WO2007037627A1

WO2007037627A1 - Pharmaceutical composition comprising polysaccharides from angelica gigas nakai for prevention and treatment of cancer disease

Info

Publication number: WO2007037627A1
Application number: PCT/KR2006/003880
Authority: WO
Inventors: Hwan Mook Kim; Sang Bae Han; Chang Woo Lee; Ki Hoon Lee; Sung Kyu Park; Ki Ho Lee; Jong Soon Kang; Won Kee Yoon
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2005-09-28
Filing date: 2006-09-28
Publication date: 2007-04-05
Also published as: KR20070048814A; KR100726006B1

Abstract

Disclosed is a pharmaceutical composition for prevention and treatment of cancers, comprising Angelan, a polysaccharide isolated from Angelica gigas Nakai, as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluents. In detail, Angelan is excellent in the inhibition of cancer cell growth and the suppression of attachment and invasion of cancer cells to extracellular matrix, thereby inhibiting metastasis of cancer cells. In addition, when Angelan is taken in combination with an existing anticancer drug such as doxorubicin, synergic anticancer effects can be obtained.

Description

[DESCRIPTION] [Invention Title]

PHARMACEUTICAL COMPOSITION COMPRISING

POLYSACCHARIDES FROM ANGELICA GIGAS NAKAI FOR PREVENTION AND TREATMENT OF CANCER DISEASE

[Technical Field]

The present invention relates to a pharmaceutical composition for prevention and treatment of cancers comprising Angelan, a polysaccharide isolated from Angelica gigas Nakai, as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluents.

[Background Art]

Cancer is one of intractable diseases humankind must deal with, and a tremendous amount of capital has been invested worldwide in developing its treatment. In Korea, cancer is the top in cause of death, and more than about 100,000 people are diagnosed with cancer each year and more than about 60,000 people actually die from cancer. Although carcinogens as risk factors contributing to causing the cancer include smoking, ultraviolet rays, chemicals, foods and other environmental factors, the causes are so diverse that it is difficult not only for developing new therapeutic agents and medicines but also for identifying their effects on different types of cancer. Unfortunately, substances currently being used as cancer therapeutic agents have a high degree of acute toxicity and cannot selectively remove cancer cells only. Therefore, there is a great need to develop an antitumor agent or an anticancer drug that can treat cancer, prevent cancerization, and is effective and less toxic.

Cancer is characterized by "uncontrollable cell growth" and those abnormally grown cancer cells usually group or clump together to form tumors. A growing tumor becomes a lump of cancer cells that break away and spread to its surrounding tissues and is likely to metastasize to other organs of the body if the cancer gets worse. In medical term, this process is called neoplasia. Even though cancers may be treated with surgery, radiation therapy and chemotherapy, these are not radical treatments to treat the intractable chronic disease, but give a lot of pain to patients in many cases, making them die eventually. There are many causes of cancers, but sometimes they are divided into internal and external causes. Although it is not accurately searched out through which mechanism a normal cell is transformed into a cancer cell, it is reported that at least 80 to 90% of cancer is influenced by an external cause such as environment. Examples of the internal cause include genetic factor, immunological factor and the like, and examples of the external cause include chemicals, radiation, viruses and the like. Genes that are related to the onset of cancer are oncogenes and tumor suppressor genes, and cancer occurs when the balance between them is broken by internal or external causes describe above.

Meanwhile, cancers are largely classified into hematologic malignancies and solid cancers, and found almost every part of the body, e.g., lung cancer, gastric cancer, breast cancer, oral cancer, hepatoma, uterine cancer, esophageal cancer, skin cancer, etc. Among the therapy methods for malignant tumors, chemotherapy except for surgery or radiation therapy is called collectively as an anticancer drug. Most anticancer drugs inhibit synthesis of nucleic acid to demonstrate anticancer activity. Chemotherapeutic agents are largely classified into antimetabolites, alkylating agents, antimitotic drugs, hormones, etc. In detail, the antimetabolites inhibit the metabolism involved in the proliferation of cancer cells, and include folate derivatives (methotrexate), purine derivatives (6- mercaptopurine and 6-thioguanine), pyrimidine derivatives (5-fluorouracil and cytarabine). The alkylating agents demonstrate the anticancer effects by introducing an alkyl into guanine in DNA molecules to cause DNA deformation and cut the chain, and include nitrogen mustard compounds (chlorambucil, and cyclophosphamide), ethyleneimine compounds (thiotepa), alkylsulfonate compounds (busulfan), nitrosourea compounds (carmustine), and triazene compounds (dacarbazine). The antimitotic drugs are mitosis specific drugs capable of suppressing cell division by interfering with mitosis, and include antineoplastic anticancer drugs, e.g., actinomycin D, doxorubicin, bleomycin, and mitomycin; plant alkaloid, e.g., vincristine and vinblastine; and mitosis inhibitors having taxane rings, e.g., taxoids. In addition, hormones like adrenal cortical hormone or progesterone and platinum containing compounds like cisplatin are used as anticancer drugs.

Chemotherapy using anticancer drugs is the most widely used method for cancer treatment, but it has shortcomings that most anticancer drugs used in the chemotherapy cannot completely remove cancer cells, are not effective for metastatic cancer cells, and have a high degree of toxicity in patients. That is to say, anticancer drugs can effectively remove 90% of cancer cells, but the remaining 10% cancer cells cannot be removed by the chemotherapy only, leaving the possibility of cancer recurrence. Moreover, even though most of the anticancer drugs are substances capable of suppressing cell proliferation, metastatic cancer cells are not easily killed by the anticancer drugs because they do not proliferate often.

In addition, one of the biggest side effects of the chemotherapy or the radiation therapy is a decrease in immunologic function. As aforementioned, anticancer drugs used in the chemotherapy consist of mainly cell-proliferation- suppressing substances, but has a shortcoming that immunocytes of a patient are also killed by them. In general, cancer cells secrete various kinds of immunosuppressive substances during their growth, resulting in very serious side effects of immunosupression.

As a complement to such shortcomings, anticancer immunotherapy has recently drawn a lot of attentions. The anticancer immunotherapy is a method for removing cancer cells by increasing immunologic function of a host, so it shows somewhat less side-effects and increases anticancer effects when used in combination with the chemotherapy. It was also reported that cancer patients' health is improved, leading to the improvement of quality of life. Although this approach has been used mainly in Asian countries including Korea, Japan, China and so on, western countries like America are now showing their interests and researches are in progress. The anticancer immunotherapy utilizes cytokine, a polysaccharide isolated from edible fungi, a polysaccharide isolated from plant and the like. Especially, anticancer effects of polysaccharides isolated from Phellinus linteus, ling chiu mushrooms, and birch mushrooms are well known. Angelicae Gigantis Radis is a naturalized plant in Korea, and also called

Angelica gigas Nakai if grown in Korean. It is a perennial plant originally from Japan. When fully grown, Angelica gigas Nakai is 60 to 90cm high and white flowers burst into bloom at the end of branches in summertime between August and September. Its fruit is in a flat, oblong shape and has a narrow wing on the edge. Actually, Angelicae Gigantis Radis is the root that is known to nourish the blood, so it can be effectively applied to people looking pale, feeling dizzy often, and being shallow-complexioned in eyes and lips. Because of its high efficacy in headaches caused by asthenia and for the maintenance and regulation of menstrual cycle, it is often used for treating many diseases before and after childbirth, menstrual irregularity (or dysmenorrheal), and abdominal pains. It is also known to be effective for the treatment of constipation caused by gastroenteric trouble (Guidelines for healthy life based on folk medicine and herb medicine, written by Ki-Whan Bae, pp. 102-103, 2003). Some of effective ingredients of Angelica gigas Nakai are coumarin derivatives, e.g., decursin, decursinol, nodakenetin, and the like, and essential oil compositions, e.g., α- pinene, limonene, β-eudesmol, elemol and the like. Therefore, it can provide uterine dysfunction regulation, sedative action, analgesic action, emmenagogue action, therapeutic action especially in diarrhea and vitamin E deficiency, and is used as an antianemic, analgesics, corroborant, emmenagogue, and gyniatrics. In springtime, people may eat young leaves of Angelica gigas Nakai as seasoned greens. It has been reported that an ether extract from Angelica gigas Nakai demonstrated excitability in an intestinal canal and uterus removed from a rabbit, and accentuate blood pressure and breathing. Meanwhile, the isolated decursin and decursinol are reported to paralyze the intestinal canal removed from the rabbit. There is another report that Angelica gigas Nakai provides an inhibitory action on a hear removed from a frog, may be used as a respiratory depressant and antihypertensive drug for a rabbit, and is capable of inhibiting automaticity or spontaneity of a rat (Published by the Korean Society of Pharmacognosy, Vol. 1, 1^st Edition, pp. 25-32, 1970).

However, no literature mentioned above teaches nor suggests that a polysaccharide isolated from Angelica gigas Nakai provides an anticancer activity. Accordingly, inventors completed the present invention by identifying the cancer cell-growth inhibitory effects and the suppression of cancer cell metastasis provided by the polysaccharide isolated from Angelica gigas Nakai.

[Disclosure]

[Technical Problem]

Therefore, an object of the invention is to provide a pharmaceutical composition for prevention and treatment of cancers comprising Angelan, a polysaccharide isolated from Angelica gigas Nakai, as an active ingredient capable of providing cancer cell-growth inhibitory effects and suppression of metastasis in cancer cells, and a pharmaceutically acceptable carrier, excipient or diluent.

The polysaccharides isolated from Angelica gigas Nakai can be used in combination with existing anticancer drugs.

[Technical Solution] Polysaccharides contained in Angelica gigas Nakai can be isolated by a method (Immunopharniacology 40, pp. 39-48, 1998) as follows.

First of all, a root of Angelica gigas Nakai is cut into little pieces, refluxed in hot water for 1 hour, and filtered through 4-layer gauze and a filter paper to produce the remainder of liquid extract. The remainder of liquid extract is added to ethanol with three times the volume, and the mixed solution is left aside at 4 "C for 3 hours for the reaction. Then, a precipitate obtained from the solution is centrifuged to yield a brown polymer fraction.

The polymer fraction can easily be obtained by precipitation with a small amount of ethanol. Discovering that no precipitate is formed by denatured proteins even after the polymer fraction was melted and boiled for 20 minutes, a conclusion may be drawn that the fraction contains a large amount of nonprotein polymers. In addition, when the fraction was adsorbed on an anion exchange resin such as DEAE-cellulose, most colored substances were adsorbed on the resin. It turned out that these are all polysaccharides containing a very small amount of proteins, and especially acid polysaccharides among them were named Angelan (molecular weight: about 10,000Da).

According to the analysis result on the composition of the fraction of Angelan that is isolated from Angelica gigas Nakai having immunological enhancement effects, Angelan fraction consisted mostly of saccharide which amounted as high as 85 to 90% (w/w), 7 to 8% proteins for each fraction, and 15.5 to 69% of uronic acid. In addition, Angelan fraction contained a relatively large amount of calcium ions and magnesium ions, and a small amount of iron, aluminum, manganese, zinc, potassium, sodium, phosphor, sulfur and so on.

The polysaccharide originated from Angelica gigas Nakai (i.e., Angelan) contains a great quantity of galacturonic acid, galactose and arabinose, and a small quantity of mannose, rhamnose, xylose and so on. In particular, Angelan, a polysaccharide isolated from Angelica gigas

Nakai, is effective for inhibiting cancer cell-growth and excellent in suppression of the attachment and invasion of cancer cells with extracellular matrix to thereby suppress metastasis of cancer cells. Moreover, Angelan enhances the antibody production by B cells and is excellent in macrophage activation, whereby it can demonstrate superior immunological enhancement effects in relation to the anticancer activity.

In view of the foregoing advantages and effects, the present invention provides a pharmaceutical composition comprising Angelan, a polysaccharide isolated from Angelica gigas Nakai, as an active ingredient capable of providing cancer cell-growth inhibitory effects and suppression of metastasis in cancer cells, and a pharmaceutically acceptable carrier, excipient or diluent for prevention and treatment of cancers.

Moreover, the polysaccharides isolated from Angelica gigas Nakai can be used in combination with existing anticancer drugs. Cancer diseases mentioned in the description include all cancer diseases in general, which is preferably selected from the group consisting of gastric cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or eye melanoma, uterine cancer, ovarian cancer, colorectal cancer, anal cancer, fallopian tube carcinoma, endometrium carcinoma, cervical carcinoma, medullary carcinoma, vulva carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid gland cancer, soft skin sarcoma, urethra cancer, penis (penile) cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, renal or hydroureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS (central nervous system) tumor, primary CNS lymphoma, spinal cord tumor, brain stem gliomatous, and pituitary adenoma.

The pharmaceutical composition for prevention and treatment of cancers according to the present invention contains 0.1 to 50% of the extract or compound by weight of a total weight of the composition.

The pharmaceutical composition containing the polysaccharides of the present invention may further comprises a pharmaceutically acceptable carrier currently used in the art, excipient and diluent.

Extracts or compounds of the present invention can be administered in form of pharmaceutically acceptable salts, singly or in combination with other pharmaceutically acceptable compounds, or in proper aggregation. The pharmaceutical composition containing extracts or compounds of the present invention may be produced in commonly used drug form for oral administration such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol and the like, external preparation, rectal suppository, and sterile injection. Examples of carriers, excipients and diluents that can be contained in the extracts or the compounds of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, arabic gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidine, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Also, they can be made in drug form with the commonly used diluent or excipient, such as filler, extender, binder, wetting agent, disintegrator and surfactant. Examples of a solid drug form for oral administration include tablet, pill, powder, granule, capsule and the like, and these solid drugs are prepared by mixing the extracts or the compounds with more than one excipient, such as starch, calcium carbonate, sucrose or lactose and gelatin. In addition, lubricants like magnesium stearate and talc can be used in addition to excipients. Examples of a liquid drug form for oral administration include suspension, internal medicine, emulsion and syrup. Diluents like water and liquid paraffin and various excipients like wetting agent, sweetener, aromatic and preservative can be included. Meanwhile, examples of a drug form for non-oral administration include sterile aqueous solution, nonaqueous solvent, suspension, emulsion, lyophilizator, and suppository. For nonaqueous solvent or suspension, propylene glycol, polyethylene glycol, plant oil like olive oil and injectable ester like ethylolate can be used. For suppository, witepsol, macrogol, tween 61, cacao oil, lauryn oil or glycerogelatin can be used.

Although an effective dose of the polysaccharides of the present invention vary depending on the condition and weight of a patient, the degree of a disease, the type of drug, and the route and period of drug administration, it can be adjusted properly by a person skilled in the art to which the present invention pertains. However, to maximize effects of the pharmaceutical composition, the polysaccharides of the present invention may be administered 0.0001 to 100 mg/kg, and preferably 0.001 to 10 mg/kg per day. It can be administered once a day, or several times a day. It should be noted that the scope of the invention is not limited by the dose in any way. The polysaccharides of the present invention may be administered to mammals including rats, mice, livestock, human beings and so on. All kinds of drug administration can be expected for use, such as, oral, endorectal-, intravenous- intramuscular- hypodermic-, intrauterine- intrathecal- and intracerebroventricular injections.

The polysaccharides of the present invention have little toxicity and almost no side effects, so they can be used at ease for a long period of time for purpose of prevention.

Reference will now be made in detail to the preferred examples of the present invention. It is to be understood that the following examples are illustrative only and the present invention is not limited thereto.

[Advantageous Effects]

The present invention provides a pharmaceutical composition for prevention and treatment of cancers comprising Angelan, a polysaccharide isolated from Angelica gigas Nakai, as an active ingredient, and a pharmaceutically acceptable carrier, excipient or and diluent. Angelan, a polysaccharide isolated from Angelica gigas Nakai as aforementioned, is excellent in the inhibition of cancer cell growth and the suppression of attachment and invasion of cancer cells to extracellular matrix, thereby inhibiting metastasis of cancer cells. In addition, when Angelan is taken in combination with an existing anticancer drug such as doxorubicin, synergic anticancer effects can be obtained.

[Description of the Drawings]

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

FIG. 1 and FIG. 2 are graphs showing cancer cell growth - inhibitory effects of Angelan;

FIG. 3 is a graph showing the inhibitory effects of Angelan involved in metastasis of cancer, which are observed after inducing the metastasis of cancer into a mouse;

FIG. 4 is a graph showing cancer cell attachment - inhibitory effects of Angelan;

FIGs. 5 and 6 illustrate cancer cell invasion - inhibitory effects of Angelan, in which FIG. 5 is a series of photographs that sequentially showing the cancer cell invasion - inhibitory effects, and FIG. 6 is a graph quantifically showing the cancer cell invasion - inhibitory effects in quantification;

FIG. 7 is a graph showing the inhibitory effects of Angelan on proliferation of B cells, T cells, and macrophage; FIG. 8 is a graph showing the effects of Angelan on antibody formation

(autoimmunity) of B cells;

FIG. 9 is a graph showing the relation between the concentration of Angelan and the nitrite formation of macrophage; and

FIG. 10 is a graph showing the relation between the concentration of Angelan and the survival rate of cancer cells, thereby demonstrating whether Angelan exerts toxicity upon cancer cells.

[Best Mode]

Example 1: Isolation of Polysaccharides from Angelica gigas Nakai Angelan, a polysaccharide originated from Angelica gigas Nakai was isolated by a method described in a paper on Immunopharmacology 40, pp. 39-48, 1998 as follows. A root of Angelica gigas Nakai was cut into little pieces, refluxed in hot water for 1 hour, and filtered through 4-layer gauze and a filter paper to produce the remainder of liquid extract. The remainder of liquid extract was added to ethanol with three times the volume, and the mixed solution was left aside at 4 "C for 3 hours for the reaction. Then, a precipitate obtained from the solution was centrifuged to yield a brown polymer fraction.

The polymer fraction can easily be obtained by precipitation with a small amount of ethanol. After observing that no precipitate was formed by denatured proteins even after the polymer fraction had been melted and boiled for 20 minutes, inventors drew a conclusion that the fraction contains a large amount of nonprotein polymers. In addition, when the fraction was adsorbed on an anion exchange resin such as DEAE-cellulose, most colored substances were adsorbed on the resin. It turned out that these are all polysaccharides containing a very small amount of proteins, and especially acid polysaccharides among them were named Angelan (molecular weight: about 10,000Da).

[Mode for Invention]

Example 2: Analysis on Composition of Polysaccharides Originated from Angelica gigas Nakai

A composition of Angelan, a polysaccharide isolated from Angelica gigas Nakai, according to the Example 1 was analyzed.

According to the analysis result, Angelan fraction consisted mostly of saccharide which amounted as high as 85 to 90% (w/w), 7 to 8% proteins for each fraction, and 15.5 to 68% of uronic acid. In addition, Angelan fraction contained a relatively large amount of calcium ions and magnesium ions, and a small amount of iron, aluminum, manganese, zinc, potassium, sodium, phosphorus, sulfur and so on.

To find out the polysaccharide composition, refined samples were hydrolyzed with 2M trifluoroacetic acid and were subjected to a composition analysis by means of a TLC and an ion exchange HPLC.

In turned out that Angelan contained a great quantity of galacturonic acid, galactose and arabinose, and a small quantity of mannose, rhamnose, xylose and so on.

Reference Example 1: Preparation of Laboratory Animal

All experiments were performed on BDFl male mice (Daehan Biolink Co., Ltd., Chungcheongbuk-do), weighing 250-260 g. The animals were kept in a cage under preset environmental conditions (temperature: 21 + 2°C under constant 12 hours light and dark cycle). Standard Lab. Feeds and water were given fully before the experiments. The animals were acclimatized and handled for 10 minutes before the experiments started.

Experimental Example 1: Cancer Cell Growth - Inhibitory Effects

1-1 Inhibitory effects of Angelan on cancer cell growth (I) 100,000 B16F10 cancer cells (American Type Culture Collection, USA) were implanted into an abdominal cavity of each mouse prepared in the Reference Example 1 to thereby induce cancer (or tumor) growth. Later, 30 mg/kg of Angelan isolated from Angelica gigas Nakai of the present invention and 0.1 mg/kg of doxorubicin (Sigma Chemical Co., USA) as an anticancer drug were administered singly or in combination to the mice by intro-abdominal injection every day, and the survival rate of the mice was observed.

As can be seen in FIG. 1, all of the mice administered with nothing (the control group) died within 20 days by the tumor growth within their abdominal cavities. On the contrary, the survival rate of the mice administered with 30 mg/kg of Angelan isolated from Angelica gigas Nakai of the present invention was increased, showing that 30% of the mice survived 60 days after the implantation of the cancer cells. An increase in the survival rate was also observed in the mice administered with 0.1 mg/kg of the anticancer drug doxorubicin. The survival rate was increased even greater in case of the mice administered with both the polysaccharides from Angelica gigas Nakai and doxorubicin at the same time, showing that about 50% of the mice survived 60 days after the implantation of the cancer cells.

1-2 Inhibitory effects of Angelan on cancer cell growth (II) Example 1-1 was repeated to observe survival rates of the cancer- induced mice, except that 30 mg/kg of Angelan isolated from Angelica gigas Nakai of the present invention and 0.3 mg/kg of doxorubicin were administered singly or in combination to the mice by intro-abdominal injection every day.

As can be seen in FIG. 2, the survival rate of the mice administered with 0.3 mg/kg of doxorubicin was increased, showing that 20% of the mice were alive after 60 days from the implantation of the cancer cells. Meanwhile, a greater increase was observed in the survival rate of the mice administered with 30 mg/kg of Angelan isolated from Angelica gigas Nakai of the present invention only. Because of the cancer cell growth - inhibitory effects of Angelan, the survival rate of the mice was substantially increased up to 30% until 60 days after the implantation of the cancer cells. In addition, a sharp increase in the survival rate was observed for the mice administered with the polysaccharides from Angelica gigas Nakai and doxorubicin in combination, showing that about 100% of the mice survived 60 days after the implantation of the cancer cells. To summarize the results from the Experimental Examples 1.1 and 1.2, when the polysaccharides of the present invention and doxorubicin were administered singly to the cancer-induced mice, the growth of the implanted cancer cells could be inhibited. The inhibitory effects were maximized, e.g., 100% of the survival rate 60 days after the implantation of the cancer cells, by administering those two substances in combination. Therefore, one can recognize the improved anticancer effects.

Experimental Example 2: Metastasis Suppression Effects 500,000 Bl 6F10 cancer cells were implanted into a tail vein of each mouse prepared in the Reference Example 1 to thereby induce metastasis. 14 days later, lung of each mouse was isolated to measure metastatic colonies. The control group was administered with physiological saline, and 30 mg/kg of Angelan isolated from Angelica gigas Nakai of the present invention and/ or and 1 - 3 mg/kg of doxorubicin singly or in combination by intro-abdominal injection every day, and the metastasis suppression effects were observed.

As can be seen in FIG. 3, in case of the mice administered with nothing, the cancer cells in the blood invaded into lung and grown fast, thereby completing metastasis. When the lung was examined after 14 days from the implantation of the cancer cells, about 270 metastatic colonies were observed. However, in case of the mice administered with Angelan, the polysaccharide of the present invention, at the contraction of 30 mg/kg, cancer cell metastasis was suppressed such that the metastatic colonies were substantially reduced. The same metastatic suppression effects were observed in the mice administered with the anticancer drug doxorubicin at the concentration of 1 - 3 mg/kg. The metastatic suppression effects were even stronger on the mice administered with both Angelan, a polysaccharide of the present invention, and doxorubicin at the same time.

Experimental Example 3: Effects on Cancer Cell Attachment It is reported that cancer cell metastasis occurs in several phases, such as a cancer cell's attachment and invasion into an extracellular matrix, migration via blood and lymph, attachment and transmigration to vascular cells, invasion into interstitial tissues and abnormal growth and so forth, in a consecutive manner. To measure the inhibitory effects of Angelan on the attachment of cancer cells to an extracellular matrix, B16F10 cancer cells were cultured for 1 hour on a culture dish coated with collagen (RPMI 1640 medium containing 10% Fetal Calf Serum, 37°C, 5% CO₂ incubator). Then, the cells were treated with the 10 to 100 μg/m# polysaccharide of the present invention, i.e., Angelan. The cells that were not attached after culturing were removed, while the cells that were attached were trypsinated and collected, to thereby count the number of cells.

As can be seen in FIG. 4, the number of cells attached to collagen for 1 hour was approximately 450, and the attachment of B16F10 cancer cells to collagen was markedly suppressed when the cells were treated with Angelan, the polysaccharide of the present invention, at the concentration of 10 to 100 μg/m#.

Experimental Example 4: Effects on Cancer Cell Invasion

A special dish was used for the invasion test. A filter having an 8 μm hole is formed at the center of the dish, dividing the dish into two parts. In this way, cells being cultured on the upper part can be transferred to the lower part by passing through matrigel (collagen) and the hole. HT-2080 cell culture fluid was added to the lower part to cause migration of the B16F10 cancer cells. The control group was administered with physiological saline, and with Angelan of the present invention at different concentrations of 10, 30 and 100 μg/mβ, separately. The B16F10 cancer cells were added onto on the upper part of the special dish coated with matrigel for culturing. After 24 hours, the cells migrated to the lower part were photographed, and the number of invaded cells was counted.

As can be seen in FIG. 5, compared with the control group treated with the physiological saline, the cancer cells treated with Angelan of the present invention were concentration-dependently and significantly inhibited from invasion.

In addition, as can be seen in FIG. 6, about 450 cells were invaded in the control group (FIG. 6A). However, about 320 (FIG. 6B), 50 (FIG. 6C), and 10 (FIG. 6D) invaded cells were observed after Angelan of the present invention had been administered at the contraction of 10 μg/m£, 30 μg/mβ, and 100 μg/m#, respectively. These results confirm that the invasion of cancer cells was excellently, concentration dependency inhibited by Angelan, the polysaccharide of the present invention.

Experimental Example 5: Immunological Enhancement Effects 5-1. Immunological Enhancement Effects (I)

Modus operandi of an anticarcinogen is known as activation of human body's immunocytes and their immunological attach against cancer cells. In particular, effects of the polysaccharide of the present invention on B cells, T cells, and macrophage were observed. B cells or T cells isolated from the spleen of the mouse were treated with

Angelan, the polysaccharide of the present invention, at different concentrations of 10, 30 and 100 μg/ml, respectively. The cells were first cultured for 60 hours, and [³H]-thymidine was added thereto for additional culturing for 12 hours. Then, radioactivity induced to DNA of the B cells or T cells were measured. As can be seen in FIG. 7, Angelan, the polysaccharide of the present invention, remarkably increased B cell proliferation, but had no influence on T cell proliferation. 5-2. Immunological Enhancement Effects (II)

On the basis of the results of the Experimental Example 5-1, B cells were separated from the spleen of the mouse, and treated with Angelan, the polysaccharide of the present invention at different concentrations of 10, 30 and 100 μg/m£, respectively. The cells were then cultured for 3 days and were subjected to an antibody-forming test of B cells by plaque forming cell assay (Han et al., International Immunopharmacology, 3(9), pp. 1301-1312, 2003).

As can be seen in FIG. 8, about 90, 140, and 170 antibody-forming cells were observed in B cells administered with Angelan, the polysaccharide of the present invention, at the concentration of 10 μg/m#, 30 μg/m-K, and 100 μg/m#, respectively. These results confirm that the polysaccharide of the present invention increased significantly and concentration-dependently the formation of antibodies of B cells.

5-3. Immunological Enhancement Effects (III)

For this experiment, macrophage was isolated from the abdominal of the mouse. Macrophage is known to be able to secrete cytokine, free radical, nitric oxide and the like when activated, and destroy cancer cells containing these components. In addition, macrophage is capable of destroy cancer cells directly. The macrophage was treated with Angelan, the polysaccharide of the present invention, at different concentrations of 10, 30 and 100 μg/M, respectively. 24 hours later, endothelial nitric oxide synthesis of the macrophage was measured by using a Griess reagent (Han et al., International Immunopharmacology, 3(9), pp. 1301-1312, 2003). As can be seen in FIG. 9, 65, 80, and 95 nmol nitrite were produced in the macrophage administered with Angelan, the polysaccharide of the present invention, at the concentration of 10 μg/m#, 30 μg/m#, and 100 μg/m£, respectively. These results confirm that the polysaccharide of the present invention increased significantly and concentration-dependently the endothelial nitric oxide synthesis of the macrophage.

To summarize the results from the Experimental Examples 5-1 to 5-3, one can draw a conclusion that Angelan, the polysaccharide of the present invention, functioned as an immunological enhancer capable of activating immunocytes, and demonstrated an excellent anticancer activity by reinforcing the functions of B cells and macrophage.

Experimental Example 6: Cytotoxicity Test

As the conventional anticancer drug doxorubicin is known as a cytotoxic drug against cancer cells, cytotoxicity of Angelan, the polysaccharide of the present invention, was subjected to the cytotoxicity test.

B16F10 cancer cells were treated directly with the polysaccharide of the present invention, and 48 hours later the number of cancer cells being still alive was measured by using sulforhodamine B assay (Han et al., International Immunopharmacology, 41(2), pp. 157-164, 1999).

As can be seen in FIG. 10, Angelan, the polysaccharide of the present invention, had no cytotoxicity against the cancer cells, but doxorubicin showed a strong cytotoxicity against the cancer cells.

[Industrial Applicability]

Angelan, a polysaccharide isolated from Angelica gigas Nakai as aforementioned, is excellent in the inhibition of cancer cell growth and the suppression of attachment and invasion of cancer cells to extracellular matrix, thereby inhibiting metastasis of cancer cells. In addition, when Angelan is taken in combination with an existing anticancer drug such as doxorubicin, synergic anticancer effects can be obtained.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

[CLAIMS] [Claim 1]

A pharmaceutical composition for prevention and treatment of cancers comprising Angelan, a polysaccharide isolated from Angelica gigas Nakai having inhibitory effects on cancer cell growth and cancer cell metastasis, as an active ingredient, and a pharmaceutically acceptable carrier, excipient or diluent.

[Claim 2]

The pharmaceutical composition according to claim 1, wherein the Angelan can be administered in combination with an existing anticancer drug.