WO2010028075A1

WO2010028075A1 - Herbal composition for treating cancer

Info

Publication number: WO2010028075A1
Application number: PCT/US2009/055770
Authority: WO
Inventors: Edith Shih Lu; Joan Young
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-09-03
Filing date: 2009-09-02
Publication date: 2010-03-11
Anticipated expiration: 2011-03-03
Also published as: DE202008011721U1

Abstract

The present invention provides an herbal composition which comprising a mixture of extracts obtain from Radix Ginseng, ganoderma lucidum, Cordyceps sinensis, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Glycyrrhiza uralensis, Hedyotis diffusa, Agastache rugosa and Prunella vulgaris. The present invention of the composition harbors anticancer effect. The present invention also provides a method of preparation an herbal composition and a method of treating cancer in a subject by administration the composition of the present invention.

Description

HERBAL COMPOSITION FOR TREATING CANCER

Field of the Invention

The present invention relates to an herbal composition and a method for treating cancer g The present invention also related to a method of preparing the composition of the present invention.

Description of the Prior Art

According to Chinese (herbal) medicine, Ginseng is reputed to effective in shock, collapse of the cardiovascular system, hemorrhage, and heart failure. Ginseng is used clinically in China, Korea and Japan for various diseases, including atherosclerosis, liver dysfunction, cerebrovascular diseases, hypertension and post-menopausal disorder. Clinical trails on volunteers show that ginseng extract can substantially slow down the heart rate and reduce oxygen demand. Ginseng is characterized by the presence of ginsenosides and the extracts rich in ginsenosides have been found to facilitate learning and memory, delay the ageing process prevent neuronal loss under hypoxia, improve the muscle oxygen utilization and physical performance in human. Ginsenosides are normally fractioned into two groups based on the types of aglycone, namely the panaxadiol group (e.g., RbI and Rc) and panaxatriol group (e.g., RgI and Re). Ginsenoside RbI and Rb2 are reported to inhibit angiogenesis (KW Leung, et. al, Br J Pharmacol. 2007, 152, 207-215), whereas ginsenoside Rb2 improved wound healing by enhancing epidermal cell proliferation (Choi, S., Arch. Pharm. Res. 2002, 25, 71 - 77.; Sato, K., et. al., Biol. Pharm. Bull. 1994, 17, 635 - 639). However, whereas some studies reported that Ginseng enhanced cardiac function, others claimed little or depressed cardiac function (Glenda I Scott, et. al., Br J Pharmacol. 2001 November; 134(6): 1159-1165.; Chang-Xi Bai, et. al., Br J Pharmacol. 2004, 142, 567-575). There are perplexing contradictions in the reported effects of various ginsenosides which derived from ginseng. The mechanisms of action of ginseng also remain largely unclear.

Ganoderma lucidum is an important medicinal fungus belonging to the Ganodermataceae family that has been studied for its health promoting properties, including anti-tumor, anti-inflammatory, and anti-platelet aggregation (Wang SY et. al., Int J Cancer. 1997 Mar 17;70(6):699-705; Vetvicka V et. al., J Immunol. 1996 JuI l;98(l):50-61). Cordyceps is prescribed for the treatment of a host of disorders, including hyposexualities, hyperglycemia, hyperlipidemia, asthenia after illness, respiratory diseases, renal disorders, liver and heart diseases (Lo, Hui-Chen et. al., Life Sciences. 2004 April 23;74(23):2897-2908; Shahed, A.R., et. al., Transplantation Proceedings 2001 pp. 2986-2987).

The Hedyotidis Diffusae belongs to the family of Rubiaceae. The entire plant is used as an herbal medicinal component. In Chinese Herbal medicine, diffuse hedyotidis clears heat and resolves dampness by promoting urination. It is useful for relieving hot painful urinary dysfunction and damp-heat jaundice.

With the development of Chinese herbal medicine, more studies have been under taken to investigate the effect of ginseng. The anticancer effect of ginseng has also been discovered. Ginsenosides were recognized as active anticancer compounds(Helms S., Altern Med Rev 2004;9:259-74). Wang CZ discovered that ginsenoside Rg3 had the most potent effect among five representative ginsenosides, RbI, Rd, Re, Rg2 and Rg3. The antiproliferative activities of red American ginseng were increased when ginsenoside Rg3 were increased(Wang CZ et. al., Planta Med. 2007 Jun;73(7):669-74).

Cancer is a malignant tumor occurred by the disorder in cell cycling resulting in abnormal differentiation. Cancer effects people at all age and result in high dead rate. Therefore, there are lots of researches under taken. The choices of the therapies and chemotherapic agents are depended on different situation such as the location of the tumor, the grade of tumor and the stage of the tumor. The chemotherapic agents limit to use because of their various adverse effect such as occurrence of intolerance to specific anticancer agent, stomach disorder, hair loss in spite of their potent anticancer activity. Therefore, developing better therapies and anticancer agents are necessity.

SUMMARY OF THE INVENTION

The present invention provides an herbal composition for treating cancer comprising a mixture of extracts obtain from Radix Ginseng, ganoderma lucidum, Cordyceps sinensis, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Glycyrrhiza uralensis, Hedyotis diffusa, Agastache rugosa and Prunella vulgaris.

The present invention also provides the method of preparing herbal composition, comprising:

(a) soaking and shaking at least on herb in an acid solution;

(b) extracting the acetic acid solution which contains herbs with an organic solvent;

(c) extracting with ethanol, H2O and an organic aprotic solvent; (d) concentrate under vacuum;

(e) extracting the extract with an etherol solvent to remove fatty components and extracting with ethanol; and

(f) filtrating the extract and concentrating under vacuum.

The present invention also provides a method of treating cancer in a subject by administration the composition of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the HPLC analysis result. The ingredients is eluted at different eluting times.

FIG. 2 shows the tumor weight taken out from the mice administration with the saline solution or the composition of the present invention.

FIG. 3 shows the tumor weight taken out from the mice administrated with the saline solution or the composition of US 6,458,361.

FIG. 4 is shows the alteration in body weight of mice in different groups administrated with the saline solution or the composition of the present invention.

FIG. 5 shows the alteration in body weight of mice in different groups administrated with the saline solution or the composition of US 6,458,361.

FIG. 6 shows the cell toxicity of the composition on HeLa cells. The cells are treated with different concentration of the composition for 48 hours and then performed with the MTT assay. The absorbance at 570 nm was measured.

FIG. 7 shows the cell morphology of HeLa cells after the treatment of the composition. The cells are treated with 0.05%, 0.1%, 0.2% or 0.4% the composition for 48 hours and then observed with microscope under 200X.

FIG. 8 shows the cell toxicity of the composition on H441GL cells. The cells are treated with different concentration of the composition for 48 hours and then performed with the MTT assay. The absorbance at 570 nm is measured. FIG. 9 shows the cell morphology and the cell number of H441GL cell after the treatment of the composition. (A)The cells are treated with 0.1%, 0.15% or 0.3% the composition for 48 hours and then observed with microscope under 200X. (B) The cells are harvested and counted after the treatment.

FIG. 10 shows the cell cycle of the treated H441GL cells. The cells are treated with 0.1%, 0.15, 0.3% or without the composition for 48 hours and then harvested. After harvesting, the cells are washed with PBS buffer solution and stained with propidium iodide (PI, 50 mg/mL). The stained cells were examined with flow cytometer, FACS, Fluorescence Activated Cell Sorter, for the cell cycle. The raw data of flow cytometry is shown in (A). The result of (A) further illustrates in table (B).

FIG. 11 shows the treated H441GL cells stained with Annexin V. The H441GL cells are treated with 0.15%, 0.3%, 0.6% or without the composition and then harvested. After harvesting, the cells are washed with PBS buffer solution and stained with Annexin V. The stained cells are examined with flow cytometer, FACS. The raw data of flow cytometry is shown in (A). The combined result of four groups is shown in B.

FIG. 12 shows the DNA fragmentation of the treated H441GL cells. The cells are treated with 0.1% the composition for 48 hours and harvested. Extract the genomic DNA of the harvested cells and analysis with electrophoresis.

FIG. 13 shows the H441GL cells infected with retrovirus carried GFP and firefly luciferase. (A)The transfected cells are observed under microscope. (B)The cells expressed GFP are observed under fluorescence microscope. (C)The intensity of the fluorescence is digitalized and illustrated in table.

FIG. 14 shows the growth of tumor in the implanted mouse. The lxl0⁵/200μL H441GL cells expressed GFP and firefly luciferase are subcutaneous injected into mouse. The growth of cells is observed with in vivo imaging system at day 0, 7, 14, 21 and 28.

FIG. 15 shows the antitumor effect of the composition in the implanted mouse. The mice are subcutaneous injected with 1x10⁵ H441GL cells and fed with ddH₂O or 100 μl the composition after 7 days from the injection of tumor cells. (A)The tumor growths of tumor cells are observed with in vivo imaging system at day 0, 7, 14, 21 and 25. (B)The software program ROI, Region of Interest, is used for obtaining more accurate luminescence intensity. The results of the groups of mice are shown in two trend curves.

FIG. 16 shows the antitumor effect of the composition at different treatment in the implanted mouse. The mice are subcutaneous injected with 1x10⁵ H441GL cells and divided into three groups. The mice in Experiment A are fed with ddF^O after injecting the tumor cells. The mice in Experiment C are fed with 100 μl the composition for 7 days and then injected the tumor cells. After the injection, the mice are fed with the composition. The mice in Experiment D are fed with 100 μl the composition after injecting the tumor cells. (A)The tumor growths of tumor cells are observed with in vivo imaging system at day 0, 7, 14 and 21. (B)The software program ROI, Region of Interest, is used for obtaining more accurate luminescence intensity. The results of the groups of mice are shown in threee trend curves.

FIG. 17 shows the growth of tumor cells in mice. The mice are subcutaneous injected with IxIO⁵ H441GL cells and divided into four groups which are as follows: Experiments A, mice are fed with water for twice a day after the injection of lung cancer cells, Experiment B, mice are fed with lOOμl the composition, Experiment C, the mice are pretreated with lOOμl the composition for one week before injection of lung cancer cells and Experiment D, the mice are fed with lOOμl the composition. (A)The tumor growths of tumor cells are observed with in vivo imaging system at day 14, 21 and 24. (B)The tumor size in mice are shown and measured by ruler.

FIG. 18 shows the body weight of the mice under different treatment which are as follows: Experiments A, mice are fed with water for twice a day after the injection of lung cancer cells, Experiment B, mice are fed with lOOμl the composition, Experiment C, the mice are pretreated with lOOμl the composition for one week before injection of lung cancer cells and Experiment D, the mice are fed with lOOμl the composition.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides the herbal composition comprising extracts from ginseng, ganoderma lucidum, Cordyceps sinensis, Hedyotis diffusa, Agastache rugosa, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Glycyrrhiza uralensis and Prunella vulgaris.

The composition of the present invention has a percentage by weight of ingredients is as follows:

7-17 % by weight of ginseng, 35-45 % by weight of Cordyceps sinensis, 10-20 % by weight of ganoderma lucidum, 5-15 % by weight of Hedyotis diffusa, 1-10 % by weight of Agastache rugosa, 1-10 % by weight of Codonopsis pilosula, 1-10 % by weight of Lycium barbarum, 0-7 % by weight of Ligustrum lucidum, and 0-7 % by weight of Glycyrrhiza uralensis and 0-7 % by weight of Prunella vulgaris.

The present invention also relates to a preparation method of an herbal composition. The herbal composition of the present invention is prepared by the following steps:

(1) dry the plant and the plant are soaking and shaking in an acid solution;

(2) extract the acetic acid solution which contains herbs with an organic solvent;

(3) extract with alcohol, H₂O and an organic aprotic solvent;

(4) concentrate under vacuum;

(5) extract the extract with an etherol solvent to remove fatty components and extracting with ethanol;

(6) filter the extract and concentrat under vacuum.

In a preferred embodiment, the acid in the step (1) is organic acid. The organic acid includes, but is not limited to: acetic acid, malic acid, maleic acid, fumaric acid, lactic acid, tartaric acid, citric acid, formic acid, gluconic acid, succinic acid, piruvic acid, oxalic acid, oxaloacetic acid, trifluoroacetic acid, benzoic acid, methanesulphonic acid, ethanesulphonic acid, benzenesulphonic acid, p-toluensulphonic acid, methanesulphonic acid and isethionic acid. Preferably, the organic acid is acetic acid.

The alcohol in step (3) refers to any Ci-Cβ alcohol, for example, methanol, ethanol, butanol or propanol.

An organic aprotic solvent is an organic solvent which does not act as a proton donor or acceptor. The organic aprotic solvent includes, but is not limited to: dimethylformamide (DMF), dimethylsulfoxide(DMSO), N-methylpyrrolidinone(NMP), acetonitrile, ethyl acetate, diethyl ether, diisopropyl ether, tert-butyl methyl ether, dioxane, tetrahydrofuran and acetone.

In a preferred embodiment, the organic aprotic solvent is dimethylsulfoxide (DMSO). In a preferred embodiment, the etherol solvent is petroleum ether.

In a preferred embodiment, the alcohol is n-butanol.

In the present composition, the ration of n-butanol, H₂O and DMSO in step (c) is 2: 1 : 1 to 1 : 1 : 2, preferably 1: 1 : 1.

The present composition shows anticancer property. The anticancer effect of the present invention is examined in vivo model with different dosage of administration. After administration of the composition, the growths of tumors in mice are inhibited.

The composition further comprises within the food or beverage or could be in the form of solution or tablet.

The present invention also provides a method of treating cancer by administration of the composition. After administering the composition, the growth of tumor is inhibited. The present invention of treating cancer further comprises one anticancer treatment selected from the group consisting of radiation therapy, chemotherapy, surgery, immunotherapy and photodynamic therapy. The combination of the present invention with other therapy can result in inhibiting the growth of cancer and better therapeutic effect.

In a preferred embodiment, the cancer is breast or lung cancer. The mice are injected with cancer cells and then orally administered with the composition. The growth of tumor in the mice administered the composition grow slower than the growth of tumor in the mice administered saline solution or water. Therefore, the composition inhibits the growth of tumor. Furthermore, the GO-Gl phase of the treated cells is decreased and the sub-Gl phase is slightly increased after the administration of the composition.

EXAMPLE

The examples below are non-limiting and are merely representative of various aspects and features of the present invention.

Example 1

Preparation of the composition of the present invention As shown in Figure 1, the composition of the present invention was prepared as follows:

(a) dried and whole plant of different herbals were treated with acetic acid solution soaking, shaking and extracts were obtained with 75% ethanol. The concentrated extract under vacuum was further extracted with n-butanol and H₂O and DMSO at a ratio of 1 : 1 : 1 and then concentrated under vacuum;

(b) mixture solution from Hedyotis diffusa, Radix Ginseng, Cordyceps sinensis, Ganoderma lucidum, Codonopsis pilosula, Agastache rugosa, Lycium barbarum, Glycyrrhiza uralensis, Ligustrum lucidum and Prunella vulgaris were treated with petroleum ether to remove fatty components and extracted with 75% ethanol;

(c) filtration the ethanol solution and concentrated under vacuum; and

(d) honey was added to the concentrated solution (5w/v: 95w/v) and then obtained the present composition, Tien Hsien Liquid PLUS.

The percentage by weight of herbs was as follows:

7-17 % by weight of ginseng, 35-45 % by weight of Cordyceps sinensis, 10-20 % by weight of ganoderma lucidum, 1-10 % by weight of Agastache rugosa, 1-10 % by weight of Codonopsis pilosula, 1-10 % by weight of Lycium barbarum, 0-7 % by weight of Ligustrum lucidum, and 0-7 % by weight of Glycyrrhiza uralensis, 5-15 % by weight of Hedyotis diffusa, and 0-7 % by weight of Prunella vulgaris.

HPLC analysis

Mobile phase • Transferring 1.2 ml of H₃PO₄ to a 1 L volumetric flask, diluting with de-ionized water to volume, and mixing. Sample preparation comprising the steps:

(a) transferring 0.5 ml extracted composition into moisture analyzer to calculate dry weight;

(b) transferring 1 ml extracted composition to a condense bottle and condense to dry, prepare in triplicate. Dissolve the condensate with adequate amount of water, 50% methanol and methanol to make a solution with a concentration of 0.1 g/mL;

(c) placing the solution from step 2 in an ultrasonic vibrator and vibrate at room temperature for 30 mins;

(d) centrifugeing the solution from step 3 at 10000 rpm for 30 mins; and

(e) filtering the supernatants obtained in step 4 through a 0.45 μm syringe filter to make water, 50% methanol and methanol samples and taking samples for HPLC analysis.

Samples were dissolved with 3 different solvents, as shown in Figure 2, the HPLC analysis results showed that the sample dissolved with water was different from the other two samples after retention time at 40 mins.

Example 2

Examination of anticancer effect on breast

The composition were test for their ability to inhibit the growth of the breast adenocarcinoma cancer cell lines in Slc:BDFi mice. The 9 weeks Slc:BDFi mice were inoculated subcutaneously in the abdominal region with breast adenocarcinoma 755 cells and administrated of the composition one day after the inoculation. The administration of a composition of the present invention was preceded to 14 days. The composition was orally administrated once a day at the dose of 0.04, 0.1 or 0.2 cc/day/mouse. The concentration of the present composition was 30 mg/ml. The mice were weighted before administration of the composition and after 14 days of the administration and compared the body weight of different groups and different time (as Table 1 and Figure 3). The mice were sacrificed after 15 days of transplantation and/or after 14 days of administration of the composition and the tumor were taken out and weighted (as Table 2 and Figure 4). The composition disclosed in the US 6,458,361 was also tested and in comparison with the composition of the present invention.

The experiment was carried out on the following 3 groups: (a) the control group administration with saline solution, (b) administration with the present invention and (c) administration of the composition disclosed in the US 6,458,361. Each group above was divided to two or three subgroups with different oral dosage of the compositions and each subgroups contained 8 mice.

Table 1

Group 1:

Administration of saline solution with 0.2 ml/Day/mouse

Group 2:

Administration of the composition of present invention

0.04 ml/Day/mouse

Group 3:

Administration of composition in the US 6,458,361

0.02 ml/Day/mouse

Table 2

The tumor weight (g) after administration of the composition

The tumor weight (g) after administration of the composition in US 6,458,361

The data shown in Figures 3 and 4 indicated that there were not significant different in the body weights of mice between experiment group and control group and none of mice died during the experiment. The result of the anticancer effect of the compositions was shown in Figure 5. The average tumor weight of control group was 1555 mg and the average tumor weights of the present invention were respectively 1314, 580, 249 mg in each dosage 0.04, 0.1, 0.2 ml/day/mouse. The anticancer effect of the composition disclosed in the US 6,458,361 was shown in Figure 6 that the tumor weights were respectively 1435, 982 mg in each dosage 0.02, 0.06 ml/day/mouse. The average tumor weight of mice between experiment group and control group was not significant different.

The values of 100-T/C (%) represent the tumor growth inhibition rates and the T/C defined as the relative size of treated and control tumors in different group of mice.

The tumor growth inhibition rates of this present invention were 15.5, 62.7, 84 % in each dosage 0.04, 0.1, 0.2 ml/day/mouse and particular inhibition were shown in the dosage of 0.1, 0.2 ml/day/mouse (as Table 3). The tumor growth inhibition rates of the composition in US 6,458,361 were 7.7 and 36.8 % in each dosage 0.02 and 0.06 ml/day/mouse and showed no statistic significant (as Table 4).

Table 3 The tumor tissue wei ht and the tumor rowth inhibition rates

The P values <0.01 was significant. ** p<0.01, under Tukey test method compare with groups.

Table 4 The tumor tissue weight and the tumor growth inhibition rates

Under Tukey test method compared groups showed no significant difference between groups.

While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention.

Example 3

Examination of anticancer effect on lung cancer

Cell culture

CGLl cell line and cervical cancer cell line, HeLa cell and HeLa-DsRed/Rluc cell, were cultured with minimum essential medium contained 10% of heat- inactivated calf serum and

Ix antibiotic in 10 cm petri dish. Lung cancer H441 cell was cultured with RPMI- 1640 contained 10% heat- inactivated fetal bovine serum and 1% NEAA (non-essential amino acid) in 10 cm² petri dish. All the cell lines were cultured in 5% CO₂ incubator at 37°C .

The medium were removed when the cell were grow to 90% confluency. The cells were washed twice with PBS. The ImI of 37°C pre-warmed 0.125% Trypsin-EDTA was added to the petri dish and incubated in incubator for 2-5 minutes. Then, additional medium was added to stop the reaction and mix the cells. 5xlO⁵ to IxIO⁶ cells were taken and add appropriate amount of medium to the new dish. The cell numbers were counted by ZI coulter counter, Beckman coulter. MTT assay for cell toxicity test

3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) was a water-soluble tetrazolium salt and formed yellow liquid when it dissolved in PBS buffering solution. The dehydrogenase in the mitochondrion could cleave the tetrazolium ring in the MTT and turned the MTT into water-insoluble blue-purple formazan. The solution which dissolves the formazan crystal, for example 10% SDS solution was added to water- insoluble formazan. The production of MTT formazan was examined by measuring the absorbance at 570 nm. The production of the formazan was higher when the activity of hydrogenase in cell is higher and thus, the value of absorbance is higher. The cell viabilities after treatment were obtained by comparing the experiment and control groups. 5 x 10³ cells were seeded on 96-well plate and the composition was added after culturing for 24 hours. The cell toxicities were examined by MTT assay after 48 hours.

HeLa cell MTT assay

The HeLa cells were treated with the composition of the present invention with two different preparations. The composition was boiled before centrifuging or was directly centrifuged without boiling. The antiproliferative activity of the composition on HeLa cell was examined and the inhibitory concentration of 50% cell viability (IC50) for the composition was determined by MTT assay. The cell viability of the treated cells was shown as Fig 6. The IC50 was about 0.2 % the composition (v/v) of present invention. There was no significant difference between two preparations in the cell viability. The examples hereafter used the concentration basis on this concentration.

Cell morphology

HeLa cells were treated with 0.05%, 0.1%, 0.2% and 0.4% the present composition and the morphology of the cells were observed after the treatment of 48 hours with microscope. The morphology was shown as Fig 7. The cell toxicity of the composition increased with the concentration of the composition. HeLa cell appeared less attach to the dish under higher concentration of the composition. Therefore, the present invention had significant toxicity on HeLa cell.

H44 IGL cell MTT assay The experiment was performed as HeLa cell. The result of MTT assay was shown as Fig 8. The IC50 was about 0.15 % the composition (v/v) of present invention. The cell viability decreased with the increasing concentration of the present invention. When the concentration reached 0.6%, the cell viability approached zero. The result indicated that the composition had high toxicity on lung cancer cells under low concentration.

Cell morphology

H441GL cells were treated with 0.1%, 0.15% and 0.3% the present composition and the morphology of the cells were observed after the treatment of 48 hours with microscope under 200X. The morphology was shown as Fig. 9A. The cell toxicity of the composition increased with the concentration of the composition. The cells were further collected and counted. The numbers of the cells treated with different concentration of the present composition were shown as Fig. 9B. The cell number was decreased with the concentration of the present composition.

Cell cycle examination Flow cytometry

In order to further examine the toxicity effect on cells, the cells were stained with PI and examined with flow cytometry to determined whether the composition affect the cell cycle and which phase of the cell cycle the composition affect. The concentration of the composition was according to the result of MTT assay which the IC50 was 0.15% the composition, which the concentration of the present compositioin was 30 mg/ml. H441GL cells were treated with 0, 0.1%, 0.15% and 0.3% the present composition, and stained with PI. The result showed that the peak of absorbance was shifted to right after the treatment of the composition (Fig. 10A). Further digitalize the selected area, the result were showed as Fig. HB. The composition affected the GO-Gl phase of H441GL cells more obviously. The GO-Gl phase of the treated cells was decreased with the raising concentration of the composition. The sub-Gl phase was slightly increased with the raising concentration of the composition.

Apoptosis examination of treated cells

Annexin V

As the result showed above, the cell cycle of H441 GL cells was affected by the composition.

The change of sub-Gl phase was particular notified for increased with concentration of the composition. The sub-Gl phase was an indication of cell apoptosis in research nowadays.

Therefore, double stained with PI and Annexin V were performed and observed. Annexin V was a dye and had a high affinity for cell membrane phosphatidylserine (PS) which apoptotic cells translocated the membrane phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. When the cells under went apoptotic pathway, the translocation of membrane phosphatidylserine (PS) was taken place. The result of the assay was shown as Fig. 11. The result showed that the fluorescence of Annexin V was increased with the concentration of the composition, in Fig HA. Fig HB was the results of the different concentrations and obviously showed that the peak was shift to right. However, the peak showed irregular pattern when the concentration of the composition reached to 0.6%. It's supposed that the cell toxicity was too high which result in cell broken into too many pieces and thus resulted in such result.

DNA fragmentation

In order to further confirmation of apoptosis, DNA fragmentation was examined. The H441GL cells were treated with 1 % the composition or without. The genomic DNA of the cells were extracted and analyzed with electrophoresis (Fig. 12). As shown in Fig. 12, the genomic DNA of the treated cells broke into many pieces, which was a character of apoptosis. This result was consistent with the result of flow cytometry.

Transfection

The H441GL cells were infected with retrovirus which expressed GFP and firefly luciferase vector. The cells were cultured and observed under microscope. The cells stably expressed green fluorescence were obtained through selection. As shown in Fig. 13 C, the cells expressed higher fluorescent intensity than control H441GL cells.

The antitumor effect on mice

Animal model

The Non Obese Diabetis Severe combined immunodeficient, NOD-SCID, mice with

C.B.17/ICR background were used for examination.The 6- to 8- week old NOD-SCID mice were purchased from the Experimental Animal Center in National Taiwan University or Tzu

Chi University.

The tumor cells, 10⁶ cells in 0.2ml,were subcutaneous injected into the hind limb of mouse.

The mice were observed with in vivo image system, IVIS 200, every three days. The weight of mice and the growth of tumor were examined. The animal behavior was also observed. The volume of tumor was calculated by the formula of LW2/2. Preliminary test

The H441GL cells expressed green fluorescence were obtained and 1x10⁵ cells were subcutaneous injected into mouse. The tumor size was observed by in vivo imaging at day 0, 7, 14 and 28. As Fig. 14 shown, although there was no tumor observed by naked eye at the first day of implantation, the luminescence signals of the cells were detected by in vivo imaging. The tumor grew larger with days and the luminescence intensity was also increased. The luminescence intensity was high at the 28th day.

Animal model experiment I

The 1x105 H441GL cells were subcutaneous injected into mouse. The mice were randomly divided into two groups after confirmation of luminescence signal under imaging at seventh day. The groups were experiment A which the mouse fed with distilled water and experiment B which fed with 100 μl the composition of the present invention. The concentration of the present compositioin was 30 mg/ml. As showed in Fig. 15, the tumor formation in mouse fed with the composition was smaller than that in mouse fed with distilled water. The tumor was increased with time and the signal was also obviously increased.

In order to further accurately analysis the luminescence signal, the analysis program ROI, Region of Interest, was used for further analysis. The result was shown in Fig. 15B. The tumor in mouse fed with distilled water was obviously increased with time. However, the tumor in the mouse fed with the composition, was not obviously increased and the trend curve kept flat. The observation was proceeded for 18 days. The examination result preliminary indicated that the composition inhibited the tumor formation.

Animal model experiment II

In the second animal model experiment, the mouse were divided into four groups and administered orally with distilled water or the composition right after injecting H440GL cells. The four groups were as follows: Experiments A, mice were fed with water for twice a day after the injection of lung cancer cells, Experiment B, mice were fed with lOOμl the composition, Experiment C, the mice were pretreated with lOOμl the composition for one week before injection of lung cancer cells and Experiment D, the mice were fed with lOOμl the composition. There were five mice in each group. After the injection of H440GL cells, the luminescence signal was captured by noninvation in-vivo imaging system every week. When the tumor formation was not observed by naked eye, the implantation of tumor cell into subcutaneous hind limb of mouse was confirmed by the in-vivo imaging system.

As shown in Fig 16A, the luminescence signal in Experiment A increased with days. The result of Experiment C showed that pretreatment of the composition didn't significantly influence the growth of cancer cells. In the Experiment D, the luminescence signal in two mice were significantly decreased with days, even was too weak to detect. The signal of one mouse in Experiment D was successfully detected at the site of injection after the injection at 0 day. The signals were disappeared at seventh and fourteenth days and were weakly detected at twenty-oneth day. The result indicated that the composition had toxicity effect on tumor cells and made the cancer cell number decreased which resulted in delaying the growth of tumor.

In addition to the in vivo imaging analysis, the tumor formation was measured with ruler. As shown in Fig. 17, the trend curve illustrated that the tumor volume in control group, Experiment A, was significant larger than the tumor volume in Experiment D.

The tumor volume in pretreatment group, Experiment C, was not significant different from that in the control group. The results were further analysis with software program, ROI, Region of Interest. The analyzed results were showed in three trend curves. As shown in Fig. 16B, the trend curve of the group fed with the present composition was obviously flatter than that of the control group. The values of the Experiment D were also lower. The body weight of the mouse were also examined (Fig. 18).

Claims

What is claimed is

1. A composition comprising a mixture of extracts obtain from Radix Ginseng, ganoderma lucidum, Cordyceps sinensis, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Glycyrrhiza uralensis, Hedyotis diffusa, Agastache rugosa and Prunella vulgaris.

2. The composition of claim 1, wherein the extracts of the composition are organic solvent extracts.

3. The composition of claim 2, wherein the organic solvent is methanol, ethanol, acetic acid, ketone, hexane, propanol, isopropanol or chloroform.

4. The composition of claim 1, which further comprises honey.

5. The composition of claim 4, wherein the composition contains 1 — 10% by weight of honey.

6. The composition of claim 1, wherein the extracts are obtained from the whole plants of Radix Ginseng, ganoderma lucidum, Cordyceps sinensis, Codonopsis pilosula, Lycium barbarum, Ligustrum lucidum, Glycyrrhiza uralensis, Hedyotis diffusa, Agastache rugosa and Prunella vulgaris.

7. The composition of claiml, wherein the percentage by weight of ingredients is as follows: 7-17 % by weight of ginseng, 35-45 % by weight of Cordyceps sinensis, 10-20 % by weight of ganoderma lucidum, 1-10 % by weight of Agastache rugosa, 1-10 % by weight of Codonopsis pilosula, 1-10 % by weight of Lycium barbarum, 0-7 % by weight of Ligustrum lucidum, and 0-7 % by weight of Glycyrrhiza uralensis, 5-15 % by weight of Hedyotis diffusa, and 0-7 % by weight of Prunella vulgaris.

8. The composition of claim 1, which is in a form of a solution, tablet, capsule, power, candy, gel or emulsion.

9. The composition of claim 8, which is within food or beverage.

10. A method of treating a cancer in a subject, comprising administering the subject in need of such treatment an therapeutically effective amount of the composition of claim 1.

11. The method of claim 10, which further comprises one anticancer treatment selected from the group consisting of radiation therapy, chemotherapy, surgery, immunotherapy and photodynamic therapy.

12. The method of claim 10, wherein the cancer is breast cancer or lung cancer.