present invention should be interpreted by the claims, and within the equivalent range All technical thoughts should be construed as being included in the scope of the present invention.

< Example  1> Honey  Leaf extract and Fraction  Produce

1-1. Honey  Leaf extract manufacturer

A hot water extract was prepared by performing hot water extraction at 110 ° C. using 10 kg of leaves of Stauntonia hexaphylla .

More specifically, after adding 200 L of distilled water to 10 kg of honey leaves washed with distilled water, hot water extraction was performed while heating at 100 ° C. for 3 hours using an electric bath. After performing the extraction, the resultant was filtered through a 400 mesh filter cloth, and the filtrate was concentrated using a vacuum concentrator. After filtration, using the same amount of distilled water again to the remaining residue was performed twice more extraction, filtration and reduced pressure in the same process.

The honey leaf hot water extract prepared through the above process was lyophilized in a freeze dryer. Through the lyophilization, 1 kg of honey leaf hydrothermal extract was obtained, and as a result, it was confirmed that the yield by the honey leaf hydrothermal extraction method was 10%.

1-2. Honey  Of leaf extract Fraction  Produce

Fraction preparation of the honey leaf hydrothermal extract was carried out by the preparation method shown in FIG.

Specifically, 250 g of the hot water extract is completely dissolved in 5 L of distilled water, and then put into a fraction filter and 5 L of hexane (Hexane) is added and mixed, and then fractionated. The hexane soluble layer hexane layer and the hexane insoluble layer are separated. The hexane fraction was prepared by separating and obtaining only the hexane layer.

5 L of chloroform was added to the remaining solution (aqueous layer), followed by mixing. The chloroform fraction was prepared by separating the chloroform layer, which is a chloroform soluble layer, and the aqueous layer, which is a chloroform insoluble layer, to obtain only a chloroform layer.

5 L of ethyl acetate was added to the remaining solution (aqueous layer), followed by mixing, fractionating, separating an ethyl acetate layer as an ethyl acetate soluble layer and an aqueous layer as an ethyl acetate insoluble layer, and obtaining only an ethyl acetate layer to prepare an ethyl acetate fraction. It was.

Butanol fraction was prepared by adding 5 L of butanol to the remaining solution (aqueous layer), followed by mixing, separating the butanol layer as the butanol soluble layer and the water layer as the butanol insoluble layer, and obtaining only butanol layer.

The butanol soluble layer was fractionated to separate the remaining butanol insoluble layer to remove the remaining organic solvent, thereby preparing a water fraction.

Each of the obtained fractions was concentrated by filtration through a vacuum filter, and then lyophilized at -20 ° C to completely remove the solvent, and then used in this experiment. Through this procedure, 0.02 g of hexane fraction (0.015%), 0.67 g of chloroform fraction (0.27%), 2g of ethyl acetate fraction (1.05%) and 68.75 g of butanol fraction (27.5%) were used as samples. Since the yield of the hexane fraction in the manufacturing process is too low can cause process problems in the industrialization, it was evaluated as inappropriate. The extracts and fractions obtained were cryopreserved until use in experiments.

< Example  2> extract and Fraction  Cytotoxicity experiment

In order to measure the cytotoxicity of fractions of the honey leaf hydrothermal extract and honey leaf hydrothermal extract prepared in Example 1, RAW264.7 cells, which are mouse macrophages, were purchased from ATCC.

DMEM / F12 (Dulbecco's modified Eagle's medium / Nutrient Mixture Ham's F12), FBS (fetal bovine serum), L-glutamine (L-glutamine) and penicillin-streptomycin used in the cell culture were Gibco / BRL (USA).

The RAW264.7 cells were cultured using a medium containing 10% FBS, 1% penicillin streptomycin and 1% L-glutamine in DMEM / F12 medium, and a 37 ° C. wet CO₂ incubator (5% CO₂ / 95% air). Incubated).

After about 80% of the cells were incubated with the cells, the monolayers of cells were washed with PBS (pH 7.4), washed, and subcultured with 0.25% trypsin and 2.56 mmol / L EDTA. Medium was changed every two days.

The cultured cells were aliquoted into 48 well-plates at a density of 50,000 cells / well and further cultured for 24 hours. After 24 hours, the LPS-treated control group without any treatment and LPS and the honey leaf extract and fraction of Example 1 were prepared at various concentrations using DMSO, which was found to have little effect on cell survival. The honey leaf extract and fractions were divided into experimental groups, and further cultured for 24 hours, the culture solution was removed, and the number of living cells was measured by MTT assay. The MTT analysis was performed in the following manner.

First, the cell culture medium was removed, and then treated with 1 ml per well of DMEM / F12 medium containing 1 mg / ml of MTT, and further incubated in a 37 ° C. humidified CO 2 incubator for 4 hours. After removing the medium, the tetrazolium bromide salt was removed, and 200 μl of DMSO was dissolved to dissolve the formazan crystal produced in each well, and the cell viability was measured by measuring absorbance at a wavelength of 540 nm in a microplate reader (BIO-RAD). Confirmed.

The result of the treatment with the honey leaf extract was performed the same three times the experiment, shown in Figure 2 as the average value of the measured value, the result of treatment with the fraction of the honey leaf hot water extract the same three times the experiment It was shown in Figure 5 as the average value of the measured value.

As shown in FIG. 2, the honey leaf hydrothermal extract prepared in Example 1-1 was treated at various concentrations, specifically, the honey leaf hydrothermal extract at a concentration of 10 μg / ml to 200 μg / ml and elapsed for 24 hours. In any case, it was confirmed that all samples did not show any effect on the proliferation of the cells as compared with the control without LPS treatment. Therefore, it was confirmed from the results that the honey leaf extract is not cytotoxic up to 200 ㎍ / ㎖.

In addition, as shown in Figure 5, in the case of the fraction of the honey leaf hot water extract prepared in Example 1-2, it was confirmed that the cell survival rate significantly decreased in the experimental group administered 25 ㎍ / ㎖ in the hexane fraction It was found to be cytotoxic. In addition, the ethyl acetate fraction was not significant in the experimental group administered with 100 ㎍ / ml, but the cell viability was slightly reduced, and further decreased in the experimental group administered with 200 ㎍ / ml, and significantly reduced to 100 ㎍ / ml. It was confirmed to be safe. In addition, the cell viability was maintained at 50 ㎍ / ㎖ or 100 ㎍ / ㎖ in the case of other solvents, there is no cytotoxicity when the 50 ㎍ / ㎖ administration of the fraction using a solvent other than hexane as a fraction solvent, It was confirmed to be safe.

< Example  3> extract and Fraction  Antipyretic effect measurement

3-1. Honey  Confirmation of antipyretic effect of leaf extract

In order to confirm the antipyretic effect of the honey leaf extract and fractions prepared in Example 1, an experimental animal was used.

The experimental animals were used to purchase 5 weeks old Sprague-Dawley (SD) male rats (Samtako, South Korea). The breeding of the experimental animals was carried out under temperature conditions of 20 ℃ to 24 ℃, humidity conditions of 50% to 60% and day and night lighting conditions of 12 hours cycle, water and diet were to be ingested freely. The diet used a solid feed (Samyang feed, South Korea). The experimental animals were bred under the above conditions for 7 days to adapt to the laboratory environment, and then used for the experiment.

Lipopolysaccharide (LPS) -induced fever experiment, which is a microbial tootoxin (bacterial endotoxin) to confirm the antipyretic effect using the experimental animal, was performed by Vilela FC et al. (Vilela FC et al. al , Anti-inflammatory and antipyretic effects of Sonchus oleraceus in rats. J Ethnopharmacol . 17; 127 (3): 737-41 (2010)).

Specifically, five of the experimental animals were randomly selected as a group, and fever was induced by intraperitoneal injection of 500 μg / kg of lipopolysaccharide (LPS, Sigma, USA). The body temperature was measured by using a rectal temperature measuring instrument (Portable Thermocouple Thermometer (Physitemp Instruments, USA) and Stainless Steel Rectal Probe for Rats (Physitemp Instruments, USA)), and measured the body temperature of the rat three times before the experiment. Temperature rise due to temperature measurement stress was minimized.

First, in order to confirm the antipyretic effect of the honey leaf hydrothermal extract, 50 mg / kg oral administration of LPS without any sample and acetaminophen (Acetaminophen, APAP, Sigma, USA) without any sample was confirmed. One positive control group (APAP) and a second positive control group (Dexamethasone) orally administered 1 mg / kg of dexamethasone (Sigma, USA) were used.

First, 500 μg / kg of exothermic material (LPS) is administered intraperitoneally to the experimental animal prepared to minimize the temperature rise due to the temperature measurement stress (ip), and then any sample is administered according to the type of the experimental group. (LPS) or honey leaf hot water extract 200 mg / kg orally (SHL-200), acetaminophen 50 mg / kg orally (APAP) and dexamethasone 1 mg / kg orally (Dexamethasone) It was. In addition, the honey leaf hot water extract 200 mg / kg after the pyrogenic substance was administered for 1 hour, and then orally administered (SHL-200 (1h)), and 1 hour, 4 hours and 8 hours for 8 hours after the exothermic substance administration Rectal body temperature was measured over time. The measurement results are shown in FIG. 3 and Table 1 below. Table 1 indicates the body temperature (° C.) measured for each hour.

Normal LPS SHL 200 SHL 200 (1h) Dexamethasone APAP 0h 37.2 37.2 37.2 37.2 37.2 37.2 1h 37.55 ± 0.21 38.2 ± 0.68 36.65 ± 0.69 38.23 ± 0.41 37.3 ± 0.52 36.58 ± 0.67 4h 37.65 ± 0.07 37.85 ± 0.33 37.20 ± 0.45 36.95 ± 0.54 37.6 ± 0.12 37.53 ± 0.59 8h 37.55 ± 0.07 37.6 ± 0.61 37.7 ± 0.14 37.73 ± 0.22 37.73 ± 0.13 37.78 ± 0.13

As shown in FIG. 3 and Table 1, when the exothermic material (LPS) was administered, the body temperature rapidly increased at about 1 ° C. to 1.8 ° C. at 1 hour. However, when the honey leaf hot water extract of the present invention (SHL-200) was administered, the body temperature was remarkably decreased, but the decrease was more excellent than the case of 1 mg / kg oral administration of dexamethasone (Dexamethasone). In addition, 50 mg / kg oral administration of acetaminophen widely used as an antipyretic agent (APAP) showed almost the same degree of antipyretic effect, and at 4 hours, 50 mg / kg of acetaminophen was obtained. It was confirmed that the body temperature was not increased compared with the oral administration (APAP), so the durability was excellent.

In addition, when oral administration of honey leaf hot water extract 200 mg / kg (SHL-200 (1h)) at 1 hour after the administration of the exothermic substance (LPS), body temperature rapidly increased as in the control group. Significantly decreased, at 4 hours, the level was similar to the 50 mg / kg oral administration of acetaminophen (APAP) with pyrogen, at which point the honey leaf hot water extract was at a certain level after the onset of exotherm. It was found to work effectively even after the body temperature was elevated.

In addition, after the administration of a heating material (LPS) to the experimental animal prepared to minimize the temperature rise due to the temperature measurement stress, do not administer any sample according to the type of the experimental group (LPS), honey leaf hot water extract 200 mg / kg orally (SHL-200) or Ivpropene (Ibupropen, Daewoong Pharm., Korea) was administered 20 mg / kg orally (Ibupropen). After the administration of the pyrogenic material and the sample, the rectal body temperature of the experimental animal was measured for 8 hours at an interval of 30 minutes or 1 hour for 8 hours. The measurement results are shown in FIG. 4.

As shown in FIG. 4, in the case of the normal group to which no sample was administered, there was almost no change in body temperature, but when the exothermic substance (LPS) was administered, the body temperature rapidly increased from 30 minutes. After 1 hour, the body temperature increased by about 1 ℃, and although there was a slight decrease, it increased again by about 1 ℃ after 7 hours. On the other hand, when the honey leaf hot water extract of the present invention (SHL-200) was administered, the body temperature was maintained even after 30 minutes, the body temperature was significantly reduced from the time point of 1 hour, somatic body temperature It was confirmed that this is not increased. Unlike the case of 20 mg / kg oral administration (Dexamethasone) of Ibuprofen, such a decrease in body temperature is not confirmed, and the body temperature is measured to be lower than that of Ivprofen in most of the sections. The honey leaf hydrothermal extract was found to be better than Ivprofen.

3-2. Honey  Of leaf extract Fraction  Check antipyretic effect

In order to confirm the antipyretic effect of the fraction of the honey leaf extract prepared in Example 1-2, experimental animals bred by the method of Example 3-1 (SD male rat of 5 weeks old (Samtako, Korea) )) Was used.

Confirming the antipyretic effect using the experimental animal As in Example 3-1, the experiment was performed by applying a method such as Vilela FC microbial toxin (Lipopolysaccharide (LPS) from E. coli 0111: B4 (Sigma, USA)) After the microbial toxin-induced fever (LPs-induced fever), the body temperature was measured by a rectal thermometer.

First, in order to confirm the antipyretic effect of the honey leaf hydrothermal extract, 20 mg / kg oral administration of the negative control group (LPS) without administration of the sample and the ibuprofen (Daewoong Pharm. Positive control group (Ibuprofen) was used. In addition, 20 mg / kg of the hexane fraction (Hx), the chloroform fraction (CHCl3), the ethyl acetate fraction (EA) and the butanol fraction (BuOH) were administered to the experimental group.

First, the body temperature of the experimental animal before the experiment using a thermometer (Portable Thermocouple Thermometer, physitemp, USA), three times by measuring the method of measuring the rectal temperature to minimize the temperature rise due to the temperature measurement stress.

Five minutes after each sample was orally administered to the experimental animal measuring the temperature, the microbial toxin (LPS), which is the pyrogenic substance, was intraperitoneally injected with 500 μg / kg of microbial toxin after 5 minutes. And the rectal body temperature of the experimental animals was measured at 30 minute intervals for 2 hours. The measurement results are shown in FIG. 6.

As shown in FIG. 6, in the case of the normal group to which no sample was administered, there was almost no change in body temperature, but when the exothermic substance (LPS) was administered, the body temperature rapidly increased from 30 minutes. The body temperature was increased by 1 ° C. or more, and the body temperature was increased by about 1 ° C. even when 1 hour had elapsed, and about 0.5 ° C. was increased even after 2 hours. On the other hand, when the hexane fraction of the honey leaf hot water extract of the present invention is administered, the temperature increase range is lower than that of the exothermic substance, but the final temperature is rather high at 2 hours, and the butanol fraction is hexane. The temperature rise was lower than the fraction, but the effect of increasing the temperature was low. On the other hand, when the chloroform fraction was administered, the temperature initially increased, but after 2 hours, the body temperature was almost returned to normal, and it was confirmed that the body temperature increased inhibitory effect or antipyretic effect, and the ethyl acetate fraction was passed for 1 hour. When the body temperature returned to normal at 2 hours, the body temperature was lower than the initial temperature at 2 hours, and it was confirmed that the body had a remarkably excellent antipyretic effect similar to that of Ivprofen, which is widely used as an antipyretic and proven effective.

Claims (7)

A composition for antipyretic, comprising honey extract of Stauntonia Hexaphylla leaf as an active ingredient. The method of claim 1,
The honey leaf extract is a composition for antipyretic, which extracts one or more selected from the group consisting of water, alcohol having 1 to 5 carbon atoms and mixtures thereof with an extract solvent. The method of claim 2,
The honey leaf extract is a composition for antipyretic which is a fraction obtained by fractionating chloroform, butanol or ethyl acetate as a fraction solvent in the honey leaf hydrothermal extract. The method of claim 3,
The honey leaf extract is a composition for antipyretic, which is a fraction obtained by fractionating solvent ethyl acetate or chloroform in the honey leaf hot water extract. 5. The method of claim 4,
The honey leaf extract is a composition for antipyretic, which is a fraction obtained by fractionating ethyl acetate with a fraction solvent in the honey leaf hot water extract. delete Honey ( Stauntonia) hexaphylla ) step of producing honey hot water extract to add water to the leaves and extract the hot water; And
Preparing ethyl acetate fraction by adding ethyl acetate to the prepared honey honey extract
Method of producing a fraction of the honey leaf extract having an antipyretic effect comprising a.

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