KR20200040701A - A composition for immune regulation or enhancement comprising Dendropanax morbifera Lev. extract or an active ingredient isolated therefrom - Google Patents

Abstract

The present invention relates to a composition for immunoregulation or immunoenhancement, comprising an extract of Dendropanax morbifera Lev. leaves, a fraction thereof, or an immunoactive component isolated therefrom. The extract of the present invention, the fraction thereof, and the immunoactive component isolated therefrom increase the proliferation of splenocytes, and the production of IL-2 and IFN-r cytokines. In particular, an ethyl acetate fraction of the extract exhibits the effect of increasing CD4^+ T cells and EL-4 T cells. In addition, a DMW-2 single component isolated from the ethyl acetate fraction dramatically increases the production of IL-2 cytokine and is significantly effective in activating transcription factors involved in T cell proliferation, IL-2, IL-4, NF-AT, AP-1, and NF-kB.

Description

A composition for immune regulation or enhancement comprising Dendropanax morbifera Lev. extract or an active ingredient isolated therefrom}

The present invention relates to a composition for immunomodulation or immune enhancement comprising a hwangchil tree extract. In addition, the present invention relates to a composition for immunomodulation or immuno-enhancement comprising the active ingredient DMW-2 or salts thereof separated from hwangchil tree leaf extract as an active ingredient.

In general, the human body protects the body and maintains homeostasis through an immune function to identify and remove when an invasion of an external pathogen occurs. Immune response is a series of reactions caused by activated immune cells against antigens, and the immune system is responsible for removing pathogens through infection, cognitive and humoral, and cellular responses to infection. In addition to the disease, it leads to various diseases.

Immunity refers to self-defense by removing the antigen by defending the host against the antigen, and is largely divided into innate immunity and acquired immunity. Innate immunity is a non-specific response to an antigen, which causes macrophage, natural killer cells (NK cells), and complements to cause an immune response. Acquired immunity remembers the first invading antigen, and when the antigen invades again, it specifically triggers an immune response to remove the antigen. Acquired immunity is divided into humoral immunity and cellular immunity, and humoral immunity involves B lymphocytes to recognize antigens, and then secrete antibodies to cause an immune response. In cellular immunity, T lymphocytes derived from the thymus recognize antigens and secrete cytokines or activate macrophages to induce an immune response.

In particular, in the immune response by T cells, an initial signal is transmitted into the cell from the TCR complex of the T cell that recognizes the antigen and another signal. This signal transduction process is shown by the action of PKC activator and ionophore, thereby increasing Ca ⁺⁺ ion in the cytoplasm of T cell. In addition, after phosphorylation of various proteins, the signal is finally transmitted to the nucleus. The initial signal transmitted from the cell membrane to the nucleus is expressed by the expression of various genes necessary for the proliferation and differentiation of T cells in the nucleus. This process is called transcriptional activation, and transcription of NF-AT, NF-kB and AP-1, which plays the largest role in T cell proliferation, occurs. In addition, transcription of IL-2 cytokines and IL-2 receptor genes appears. Through this, new cytokines are secreted to proliferate and differentiate T cells. T cell activity is linked to the synthesis of IL-2, and secreted IL-2 induces T cell proliferation. IL-2 is a T cell growth factor and activates immune cells such as T cells, B cells, and natural killer cells to display an immune response. IL-2 binds to IL-2R present on the surface of T cells and induces various intracellular responses. When IL-2 binds to IL-2R of T cells, T cell differentiation occurs and cytokines such as IFN-r (interferon-gamma) and TNF (tumor necrosis factor) are liberated, affecting the immune response. It also increases IL-1, IL-6, and TNF secretion in monocytes.

However, if the immune function is lowered, disorders in immune regulation occur due to causes of imbalance of cytokines in the body and proliferation imbalance of T / B cells. Accordingly, various immune diseases such as asthma, rhinitis, allergic rhinitis, atopic dermatitis, irritable dermatitis, cold, and chronic fatigue may occur. Therefore, the immune response of the human body must be balanced and regulated normally to maintain health, so prevention of immune regulation is more important than treatment. However, since the currently available therapeutic agents are immunopotentiators that have the potential for side effects to be taken for prevention, a substance capable of preventing and treating immune dysfunction is required.

To this end, research is being actively conducted to find a component capable of modulating immune activity from natural products with few side effects and to develop a formulation capable of modulating immune activity using the same. Korean Patent Publication No. 10-2015-0051174 discloses a composition for enhancing immunity comprising an alcohol extract of elm bark as an active ingredient, and Korean Patent Registration No. 10-1536652 contains a composition for boosting immunity including turmeric extract This is disclosed.

Since ancient times, the hwangchil tree ( Dendropanax morbifera Lev. ) Is a genus of the elmaceae ( oralaceae ). As a Korean native species, it is an evergreen large tree growing in the shady and humid broad-leaved forests of Jeollanam-do and Jeju islands and the hillsides of the island. The leaves are hairless, smooth, and misaligned on the surface, and the oval or elliptical leaves do not crack at all, or they may split deeply like a finger with 3-5 branches. If you look at the distribution of Hwangchil trees in Korea, it grows a lot in the beaches such as Jeju Island, Wando, Jeonnam, Daeheksando, and Geomundo. Dendropanax , the generic name for Hwangchil, is a compound word of the Greek word dendro (tree) and panax (all-powerful medicine). Hwangchil tree's scientific name, dendropanax mobifer, has the meaning of a panacea and is also called tree ginseng. Also, the name Hwangchil is a name given that yellow liquid appears as lacquer of lacquer when it is cut on the bark of Hwangchil. Hwangchil is used to process or color the surface in the process of making crafts. It has been used in various ways, such as lacquerware, Goryeo Buddha, and various other furniture and metals. According to the Korean native book, the roots and stems of Hwangchil tree have a sweet taste, warm properties, and are effective in Geopung-pung and Hwanghyeol-maek.

In addition, according to a recent study, a single component of Hwangchil and Hwangchil is used as a material with more interest as pharmacological effects such as antibacterial, anticancer, hyperlipidemia, antithrombotic anti-inflammatory and antioxidant effects are revealed. In relation to the immune enhancing effect of hwangchil, Korean Patent Publication No. 10-2015-0144061 discloses a composition for immunomodulation or immunopotentiation containing hwangchil tree stem extract as an active ingredient. However, there has been no exact report on the immunomodulatory effect of single components in hwangchil tree.

Hwangchil tree fractions and hwangchil tree single component has helped to proliferate T cells related to acquired immunity and increase immune activity. Accordingly, the present inventors tried to confirm whether hwangchil tree helps immune enhancement by activating the immune function through the proliferation of T cells.

The present inventors have made extensive efforts to develop a plant-derived immune-activating composition for immunomodulation or immune enhancement. As a result, the present inventors have developed a composition for immunomodulation or immuno-enhancement comprising DMW-2, an active substance isolated from Hwangchil tree leaf extract or Hwangchil tree leaf extract.

The present inventors have shown that the ethylacetate fraction isolated from the extract of hwangchil tree leaf extract increases the activation of T cells, increases the transcription factors NF-AT, AP-1, NF-kB, and increases IL-2 cytokine production. As an effect, it was found that it is very effective for immunomodulation or immune enhancement.

Meanwhile, DMW-2 ((10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol), a compound isolated from the ethyl acetate fraction, is T cell Although proliferation was not activated, IL-2 cytokine production was significantly increased. In addition, the effect of activating transcription factors IL-2, IL-4, NF-AT, AP-1, and NF-kB involved in T cell proliferation was excellent.

Accordingly, an object of the present invention is to provide a composition for immunomodulation or immune enhancement.

Another object of the present invention is to provide an anti-tumor immunity (antitumor immunity) composition or anti-cancer composition.

Another object of the present invention is to provide a method for separating (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol compounds.

One aspect of the present invention relates to a composition for immunomodulation or immune enhancement, comprising an extract of hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf).

The present inventors have made extensive efforts to develop a plant-derived immune-activating composition for immunomodulation or immune enhancement. As a result, the present inventors have developed a composition for immunomodulation or immuno-enhancement comprising DMW-2, an active substance isolated from Hwangchil tree leaf extract or Hwangchil tree leaf extract.

The present inventors show that the hwangchil tree leaf extract and the ethyl acetate fraction isolated from the extract activate the proliferation of T cells, increase the transcription factors NF-AT, AP-1, NF-kB and IL-2 cytokines involved in T cell proliferation As an increasing effect, it has been found to be very effective for immunomodulation or immune enhancement.

On the other hand, the immune enhancing active material isolated from the ethyl acetate fraction is as follows.

DMW-2: (10E)-(-)-10,17-Octadecadiene-12,14-diyne-1,9,16-triol; (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol.

DMW-2 did not activate T cell proliferation, but significantly increased IL-2 cytokine production. In addition, the transcription factor IL-2, IL-4, NF-AT, AP-1, and NF-kB activation effects involved in T cell proliferation were excellent.

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

The hwangchil tree leaf extract of the present invention is dried and shredded hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf), and then added a polar solvent (for example, distilled water, methanol, ethanol, propanol, butanol, etc.) as an extraction solvent, 80 It can be extracted by heating to ℃ -120 ℃. According to one embodiment of the invention, it can be extracted by heating to 80 ℃ -120 ℃ for 2-8 hours.

The polar solvent used in the extraction method of the present invention is (i) water, (ii) alcohol (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol), (iii) Acetic acid, (iv) dimethyl-formamide (DMFO) and (v) dimethyl sulfoxide (DMSO). According to an embodiment of the present invention, the polar solvent is one or more solvents selected from the group consisting of water and lower alcohol having 1 to 3 carbon atoms. According to another embodiment of the invention, the polar solvent is water, methanol or ethanol.

According to one embodiment of the present invention, a polar solvent may be added to the hwangchil tree leaves to extract at 90 ° C-110 ° C for 3-5 hours.

According to a specific embodiment of the present invention, a polar solvent may be added to hwangchil leaves to extract at 95 ° C-105 ° C for 3-5 hours.

According to a specific embodiment of the present invention, it can be extracted for 4 hours at 95 ℃ -105 ℃ by adding a polar solvent to the hwangchil tree leaves.

In the present invention, when extracting hwangchil tree leaves, hot water extraction, reflux extraction, room temperature extraction, or ultrasonic extraction may be used. According to one embodiment of the present invention, a hot water extraction method or a reflux extraction method may be used.

According to a specific embodiment of the present invention, the hwangchil tree leaves were extracted with distilled water, filtered, concentrated under reduced pressure, and dried.

Meanwhile, the composition of the present invention may include an ethyl acetate extract fraction obtained by purifying only an extract soluble in an ethyl acetate solvent from a hwangchil tree leaf water extract or an alcohol extract.

For example, after adding water of about 0.001 to 100 times, preferably 1 to 50 times the volume (v / w%) of water by weight of hwangchil tree leaf water extract or alcohol extract, hexane, chloroform, and ethyl acetate are used as usual. By performing the fractionation process, a non-polar solvent-soluble extract fraction soluble in a non-polar solvent can be obtained.

Subsequently, a polar solvent-soluble extract fraction soluble in a polar solvent such as butanol and water can be obtained.

The present inventors confirmed that through the EL-4 T cell proliferation capacity reactivity measurement experiment, the hwangchil tree leaf water extract and five fractions (hexane, chloroform, ethyl acetate, butanol, and water fraction) activate T cell proliferation. Among them, it was confirmed that the ethyl acetate fraction was excellent in proliferation of CD4 ⁺ T cells and EL-4 T cells.

The composition of the present invention may be implemented as a pharmaceutical composition or a food composition comprising hwangchil tree leaf extract or a fraction thereof as an active ingredient.

Another aspect of the present invention relates to a composition for immunomodulation or immuno-enhancement comprising at least one compound selected from the group consisting of a compound represented by Formula 1 or salts thereof:

[Formula 1]

.

.

The compound of Formula 1 isolated from the ethyl acetate fraction of the above-mentioned extract of Dendropanax morbifera Lev. Leaf did not activate T cell proliferation, but significantly increased IL-2 cytokine production. In addition, the transcription factor IL-2, IL-4, NF-AT, AP-1, and NF-kB activation effects involved in T cell proliferation were excellent. Based on this, the composition of the present invention can be used as a composition for immunomodulation or immune enhancement comprising the compound of Formula 1.

The compound name of Formula 1 is as follows:

DMW-2: (10E)-(-)-10,17-Octadecadiene-12,14-diyne-1,9,16-triol; (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol.

The upper limit of the amount of the compound or salts included in the composition of the present invention can be carried out by a person skilled in the art within a suitable range.

The composition of the present invention is a drug containing (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol, or salts thereof as an active ingredient It can be implemented as a pharmaceutical composition or a food composition.

As used herein, the term "comprising as an active ingredient" means to include an amount sufficient to achieve the efficacy or activity of a hwangchil tree leaf extract, a fraction thereof, a compound of Formula 1 separated from the fraction, or a salt thereof. do. Quantitative upper limit of the hwangchil tree leaf extract, its fraction or compound contained in the composition of the present invention can be carried out by a person skilled in the art within a suitable range.

The compound used as an active ingredient in the present invention may be used by itself or in the form of salts, preferably in the form of pharmaceutically acceptable salts. The salt is preferably an acid addition salt formed by free acid. As the free acid, an organic acid and an inorganic acid can be used. The organic acid is citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, glutanoic acid and aspartic acid, etc. It includes, but is not limited to. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

The (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol contained in the composition of the present invention is a natural plant material, Hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf). Hwangchil tree leaves have been used for edible and folk medicine for a long time. Since it has come, it can be expected that the compound of the present invention separated therefrom will also have no problems such as toxicity and side effects. Thus, (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol, or salts thereof, can be used as a pharmaceutical or food composition.

According to one embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and for parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like.

Suitable dosages of the pharmaceutical compositions of the present invention vary by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity, Usually, a skilled physician can easily determine and prescribe a dose effective for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10000 mg / kg.

The pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of ex, powder, granule, tablet, or capsule, and may further include a dispersant or stabilizer.

According to another embodiment of the present invention, the composition of the present invention may be a food composition.

When the composition of the present invention is a food composition, it may be prepared in the form of powder, granule, tablet, capsule or beverage. For example, there are various foods such as candy, beverages, gum, tea, vitamin complexes, and health supplements.

Food composition of the present invention, as an active ingredient hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf) extract, ethyl acetate fraction, (10E)-(-)-10,17-octadecadiene-12,14-diyne-1, 9,16-triol, or salts thereof, as well as ingredients commonly added in food preparation, may include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents do. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose, sucrose, oligosaccharides, etc .; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizine, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink agent, in addition to the extract or the compound of the present invention, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, worm extract, jujube extract, licorice extract, etc. You can.

Another aspect of the present invention relates to a method for immunomodulation or immunopotentiation, comprising administering to a subject an immunomodulatory or immunopotentiating composition comprising an extract of hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf). .

Another aspect of the invention is in the group consisting of (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol, and salts thereof. It relates to a method of immunomodulation or immune enhancement comprising the step of administering to a subject a composition comprising one or more compounds selected.

The term "administration" as used herein means providing a given substance to a subject in any suitable way. The route of administration of the composition for treating, inhibiting or improving skin pigmentation of the present invention can be administered orally or parenterally through any general route as long as it can reach the target tissue. In addition, the composition of the present invention may be administered using any device capable of delivering an active ingredient to target cells.

The term "subject" herein is not particularly limited, for example, human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, or Guinea pigs, preferably mammals, more preferably humans.

Another aspect of the present invention relates to a composition for enhancing anti-tumor immunity, comprising an extract of hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf).

Another aspect of the invention, (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol ((10E)-(-)-10, 17-Octadecadiene-12,14-diyne-1,9,16-triol) or a salt thereof, it relates to a composition for enhancing anti-tumor immunity (antitumor immunity).

Extracts, fractions and immune active components isolated therefrom increase the proliferative capacity of splenocytes and the production of IL-2, IFN-γ cytokines. In particular, the ethyl acetate fraction has CD4 ⁺ T cell and EL-4 T cell proliferation effects. In addition, the DMW-2 single component isolated from the ethyl acetate fraction greatly increases IL-2 cytokine production and transcription factors IL-2, IL-4, NF-AT, AP-1, NF involved in T cell proliferation -kB activation effect was very good.

IL-2 is a T cell growth factor that activates immune cells such as T cells, B cells, natural killer cells (NK cells), or lymphokine-activated killer cells to enhance immune and anticancer effects. Indicates.

Another aspect of the present invention relates to an anticancer composition comprising an extract of Dendropanax morbifera Lev. Leaf.

Another aspect of the invention, (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol ((10E)-(-)-10, 17-Octadecadiene-12,14-diyne-1,9,16-triol) or a salt thereof.

The cancer is gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, larynx cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, skin cancer, kidney cancer, drainage Polyploid carcinoma, thyroid cancer, parathyroid cancer and ureteral cancer.

The anti-tumor immunity enhancing or anti-cancer composition of the present invention may be administered orally or parenterally, and for parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, intratumorally administration, etc. Can be administered.

The anti-tumor immunity enhancement or anti-cancer composition of the present invention is the above-mentioned hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf) extract or (10E)-(-)-10,17-octadecadiene-12,14-die Phosphorus-1,9,16-triol ((10E)-(-)-10,17-Octadecadiene-12,14-diyne-1,9,16-triol) is used. In order to avoid excessive complexity of the present specification, description thereof is omitted.

Another aspect of the present invention relates to a method for separating a (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol compound comprising the following steps: will be:

(a) extracting hwangchil tree leaves ( dendropanax morbifera lev . leaf) with a polar solvent;

(b) purifying only the extract soluble in the ethyl acetate solvent from the hwangchil tree leaf extract of step (a) to obtain an ethyl acetate extract fraction; And

(c) Purifying the ethyl acetate extract fraction to obtain (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol.

Hereinafter, the method for separating the (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol compound will be described in detail.

Step (a): Hwangchil tree leaves ( Dendropanax morbifera Lev. leaf)

First, a polar solvent is added to the hwangchil tree leaf ( Dendropanax morbifera Lev. Leaf) to extract it.

The method for obtaining the hwangchil tree leaf extract is as described above.

According to one embodiment of the present invention, the polar solvent is water or a low alcohol having 1 to 3 carbon atoms.

According to a specific embodiment of the present invention, water or methanol may be added to the hwangchil tree leaves and heated to 80 ° C-120 ° C to obtain an extract.

Step (b): Preparation of ethyl acetate extracted fraction

Subsequently, by extracting only the extract soluble in the ethyl acetate solvent from the hwangchil tree leaf extract of step (a), an ethyl acetate extract fraction is obtained.

According to one embodiment of the present invention, after adding water of about 0.001 to 100 times, preferably 1 to 70 times, more preferably 50 times the volume (v / w%) of the weight of hwangchil tree leaf extract, hexane, chloroform , By performing a conventional fractionation process using a non-polar solvent such as ethyl acetate, a non-polar solvent-soluble extract fraction soluble in the non-polar solvent can be obtained.

The non-polar solvent used in the separation method of the present invention is hexane, chloroform, ethyl acetate, acetone, acetonitrile, methyl acetate, fluoroalkane, pentane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane , Diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether , Diethyl sulfide, dichloromethane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride and THF. According to one embodiment of the invention, the non-polar solvent is hexane, chloroform, ethyl acetate or dichloromethane. According to a specific embodiment of the present invention, in step (b), as a non-polar organic solvent, (i) hexane, chloroform, and ethyl acetate are sequentially used to obtain a distribution fraction; Or (ii) dichloromethane and ethyl acetate are used sequentially to obtain a partitioned fraction.

On the other hand, after obtaining a fraction using a non-polar solvent, a polar solvent-soluble extract fraction soluble in a polar solvent (for example, butanol, water, etc.) can be obtained from the remaining hwangchil tree leaf extract.

The polar solvent used in the separation method of the present invention is (i) water, (ii) alcohol (preferably, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol , 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) DMFO (dimethyl-formamide) and (v) DMSO (dimethyl sulfoxide). According to one embodiment of the invention, the polar solvent is water or alcohol. According to a specific embodiment of the present invention, after adding butanol to the hwangchil tree leaf extract, a fraction of the butanol layer and the aqueous layer was obtained.

According to one embodiment of the invention, step (b) may be carried out as follows:

(b-1) separating and removing extract fractions containing only extracts soluble in at least one non-polar solvent selected from the group consisting of dichloromethane, hexane, and chloroform from the hwangchil tree leaf extract of step (a); And

(b-2) Separating the extracted fraction of step (b-1) and remaining hwangchil tree leaf extract

The step of obtaining an ethyl acetate extract fraction containing only the extract soluble in the ethyl acetate solvent.

Step (c): (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol purification

Then, the ethyl acetate extract fraction obtained in step (b) is purified to obtain (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol.

According to an embodiment of the present invention, in the purification step (c), various known purification methods can be used, for example, column chromatography and thin layer chromatography.

According to an embodiment of the present invention, after the ethyl acetate extract fraction of step (b) is loaded onto a column, column chromatography purification may be performed using a solvent gradient of a non-polar solvent-polar solvent.

According to one embodiment of the present invention, a purified ethyl acetate fraction can be obtained using a solvent gradient of chloroform-methanol.

The purified ethyl acetate fraction can be separated from the compound DMW-2 by repeating column chromatography with a non-polar solvent as a mobile phase. According to one embodiment of the present invention, the purified ethyl acetate fraction was subjected to silica gel and YMC RP-18 column chromatography repeatedly to separate compound DMW-2.

According to one embodiment of the present invention, hexane-acetone may be used as a non-polar organic solvent.

On the other hand, the purified ethyl acetate fraction is a compound DMW-1 (methyl (10E) -9,16-dihydroxyoctadeca-10,17-dien-12,14-diynoate) by repeating column chromatography with a mobile phase in a polar solvent. Can be separated. According to one embodiment of the present invention, the purified ethyl acetate fraction was subjected to YMC RP-18 column chromatography repeatedly to separate compound DMW-1.

According to an embodiment of the present invention, methanol-water may be used as a polar organic solvent.

The polar solvent or non-polar solvent usable in step (c) is as described above.

The features and advantages of the present invention are summarized as follows:

The present invention relates to a composition for immunomodulation or immune enhancement comprising an extract of hwangchil tree ( Dendropanax morbifera Lev. Leaf), a fraction thereof, or an immune active ingredient isolated therefrom.

Extracts, fractions and immune active components isolated therefrom increase the proliferative capacity of splenocytes and the production of IL-2, IFN-γ cytokines.

In particular, the ethyl acetate fraction has CD4 ⁺ T cell and EL-4 T cell proliferation effects. In addition, the DMW-2 single component isolated from the ethyl acetate fraction greatly increases IL-2 cytokine production and transcription factors IL-2, IL-4, NF-AT, AP-1, NF involved in T cell proliferation -kB activation effect is very good.

Figure 1 shows the effect on the proliferative capacity of T cells in mouse spleen after administration of hwangchil tree leaf extract for 3 weeks and the degree of IL-2 and IFN-γ cytokine production.
Figure 2a shows the effect of hwangchil tree leaf extract and fractions on EL-4 T cell proliferation capacity.
Figure 2b-2c shows the effect of hwangchil tree leaf extract and fractions on the proliferative capacity of splenocytes or mouse CD4 ⁺ T cells, respectively.
W-Ex: Hwangchil tree leaf extract
W-Hex: n-hexane fraction obtained from hwangchil tree leaf extract
W-CH: chloroform fraction obtained from hwangchil tree leaf extract
W-EA: ethyl acetate fraction obtained from hwangchil tree leaf extract
W-Bu: n-butanol fraction obtained from hwangchil tree leaf extract
WW: Water fraction, excluding W-Hex, W-CH, W-EA, and W-Bu
Figure 3a shows the structure of DMW-2, a single component in hwangchil tree leaf extract (W-Ex) and ethyl acetate fraction (W-EA).
Figure 3b-3c shows the results of LC / MS / MS analysis of DMW-2, a single component in hwangchil tree leaf extract W-Ex) and ethyl acetate fraction (W-EA).
Figure 4a shows the effect of hwangchil tree leaf extract (W-Ex), ethyl acetate fraction (W-EA), and DMW-2, a single component of hwangchil tree leaf extract, on T cell proliferation.
Figure 4b-4c is hwangchil tree leaf extract (W-Ex), ethyl acetate fraction (W-EA), and hwangchil tree leaf extract, a single component, DMW-2, produces IL-2 and IFN-γ cytokines of T cells It shows the effect on the level.
Figure 5a-5e shows the effect of a single component in hwangchil tree leaf extract on the activation of transcription factors NF-AT, AP-1, NF-kB and IL-2, and IL-4 involved in T cells.

Hereinafter, the present invention will be described in more detail by examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, "%" used to indicate the concentration of a specific substance, unless otherwise specified, solids / solids (weight / weight)%, solids / liquids (weight / volume)%, and The liquid / liquid is (volume / volume)%.

Example 1: Preparation of hwangchil tree leaf extract

Hwangchil tree leaves were purchased and used from Jangheung origin, Jeollanam-do, Korea. 10 L of distilled water was added to 1 kg of dried hwangchil tree leaves, heated at 100 ° C., extracted once, filtered with filter paper, and concentrated under reduced pressure (~ 40 ° C., 670 mmHg). The hwangchil tree leaf extract concentrated under reduced pressure was freeze-dried and stored at -20 ° C (hereinafter referred to as "W-Ex").

Example 2: Preparation of soluble fraction of polar solvent and non-polar solvent from water extract of hwangchil tree leaves

2-1. Preparation of n-hexane fraction from hwangchil tree leaf water extract

20 g of Hwangchil tree leaf water extract (W-Ex) obtained in Example 1 was suspended in 1 L of distilled water. Subsequently, the suspension was placed in a separatory funnel, 1 L of n-hexane was added, shaken well, and then fractionated. After about 12 hours, the supernatant extracted with n-hexane fraction was taken to obtain n-hexane fraction (hereinafter referred to as "W-Hex"). The fractionation process was repeated twice, after which all the supernatant was concentrated under reduced pressure and freeze-dried to obtain 533 mg.

2-2. Preparation of chloroform fraction from water extract of hwangchil tree leaves

After separating the n-hexane layer from Example 2-1, 1 L of chloroform was added to the remaining hwangchil tree leaf water extract suspension, and the fractions were extracted by shaking well. A chloroform fraction extract was obtained after about 12 hours (hereinafter referred to as "W-CH"). The fractionation process was repeated 3 times, after which all fractions were concentrated under reduced pressure and freeze-dried to obtain 62.2 mg.

2-3. Preparation of ethyl acetate fraction from water extract of hwangchil tree leaves

In Example 2-2, the chloroform layer was separated and 1 L of ethyl acetate was added to the remaining hwangchil tree leaf water extract suspension, followed by shaking well to extract fractions. After about 12 hours, ethyl acetate fraction extract was obtained (hereinafter referred to as "W-EA"). The fractionation process was repeated 3 times, after which all fractions were concentrated under reduced pressure and freeze-dried to obtain 350 mg.

2-4. Preparation of n-butanol fraction from hwangchil tree leaf water extract

In Example 2-3, the ethyl acetate layer was separated and 1 L of n-butanol was added to the remaining hwangchil tree leaf water extract suspension, followed by shaking well to extract fractions. After about 12 hours, an n-butanol fraction extract was obtained (hereinafter referred to as "W-Bu"). The fractionation process was repeated twice, after which both the fraction and the remaining aqueous layer were concentrated under reduced pressure and freeze-dried to obtain 2.08 g of n-butanol layer and 13 g of aqueous layer.

Example 3: Separation of compounds

3.0 kg of dried and shredded hwangchil leaves ( Dendropanax Morbifera Leveille leaf ) were extracted with reflux three times for 3 hours with 10 L of 100% methanol. Then, the extract obtained by filtration was concentrated under reduced pressure to obtain 470 g of methanol X.

2 L of distilled water and 2 L of dichloromethane were added to 470 g of the obtained methanol extract, and shaken to fractionate into a dichloromethane layer (A) and an aqueous layer (B). Thereafter, the dichloromethane layer was concentrated under reduced pressure to obtain 159 g of a dichloromethane fraction.

After adding 2 L of ethyl acetate to 2 L of the fractionated aqueous layer (B), shaking and dividing it into an ethyl acetate layer and a water layer (C), the ethyl acetate layer was concentrated under reduced pressure to obtain 21 g of ethyl acetate fraction.

After adding 2 L of butanol to 2 L of the fractionated aqueous layer (B), shaking and dividing it into a butanol layer and a water layer, the butanol layer and the water layer were respectively concentrated under reduced pressure to obtain 56 g of butanol fraction and 145 g of water-soluble fraction.

After loading the obtained ethyl acetate fraction on a silica gel column, a solvent gradient using chloroform and methanol as the mobile phase (chloroform-methanol; 1: 0, 60: 1, 30: 1, 15: 1, 10: 1, 7: Elution with 1, 5: 1, 3: 1, 1: 1, 0: 1) yielded 10 ethyl acetate fractions (EA1 to EA10).

After loading 900 mg of the obtained EA3 fraction on a silica gel column, 8 fractions (EA3A to EA3H) were eluted by gradient elution with a solvent gradient (hexane-ethyl acetate; 3: 1 to 1: 6) using hexane and ethyl acetate as mobile phases. Got.

The obtained EA3C fraction 70 mg was repeatedly subjected to YMC RP-18 column chromatography with MeOH-water (3: 1) to obtain 5.3 mg of compound DMW-1.

238 mg of the obtained EA3D fraction was repeatedly subjected to silica gel and YMC RP-18 column chromatography with n-hexane-acetone (1: 1) to obtain 6.2 mg of compound DMW-2.

Example 4: Structural analysis of compounds DMW-1 and DMW-2

The HR-MS spectrum (Agilent 6530 accurate mass QTOF LC / MS) of the compound DMW-1 obtained in Example 3 was measured ([M ⁺ NH ₄ ] ⁺ m / z 336.2167, calcd for 336.2175), C ₁₉ H The molecular formula of ₂₆ O ₄ was estimated. ^{Through the 1} H-NMR, ¹³ C-NMR, HMBC, HSQC, and COSY spectra, the compound DMW-1 was identified as a novel substance that was first isolated from nature. DMW-1 was identified as methyl (10E) -9,16-dihydroxyoctadeca-10,17-diene-12,14-diinoate, a new polyacetylene compound.

By measuring the HR-MS spectrum of compound ^{DMW-2 ([M + HCOO} ] - m / z 335.1872, calcd for 335.1858), estimated a molecular formula of C ₁₈ H ₂₆ O _3. (10E)-(-)-10,17-octadecadiene-12,14-diyne-1,9,16-triol via ¹ H-NMR, ¹³ C-NMR, HMBC, HSQC and COSY spectra, That is, it was identified as being fruticotriol.

Table 1 below shows the physical properties of the compounds DMW-1 and DMW-2.

- - Compound 1 - Compound 2 location δ _C δ _H ( J in Hz) δ _C δ _H ( J in Hz) One 176.0 - 63.0 3.56, t (6.7) 2 34.8 2.34, t (7.5) 33.7 1.59 *, m 3 26.0 1.62, m 26.9 1.37 *, m 4 30.4 1.35 *, m 30.7 1.37 *, m 5 30.3 1.35 *, m 30.6 1.37 *, m 6 30.1 1.35 *, m 30.5 1.37 *, m 7 26.3 1.43 *, m 26.4 1.54 *, m 8 37.8 1.51, dt (11.7, 3.6) 37.8 1.53 *, m 9 72.4 4.13, dtd (7.2, 5.7, 1.6) 72.5 4.14, m 10 151.9 6.34, dd (15.9, 5.7) 151.9 6.34, dd (15.9, 5.6) 11 108.4 5.79, ddd (15.9, 1.6, 0.9) 108.4 5.79, ddd (15.9, 1.6, 0.8) 12 78.0 - 78.0 - 13 74.0 - 74.0 - 14 70.6 - 70.6 - 15 82.2 - 82.2 - 16 64.0 4.96, dd (5.4, 1.1) 64.0 4.93, dd (5.5, 1.1) 17 138.1 5.94, ddd (17.1, 10.2, 5.4) 138.1 5.95, ddd (17.0, 10.2, 5.5) 18 116.6 5.42, d (17.1) 5.22, d (10.2) 116.6 5.43, d (17.1)
5.22, d (10.2) OCH ₃ 52.0 3.67, s - -

* Signals overlap.

Example 5: Experimental animals and breeding conditions

In the present invention, magnetic BALB / c mice of 5 weeks and 7 weeks of age were purchased from Damul Science, adapted for 1 day, and used in the experiment. The environment of the laboratory animal room is a standard breeding condition, the temperature is 23 ± 3 ℃, the humidity is 40-60%, and the contrast cycle is maintained for 12 hours. All animal experiments of the present invention were performed under the approval of the Chonnam National University Animal Experiment Ethics Committee.

Example 6: Effect of hwangchil tree water extract on cellular immune response

After the periodical administration of Hwangchil tree water extract for 3 weeks, the effect on the cellular immune response by T cells was investigated in order to investigate whether it exhibits an immune enhancing effect. In this example, 10 magnetic BALB / c mice, 5 weeks of age, were purchased from Multi-Muse Sciences, adapted for 1 day, and used in the experiment. Experimental animals and breeding conditions are the same as in Example 3 mentioned above.

In the present invention, the cellular immune response of T cells in the mouse spleen was investigated in order to investigate whether it exhibits an immune enhancing effect when a water extract of Hwangchil tree is periodically administered. This experiment consisted of two groups, the normal control group and the 200 mg / kg hwangchil tree leaf extract group, and five experiments were conducted per group. All diets and negatives were provided freely. The experiment was conducted for a total of 3 weeks, and sacrificed after 3 weeks. Hwangchil tree leaf extract was injected intraperitoneally every day for 3 weeks.

At the third week, the mouse spleen was removed, the spleen tissue was transferred to a sterile petri dish, and the spleen was changed using a 50 μm cell strainer to separate cells from the tissue. All contents were transferred to a 15 mL tube, filled with RPMI medium, and centrifuged at 1200 RPM for 5 minutes. Thereafter, 3 mL of red blood cell lysis buffer was added to the pellet from which the supernatant was removed, and the red blood cells were lysed by mixing well, and then centrifuged at 1500 RPM for 15 minutes. After washing the pellet from which the supernatant was removed twice with PBS, the spleen cells were isolated by being suspended in RPMI medium.

The isolated splenocytes were dispensed into a 24 well plate at 2 X 10 ⁶ cells / mL and a 96 well plate at 0.5 X 10 ⁵ cells / mL and treated with ConA (5 μg / mL) for 48 hours. After that, in the case of a 96-well plate, MTT reagent was treated to confirm the proliferation ability of the cells, and after taking the supernatant from the 24-well plate, the supernatant was measured using an R & D system ELISA kit (Minneapolis, MN, USA).

As a result, as shown in Figure 1a, it was confirmed that the proliferation capacity of the cells was significantly increased than the normal control group in the hwangchil tree leaf extract 200 mg / kg administration group. In addition, as shown in Figures 1b and 1c, compared to the control group, IL-2 and IFN-r were significantly increased in the group treated with 200 mg / kg of hwangchil tree leaf extract.

Example 7: Experimental animal spleen cells and CD4 ⁺  T cell separation and proliferation

7-1. Isolation of spleen cells from mouse spleen

To confirm the splenocyte culture and the proliferation capacity of T cells in animals, spleens isolated from animals were washed with PBS to make single cell suspension using a 50 μm cell strainer (BD Falcon, San Diego, CA, USA). Single cell suspension with RPMI-1640 (Welgene, Daegu, Korea) with 10% FBS (Gibco BRL, Grand Island, NY, USA) and 0.1% Penicillin / Streptomycin (Gibco BRL, Grand Island, NY, USA) After washing, erythrocytes were lysed with red blood cell lysis buffer to make a spleen cell suspension, and 150 μL of 1 X 10 ⁶ cells / mL was dispensed into each well in a 96-well plate.

7-2. Isolation of CD4 ⁺ T cells from mouse spleen and lymph nodes

To confirm the CD4 ⁺ T cell culture and the proliferation capacity of T cells in animals, the spleen and lymph nodes isolated from the animals are separated and washed with PBS, and then using a 50 μm cell strainer (BD Falcon, San Diego, CA, USA). A single cell suspension was made. Single cell suspension with RPMI-1640 (Welgene, Daegu, Korea) with 10% FBX (Gibco BRL, Grand Island, NY, USA) and 0.1% penicillin / streptomycin (Gibco BRL, Grand Island, NY, USA) After washing, red blood cells were lysed with red blood cell lysis buffer to make a suspension. CD4 ⁺ T cells were isolated from the suspension using CD4 ⁺ beads.

The isolated T cells were previously dispensed with 150 μL of 1 × 10 ⁷ cells / mL per well in a 96-well plate coated with CD3 ⁺ antibody and CD28 ⁺ antibody, and incubated at 37 ° C for 48 hours. After the cultivation was completed, 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich Co., St. Louis, MO, USA) was treated and cultured for 4 hours. The absorbance was measured at 570 nm.

7-3. Confirmation of the proliferation capacity of splenocytes and CD4 ⁺ T cells

After measuring 1 μg / mL of concanavalin A (Concanavalin A, ConA, Sigma-Aldrich Co., St. Louis, MO, USA) on spleen cells for measuring the proliferation capacity of T cells, at 37 ° C. for 48 hours. Cultured. In addition, CD3 ⁺ and CD28 ⁺ CD4 ⁺ T cells were dispensed into a 96-well plate coated and cultured for 48 hours. After the cultivation was completed, the MTT was treated to incubate for 4 hours, and absorbance was measured at 570 nm.

As a result, the proliferation effect was not found in cells with different fractions of the ethylacetate layer of hwangchil tree leaves, but it was found that the proliferation effect was particularly excellent for T cells (FIGS. 2B and 2C). In addition, it was confirmed that DMW-2, a single component, had no significant effect on the proliferation of T cells (FIG. 4A).

Example 8: LC / MS / MS analysis of DMW-2, a single component in hwangchil tree leaf water extract

LC / MS / MS analysis was performed to confirm the content of DMW-2 in the hwangchil tree leaf water extract and ethyl acetate fraction. LC / MS / MS analysis was performed under the following conditions, and the results are shown in FIG. 3.

1.MS condition: Turbo Ion Spray MRM scan type DMW-2 m / z 273.213 / 55.1 Spray voltage 5500V, temperature 500 ℃, positive mode, CG 20, GS1 60, GS2 60.

2. LC conditions:

1) Column: Gemini C18 5 μm, 50 mm Х 2.0 mm Gemini C18 (4.0 mm Х 2.0 mm) guard cartridge, 2) Column oven: 40 ℃, 3) Autosampler: 15 ℃, 4) Mobile phase-A: Water, 0.1 % Formic Acid B: Acetonitrile, 0.1% Formic Acid, 5) Gradient: 1-4 minutes 15-60% B, 5 minutes 60% B, 5.1 minutes 15% B, 6) Flow rate: 0.3 mL / min, 7) Injection volume: 10 μL

Experimental Example 1: IL-2, IFN-γ cytokine concentration measurement using ELISA

IL-2 and IFN-γ cytokines related to T cell growth were measured using an R & D system ELISA kit (Minneapolis, MN, USA). The splenocyte suspension isolated from the mouse was previously dispensed at a concentration of 500 μL of 5 X 10 ⁶ cells / mL per well into a 48-well plate. Subsequently, Hwangchil tree water extract (W-Ex: 100 μg / mL), ethyl acetate fraction (W-EA: 10 μg / mL), and DMW-2 (1 μM or 3 μM) were treated for each concentration for 72 hours. Cultured. After that, the supernatant (culture) was centrifuged at 1200 RPM for 15 minutes, and then the supernatant was taken again to conduct the experiment.

When treated with hwangchil tree water extract and ethyl acetate fraction, the effect of increasing IL-2 and IFN-γ cytokines was excellent. In addition, DMW-2, a single component, had a good effect of increasing IL-2 cytokine production and also increased the production of IFN-γ at low concentrations (FIGS. 4B and 4C).

Experimental Example 2: Confirmation of NF-AT, NF-kB, and AP-1 factors involved in T cells and IL-2, IL-4 transcription factors using reporter assay

After dispensing the mouse (Mus musculus) lymphoma cell line EL-4 cell line into a 6-well plate at 5 X 10 ⁵ cells / well, 4 μg of each reporter (Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) ) Was used for transfection and cultured in a 37 ° C., CO ₂ incubator for 48 hours.

Then, Hwangchil tree water extract (W-Ex: 100 μg / mL), ethyl acetate fraction (W-EA: 10 μg / mL), and DMW-2 (1 μM or 3 μM) were treated, respectively. After 4 hours, PMA / Io was treated and incubated in a CO ₂ incubator at 37 ° C. for 24 hours. After 24 hours, the plate was centrifuged at 1200 RPM for 15 minutes to dissolve and measured using a luminometer using a luciferase reporter system (Promega).

As a result, in the hwangchil tree water extract and ethyl acetate fraction treatment group, NF-AT, AP-1 transcription factor expression increase effect was observed, and NF-kB did not show a large effect (FIGS. 5B-5D). In addition, it also showed an increase in IL-2 transcription level (Fig. 5a).

Meanwhile, DMW-2, a single component, significantly increased the transcription factor expression of NF-AT, NF-kB, and AP-1 (FIGS. 5B-5D). In addition, when DMW-2 low concentration (1 μM) treatment, IL-2 and IL-4 transcription levels were significantly increased (FIGS. 5A and 5E).

Claims (12)

Dendropanax morbifera Lev. Leaf extract, pharmaceutical composition for immunomodulation or immune enhancement. The method of claim 1, wherein the hwangchil tree leaf extract is made of water and one or more solvents selected from the group consisting of lower alcohols having 1 to 3 carbon atoms as an extraction solvent, immunomodulatory or immune enhancing pharmaceutical composition. The pharmaceutical composition for immunomodulation or immune enhancement according to claim 2, wherein the composition comprises an ethyl acetate extract fraction obtained by purifying only an extract soluble in an ethyl acetate solvent from a hwangchil tree leaf water extract or an alcohol extract. . Food composition for immunomodulation or immune enhancement, including extract of hwangchil tree leaves ( dendropanax morbifera lev . Leaf). The method of claim 4, wherein the hwangchil tree leaf extract is water and one or more solvents selected from the group consisting of lower alcohols having 1 to 3 carbon atoms as an extraction solvent, immunomodulation or immune enhancement food composition. The food composition for immunomodulation or immune enhancement according to claim 5, wherein the composition comprises an ethyl acetate extract fraction obtained by purifying only extracts soluble in ethyl acetate solvent from water extract or alcohol extract of hwangchil tree leaves. Pharmaceutical composition for enhancing antitumor immunity, including extract of hwangchil tree leaf ( dendropanax morbifera lev . Leaf). Food composition for promoting antitumor immunity, including extract of hwangchil tree leaf ( dendropanax morbifera lev . Leaf). Anti-cancer pharmaceutical composition comprising hwangchil tree leaf ( Dendropanax morbifera lev . Leaf) extract. The method of claim 9, wherein the cancer is gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, Skin cancer, kidney cancer, drainage cancer (polyploid carcinoma), thyroid cancer, parathyroid cancer, or ureter cancer. Anti-cancer food composition comprising hwangchil tree leaf ( dendropanax morbifera lev . Leaf) extract. The method of claim 11, wherein the cancer is gastric cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, colon cancer, colon cancer, cervical cancer, brain cancer, prostate cancer, bone cancer, head and neck cancer, Skin cancer, kidney cancer, drainage cancer (polyploid carcinoma), thyroid cancer, parathyroid cancer, or ureter cancer.

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