WO2002064153A1

WO2002064153A1 - Ixeris dendata extracting having biological activities and immune enhancing effects

Info

Publication number: WO2002064153A1
Application number: PCT/KR2001/001653
Authority: WO
Inventors: Dong-Myong Jeong
Original assignee: Dong-Myong Jeong
Priority date: 2001-01-26
Filing date: 2001-10-04
Publication date: 2002-08-22
Also published as: KR20020063038A; KR100411370B1

Abstract

This invention is concerned about Ixeris dendata(sowthistle) extract which have anti-cancer, anti-oxidative, anti-stress, anti-bacteria, anti-microbial, anti-allergy and cholesterol reduce effects. More in detail, it is a invention about pharmacological composition mateial and health food which are obtained soluble fraction with methanol, ethanol, n-hexane, EtOAc, n-BuOH or water, It could be used widely in prevention and treatment of cancer, adult diseases, cerebral apoplexyl, allergy, stress diseases, inflammation diseases and cardiovascular disease.

Description

Ixeris Dendata extract having biological activities and immune enhancing effects

[ Purpose of the invention ]

The field that the invention belongs to and hitherto technique of the field

This invention is concerning Ixeris dentata(sowthistle) extract which has anti-cancer, anti- oxidization, anti-stress, anti-bacteria, anti-microbial, anti-allergy and cholesterol reduce effects and the manufacturing process of it. To be more detail, it is a invention about pharmacological constitution material and health food which are obtained soluble fraction by methanol, hexane, ethyl acetate, Buthane or water. It could be used widely in prevention and treatment of cancer, adult diseases, cerebral apoplexy, allergy, stress diseases, inflammation diseases and cardiovascular disease. Ixeris dentata belongs to composite and it is a perennial plant. It is widely acknowledged that it composes of more than 80 volatile flavor oil refining ingredients including hexenol. It is described as bitter herbs in the oriental medicine. According to ancient writings, it is efficacious against fever, hemotooiesis, indigestion, pneumonia, hepatitis, bruise and swelling. In addition to it, the books said that it functions sedation, hypnosis and promotion of appetite.

Meanwhile, some of the oxygen coming in body can't be resolved into water and it remains in the condition which keeps the electrons not accompany with the pair. These electrons are called active oxygen. This is caused by stress, the ultraviolet rays, radiation and the chemical material (agricultural chemical, insecticide, medical supply and nitrogen chemical compound).

The active oxygen is not stable so it try to stabilize its state. It refuses to deal with the electrons which do not accompany with the pair or it tries to get rid of the electron. This active oxygen causes cancer, stroke, artery stiffening, diabetes, adult diseases and arthritis since it changes the surrounding systems. An antioxidizer is free radical that is material which neutralizes the action of the active oxygen. It includes various enzymes produced in the body(SOD, POD, catalase, glutathione, peroxidase) and some nutrition materials which should be absorbed from the outside of the body. These are low molecular compounds and they help high molecular antioxidizer or remove the active oxygen for themselves. β-carotene, vitamin A, vitamin C (ascorbic acid), vitamin E (tocophrol) and flavonoids are the representative antioxidizing ingredients and they are strong antioxidizer. Phytonutrients are regarded as antioxidizing ingredients and researched lively. However, any studies have done on antioxidizing activity with the extract of Ixeris Dendata so far. Also macrophage inside of the organism is participating in anti-microbe action, anti-virus action and anti-cancer action. That is, the lymph(T lymph and B lymph) protect the organism in means of cooperating with other immunity cell. However, the cell which has the closest relationship with lymph is the macrophage. Once the macrophage is activated with various stimuli, it moves rightly and performs its role.

The macrophage is activated with interferon-γ(IFN-γ) which is produced by T-lymph activated by antigens and promote immunity. The macrophage participates immunity and at the same time it controls unique immunity with lymph. However it has never been studied that how Ixeris dentata influences the macrophage which play the important role in immunity so far.

Futhermore, the anti-allergy effect of Ixeris dentata has never been studied. The 9 ~ 20 μ size of mast cell is known that it is widely distributed around body organs; skin .respirator, mucosa of camouflage , blood vessels, brain etc. and it cause many allegies. The mast separates chemical carriers from cell tissues.

Especially histamine is separated most quickly and it extends peripheral blood vessels . In addition, it cause allergic reactions immediately through smooth muscle contraction. It hasn't been researched that thowlxeris Dendata act on allegic reactions to suppress that so far.

In fact, the research on Ixeris dentata extract has just begun. Any anti-cancer, anti- oxidization, anti-stress and anti-allergy component with Ixeris dentata extract has been reported in the inside and outside of the country yet. The inventor started the research on Ixeris dentata extract to confirm the medical activation then discovered that it has anti-cancer, anti-oxidization, anti-stress, anti- bacteria and anti-allergy functions. The inventor completed this research with applying this component to medicine, drinking, food and so on widely.

[ Invention accomplishes and technical subject]

This invention intends to provide Ixeris dentata extract which has physiology activation function to promote immunity against cancer, oxidization, stress, bacteria and allergy and also reduce cholesterol. In addition to it, it is expected to be applied to medical supply and health assistance foodstuff. [ Composition of invention ]

In order to accomplish all the purpose mentioned above, this invention provides Ixeris dentata extract which is extracted with pure alcohol since the extract does anti-cancer, anti-oxidization, anti-stress, anti-bacteria and anti-allergy function. In the process above, it can be separated into hexane, ethylacetate, buthanol or water again. This invention provides the manufacturing process of Ixeris dentata extract by means of extracting Ixeris dentata extract with low-grade alcohol then this diluting this alcohol extract with diluted water. Futhermore, it provides pharmacological composition against cancer which consists mainly Ixeris dentata extract.

In addition, the invention provides pharmacological composition against allergy, stress, oxidization and bacteria with Ixeris dentata extract. It is also effective pharmacological composition for reducing cholesterol. This invention can be applied to functional drinks and health assistance foodstuff which are made up of Ixeris dentata extract. The below explains the invention in detail.

The invention provides Ixeris dentata extract which promotes immunity and physiology activation. This Ixeris dentata extract can be used in various forms. It is used after extracting the freeze-dried powder with low-grade alcohol or its juice it self. It is advised to use ethanol or methanol as the low-grade alcohol. Generally when it is used for food, it is advisable to use not methanol but pure ethanol or extracts which is extracted with diluted water. This diluted extract tent to have low gaining ratio. In the mean time, when Ixeris dentata is juiced it can be used as itself. Such extract or juice can be used again after extracting with water. In this case, it is advised to use hexane, ethylacetate or butanol as a solvent.

Ixeris dentata extract produced through the process above and solvent do anti-cancer, anti-oxidization, anti-stress, anti-bacteria, anti-allergy and cholesterol suppression functions.

Especially methanol extract, ethylacetate fraction and butanol fraction are relatively effective for anti-oxidization activation and ethylacetate fraction and butanol fraction are good for anti-lipid hyperoxidation activation. Moreover, hexane fraction, ethylacetate fraction are excellent for anti-microbe activation.

The anti-oxidation activation is proved with 1 ,1 - dipheynil-2- crylhydrazyl and the anti- lipid hyperoxidation activation is researched with microgerm in the ver of a mouse. Macrophage's NO synthesis was researched regarding the anti-cancer activation since macrophage is known to play an important role in anti-cancer, anti-microbe and anti- virus, macrophage in the human body participates in anti-microbe, ant-virus and anti- cancer functions and it is acknowledged that NO acts as an agency in the functions above. One of important things produced as a result of the immunity reactions is NO. NO is very unstable and reacts very easily so it participate in various functions in the human body. It is examined that macrophage is activated by recompounding interferon-γ(rlFN-γ) and LPS and generates nitrite and nitrate after it is revealed that many experimental animals which are injected with lipopolysaccharide, LPS) excrete a big amount of NO. Therefore, it is studied that whether sowthistle derives the genesis of NO in macrophage which plays important role in immune-cell activation. As a result of it, it is proved that sowthistle has anti-cancer and anti-virus functions by means of promoting immunity. Therefore, it is expected that it could be used for various kinds of cancer. An appropriate amount of compound 48/80 has been used as a direct and convenient reagent to study the mechanism of anaphylactic reactions. In the present study, we tried to examine the effects of the IXD on mast cell-mediated anaphylactic reaction. As efforts for this we showed that IXD inhibited compound 48/80-induced anaphylaxis-like fatal response. We also showed that IXD inhibited anti-dinitrophenyl (DNP) IgE antibody- mediated PCA and compound 48/80-induced histamine release from the rat peritoneal mast cells (RPMCs). So we confirmed that IXD had an anti-allergic effect. As it has been explained, this Ixeris dentata extract has excellect anti-cancer, anti-stress, anti-bacteria, anti-allergy and cholesterol suppression functions so that it is expected that it can be applied to pharmacologic compositions, cosmetics, food and drinks. Effective dose of Ixeris dentata(sowthistle) extract can be selected as inactivation and secretion rate, age, sex and station of patients, the seriousness of an illness, and absorption degree activity component in body Also, serveral dosage form of Ixeris dentata extract can be used as main facter of health assistance food, and drinking.

Below, Explanation for preparation method and test of pharmacological action of Ixeris dentata extract

Execution example 1 manufacture of the Ixeris dentata extract

(preparation of the IXD powder) after washing the IXD herb, it was dried at 35°C and then lyophilized and pulverized.

We got the 205.8g of methanol extracts which is extracted by 1 kg dried IXD using 100% cold methanol for one week, then we added 100 ml distilled water and 100 ml hexane so it was separated water and hexane extract, and we added 1000 ml acetealdyhyde to the water extract so it was separated acetaldehyde and water extract, finally, we added 500 ml butanol to the water extract so it separated butanol and water extract. We repeated each step for 3 times. We got 33 8g hexane, 5.8g acetaldehyde, 15.66g butanol and 141 .7g water extracts, respectively. while, we obtained 2>.2l IXD extract after IXD juice, which was obtained using general method, filtering, then we got 0.32g hexane, 2.66g ethylacetate, 5.1 g buthanol and 77 7g water extract, respectively.

Test example 1 investigation of antioxidant activity on IXD extract

We investigated IXD extracts which were produced by application 1 on antioxidant effect using antioxidant detection method (Blois, Nature, 188: 1 199, 1958; Choi, et al., Kor. J. Phamacogn, 24: 299-303, 1993) by free radical , DPPH. We added 4ml methanol in test tube and added various concentration compounds (1 .5- 30ul). we added 1 ml DPPH in the tube and incubated for 30 minute and then estimated absorbance on 517nm. RC50(/€/ml) meaned the compound concentration which reduced 50% of comparative group, adding nothing. We described the result in table 1 and table 2.

[table 1 ]

DPPH free radical scavenging activities of Ixeris dentata juice expressed by RC50( g/^)a in different extracting conditions.

[table 2]

DPPH free radical scavenging activities of Ixeris dentata powder expressed by RC50( g/ m a in different extracting conditions.

As shown in table 1 and 2, it had strong antioxidant activity in ethyl acetate fraction and buthanol fraction and weak activity in water fraction on the whole, it was the best activity in powder methanol fraction.

Test example 2 investigation of IXD on lipid peroxidation inhibitory activity

In order to investigate lipid peroxidation inhibitory activity of IXD, we investigated using microsome of mouse as Ohkawa method(Ohkawa, et al., Anal. Biochem., 95: 351 , 1979) backs and it . First, we fractionize microsome to liver of mouse, then mixed with 100ml Tris-HCI buffer)(pH 7.4). after mixed the got microsome fraction 0.3% and a compound, we added 500 μM FeS04-7H20 to the mixed liquid, then make shacking incubation for 30 minute at 37 °C . and then stopped reaction adding 20% TCA(Trichloroacetic acid :2.5M ammonium chloride=1 : 1 )

lipid peroxidation inhibitory prevention activity

1 -(T-B)/C-B x 100 where, the T is the test group where the oxidation reaction happens which added the chemical compound, the C is the test group where the oxidation reaction happens which without added the chemical compound, the B is intensity of light in the comparative group where the oxidation reaction didn't happens [table 3]

Inhibitory activity on lipid peroxidation of Ixeris dentata juice expressed by RC50( g/"^)a in different extracting conditions.

[table 4]

Inhibitory activity on lipid peroxidation of Ixeris dentata powder expressed by RC50(/g/ mi) a in different extracting conditions.

lipid peroxidation inhibition activity was best in EtOAc fraction and weak effect in butanol fraction. Test example 3

Antimicrobial activities of Ixeris dentata

[table 5]

Antimicrobial activities of Ixeris dentata juice (MIC : "g/ml)

[table 6] Antimicrobial activities of Ixeris dentata powder(MIC : £g/ml)

The original stock of ICR mice and Sprague-Dawley rats were purchased from Dae-Han

Experimental Animal Center (Taejeon, Republic of korea). the animals were kept in a laminar air flow room maintained under a temperature of 22±2°C and relative humidity of

55+10% throughout the study. All reagents which were used in this test were purchased from Sigma Chemical Co. (St. Louis, MO, USA)

Green sap of IXD was prepared by mashing the raw herbs with a mortar. The extract was filtered, lyophilized, and kept at 4C. The powdered extract was dissolved in sterile saline or Tyrode buffer A (10 mM HEPES, 130 mM NaCI, 5 mM KCI, 1.4 mM CaCI2, 1 mM MgCI2, 5.6 mM glucose, 0.1 % bovine serum albumin), and then the extract was filtered through a 0.20/™ filter in order to use in cell experiment.

inhibitory effect of IXD on compound 48/80-induced systemic anaphylaxis>

We used compound 48/80 as a non-immunologic stimulator. Mice were given an i.p. injection of 8mg/kg of compound 48/80. the IXD was dissolved in saline and administered orally 1 h before the injection of compound 48/80. Mortality was monitored for 1 h after induction of the anaphylaxis-like fatal response. We used 8 mice in each experiment group.

The mortality was calculated using following equation.

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

As a result, the oral administration of saline as a control proved 100% fatal. When the IXD was orally administered before compound 48/80 injection, the mortality was significantly reduced, as shown table 7.

[table 7] the IXD inhibited the systemic anaphylactic reaction.

inhibitory effect of Ig-E dependent cutaneous reaction >

Next, to assess the contribution of IXD in IgE-rmediated anaphylactic reaction, we used the in vivo model of cutaneous anaphylaxis. the cutaneous reaction could be quantified by measuring the concentration of leaked dye. As explain in detail, An IgE-dependent cutaneous reaction was generated by sensitizing the skin with an id injection of anti-DNP IgE followed 24 h later with an injection of DNP- HSA into the tail vein of mouse. The DNP-HSA was diluted in phosphate-buffered saline (PBS). The mice were injected intradermally with 100 ng of anti-DNP IgE into each of two dorsal skin sites that had been shaved 24 h earlier. The sites were outlined with a water- insoluble red marker. Twenty-four hours later, each mouse received an injection of 200 I of the 1 : 1 mixture of 1 mg/ml DNP-HSA in PBS and 4% Evans blue via the tail vein. IXD was orally administered 1 h before the challenge. Forty minutes after the challenge, the mice were killed and the dorsal skin was removed for measurement of the pigment amount of area. The amount of dye was then determined colorimetrically after extraction with 1 ml of 0.1 N KOH and 9 ml of a mixture of acetone and phosphoric acid (5:13) based on the method of Katayama et al. (14). The absorbance at 620 nm of the extract was measured in spectrofluorometer and the amount of dye was calculated with the standard line of Evans blue. We showed the calculated leaked dye in table 8.

[table 8]

Each value represents the mean + SEM of three independent experiments(n=8). *P<0.05; significantly different from the saline value.

As shown table 8, the IXD inhibited PCA reaction in does dependent manner, and 0.1 g/kg of IXD significantly inhibited PCA reaction.

inhibitory effect of histamine release from rat peritoneal mast cells. >

Next, we tested the inhibitory effects of IXD on compound 48/80-induced histamine release from RPMCs, rats were anesthetized by ether and injected with 20 ml of Tyrode buffer B (137 mmol/L NaCI, 5.3 mmol/L glucose, 12 mmol/L NaHC03, 1.1 mmol/L KCI, 0.3 mmol/L NaH2P04, pH 7.4) containing 0.1 % gelatin (Sigma Chemical Co.) into the peritoneal cavity and the abdomen was gently massaged for about 90 s. The peritoneal cavity was carefully opened and the fluid containing peritoneal cells was aspirated by a Pasteur pipette. Thereafter, the peritoneal cells were sedimented at 150 x g for 10 min at room temperature and resuspended in Tyrode buffer B. peritoneal cells suspended in 1 ml of Tyrode buffer B were layered on 2 ml of 22.5% w/v metrizamide (density, 1 .120 g/ml, Sigma Chemical Co.) and centrifuged at room temperature for 15 min at 400 x g. The cells remaining at the buffer-metπzamide interface were aspirated and discarded. Mast cell suspensions (2 X 105 cells/mL) were pre-incubated for 20 min at 37C for stabilization before adding IXD. After that the cells were pre-incubated (15 min) with the IXD, and then incubated (10 mm) with compound 48/80. The inhibition percentage of histamine release was calculated using the following equation.

The inhibition percentage of histamine release was calculated using the following equation. Suppression (%) = (Histamine release without IXD -Histamine release with IXD) x 100/Histamine release without IXD. The result was shown in table 9.

[table 9]

Each value represents the mean ±SEM of three independent experiments(n=8). *P<0.05; significantly different from the saline value.

As shown in Table 9, IXD dose-dependently inhibited compound 48/80-induced histamine release at concentrations from 0.005 to 0.1 mg/ml.

Test example 5

Investigation of activation of immune cells by IXD

We tested whether macrophages which are important as immune cells could induce NO production.

Male C57BL/6 mice were purchased from Dae Han Experimental Animal Center (Taejeon, Republic of Korea). <Peritoneal macrophage cultures >

TG(Dιfco Laboratorιes(Detroιt, Ml, USA)-elιcιted macrophages were harvested 3~4 days after i p injection of 2 5 ml TG to the mice and isolated Using RPMI 1640 (Sigma Chemical Co (St Louis, MO, USA) containing 10 U/ml heparin, peritoneal lavage was performed Then, the cells were distributed in DMEM(Lιfe Technologιes(Grand Island, NY, USA), which was supplemented with 10% FBS(Lιfe Technologιes(Grand Island, NY, USA), in 4-well tissue culture plates incubated for 3 h at 37C in an atmosphere of 5% C02, washed three times with HBSS to remove non-adherent cells, and equilibrated with DMEM that contained 10% FBS before treatment

Cells were cultured with rlFN-γ(Genzyme(Munchen, Germany) or alone The cells were then stimulated with various concentratιons(10ug/m , I 00ug/π^, 1000ug/πι«) of IXD The resultant NO production was determined by detecting nitrite concentrations in the cell supernatants after 48 h of treatment the comparative group was LPS-treated group To measure nitrite, 100 ul aliquots were removed from conditioned medium and incubated with an equal volume of Gπess reagent (1 % sulfanιlamιde/0 1 % Λ/-(1 -naphtyl)- ethylenediamine dιhydrochlorιde/2 5% H3P04) at room temperature for 10 mm The absorbance at 540 nm was determined in a Titertek Multiskan (Flow Laboratories, North Ryde, Australia) N02- was determined by using sodium nitrite as a standard The results were shown in table 10

[table 10]

Values are the mean ± S E M of four independent experiments duplicate in each run statistical analysis was performed by the Student's t-test to express the difference between two groups.

IXD had weak effect on NO production in resting mouse peritoneal macrophages. However, when mouse peritoneal macrophages were primed for 6 h with murine rlFN-γ and then treated with IXD, NO production was highly increased. IXD had LPS like effect but it could produce NO by stimulating macrophages innoxiously. The results of this study suggest that IXD may provide anti-cancer and anti-virus effects by stimulating immune cells.

[ Effect of invention ]

In this invention, alcohol and water extract, hexane fraction, ethyl acetate fraction, buthanol fraction, and water fraction of Ixeris dentata have excellent Anti-cancer, anti- oxidization, anti-stress, anti-bacteria, anti-allergy and cholesterol suppression functions. Therefore, Ixeris dentata extraction can be used prevention and medical treatment of cnacer, apoplexy, stress, inflammation disease, cardiovascular disease etc. It can be used as various functional food and health assistance food.

Claims

[Claims]

[Item 1 ]

Ixeris dentata extract produced by means of extracting sowthistle with pure low-grade alcohol or water. It has Anti-cancer, anti-oxidization, anti-stress, anti-bacteria, anti- allergy and cholesterol suppression functions.

[Item 2]

Ixeris dentata extract which fractionize with hexane, ethyl acetate, buthanol or water in addition to the process above.

[Item 3] The manufacturing process of Ixeris dentata extract which is characterized by extracting sowthistle with low-grade alcohol to get alcohol extract and diluting this alcohol extract with distilled water then fraction into organic solvent to get various solvent fraction over and over again.

[Item 4]

Pharmacological composition which consists Ixeris dentata extract from the 1 st or 2nd item anti-cancer.

[Item 5]

Pharmacological composition which consists Ixeris dentata extract from the 1 st or 2nd item Anti-allergy.

[Item 6]

Pharmacological composition which consists Ixeris dentata extract from the 1 st or 2nd item anti-stress.

[Item 7]

Pharmacological composition which consists Ixeris dentata extract from the 1 st or 2nd item anti-oxidization.

[Item 8] Pharmacological composition which consists Ixeris dentata extract from the 1 st or 2nd item for curing inflammation.

[Item 9]

Pharmacological composition h consists Ixeris dentata extract from the 1 st or 2nd item for suppressing cholesterol.

[Item 10]

Functional drinks which consist Ixeris dentata extract.

[Item 1 1 ] Functional food which consists Ixeris dentata extract.

[Item 12] Functional cosmetics which consist Ixeris dentata extract.