It is known that many compositions are derived from Chinese herbal medicines, medicinal plants and extracts thereof for treating cancers.

Nervertheless, a single plant extract without being conjugated with other effective ingredients, its anti-tumor effect is still unsatisfactory.

The present inventor has found these phenomena and invented the present anti-tumor composition by conjugating Canavalia ensiformis-extracted protein with metal ions.

Disclosure of Invention: The object of the present invention is to provide an anti-tumor pharmaceutical composition comprising the conjugration of Canavalia ensiformis-extracted protein with metal ions to form a metalloprotein complex for inhibiting the growth of tumor with enhanced activity and stability, but without toxicity.

Brief Description of Drawings: Fig. 1 shows a SW-480 cell proliferation assay, in which the ordinate indicates cell number as percent of control, and the abscissa indicating the concentration of crystallized complex (mg/ml) ; IC 50: 0.375 mg/ml.

Fig. 2 shows a TSGH cell proliferation assay, in which the ordinate

indicates cell number as percent of control, and the abscissa indicating the concentration of crystallized complex (mg/ml) ; IC 50: 0. 1875 mg/ml.

Fig. 3 shows a MCF-7 cell proliferation assay, in which the ordinate indicates cell number as percent of control, and the abscissa indicating the concentration of crystallized complex (mg/ml) ; IC 50 : 0. 25 mg/ml.

Fig. 4 shows a LLC cell proliferation assay, in which the ordinate indicates cell number as percent of control, and the abscissa indicating the concentration of crystallized complex (mg/ml) ; IC 50: 0.5 mg/ml.

Fig. 5 shows the relationship between the tumor size and time for untreated and treated animals.

Best Mode for Carring Out the Invention: For preparing the anti-tumor pharmaceutical composition of the present invention, the following steps of the preparation procedure may be applied : A. Pre-treatment of Canavlia ensiformis Seeds: 1. Fresh seeds of Canavalia ensiformis as planted in Taiwan, 10 grams, are obtained for water-washing, soaking in the water and drying in air.

2. The dried seeds are then ground into fine powder, and lipids were extracted with hexane. The solvent is then decanted and the precipitate is collected for next treatment.

3. The precipitate is then dissolved in 100 ml of pure water (pH: 7) and stirred for 30 minutes at the temperature ranging from 0°C to room temperature to obtain an aqueous solution containing the Canavalia ensiformis protein.

B. Formation of Metalloprotein Complex: 1. The aqueous solution is settled for 30 60 minutes to precipitate any impurity residue which is then removed. Such an aqueous solution is then filtered to remove any residue through a filtration membrane, e. g. , 0. 22 m milipore membrance to obtain a crude solution of Canavalia ensiformis-extracted protein from the filtrate.

2. MgO or Cacl2 or A1 (OH) 3 were mixed with the solution of crude water extract of Canavalia ensiformis protein under agitation for four hours to obtain the metalloprotein complex solution. Pure water was added to adjust protein concentration to 1 mg protein/lml of metalocomplex solution for oral administration. MgO, Cacl2 and Al (OH) 3 can also be used when mixed in a weight ration 1: 1: 1 Such group of metallic-ion containing compounds include metallic ions of aluminum ion (Al+++), magnesium ion (Mg++) and calcium ion (Ca++). When mixing the metallic-ion containing compounds (of which the weight may be designated as Wp) for making the complex solution, the following mixing ratio may be considered: Wi: Wp=1 : 50 3. The metalloprotein complex solution is lyophilized by a lyophilizer to obtain crystallized complex. An excipient such as lactose may be added into the crystallized complex with a weight ratio of 3: 1, i. e, 3 parts of excipient with one part of crystallized complex, thereby producing pharmaceutically acceptable power, tablets and capsules of the Canavalia ensiformis-extracted protein conjugated with metal ions.

Conjugating the Canavalia ensiformis extract with metallic ions increase the activity and stability of the crude extract.

When the conjugate (pH>7) of this invention passing through the stomach filled with gastric acid into the intestinal canal, the metallic ions will render an active transport in cooperation with the excipient or carrier (such as lactose) to thereby increase the absorptivity of the effective increatly inhibit the tumor growth for enhancing the anti-tumor effect.

Besides the excipient of lactose other protein carriers such as the protein carrier existing in milk or other natural foods may be served for an effective"carrier"for carrying the effective ingredients of the present invention to the patient's target organs or cells for treating his or her cancer.

Although the metallic-ion compounds containing group ions of aluminum, magnesium and calcium ions as above-mentioned may be together conjugates with the Canavalia ensiformis extracted protein to effectively treat a patient's tumor, other metallic-ion compound containing single ion (without forming group of ions. or plural ions) may also be conjugates with the extracted protein, not limited in this invention.

The above-mentioned examples are given for describing the present invention only, but not to limit the claiming scope of this application within the examples as described herewith. If merely feeding natural beans of Canavalia ensiformis, no anti-tumor effect has been proven.

In order to prove the medicinal effect of the present invention, a plurality of tests are performed and reported as fllows:

In vitro test: Four different cancer cells as listed below are respectively tested for checking their proliferation by adding therein with the aqueous solution of the crystallized complex of the present invention: a. Cells from a primary adenocarcinoma of the human colon, SW-480 according to ATCC of USA ; b. Cells from human bladder cancer, TSGH-8301 ; c. Cells from mammary gland, breast adenocarcinoma MCF-7 (ATCC, USA); And d. Cells from Lewis lung carcinoma, LLC (ATCC, USA).

Example 1: Five tubes are provided each filled with 1 ml of aqueous solution of crystallized complex of the present invention and their concentrations have been adjusted for five groups as follows: (1) Original concentration solution: 1 mg crystallized complex/l ml solution ; (2) diluted solution by adding 1 ml phosphate buffer solution (PBS) into original-concentration. solution: 0.5 mg complex/ml ; (3) diluted solution (added with 3 ml PBS): 0.25 mg/ml ; (4) diluted solution (added with 5 ml PBS): 0.125 mg/ml ; (5) diluted solution (added with 7 ml PBS): 0.625 mg/ml ; The SW-480 (colon cancer) cells, with cell number 1x104 cell/ml, are transferred by pipet into wells of several well plates. Each well is then added therein with the above-prepared aqueous solution of crystallized complex of different concentrations, i. e. , 1 mg/lml, 0.5 mg/ml, 0.125 mg/ml

and 0.0625 mg/ml respectively for incubation of the cancer cells. After 3 days, each group of the aqueous solution is analyzed by adding crystalviolet (0. 1%) therein and by an analysis instrument such as ELISA reader to obtain the test result as shown in Fig. 1, from which the solution with increased concentration of the crystallixed complex of the present invention results in a smaller cell number, therby proving the tumor-inhibition effect of the present invention.

Example 2: The testing procedures of Example 1 are repeated by checking the cells of TSGH-8301 (bladder cancer) to obtain the test result as shown in Fig. 2.

Example 3: Testing procedures of Example 1 are repeated, but cells of MCF-7 (breast cancer) are tested to obtain the test result as shown in Fig. 3.

Example 4: Example 1 is repeated, except that the LLC (lung cancer) are tested to have the result as shown in Fig. 4.

Anti-tumor animal module: C57BL/6 mice are divided into three groups, 10 mice for each group (5 male and 5 female) to be tested as follows: Each mouse ready for test has an average weight of. 20 grams.

1. Control group: each C57BL/6 mouse fed with PBS 2 ml every day.

2. Low dose group: each C57BL/6 mouse fed with 10 mg crystallized complex in 2 ml PBS every day.

3. High dose group: each C57BL/6 mouse fed with 40 mg crystallized complex in 2 ml PBS every day.

Injection with Lewis Lung Carcinoma cell: 4 x 106/ml After feeding Group 1,2, 3 from the first day through the 35th day, the respective tumor size and weight of mice are observed as follows: a. Average weight (g): There is showing no appreciable weight variation even fed with high dose.

Control low dose group high dose group 1"day 19.5 20 18.6 7'"day 19 17 18.5 14th day 20.1 20.5 20.1 21"day 20.4 21.1 20.3 28th day 20.3 20.8 20.1 35th day 20.2 21.0 20.4 b. Average tumor size (mm3) : Control low dose group high dose group 1"day 0 0 0 7th 90 44 30 14th day 190 107 57 21th day 539 388 190 28th day 880 557 208 35th day 1545 685 188 The tumor size vs. days after dosage is shown in Fig. 5. Especially reviewing the high dose curve (with logenze dots), the period after 21 days through 28 days and 35 days indicates a flat (not sharp upwardly) growth rate, proving the remarkable tumor inhibition as effected by present invention.

Toxicity Test 10 rats (5 male, 5 female) average 200-250g for each rat, 8 rats fed with 4g crystallized complex in 4 ml normal, 2 rats fed with 4ml normal saline as control group.

Observation of survial ; changes in hair, skin, weight, and toxicity syndrome, provide that administration of composition of the present invention, has no toxic effects. Table 1 shows the result.

Table 1 WBC RBC Hb platelet Hair& Weight Toxic (xlO'/, 1) (xlO3/ß 1) (g/dl) (x103/ß 1) skin (g) syndrome (1) the 1th day Average 4.25 8. 68 11. 15 646. 3 Normal 235 None Control group average 4.65 6 12.45 542 Normal 235.5 None (2) the 14th day Average 4.85 6.3 12.1 786. 6 Normal 239 None Control group average 4.85 6. 15 17. 45 675 Normal 235 None (3) the 28th day Average 5.75 6.5 12.3 637. 5 Normal 240 None Control group average 4.5 6.3 15.25 516 Normal 235. 5 None (4) the 90th day Average 6.24 6.59 11.71 665.12 Normal 242. 6 None Control group average 4.5 6.55 14.75 485.5 Normal 235.5 None

Accordingly, the present invention has shown its effect for inhibiting tumor, but without any toxicity.

Industrial Applicability: The present invention is prepared from a plant extract for treating cancer, causing no side effect as commonly resulted from chemotherapy or radiation therapy.

The present invention may be modified without departing from the spirit and scop of the present invention.

The basic composition may be optionally adjusted for obtaining a proper conjugating proportion range as follows: Canavalia ensiformis extracted protein..................... 60% to 98% (by weight); Metallic-ion containing compound.............................. 2% to 10% (by weight).

Other excipients or carriers may be further added into the basic composition as above-mentioned.