TW201201821A - Leaf juice of Plectranthus amboinicus for treating cancer and/or tumor - Google Patents

Abstract

The present invention relates to a composition for treating tumor comprising an effective amount of leaf juice of Plectranthus amboinicus. The present invention also provides a method for producing the composition.

Description

201201821 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a plant composition for treating cancer and/or tumor. [Prior Art] Plectranthus amboinicus, Plectranth.us amboinicus (Lour.) SpTeng., Coleus amboinicus Lour., Coleus aromaticus Benth., Coleus crassifolius Benth., Plectranthus aromaticus (Benth.) Roxb., Coleus suganda Blanco, Coleus carnosus Hassk., ^Majana amboinica (Lour.) Kuntze) » A plant belonging to the family Lamiaceae and having the following common names: country borage, Cuban oregano (Cuban 〇regan〇) ), Indian borage, Amb〇ini c〇ieus, French thyme, Mexican mint, Spanish thyme, to the hand scent (Ta〇) _sh〇u_hsiang) and soup mint (soup mlnt). It is used as a medicinal plant, wild vegetable and seasoning plant in the tropics (part of Africa, parts of Africa, Southeast Asia, West Indies, Mexico, and recently in the southern United States) for meat, soups, fish and local beer. Condiments, and can be used for vegetables and for washing clothes and hair. In the Far East, people also planted hand fragrances in order to obtain the essential oils of hand-flavored fragrances and use them as ornamental plants. The hand fragrance is also used in traditional therapies. For example, 诖4- + its leaves are changed to juice (sweetened with honey) month b to reduce cough and cold. Another ^ ^ ± force the friction of the blades to help eliminate the nasal congestion. It can be used for external application in the case of 157798.doc 201201821 such as burns, mosquito bites, sputum and edema, or as a wind-driven and anti-asthmatic drug for internal use. In addition, for many decades, the hand scent has been known for its antibacterial and antifungal properties. Recently, it has been proved that 'from the fragrance of the two @ 内脂类, three @ (Cai (4) and oleanolic acid can inhibit the inflammatory reaction of animals by inhibiting the activity of Coy and c〇x 2 which can induce inflammatory reactions. (U.S. Patent Publication Nos. 2002-8-No., No. 2007-007645, No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. No. The present invention provides a composition for treating cancer and/or tumor comprising an effective amount of juice to the hand scented leaf. The present invention simultaneously provides a treatment for cancer and/or tumor A method comprising administering to a patient in need of such treatment a composition of the invention. The invention further provides a method for preparing a composition of the invention, wherein the leaf juice is prepared using a method comprising the following steps: 0) Harvesting (b) washing the leaves obtained in step (a) with distilled water; (c) removing water from the leaves; and (d) obtaining leaf juice from the leaves obtained in step (c). One embodiment of the present invention is a composition for treating cancer and/or tumor comprising an effective amount of molecular weight greater than 50 kD to the hand geranium juice group 157798.doc 201201821 portion.

Accordingly, the present invention also provides a method for the preparation of a composition for treating cancer or tumors, the composition comprising an effective amount of a molecular weight greater than 5 〇 kD to the hand geranium juice component, and the smear molecule 4 is greater than 5 〇 The preparation method of the fragrant leaf juice component of (10) comprises the steps of: (a) harvesting the leaves of the hand scent; (b) washing the leaves obtained in the step (a) with distilled water; (c) removing the water from the leaves; (d) self-step (c) obtaining the leaf juice from the obtained blade; and (e) passing the leaf juice obtained in the step (d) through a filter to remove the component having a molecular weight of less than 50 kD and obtaining a component having a molecular weight of more than 5 〇 kD. [Embodiment] According to the present invention, it has been surprisingly found that hand-scented leaf juice has a significant effect on the treatment of cancer and/or tumor. According to the present invention, there is provided a composition for treating cancer and/or tumor comprising an effective amount of scented leaf juice. The term "leaf juice" as used herein is also referred to as "leaf extract", which refers to the natural juice of 3 in the leaves. Leaf juice can be obtained by removing tissue fragments and/or residues. As used herein, the term "tumor" refers to pathological protuberances, canines or hyperplasia in any part of the body, especially the proliferation formed by new tissue deposits, or the term "malignant tumor" as used herein. Malignant neoplasms "cancerous cancer" is the growth of tumors caused by abnormal and uncontrolled cell division that can spread through the lymphatic system or bloodstream to other parts of the body. 157798.doc 201201821 As used herein, the term "effective amount" refers to an amount which, when administered to an animal, produces the desired effect on the animal. For example, an effective amount of a composition for treating a tumor refers to an amount that can be used to control tumor growth in an animal and/or to remove tumors in an animal. In one embodiment of the invention, the compositions of the invention are used to inhibit the growth of malignant tumors. In another embodiment of the invention, the number of malignant tumor cells, such as HepG2, Huh7 and Bowes, can be reduced or even eliminated when treated with the compositions of the invention. During screening of tumor cell lines that responded to the compositions of the invention, the composition was found to control tumor growth by the P53 network. The composition of the present invention inhibits the growth of tumor cells functional of the ρ53 gene (including wild type, partially functional type, and mutant active form). Therefore, the tumor line treated according to the present invention is a tumor associated with ρ53. The ρ53 network is a molecular detector of the G1 checkpoint in the cell cycle and monitors DNA damage, nucleotide library levels (p〇〇1 丨), mitotic spindle status, and genotoxic stress responses. The p53 network also controls cell cycle progression, cell death programs, periodic senescence, and possible differentiation. Therefore, p53 is considered to be a tumor suppressor gene. In fact, more than half of human cancers are associated with one or more changes in p53. In addition, the mutant p53 protein may have acquired a novel tumor-promoting activity independent of wild-type p53. Methods for the treatment of malignant tumors have been developed for the p53 network (U.S. Patent Nos. 5,382,51, 5,84,579, 6,183, 964, 6,472,385, and 6,531,512). In one embodiment of the invention, the tumor treated comprises hepatocellular carcinoma and black 157798.doc 201201821 cytoma. More preferably, the tumor to be treated includes hepatocellular carcinoma and melanoma associated with the p53 network. The geranium leaf juice of the present invention can be analyzed and identified by high performance liquid chromatography (HPLC). For example, '50 microliters to hand-flavored leaf juice is subjected to high performance liquid chromatography using a ZORBAXTM C18 column with an inner diameter (ID) of 46 mm and a length (L) of 15 cm, and a map is obtained at 2 14 nm. The map includes peaks with residence times of 1.756, 2.573, 7.118, 7.85, 9.615, 10.278, 10.864, Π.212, 12.287, 12.799, 13.178, 13.413, 14.027, 14.794, 16 253, and 18.742 minutes; wherein the leaf juice is used 〇_3〇% of acetonitrile and 〇1% of TFA were separated by linear gradient elution in 30 minutes at a flow rate of 1 ml/min. When the effects of the components of the hand-scented leaf juice were studied, it was found that the component having a molecular weight of more than 5 效果 was more effective than other components in treating cancer and/or tumor. In one embodiment of the invention, a component having a molecular weight greater than 5 〇 kD of 〇〇〇1% exhibits a strong ability to inhibit the growth of hepatocellular carcinoma (HepG2), while other components have a dose of 0.5%. The same effect. The component of the present invention having a molecular weight of more than 5 〇 kD can be analyzed and identified by high performance liquid chromatography. For example, 50 μl of a component having a molecular weight greater than 5 〇 kD is subjected to a chromatographic liquid chromatography using a z〇rbAXtm C18 column having an inner diameter U D.) of 4.6 mm and a length (£) of 15 cm, and at 2 14 A map was obtained at the nanometer, which included retention times of 2.574, 4·798, 5·728, 7.1 (Η, 9.70), 10.279, 10.808, 11 189, 12.235, 13.35, 13.5 84 ' 13.903, respectively. Peaks of 14.113, 15.114, 16.206, 16.736, 18.619, 22.137, 24.676 and 26.902 knives; where the molecular weight greater than 50 kD uses 0-30% acetonitrile 157798.d. 201201821 and 0.1% TFA to implement a linear gradient in 30 minutes The elution was carried out for separation at a flow rate of 1 ml/min. In one embodiment of the invention, the composition further comprises an anti-tumor agent. Because tumor growth involves a complex network interaction, an anti-tumor against different networks is used in combination. The agent can reasonably improve the therapeutic effect. In a preferred embodiment of the invention, the anti-tumor agent is paclitaxel. Pacific paclitaxel (also known as taxol) is a clinical anticancer drug with strong inhibition of G2M late phase. Silky The properties of the cleavage show strong anti-tumor properties by mediating Bci_2 phosphorylation and paclitaxel-induced apoptosis. In addition, it has been found that paclitaxel can block mitosis by binding to microtubules. Role (Lanni s. jennifei·, L()we w

Scott, Licitra J. Edward, Liu Jun Jun, Jacks Tyler P53-independent aopotosis induced by paciltaxel through an indirect mechanism. Proc. Natl. Acad. Sci. USA Vol 94 » September Ϊ997:9979-9683). In one embodiment of the invention, a composition comprising 5% to hand scented leaf juice and 50 nM of paclitaxel has a greater inhibitory effect on tumor growth than a composition comprising only scented leaf juice or paclitaxel. This - the results suggest that cross-talking of the P53 and Bci-xL cell pathways is an effective method for treating cancer and/or tumors. For ease of administration, the compositions of the present invention are formulated into a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" includes any solvent, dispersion medium, isotonicity agent, absorption inhibitor, and the like. These pharmaceutically acceptable carriers can be prepared from a variety of materials including, but not limited to, dilute 157798.doc 201201821 agents, binders and binders, lubricants, colloids, lunar agents, colorants, bulking agents And the preparation of the special > medicinal composition 雹鸯 _ _ _. Other materials needed for the material, such as mild absorbents. The use of such media and agents for the medicinal use of sputum is well known to those skilled in the art. The present invention encompasses that it can be used in the compositions of the present invention unless the active ingredients of any of the commonly used media or reagent disks are not identical. More specifically, the composition of the present invention can be administered to a patient by injection, oral or topical administration in a number of ways. The present invention provides a method of preparing a composition of the present invention, which is prepared by a method comprising the steps of: (a) harvesting a hand-scented leaf; (b) washing the leaf obtained in the step (a) with distilled water; (c) removing the blade Moisture, preferably by natural air drying; and (d) obtaining leaf juice from the leaves obtained in step (c). In the embodiment of the invention, the blades can be processed into small pieces by a number of conventional methods such as rigid grinding, agitation, agitation, cutting or chopping. The leaf juice preparation method may further comprise the step (d2), i.e., the leaf juice obtained in the centrifugation step (d) to remove the tissue fibers, as needed. In one embodiment of the invention, centrifugation is carried out at 15,000 rpm & 24 ° C for 5 minutes each time. The leaf juice preparation method may further include a leaf juice sterilization step as needed. In one embodiment of the invention, the 'leaf juice is sterilized by filtration. The leaf juice preparation method may further include a leaf juice concentration step as needed. The invention also provides a composition for treating cancer and/or tumor comprising an effective amount of a palm berry juice component having a molecular weight greater than 50 kD. 157798.doc 201201821 The present invention also provides a method for preparing a group of people for cancer and/or tumor treatment, the composition comprising " an effective amount of sheep scented leaf juice having a molecular weight greater than 50 kD a component, which is prepared by a method comprising the following steps: a (a) harvesting a hand-scented leaf; (b) using a component of the scented leaf juice containing more than 50 kD The leaves obtained in the step (a) are washed with distilled water; (c) the water on the leaves is removed, (d) the leaves obtained from the step (c) are obtained; and (4) the leaves obtained in the step (4) are passed through the filter to obtain the self-generated The leaf juice removes the component having a molecular weight of less than 50 kD and obtains a component having a molecular weight of more than 5 〇 kD. In addition to the step (e), the method for preparing the fragrant leaf juice component having a molecular weight of more than 50 kD and the above preparation to the scented leaf juice The method is the same. The method of separating the components according to the molecular weight range is well established in the art. One embodiment of the present invention uses Amicon® ultracentrifugal filtration devices having pore sizes of different sizes, which can be determined by molecular weight. To separate the various groups The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention.Example 1: Preparation of hand-scented leaf juice to hand-flavored from the Taiwan Endemic Species Research Institute (Taiwan Endemic Species Research Institute, located in Taiwan) Nantou). After harvesting the leaves of the hand, wash the leaves with distilled water and air dry to remove the residual moisture on the leaves. The leaf tissue is obstructed into fine particles and leaf juice. The leaf juice is at 15 ° C at 15 ° rpm Centrifuge for 5 minutes to concentrate the leaf juice. The supernatant was collected, 157798.doc -10- 201201821 and centrifuged again at 15,000 rpm for 5 minutes at 24 ° C to remove tissue fibers as much as possible. The resulting leaf juice was filtered through a 0.22 μm syringe filter. Keep fresh leaf juice in the dark at 4 ° C. All plant crude extracts should be used within 3 days or stored at -80 ° C for a long time. Perform chromatography on Agilent® 100 Series Liquid Chromatography System Inspection, the system consists of 1100 four sets of pumps (with degasser), 1 〇〇 variable wavelength detector and i 〇〇 示 准 automatic sample collector. Implemented using ChemStation® software Step analysis. A large amount of solvent and mobile phase were transferred using a Mjllip® re8 solvent filtration unit (MUHP® Re®, Bedford, MA) by a 〇22 μm nylon membrane filter (Alltech® Associates, Pty. Ltd). % TFAg fluoroacetic acid) equilibration column, the helium flow rate is 1 ml / min. Inject 5 l of microliters of lx crude sample dissolved in PBS into Z0RBAX®

The matter is separated. Analytes were detected at 214 nm. (: 18 column (4.6 mm inner diameter >< 15 cm), and the column was placed in a column oven at 25 ° C. The injection results are shown in Figure 1. The recorded peaks are shown. The data is shown in Table 1.

157798.doc 201201821 8 11.212 1983.91 1.39 114 65 9 12.287 9357.61 6.54 167 46 10 12.799 7150.80 5.00 351 98 11 13.178 11427.11 7.99 723 62 12 13.413 6133.80 4.29 324 05 13 14.027~ 3307.24 2.31 111 19 14 14.794 2816.79 1.97 107 32 15 16.253 5357.76 3.74 187 43 16 18.742 5725.88 4.00 121.51 Example 2: Screening cell strains of cell lines that respond to leaf juice. Unless otherwise specified, the following 9 human tumor cell lines of different tissue origin were purchased from commercial cell culture collection centers: food Industrial Development Institute. Hep(}2 and HeP3B cells were cultured in depleted essential medium (with 1 〇./ fetal calf serum, 2 L-glutamine, 50 IU/mL penicillin, and 50 μg/ml streptomycin). In RPMI_1640 U937 and K562 cells were cultured in medium (with 1% fetal calf serum, 2 mM L-prolinamide, 50 IU/mL penicillin, and 50 μg/ml streptomycin). Essential medium in high sugar Dalke ( Calu-Ι and TL cells were cultured with 10% fetal bovine serum, 2 mML-prolinamide, 5〇11; /^^ penicillin and 50 μg/ml streptomycin. The culture was stored at 5%. C〇2, a temperature of 37 ° C and a certain humidity in the ring brother. All cell culture reagents were purchased from Gibco-Invitrogen Co., Ltd. (Grand Island, NYUSA) and HyCl〇ne (L〇gan, Utah, USA) Treatment: In all in vitro tests, '3 % of the leaf juice prepared according to Example 1 was added directly to the subcultured cells. Cell Count: 20 μl of cell suspension was added to 2 μL of microliters 4 In the cone blue, the total cell count was performed by phase contrast microscopy. 157798.doc -12- 201201821 The results are shown in Table 2. Ο representative of "leaf juice has a cellular response (i.e., reducing the number of cells after treatment); the representative" X "of the leaf juice unresponsive cells (i.e., increasing the number of cells after treatment) Table 2:

Cell line derived from p53 Response to leaf juice U937 Bone marrow monocytic leukemia/mesodermal inactive mutant X Κ562 Erythroblast leukemia/mesodermal negative X Calu-1 Lung squamous cell carcinoma/endodermal loss X TL Trophoblastoma/inside Germ-negative X Jurkat human T lymphocyte strain negative X Bowes human melanoma cell fractional active mutant 0 HepG2 hepatocellular carcinoma / ectodermal wild type 0 Huh7 hepatocellular carcinoma / ectodermal activity mutant 0 Hep3B hepatocellular carcinoma / ectoderm zero X According to the results shown in Table 2, the functional cell line of ρ53 gene responded to leaf juice, demonstrating that leaf juice can control tumor growth by ρ53 network. Example 3: Treatment of hepatocellular carcinoma with leaf juice The hepatocellular carcinoma cell lines used in this example were HepG2 and Huh7, and the cell culture and treatment operations were as described in Example 2. The cells were divided into a control group (untreated) and a test group (treated with 1%, 3%, 5%, 7% or 9% of leaf juice, respectively). Each group was repeated 8 times (HepG2) or 4 times (Huh7). Each group of HepG2 cell counts was determined on day 1, day 3, and day 5, respectively, and the results are shown in Figures 2, 3, and 3. As can be seen from Fig. 2 and Fig. 3, the inhibition effect of the leaf juice on the cells was observed on the first day (P<0.001), the third day (Ρ<0·001), and the fifth day (Ρ<0·001). The dose is increased and enhanced with dose dependence. In addition, the inhibition of leaf juice on the first day (suppressed cell count / leaf juice percentage: 1.2 χ 104), day 3 (suppressed cell count / leaf juice percentage: 6.18 χ 104) 157798.doc -13 - 201201821 and day 5 (inhibited cell count / percentage of leaf juice: ι〇44χι〇4) increased with increasing treatment time. Table 3: Group - Cell growth rate (104 cells/day plant disassembly/system effect #4·na in M, control group 27.59 (P<0.001) - for group M compared to J 1% leaf juice 16.75 ( P<0.001) "〇607l~~~~ 3% leaf juice 11.66 (P<0.001) 〇.4226~~^ 5% leaf juice 8.66 (P<0.01) ~〇3139~~--- 7% leaf juice 6.61 (P<0.01) ^2396 9% leaf juice 3.13 (P=0.05) ~ - Each group of Huh7 cell counts was determined on day 1, day 3 and day 5, respectively. Results are shown in Figure 4, Figure 5 and Table 4. As can be seen from Fig. 4 and Fig. 5, the inhibition of leaf juice on cells was on day 1 (P<〇.〇5), day 3 (P<0_001) and R (p<〇〇〇1) Both increased with dose increase, with dose dependence. In addition, the inhibition of leaf juice on the first day (inhibited cell count / leaf juice percentage: 0 _49 X 1 〇 4), day 3 (suppressed Cell count/leaf percentage: 2 47 χ 1 〇 4) and day 5 (inhibited cell count / leaf juice percentage: 3 63 X1 〇 4) increased with increasing treatment time. Table 4: Group cell growth rate (104 cells/day) Inhibition (compared with control group) Control group 8 .44 (P<0.001) 1% leaf juice 6.41 (Ρ<0·01) 0.7595 3% leaf juice 3.09 (Ρ<0_01) 0.3661 5% leaf juice 2.38 (Ρ<0.05) 0.2820~~ 7% leaf juice 2.97 ( Ρ<0.001) 0.3519 - 9% leaf juice 0.19 (Ρ=0_614) 0.0225 Example 4: Treatment of melanoma with leaf juice The melanoma cell line used in this example is B 〇wes, cell culture and processing operations as in the case 2. The cells were divided into control group (untreated) 157798.doc -14-201201821 and test group (treated with 1.0% or 3% leaf juice). Each group was repeated 5 times. On the first day, On the 3rd and 5th day, each group was counted as the cell count, and the results are shown in Fig. 6. As can be seen from Fig. 6, the inhibition of leaf juice on day 1 (Ρ <0.01) and day 5 (ρ<〇·〇 ι) both increased with increasing dose, showing strong dose dependence. In addition, sputum inhibition was observed on day ( (suppressed cell count / leaf juice percentage 2 · 22 X 1 〇), day 3 (Suppressed cell count/leaf percentage: 3.26x10)' and day 5 (inhibited cell count/leaf percentage: 25·8χ104) were reluctant with increasing treatment time. Example 5: Treatment of hepatocellular carcinoma with leaf juice and/or paclitaxel The hepatocellular carcinoma cell line used in this example was as in Yang, and the cell culture and treatment operations were as described in Example 2. The cells were divided into a control group (untreated) and a test group (treated with a mixture of 5% leaf juice, 5 〇 nM paclitaxel, and 5% leaf juice and 5 〇 nM paclitaxel, respectively). Each group was repeated 6 times. The results of each group of HepG2 cells measured on day 3 are shown in Figure 7. As can be seen from Fig. 7, the cell growth of all the test groups was significantly inhibited. The inhibition rates of each test group were 52_13% (5% leaf juice: p < 0 〇 5), 43 77% (5 〇 paclitaxel: Ρ = 0. 052), or 76.57% (5% leaf juice and % nM paclitaxel). a mixture of .P < 0_001). The above results indicate that the combination of the leaf juice of the present invention and paclitaxel has a stronger inhibitory effect on cell growth than leaf juice or paclitaxel alone. The Huh7 cell counts of each group were determined on the third day, and the results are shown in Fig. 8. As can be seen from Fig. 8, the cell growth of all the test groups was significantly inhibited. The inhibition rates of the test groups were 50.21% (5% leaf juice: ρ=〇.ι 1〇), 66 〇9% (5〇nM paclitaxel: P<0.05), and 74.68% (5% leaf juice and 50 nM paclitaxel). Mixture: p 157798.doc 15 201201821 < 0,01). The above results indicate that the combination of the leaf juice of the present invention and paclitaxel has a stronger inhibitory effect on cell growth than leaf juice or paclitaxel alone. Example 6: Animal model for treatment of hepatocellular carcinoma with leaf juice Eight species: Huh7 cells were cultured under the conditions described in Example 2 to obtain a total of 3 x 10 cells. The resulting cells were then suspended in a dm read to give a cell density of 2 x 108 cells/ml. A 5 to 6 week old baby-heart mouse weighing about 20 g was taken and 1-1 ml of the above cell solution was inoculated into the subcutaneous region of the right back of the mouse. Control mice were inoculated with 1 ml of DMEM. Treatment: large tumors (size greater than 1 mm mm χ 1 mm, one mouse), medium tumors (between 7 mm χ 7 mm to 1 mm mm mm '- mice) and small Mice of tumors (size less than 7 mm χ 7 mm, two mice) were treated with leaf juice prepared as described in Example (10), respectively. The leaf juice dose is 10% of the body weight of the mouse. The leaf juice is placed in a syringe, the needle is inserted into the subcutaneous part around the tumor, and the leaf juice is injected into the inside of the tumor. Tumor size was measured weekly, Wednesday, and Friday and weighed small _ continue to be treated with leaf juice. The blank group was injected with 1% pBS. f fruit: The results are shown in Figure 9. After treatment with leaf juice, the area of small tumors was reduced by 2G9 mm 2 /day '(4); the middle tumor was i (R-0.834, =6.19 mm 2 /day, p < G Qi) was inhibited, only 27.1 of the control group %; however, the growth of large tumors (r2 = 〇995, b = 4.38 square millimeters/day, Ρ<〇·〇1) was not inhibited. In addition, all mice that received no mice had decreased body weight (unvaccinated mice: r2=., two '...therapy® y B=-〇.13^/day, P<0_05; mice with small tumors :r2 = 〇,Β = -〇.〇9 έ / day' Ρ=0_051; mice with intermediate tumors: r2 * Β = -〇.ι1^/ 157798.doc •16·201201821 days, P<0_05; A mouse with a large tumor··r2=〇.21〇, Bs-Ou*/day, ρ=0·302). Only mice that were not treated with leaf juice gained weight (R2 = 0.730, Β = 0.24 g/day 'ρ< 〇 〇〇ι) (see Figure 10). Recurrence: The small tumor group stopped taking the drug on the 13th day, but continued to monitor the tumor area. The results are shown in Figure 11. The results showed that the growth rate of small tumors (R2 = 0_558 'Β = 0·72 mm 2 / day, Ρ < 〇〇 5) was only 2.4o/〇 of the control group (R2 = 0_622 ' Β = 30.62 mm 2 / Day, Ρ <〇〇1). In addition, the weight of all mice increased after discontinuation of administration (blank group: R2 = 〇743 'B-0.53 g/day, Ρ<〇·〇〇1; small tumor group: r2=〇〇45, g/day , Ρ <0·615; control group: r2 = 〇 214, B = 〇 ^ g / day, p < 〇〇 5) (see Figure 12) 〇 Example 7. Animal model of melanocytoma treated with leaf juice: The Β〇_ cells were cultured under the conditions described in Example 2 to obtain a total of 3 X 1 cells. The obtained cells were then suspended in the medium so that the fineness reached 2 x 10 cells/ml. The mice were treated with a cell weight of about 2 g at 5 to 6 weeks and the cells were taken from the subcutaneous region of the right back of the mouse. The control mice were inoculated with 0.1 ml of 1)]^^;;^. Treatment: For mice with large tumors (size greater than 1 mm mm χ 1 mm, one mouse) and small tumors (small size less than 〇^10 Χ10 mm, two mice) The leaf juice prepared by the method described in Example 1 was treated. The leaf juice dose is 10/〇 of the mouse body weight. The leaf juice is placed in a syringe, and the needle is inserted into the subcutaneous part around the tumor, and then the leaf juice is injected into the inside of the tumor. On a weekly-, 1-week basis, the tumor size was measured and the mouse body was weighed for $m continued treatment with leaf juice. The blank group was injected with 10% PBS. 157798.doc 201201821 Results: The results are shown in Figure 13. The area of large tumor after treatment with leaf juice (R2=0 924, B=-3 G8 mm 2 /day, 卩<〇.叩 small tumor birth^ (R2 = 0.564 ' Β = 1.50 mm 2 / day) ' p < 〇〇 1 ) was inhibited, only 37.7% of the control group. In addition, except for mice that did not receive leaf juice treatment, the body weight increased significantly (R2 = 0.897, B = 0.09 g / day, p < 1) All mice receiving leaf juice treatment had a slight increase in body weight (see Figure 14). Recurrence: The small tumor group stopped taking the drug on day 24' but continued to monitor the tumor area. The results are shown in Figure 15. The results indicate that the The growth rate of small tumors after administration (R2=0.750, B=4.1 mm 2 /day, p<〇〇〇1) compared with administration (R2=0.564, Β=1.50 mm 2 /day, p<〇〇1 ) Faster than 173%. In addition, the tumor growth rate (R2 = 〇32〇, Β = 5.13 mm 2 / day, P < 0 〇〇 1) of the blank group mice after stopping the PBS injection was more than when injected with pBS ( R2 = 〇6〇8, B=3.98 mm 2 /day, P < 0 〇〇 1) is 29% faster. The small tumor group and the blank group have similar growth rates after stopping the administration. In addition, the leaf juice is stopped. The body weight of all mice increased slightly after treatment, but the weight gain of the blank group (R = 0.315, B = -〇.〇3 g / day, p < 〇〇 5) was significantly higher than that of the small tumor group (see Figure 16). Example 8: Inhibitory effect of leaf juice component on HepG2 cell growth Leaf/ten component. Amicon® ultracentrifugal filter unit (Amicon Ultra PL-10 ' 1〇, 〇〇〇 nominal molecular weight limit (Nmwl) Amicon Ultra PL-30 '30, 〇〇〇NMWL; Amicon Ultra PL-50, 50,000 NMWL, Millipore®) The leaf juice prepared in Example i was divided into the following components: #iComponent (molecular weight <10) K, 25X leaf juice), #2 component (molecular weight < 10 kD, IX leaf juice), #3 component (molecular weight between 10 kD and 30 kD, 25X leaf juice), #4 component 157798. Doc -18- 201201821 (molecular weight between 10 kD and 30 kD, IX leaf juice), #5 component (molecular weight between 30kD and 50kD '25X leaf juice), #6 component (molecular weight between 30 'IX leaf juice between kD and 50kD, #7 component (molecular weight <5()1<1), 25) (leaf juice), #8 component (molecular weight > 50 kD '25X leaf juice), group Serving (molecular weight > 5〇kD, IX leaf juice) . Treatment: The HepG2 cell line was used to screen the leaf juice fraction. Cell line manipulations were as described in Example 2 and cell counts were performed on day 4.

The viability of HepG2 cells on day 4 is shown in Table 5. Table 5: 1% dose 5% dose control group 100 100 IX leaf juice 88 47 25 Χ leaf juice 33 35 #1 component 11 14 #2 component 59 18 #3 component 74 27 #4 component 92 96 # 5 components 57 57 #6 components 90 100 #7 components 98 33 #8 components 80 0 #9 components 13 0 The results indicate '#1, #2, #3, #7, #8 and #9 groups The inhibition of tumor growth has a dose dependency. Moreover, the #2 and #9 components inhibited the growth of HepG2 cells more strongly than the other components. Use IX and 25X leaf juice, 0%, 0.01〇/〇, 〇.1〇/. HepG2 cells were treated with 〇.5〇/0, 1〇/〇 and 3% of #1, #2 and #9 components. Each group is repeated 4 to 7 times. The results showed that 〇5% (P < 0.05), 1% (Ρ < 0·001) and 3% (Ρ <〇·〇〇1) of the #2 component, 〇01% of the #9 component (P<; 0 001), i% leaf juice (ρ < 〇〇 5) 157798.doc • 19- 201201821 and 1% (P < 0.01) and 3% (P < 0_001) 25X leaf juice can significantly inhibit HepG2 growth. On the other hand, 1% of IX leaf juice (Ρ=〇·848) and 1% of #1 component did not have a significant inhibitory effect. This proves that the #9 component has the strongest inhibitory effect, and its effective dose is only 〇1%; the #2 component also has a strong inhibitory effect, and its effective dose is 0.5%; the effective dose of 25 Χ leaf juice is 1%. The inhibition rates of #5%, 1% and 3〇/〇#2 components ' 001% of the ingredients, 3% of IX leaf juice and 1% and 3% of 25 Χ leaf juice were 34.3% and 62.8%, respectively. 98.7%, 39.7. /. , 54.8%, 53.6% and 83.8%. The ratio of the number of inhibited cells to the percentages of the 1 χ leaf juice, 25 χ leaf juice, #1 component, #2 component, and #9 component is shown in Fig. 17. Figure 17 shows that 1Χ(Ρ<0·001,r2 = 〇525) and 25χ(ρ=〇〇〇ι, R =〇·689) leaf juice and #1 component (ρ<0·001, R2=0.333 The #2 component (ρ<ο·οοι 'r2=〇792) and the #9 component (p<〇〇〇1, r2=〇498) significantly inhibited HepG2 growth with dose dependence. The ratio of the percentage of cells inhibited on day 4 to the percentages of IX leaf juice, 25X leaf juice, component, #2 component, and #9 component was 1〇2χ1〇4, 15 7χ1〇4, respectively. 9 22χ1〇4, ΐ8 4χΐ〇4 and 43·7χ1〇4. The inhibitory effect is from large to small as #9Component>#2 component eucalyptus juice> 1x leaf juice>#1 component. In addition, the inhibitory effects of the same doses of iota leaf juice, 25 χ leaf juice, #1 component, #2 component, and #9 component were also analyzed. The results are shown in Fig. 18. When the dose was 0.1% and 0.5 〇/〇, the inhibitory effect of the #9 component was significantly stronger than that of the 25 Χ leaf juice, the #1 component, and the #2 component (both p<〇 〇〇 j). When the dose was 1%, the inhibitory effect of #9 component was significantly stronger than that of χ χ leaf juice (Ρ=0·001), 25 Χ leaf juice, #1 component (ρ=〇〇59), and #2 component...〇 〇 72). 157798.doc -20- 201201821 In addition, the inhibitory effect of #2 component was significantly stronger than that of IX leaf juice (P=0.05). When the dose was 0.001%, the inhibitory effect of the #9 component was significantly stronger than that of the 25X leaf juice (P = 0_063). When the dose was 0.1%, the inhibitory effect of the #9 component was significantly stronger than that of the IX and 25X leaf juice, the #1 component, and the #2 component (both P<0_001). Example 9: Treatment of hepatocellular carcinoma HepG2 with #9 component of leaf juice The #9 component was subjected to chromatographic analysis as described in Example 1. The results of the chromatographic analysis are shown in Figure 19. The data of each peak recorded are shown in Table 6. Table 6: Peak number retention time (minutes) Peak area (mAU) Peak height (mAU) 1 2.574 2729.02 161.45 2 4.798 17088.54 283042 3 5.728 10144.49 174.31 4 7.101 4715.61 137.99 5 9.701 7839.76 282.37 6 10.279 5654.36 251.75 7 10.808 2310.79 108.42 8 11.189 1406.56 81.95 9 12.235 4973.73 92.76 10 13.35 6341.88 177.22 11 13.584 2012.7 156.81 12 13.903 1359.22 88.11 13 14.113 1046.2 84.5 14 15.114 9445.97 105.98 15 16.206 3847.13 114.97 16 16.736 4171.49 77.61 17 18.619 7938.15 112.43 18 22.137 13159.29 139.24 19 24.676 3591.43 35.14 20 26.902 7333.99 27.14 The hepatocellular carcinoma cell line used in this example was HepG2, and the cell culture and processing operations were as described in Example 2. The cells were divided into a control group (untreated) and a test group (as described in Example 8, using IX leaf juice, 0%, 0.001%, 0.01%, 157798.doc -21 - 201201821 0.1%, 0.5% or 1%, respectively). #9Component treatment. Each group was repeated 4 to 8 times. The HepG2 cell counts of each group were measured on the 4th day, and the results are shown in Fig. 2〇. From Fig. 2〇, it can be seen that '1〇/〇(Ρ<0·001 ), 〇.5% (Ρ<0·001), 〇1% (p<〇〇〇1), and 0.01% (P<0_001) of the #9 component have a significant inhibitory effect. In addition, 0.001% (P The #9 component of =0.092) also significantly inhibited cell growth. The inhibition rates of #9, 0.5%, 0.1%, 0.01%, and 〇_001% of #9 components were 93.6%, 94.7%, and 93.8, respectively. %, 39.7%, and 18.5%. The ratio of the number of cells inhibited to the percentage of component #9 was 43.65x1ο4. Example 10: Inhibitory effect of leaf juice component on growth of Huh7 cells The leaf juice component was as in Example 8. Preparation: Treatment of the leaf juice fraction using the Huh7 cell line. Cell line manipulation As described in Example 2, cell counts were performed on day 4.

The survival rate of Huh7 cells on day 4 is shown in Table 7. Table 7: 1% dose 5% dose control group 100 100 IX leaf juice 100 39 25X leaf juice 33 0 #1component 83 39 #2Component 0 — 6 #9Component 13 ^ 0 The result indicates '# 1 The #2 and #9 components inhibit tumor growth and are dose dependent. Further, Huh7 cells were further treated with IX and 25X leaf juice, 〇%, 〇1%, 〇1〇/〇, 0.5%, 1%, and 3% of #1, #2, and #9 components. Each group is repeated 4 to 7 times. 157798.doc -22- 201201821 The results show that '1% (P<0_01) and 3% (p=0.001) of #1 component, 1% (Ρ<0·05) and 3% (P<0.05)# 2 components, 0.01% of #9 components (P=〇.01) and 0.5% (Ρ<0.05), 1% (ρ<〇·〇5) and 3% (p<0 01) of 25X leaf juice Can significantly inhibit the growth of 1111117. On the other hand, 1% (? = 937) and 3 〇 / 〇 (? = 0.666) 1 乂 leaf juice showed no significant inhibitory effect. This proves that the #9 component has the strongest inhibitory effect's effective dose is only 0.01%; 25X leaf juice also has a strong inhibitory effect' effective dose is 0 · 5%; effective dose of #1 and #2 components It is 1 〇/〇. The inhibition rates of 1% and 3% of #1 component, 1% and 3% of #2 component, 〇〇]% of #9 component, and 0.5%, 1%, and 3% of 25X leaf juice were respectively 59 3%, 76 2%, 53.9%, 64.2%, 53.7%, 49.9%, 53.9% and 67,10/0. The ratio of the number of inhibited cells to the percentage of IX leaf juice, 25X leaf juice, #1 component, #2 component, and #9 component is shown in Figure 2 j. Figure 21 shows '1X leaf juice (P=0 〇〇), r2=〇346) and #1 component (p<o.ooi 'r2=0.429), #2 component (P=0 〇〇1, r2 The =〇354) and #9 components (P<0.001, R2 = 0.440) significantly inhibited HepG2 growth with dose dependence. The ratio of the number of inhibited cells to the percentage of 25 χ leaf juice, #ι component, component and #9 component on day 4 was 4 4 χΐ〇4, 5·22χ1〇4, 4 52χ1〇4 , 15 8χ1〇4. The suppression effect is from large to small for the component >#1 component>#2 component>25 eucalyptus juice. In addition, the inhibitory effects of the same dose of 1) (leaf juice, 25 χ leaf juice, #1 component, #2 component, and #9 component were also analyzed, and the results are shown in Fig. 22. When the dose is 1% (1>< When 0.01) and 0·05% (Ρ<〇〇1), the inhibitory effect of #9 component is significantly stronger than that of X-leaf juice. In addition, when the dose is 〇5./, the inhibition of #9 component The effect was significantly stronger than #2 component (Ρ = 0.05). 157798.doc •23· 201201821 Example 11: Treatment of hepatocellular carcinoma Huh7 with #9 component of leaf juice The hepatocellular carcinoma cell line used in this example was Huh7, Cell culture and treatment procedures were as described in Example 2. Cells were divided into control (untreated) and test groups (using IX leaf juice, 0%, 0.001%, 〇.〇1 〇/〇, respectively, as described in Example 10). 0.1%, 0.5%, or 1% of #9 components.) Each group was repeated 4 to 6 times. The Huh7 cell counts of each group were determined on the 4th day, and the results are shown in Fig. 23. As can be seen from Fig. 23. '1%(Ρ<0·001), 0.5%(Ρ<〇.〇〇ΐ), 〇·ι〇/0(ρ<0 〇5), and 0_01°/〇(Ρ<0.05)# The 9 components had significant inhibitory effects. In addition, the #9 component of 0·001% (Ρ=0.067) was also significantly inhibited. Cell growth: 1%, 0.5%, 0.1%, 0.01%, and 〇. 〇〇1% of the #9 components were inhibited at 90.8%, 88.6%, 58.5%, 53.7%, and 48.5%, respectively. The ratio of the amount to the percentage of the #9 component is 15 8χ1〇4. Although the embodiments of the present invention have been illustrated and described above, various modifications and improvements can be made thereto by those skilled in the art. The examples are for illustrative purposes only and are not intended to be limiting, and the invention is not intended to be limited to the specific forms disclosed, and all modifications are included in the scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows an HPLC chromatogram of the leaf juice of the hand. Fig. 2 is a graph showing the relationship between the number of leaves described in Example 3. Effect of the percentage of juice on the leaf juice of the juice treated by HepG2 cells Days Figure 3 shows the relationship between leaf-to-HepG2 cell counts as described in Example 3. Figure 4 shows the percentage of leaf juice content in Example 3 versus Huh7 cell count 157798.doc -24.201201821 Figure 5 shows the embodiment 3 The relationship between the days of action of the leaf juice treated with the leaf juice on the Huh7 cell count. Figure 6 is a graph showing the relationship between the days of action of the leaf juice treated with the leaf juice and the Bowes cell count as described in Example 4. 7 shows the results of changes in cell count of HepG2 cells treated with leaf juice and/or paclitaxel on day 3 as described in Example 5. Figure 8 is a graph showing the results of changes in cell count of Huh7 cells treated with leaf juice and/or paclitaxel on day 3 as described in Example 5. Figure 9 is a graph showing the results of changes in tumor area of mice inoculated with Huh7 cells after treatment with leaf juice as described in Example 6. Fig. 10 is a graph showing the results of changes in body weight of mice inoculated with Huh7 cells after treatment with leaf juice as described in Example 6. Figure 11 is a graph showing the results of changes in tumor area of the Huh7-inoculated mice described in Example 6 after stopping the treatment of the leaf juice. Fig. 12 is a graph showing the results of changes in body weight of the Huh7-inoculated mice described in Example 6 after stopping the treatment of the leaf juice. Figure 13 is a graph showing the results of changes in tumor area of mice inoculated with Bowes cells after treatment with leaf juice as described in Example 7. Figure 14 is a graph showing the results of changes in body weight of mice inoculated with Bowes cells after treatment with leaf juice as described in Example 7. Fig. 15 is a graph showing the results of changes in tumor area of the mice inoculated with Bowes cells after stopping the treatment of the leaf juice as described in Example 7. Figure 16 is a graph showing the results of changes in body weight of mice inoculated with Bowes cells as described in Example 7 after the treatment of 157798.doc •25-201201821 leaf juice. Figure 17 shows a graph of the percentage of leaf juice and its components as described in Example 8 versus HepG2 cell count. Figure 18 shows the results of cell count changes after treatment of HepG2 cells with leaf juice and its components as described in Example 8. Figure 19 shows an HpLc map of the component of the palm leaf juice having a molecular weight greater than 50 kD. Fig. 20 is a graph showing the results of cell count change after treatment of HepG2 cells with fluorescein, + ganto tea juice and its components as described in Example 9. Figure 21 shows a line graph described in Embodiment 10. Treatment of Huh7 fines with leaf juice and its components, treatment of Huh7 with leaf juice and its components

The relationship between HepG2 cell counts Figure 22 shows the results of cell count changes after the cells in Example 10. Figure 23 shows the change in cell count after the cells in Example 11 and the percentage of leaf juice and its components to 157798. Doc * 26 -

Claims (1)

201201821 VII. Patent Application Range: 1 · A composition for treating cancer and/or tumor, comprising an effective amount of ZORBAXTM C18 tube to hand scented leaf juice, 50 microliters to hand millimeters and length (L) 15 cm The column was subjected to high performance liquid chromatography (HPLC) ' and a map was obtained at 214 nm, which included retention times of 1.756, 2.573, 7.118, 7.851, 9.715, 10.278, 10.864, 11.212, 12.287, 12.799, 13.178, 13.413, respectively. , peaks of 14.027, 14.794, 16.253, and 18.742 minutes; wherein the hand-flavor (P/eciraW/^'(10)~/(3) uniform leaf juice uses 0-30 〇/〇 acetonitrile and 0.1% TFA to perform a linear gradient in 30 minutes The elution is carried out for separation at a flow rate of 1 ml/min. 2. The composition of claim 1 for use in the treatment of hepatocellular carcinoma and/or melanoma. 3. The composition of claim 1 further comprising a 5. The anti-tumor agent. The composition of claim 3, wherein the anti-tumor agent is paclitaxel, wherein the anti-tumor agent is formulated as a pharmaceutically acceptable carrier for oral or topical administration. 6. The composition of claim 1 'by injecting the patient. 7. A method for preparing the composition of claim 1 is prepared using a method comprising the steps of: wherein the leaf juice (4) is harvested with a hand ( (b) washing the leaves obtained in the step (a) with distilled water; 157798.doc 201201821 (C) removing water from the leaves; and (d) obtaining leaf juice from the leaves obtained in the step (c). 11. The method of claim 7 wherein the method for preparing leaf juice further comprises the step (4), wherein the leaf juice obtained from the step (4) is centrifuged to remove tissue fibers. The method of claim 7 wherein The method for preparing a leaf juice further comprises a leaf juice sterilization step. The method of claim 9, wherein the leaf juice is sterilized by filtration. The method of claim 7, wherein the method for preparing the leaf juice is further The step comprises a leaf juice concentration step. 〆I A use of a composition as claimed in the manufacture of a medicament for the treatment of hepatocellular carcinoma and/or melanoma. ", 157798.doc