TWI399210B - Used to treat cancer and/or tumors - Google Patents

Description

Used to treat cancer and/or tumors

The present invention relates to a plant composition for treating cancer and/or tumor.

Hand incense (Plectranthus amboinicus, also known as Plectranthus amboinicus (Lour.) Spreng. , Coleus amboinicus Lour., Coleus aromaticus Benth., Coleus crassifolius Benth., Plectranthus aromaticus (Benth.) Roxb., Coleus suganda Blanco, Coleus carnosus Hassk. , and Majana amboinica (Lour.) Kuntze), a plant belonging to the family Lamiaceae, and has the following common names: country borage, Cuban oregano, Indian borage (Indian borage) ), Amboini coleus, French thyme, Mexican mint, Spanish thyme, Tao-shou-hsiang and soup mint. It is cultivated as a medicinal plant, wild vegetable and seasoning plant in the tropics (part of Africa, India, Southeast Asia, West Indies, Mexico, and recently in the southern United States). Its aromatic leaves can be used as a seasoning for meat, soups, fish and local beer, and can be used as a vegetable 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, its leaf dip (sweetened with honey) can reduce cough and colds. In addition, the friction generated by the friction of the blades helps to eliminate the nasal congestion. The hand fragrance is widely used in Taiwan, and can be used for external application in the case of, for example, burns, mosquito bites, sputum and edema, and can also be used for internal administration as, for example, a wind-driven agent and a asthma drug. In addition, for many decades, the hand scent has been known for its antibacterial and antifungal properties. It has recently been demonstrated that diterpene, triterpene and oleanolic acids from the scent of scent can inhibit the inflammatory response of animals by inhibiting the activity of COX-1 and COX-2, which can induce inflammatory responses. U.S. Patent Publication Nos. 2006068098, 20020076452, 20020077350, 20020110604, and 20030108628, and U.S. Patent No. 6,629,835.

However, prior art literature has never taught or implied that the hand fragrance has the utility of treating cancer and/or tumors.

The present invention provides a composition for treating cancer and/or tumor comprising an effective amount of scented leaf juice.

The invention also provides a method of treating cancer and/or tumor comprising administering to a patient in need of such treatment a composition of the invention.

The invention further provides a process for the preparation of a composition of the invention wherein the leaf juice is prepared using a process comprising the following steps:

(a) harvesting the leaves of the hand;

(b) washing the leaves obtained in step (a) with distilled water;

(c) removing water from the blade; and

(d) Leaf juice obtained from the leaves obtained in step (c).

One embodiment of the invention is 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.

Accordingly, the present invention also provides a method for the preparation of a composition for the treatment of cancer and/or tumor, comprising an effective amount of a component having a molecular weight of more than 50 kD to a hand-shake leaf juice, wherein the molecular weight is greater than 50 kD. The preparation method of the leaf juice component comprises the following steps:

(a) harvesting the leaves of the hand;

(b) washing the leaves obtained in step (a) with distilled water;

(c) removing water from the blades;

(d) obtaining leaf juice from the leaves obtained in step (c); 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 50 kD.

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 tumors.

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 contained in the leaves. Leaf juice can be obtained by removing tissue fragments and/or residues.

As used herein, the term "tumor" refers to a pathological protuberance, protrusion, or hyperplasia of any part of the body, particularly a hyperplasia formed by new tissue deposits, or a tumor. The term "malignant tumor" as used herein, also referred to as "malignant tumor disease" or "cancer", refers to tumor growth caused by abnormal and uncontrolled cell division, which can spread to other parts of the body through the lymphatic system or blood flow. .

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 eliminate tumors in an animal.

In one embodiment of the invention, the compositions of the invention are used to inhibit malignant tumor growth. 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 functionalizing the p53 gene (including wild type, partially functional type and mutant active form). Thus, a tumor line treated in accordance with the present invention is a tumor associated with p53.

The p53 network is a molecular detector of the G1 checkpoint in the cell cycle and monitors DNA damage, pool level, 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. A number of methods have been developed for the treatment of malignant tumors by the p53 network (U.S. Patent Nos. 5,382,510, 5,840,579, 6,183,964, 6,472,385 and 6,531,512).

In an embodiment of the invention, the tumor treated comprises hepatocellular carcinoma and melanoma. More preferably, the tumors treated include 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 hand juice geranyl inner diameter (ID) of 4.6 mm and a length (L) of 15 cm ZORBAX ^TM C18 column for high performance liquid chromatography, and the pattern is obtained at 214 nm, the pattern Including retention peaks 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, 14.027, 14.794, 16.253, and 18.742 minutes; wherein the leaf juice uses acetonitrile and 0.1% The TFA mixture was subjected to elution in 30 minutes for separation, wherein the concentration of acetonitrile in the mixture was a linear gradient of 0-60% at a flow rate of 1 ml/min.

When studying the efficacy of the components of the hand-scented leaf juice, it was found that the component having a molecular weight of more than 50 kD was more effective in treating cancer and/or tumor than other components. In one embodiment of the invention, 0.001% of the component having a molecular weight greater than 50 kD exhibits a strong ability to inhibit the growth of hepatocellular carcinoma (HepG2), while other components have the same effect at a dose of 0.5%.

The components of the present invention having a molecular weight greater than 50 kD can be analyzed and identified by high performance liquid chromatography. For example, 50-kd molecular weight greater than 50 [mu] l using the ingredients of the inner diameter (ID) of 4.6 mm and a length (L) of 15 cm ZORBAX ^TM C18 column for high performance liquid chromatography, and the pattern is obtained at 214 nm The map includes peaks with retention times of 2.574, 4.798, 5.728, 7.101, 9.701, 10.279, 10.808, 11.189, 12.235, 13.35, 13.584, 13.903, 14.113, 15.114, 16.206, 16.736, 18.619, 22.137, 24.676 and 26.902 minutes, respectively. The fraction having a molecular weight greater than 50 kD was separated by elution in 30 minutes using a mixture of acetonitrile and 0.1% TFA, wherein the concentration of acetonitrile in the mixture was a linear gradient of 0-60%, and the flow rate was 1 ml/min. .

In an embodiment of the invention, the composition further comprises an anti-tumor agent. Because tumor growth involves a complex network of interactions, the combined use of anti-tumor agents for different networks can reasonably improve the therapeutic effect. In a preferred embodiment of the invention, the anti-tumor agent is paclitaxel. Paclitaxel (also known as taxol) is a clinical anticancer drug with strong inhibition of G ₂ M late mitosis by mediated Bcl-2 phosphorylation and paclitaxel-induced apoptosis. Shows strong anti-tumor properties. In addition, paclitaxel has been found to increase mitotic blocking by binding to microtubules (Lanni S. Jennifer, Lowe W. Scott, Litica J. Edward, Liu O. Jun, Jacks Tyler. P53-independent aopotosis induced By paciltaxel through an indirect mechanism. Proc. Natl. Acad. Sci. USA Vol 94, September 1997: 9979-9683). In one embodiment of the invention, a composition comprising 5% to hand geranium juice and 50 nM paclitaxel has a greater inhibitory effect on tumor growth than a composition comprising only scented leaf juice or paclitaxel. This result suggests that cross-talking of the apoptotic pathways of p53 and Bcl-xL cells 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 carrier" 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, diluents, binders and binders, lubricants, disintegrants, colorants, bulking agents, and the preparation of particular therapeutic compositions. Other materials such as buffers and absorbents. The use of such media and agents for pharmaceutically active substances is well known to those skilled in the art. Unless any conventional media or agent is incompatible with the active ingredient, the invention encompasses that it can be used in the compositions of the invention.

The compositions of the invention can be administered to a patient in a number of ways. Preferably, the composition is administered to the patient by injection, orally or topically.

The present invention provides a method of preparing the compositions of the present invention. Specifically, the leaf juice is prepared by a method comprising the following steps:

(a) harvesting the leaves of the hand;

(b) washing the leaves obtained in step (a) with distilled water;

(c) removing moisture from the leaves, preferably by natural air drying;

(d) Leaf juice obtained from the leaves obtained in step (c).

In one embodiment of the invention, the blades can be processed into small pieces by some common methods such as milling, agitation, agitation, cutting or chopping.

The leaf juice preparation method may further include the step (d2), that is, centrifuging the leaf juice obtained in the step (d) to remove the tissue fibers. In one embodiment of the invention, centrifugation is performed at 15,000 rpm and 24 ° C for 5 minutes each time.

The leaf juice preparation method may further include a leaf juice sterilization step as needed. In an 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 component of a palm leaf juice having a molecular weight greater than 50 kD.

The present invention also provides a method of preparing a composition for treating cancer and/or tumor, the composition comprising an effective amount of a hand-flavored leaf juice component having a molecular weight of more than 50 kD, wherein the molecular weight is greater than 50 kD to the hand-scented leaf juice The components are prepared by a method comprising the following steps:

(a) harvesting the leaves of the hand;

(b) washing the leaves obtained in step (a) with distilled water;

(c) removing moisture from the leaves;

(d) obtaining leaf juice from the leaves obtained in step (c);

(e) passing the leaf juice obtained in the step (d) through a filter to remove a component having a molecular weight of less than 50 kD from the obtained leaf juice, and obtaining a component having a molecular weight of more than 50 kD.

In addition to the step (e), the method of preparing the palm berry juice component having a molecular weight of more than 50 kD is the same as the above-described method of preparing the scented leaf juice. Methods for separating components based on molecular weight ranges are well established in the art. An embodiment of the invention uses Amicon Ultracentrifugal filtration devices having pore sizes of different sizes to separate various components by molecular weight.

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

The hand fragrance comes from the Taiwan Endemic Species Research Institute (located in Nantou, Taiwan). After harvesting the leaves of the hand, the leaves were washed with distilled water and air dried to remove residual moisture from the leaves. The leaf tissue is milled into fine particles and leaf juice. The leaf juice was centrifuged at 15,000 rpm for 5 minutes at 24 ° C to concentrate the leaf juice. The supernatant was collected 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 sterilized by filtration through a 0.22 micron syringe filter. Store fresh leaf juice in a dark place at 4 °C before use. All plant crude extracts should be used within 3 days or stored at -80 °C for long periods of time.

At Agilent Chromatography was performed on a 1100 Series Liquid Chromatography System consisting of a 1100 four-stage pump (with degasser), a 1100 variable wavelength detector, and a 1100 standard automatic sample collector. Use ChemStation The software implements further peak analysis.

Large amounts of solvent and mobile phase using Millipore Solvent filtration equipment (Millipore , Bedford, MA) with 0.22 micron nylon membrane filter (Alltech Associates, Pty. Ltd) Filtration.

The column was equilibrated with 0.1% TFA (trifluoroacetic acid) and the Milli-Q flow rate was 1 mL/min. Inject 50 μl of 1× crude sample dissolved in PBS into ZORBAX A C18 column (4.6 mm inner diameter x 15 cm) was placed in a column oven thermostat set at 25 °C. After injection, the analyte was subjected to a linear gradient elution with 0-60% acetonitrile and 0.1% TFA over 30 minutes to separate the analyte. Analytes were detected at 214 nm.

The chromatographic results are shown in Figure 1. The data of each peak recorded are shown in Table 1.

Example 2: Screening for cell lines that respond to leaf juice

Cell lines: Unless otherwise specified, the following nine human tumor cell lines of different tissue origin were purchased from the Commercial Cell Culture Collection Center: Food Industry Development Institute.

HepG2 and Hep3B cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 μg/ml streptomycin. U937 and K562 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 μg/ml streptomycin. Calu-1 and TL cells were cultured in a high-dose Dalke essential medium (plus 10% fetal bovine serum, 2 mM L-glutamine, 50 IU/mL penicillin, and 50 μg/ml streptomycin).

The culture was stored in an environment containing 5% CO ₂ at a temperature of 37 ° C and a certain humidity. All cell culture reagents were purchased from Gibco-Invitrogen Co., Ltd. (Grand Island, NY USA) and HyClone (Logan, Utah, USA).

Treatment: 3% of the leaf juice prepared according to Example 1 was added directly to the subcultured cells in all in vitro experiments.

Cell count: 20 microliters of cell suspension was added to 20 microliters of 0.4% cone blue and total cell counts were performed using phase contrast microscopy.

The results are shown in Table 2. "O" represents cells that respond to leaf juice (ie, the number of cells after treatment is reduced); "X" represents cells that do not respond to leaf juice (ie, the number of cells after treatment increases).

According to the results shown in Table 2, the p53 gene-functional cell line responded to leaf juice, demonstrating that leaf juice can control tumor growth by the p53 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 processing operations were as described in Example 2. The cells were divided into control (untreated) and experimental groups (treated with 1%, 3%, 5%, 7% or 9% 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. It can be seen from Fig. 2 and Fig. 3 that the inhibition of the leaf juice on the cells increased with the dose on the first day (P<0.001), the third day (P<0.001) and the fifth day (P<0.001). Enhanced with dose dependence. In addition, the inhibition of leaf juice was on the first day (inhibited cell count / leaf juice percentage: 1.2 × 10 ⁴ ), day 3 (inhibited cell count / leaf juice percentage: 6.18 × 10 ⁴ ) and Day 5 (inhibited cell count/leaf percentage: 10.44 x 10 ⁴ ) increased with increasing treatment time.

The Huh7 cell counts of each group were determined on Day 1, Day 3, and Day 5, respectively, and the results are shown in Figures 4, 5, and 4. It can be seen from Fig. 4 and Fig. 5 that the inhibition of the leaf juice on the cells increased with the dose on the first day (P<0.05), the third day (P<0.001) and the fifth day (P<0.001). Enhanced with dose dependence. In addition, the inhibition of leaf juice was on the first day (suppressed cell count / leaf juice percentage: 0.49 × 10 ⁴ ), day 3 (suppressed cell count / leaf juice percentage: 2.47 × 10 ⁴ ) and Day 5 (inhibited cell count/leaf percentage: 3.63 x 10 ⁴ ) increased with increasing treatment time.

Example 4: Treatment of melanoma with leaf juice

The melanoma cell line used in this example was Bowes, and the cell culture and processing operations were as described in Example 2. Cells were divided into control (untreated) and test groups (treated with 1% or 3% leaf juice). Each group was repeated 5 times.

Each group of Bowes cell counts was determined on day 1, day 3, and day 5, and the results are shown in FIG. It can be seen from Fig. 6 that the inhibition of leaf juice on day 1 (P<0.01) and day 5 (P<0.01) increased with increasing dose, showing strong dose dependence. In addition, the inhibition of leaf juice on the first day (inhibited cell count / leaf juice percentage: 2.22 × 10 ⁴ ), day 3 (inhibited cell count / leaf juice percentage: 3.26 × 10 ⁴ ), And on the fifth day (inhibited cell count / leaf juice percentage: 25.8 × 10 ⁴ ), both increased with the increase of treatment time.

Example 5: Treatment of hepatocellular carcinoma with leaf juice and/or paclitaxel

The hepatocellular carcinoma cell lines used in this example were HepG2 and Huh7, and the cell culture and processing operations were as described in Example 2. Cells were divided into control (untreated) and experimental groups (treated with 5% leaf juice, 50 nM paclitaxel, and 5% leaf juice and 50 nM paclitaxel, respectively). Each group was repeated 6 times.

The HepG2 cell counts of each group were determined on the third day, and the results are shown in Fig. 7. As can be seen from Fig. 7, the cell growth of all the test groups was significantly inhibited. The inhibition rates of the test groups were 52.13% (5% leaf juice: P < 0.05), 43.77% (50 nM paclitaxel: P = 0.052), or 76.57% (mixture of 5% leaf juice and 50 nM paclitaxel: 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 each test group were 50.21% (5% leaf juice: P=0.110), 66.09% (50 nM paclitaxel: P<0.05), 74.68% (mixture of 5% leaf juice and 50 nM paclitaxel: P<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 of hepatocellular carcinoma treated with leaf juice

Inoculation: Huh7 cells were cultured under the conditions described in Example 2 to obtain a total of 3 × 10 ⁸ cells. The obtained cells were then suspended in DMEM to have a cell density of 2 × 10 ⁸ /ml. NOD-scid mice weighing about 20 grams at 5-6 weeks old were selected, and 0.1 ml of the above cell solution was inoculated into the subcutaneous region of the right back of the mouse. Control mice were inoculated with 0.1 ml of DMEM.

Treatment: large tumors (larger than 10 mm × 10 mm, one mouse), medium tumors (between 7 mm × 7 mm to 10 mm × 10 mm, one mouse) and small tumors Mice (size less than 7 mm x 7 mm, two mice) were treated with leaf juice prepared as described in Example 1, respectively. The leaf juice dose is 10% of the mouse body weight. The leaf juice is placed in a syringe, the needle is inserted into the subcutaneous portion around the tumor, and the leaf juice is injected into the inside of the tumor. Tumor size was measured every Monday, Wednesday and Friday and the body weight of the mice was weighed while continuing treatment with leaf juice. The blank group was injected with 10% PBS.

Results: The results are shown in Figure 9. The area of small tumors was reduced after treatment with leaf juice (R ² =0.564, B=-2.09 mm 2 /day, P<0.01); growth of tumors in the middle (R ² =0.834, B=6.19 mm 2 /day, P< 0.01) was inhibited, only 27.1% of the control group; however, growth of large tumors (R ² =0.995, B = 34.38 mm 2 /day, P < 0.01) was not inhibited. In addition, all mice receiving leaf juice treatment lost weight (unvaccinated mice: R ² = 0.178, B = -0.13 g / day, P <0.05; mice with small tumors: R ² = 0.281, B =-0.09 g/day, P=0.051; mice with intermediate tumors: R ² =0.576, B=-0.11 g/day, P<0.05; mice with large tumors: R ² =0.210, B=- 0.11 g/day, p=0.302). Only mice that did not receive leaf juice treatment gained weight (R ² = 0.730, B = 0.24 g/day, P < 0.001) (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 (R ² =0.558, B=0.72 mm 2 /day, P<0.05) was only 2.4% of the control group (R ² =0.622, B=30.62 mm 2 /day, P<0.01). . In addition, after stopping the administration, the body weight of all mice increased (blank group: R ² = 0.743, B = 0.53 g / day, P <0.001; small tumor group: R ² = 0.045, B = 0.02 g / day, P <0.615; control group: R ² = 0.214, B = 0.11 g/day, P < 0.05) (see Fig. 12).

Example 7: Animal model of melanoma treated with leaf juice

Inoculation: Bowes cells were cultured under the conditions described in Example 2 to obtain a total of 3 x 10 ⁸ cells. The obtained cells were then suspended in DMEM to have a cell density of 2 × 10 ⁸ /ml. NOD-scid mice weighing about 20 grams at 5-6 weeks old were selected, and 0.1 ml of the above cell solution was inoculated into the subcutaneous region of the right back of the mouse. Control mice were inoculated with 0.1 ml of DMEM.

Treatment: Mice with large tumors (size greater than 10 mm x 10 mm, one mouse) and small tumors (less than 10 mm x 10 mm in size, two mice) were prepared as described in Example 1 The leaf juice is used for treatment. 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 portion around the tumor, and the leaf juice is injected into the inside of the tumor. Tumor size was measured on Monday, Wednesday, and Friday and the body weight of the mice was weighed while continuing treatment with leaf juice. The blank group was injected with 10% PBS.

Results: The results are shown in Figure 13. The area of large tumors was reduced after treatment with leaf juice (R ² =0.924, B=-3.08 mm 2 /day, P<0.01); growth of small tumors (R ² =0.564, B=1.50 mm 2 /day, P< 0.01) 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 (R ² =0.897, B = 0.09 g / day, P < 0.001), and all mice receiving leaf juice treatment showed a slight increase in body weight (see Figure 14). ).

Recurrence: The small tumor group stopped taking the drug on the 24th day, but continued to monitor the tumor area. The results are shown in Figure 15. The results showed that the growth rate of small tumors after discontinuation of administration (R ² =0.750, B=4.1 mm 2 /day, P < 0.001) was faster than when administered (R ² =0.564, B=1.50 mm 2 /day, P<0.01). 173%. In addition, the tumor growth rate (R ² =0.320, B = 5.13 mm 2 /day, P < 0.001) of the blank group mice after stopping the PBS injection was higher than that of the injection with PBS (R ² =0.608, B = 3.98 mm 2 / Days, P < 0.001) is 29% faster. The small tumor group and the blank group had similar growth rates after stopping the administration. In addition, the body weight of all mice increased slightly after stopping the treatment of leaf juice, but the weight gain of the blank group (R ² =0.315, B=-0.03 g/day, P<0.05) was significantly higher than that of the small tumor group (see Figure 16).

Example 8: Inhibitory effect of leaf juice component on growth of HepG2 cells

Leaf Juice Components: Using Amicon Ultracentrifugal filtration unit (Amicon Ultra PL-10, 10,000 nominal molecular weight limit (NMWL); Amicon Ultra PL-30, 30,000 NMWL; Amicon Ultra PL-50, 50,000 NMWL, Millipore The leaf juice prepared in Example 1 was divided into the following components: #1 component (molecular weight <10 K, 25X leaf juice), #2 component (molecular weight <10 kD, 1X leaf juice), #3 component ( Molecular weight between 10 kD and 30 kD, 25X leaf juice), #4 component (molecular weight between 10 kD and 30 kD, 1X leaf juice), #5 component (molecular weight between 30 kD and 50 kD) Between, 25X leaf juice), #6 component (molecular weight between 30 kD and 50 kD, 1X leaf juice), #7 component (molecular weight <50 kD, 25X leaf juice), #8 component (molecular weight >50 kD, 25X leaf juice), #9 component (molecular weight > 50 kD, 1X 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.

The results showed that the #1, #2, #3, #7, #8, and #9 components had dose-dependent inhibition of tumor growth. Moreover, the growth inhibitory effects of #2 and #9 components on HepG2 cells were stronger than those of the other components.

HepG2 cells were further treated with 1X and 25X leaf juice, 0%, 0.01%, 0.1%, 0.5%, 1%, and 3% of #1, #2, and #9 components. Each group is repeated 4 to 7 times.

The results showed that 0.5% (P < 0.05), 1% (P < 0.001) and 3% (P < 0.001) of #2 components, 0.01% of #9 components (P < 0.001), 1% of leaf juice (P<0.05) and 1% (P<0.01) and 3% (P<0.001) of 25X leaf juice significantly inhibited HepG2 growth. On the other hand, 1% of 1X leaf juice (P = 0.884) 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 0.01%; the #2 component also has a strong inhibitory effect, and the effective dose is 0.5%; the effective dose of 25X leaf juice is 1%. The inhibition rates of 0.5%, 1%, and 3% of #2 components, 0.01% of #9 components, 3% of 1X leaf juice, and 1% and 3% of 25X leaf juice were 34.3%, 62.8%, and 98.7, respectively. %, 39.7%, 54.8%, 53.6% and 83.8%.

The ratio of the number of inhibited cells to the percentage of 1X leaf juice, 25X leaf juice, #1 component, #2 component, and #9 component is shown in Figure 17.

Figure 17 shows that 1X (P < 0.001, R ² = 0.525) and 25X (P = 0.001, R ² = 0.689) leaf juice and #1 component (P < 0.001, R ² = 0.333), #2 component ( P < 0.001, R ² = 0.792) and #9 components (P < 0.001, R ² = 0.498) significantly inhibited HepG2 growth with dose dependence. The ratio of the number of cells inhibited on day 4 to the percentages of 1X leaf juice, 25X leaf juice, #1 component, #2 component, and #9 component was 10.2x10 ⁴ , 15.7x10 ⁴ , 9.22x10, respectively. ⁴ , 18.4x10 ⁴ and 43.7x10 ⁴ . The inhibition effect is from large to small as #9component>#2 component>25 times leaf juice>1 times leaf juice>#1 component.

In addition, the inhibitory effects of the same dose of 1X leaf juice, 25X leaf juice, #1 component, #2 component, and #9 component were also analyzed, and the results are shown in Fig. 18.

When the dose was 0.1% and 0.5%, the inhibitory effect of the #9 component was significantly stronger than that of the 1X and 25X leaf juice, the #1 component, and the #2 component (both P < 0.001).

When the dose was 1%, the inhibitory effect of the #9 component was significantly stronger than that of the 1X leaf juice (P=0.001), the 25X leaf juice, the #1 component (P=0.059), and the #2 component (P=0.072). In addition, the inhibitory effect of #2 component was significantly stronger than that of 1X 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 1X 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.

The hepatocellular carcinoma cell line used in this example was HepG2, and the cell culture and processing operations were as described in Example 2. Cells were divided into control (untreated) and test groups (treated as 1X leaf juice, 0%, 0.001%, 0.01%, 0.1%, 0.5% or 1% of #9 components, respectively, as described in Example 8. The group is repeated 4 to 8 times.

The HepG2 cell counts of each group were determined on the fourth day, and the results are shown in Fig. 20. As can be seen from Fig. 20, the #9 component of 1% (P < 0.001), 0.5% (P < 0.001), 0.1% (P < 0.001), and 0.01% (P < 0.001) had a remarkable inhibitory effect. In addition, 0.001% (P = 0.992) of the #9 component also significantly inhibited cell growth. The inhibition rates of #1, 0.5%, 0.1%, 0.01%, and 0.001% of the #9 component were 93.6%, 94.7%, 93.8%, 39.7%, and 18.5%, respectively. The ratio of the number of cells inhibited to the percentage of component #9 was 43.65 x 10 ⁴ .

Example 10: Inhibitory effect of leaf juice component on growth of Huh7 cells

The leaf juice fraction was prepared as described in Example 8.

Treatment: Huh7 cell line was used to screen for leaf juice components. Cell line manipulations were performed as described in Example 2, and cell counts were performed on day 4.

The survival rate of Huh7 cells on day 4 is shown in Table 7.

The results indicate that the #1, #2, and #9 components inhibit tumor growth and have dose dependence.

In addition, Huh7 cells were further treated with 1X and 25X leaf juice, 0%, 0.01%, 0.1%, 0.5%, 1%, and 3% of #1, #2, and #9 components. Each group is repeated 4 to 7 times.

The results showed that 1% (P < 0.01) and 3% (P = 0.001) of #1 component, 1% (P < 0.05) and 3% (P < 0.05) of #2 component, 0.01% of #9 Components (P = 0.01) and 0.5% (P < 0.05), 1% (P < 0.05) and 3% (P < 0.01) of 25X leaf juice significantly inhibited Huh7 growth. On the other hand, 1% (P = 0.937) and 3% (P = 0.666) 1X leaf juice showed no significant inhibitory effect. This proves that the #9 component has the strongest inhibitory effect, and its effective dose is only 0.01%; 25X leaf juice also has a strong inhibitory effect, and its effective dose is 0.5%; the effective dose of #1 and #2 components is 1 %. The inhibition rates of 1% and 3% of #1 component, 1% and 3% of #2 component, 0.01% of #9 component, and 0.5%, 1%, and 3% of 25X leaf juice were 59.3%, respectively. 76.2%, 53.9%, 64.2%, 53.7%, 49.9%, 53.9% and 67.1%.

The ratio of the number of inhibited cells to the percentage of 1X leaf juice, 25X leaf juice, #1 component, #2 component, and #9 component is shown in Figure 21.

Figure 21 shows 1X leaf juice (P = 0.001, R ² = 0.346) and #1 components (P < 0.001, R ² = 0.429), #2 components (P = 0.001, R ² = 0.354) and #9 The components (P < 0.001, R ² = 0.440) significantly inhibited HepG2 growth with dose dependence. The ratio of the number of inhibited cells to the percentage of 25X leaf juice, #1 component, #2 component, and #9 component on day ⁴ was 4.41x10 ⁴ , 5.22x10 ⁴ , 4.52x10 ⁴ , 15.8x10 ⁴ , respectively. . The inhibition effect is from large to small as #9component>#1 component>#2 component>25X leaf juice.

In addition, the inhibitory effects of the same dose of 1X leaf juice, 25X leaf juice, #1 component, #2 component, and #9 component were also analyzed, and the results are shown in Fig. 22.

When the dose was 1% (P < 0.01) and 0.05% (P < 0.01), the inhibitory effect of the #9 component was significantly stronger than that of the 1X leaf juice. In addition, when the dose was 0.5%, the inhibitory effect of the #9 component was significantly stronger than that of the #2 component (P=0.05).

Example 11: Treatment of hepatocellular carcinoma Huh7 with #9 component of leaf juice

The hepatocellular carcinoma cell line used in this example was Huh7, and the cell culture and processing operations were as described in Example 2. Cells were divided into control (untreated) and test groups (treated as 1X leaf juice, 0%, 0.001%, 0.01%, 0.1%, 0.5% or 1% of #9 components as described in Example 10) . Each group is repeated 4 to 6 times.

The Huh7 cell counts of each group were determined on the fourth day, and the results are shown in Fig. 23. As can be seen from Fig. 23, 1% (P < 0.001), 0.5% (P < 0.001), 0.1% (P < 0.05), and 0.01% (P < 0.05) of the #9 component had a remarkable inhibitory effect. In addition, 0.001% (P = 0.067) of the #9 component also significantly inhibited cell growth. The inhibition rates of #9, 0.5%, 0.1%, 0.01%, and 0.001% of the #9 components were 90.8%, 88.6%, 58.5%, 53.7%, and 48.5%, respectively. The ratio of the number of inhibited cells to the percentage of the #9 component was 15.8 x 10 ⁴ .

Although the embodiments of the present invention have been illustrated and described above, various modifications and improvements can be made by those skilled in the art. Therefore, the embodiments of the present invention are for illustrative purposes only and not limiting. The present invention is not intended to be limited to the specific forms disclosed, and all modifications are intended to be included within the scope of the appended claims.

Figure 1 shows an HPLC chromatogram of the scented leaf juice.

Figure 2 is a graph showing the relationship between the percentage of leaf juice and the HepG2 cell count as described in Example 3.

Figure 3 is a graph showing the relationship between the number of days of action of leaf juice treated with leaf juice and HepG2 cell count as described in Example 3.

Figure 4 is a graph showing the relationship between the percentage of leaf juice and the Huh7 cell count as described in Example 3.

Figure 5 is a graph showing the relationship between the number of days of action of leaf juice treated with leaf juice and Huh7 cell count as described in Example 3.

Figure 6 is a graph showing the relationship between the number of days of action of leaf juice treated with leaf juice and the count of Bowes cells as described in Example 4.

Figure 7 shows the results of cell count changes on day 3 of HepG2 cells treated with leaf juice and/or paclitaxel as described in Example 5.

Figure 8 shows the results of cell count changes on day 3 of Huh7 cells treated with leaf juice and/or paclitaxel 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.

Figure 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.

Figure 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 shows the results of changes in body weight of mice inoculated with Bowes cells after treatment with leaf juice as described in Example 7.

Figure 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 shows the results of changes in body weight of the mice inoculated with Bowes cells after stopping the treatment of the leaf juice as described in Example 7.

Figure 17 is a graph showing the relationship between the percentage of leaf juice and its components 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 profile of the palm berry juice component having a molecular weight greater than 50 kD.

Figure 20 shows the results of cell count changes after treatment of HepG2 cells with leaf juice and its components as described in Example 9.

Figure 21 is a graph showing the relationship between the percentage of leaf juice and its components described in Example 10 versus HepG2 cell count.

Figure 22 shows the results of cell count changes after treatment of Huh7 cells with leaf juice and its components as described in Example 10.

Figure 23 shows the results of cell count changes after treatment of Huh7 cells with leaf juice and its components as described in Example 11.

(no component symbol description)

Claims (10)

A use of leaf juice of Plectranthus amboinicus for the manufacture of a medicament for the treatment of hepatocellular carcinoma and/or melanoma, wherein 50 μl of the leaf juice ( Plectranthus amboinicus ) leaf juice system uses an inner diameter (ID) 4.6 mm, length (L) of 15 cm ZORBAX ^TM C18 column for high performance liquid chromatography (HPLC), using acetonitrile and 0.1% TFA elution mixture separated within 30 minutes, wherein the mixture of acetonitrile The concentration is a linear gradient of 0-60%, the flow rate is 1 ml/min, and the spectrum is obtained at 214 nm, which includes retention times of 1.756, 2.573, 7.118, 7.851, 9.715, 10.278, 10.864, 11.212, respectively. Peaks of 12.287, 12.799, 13.178, 13.413, 14.027, 14.794, 16.253 and 18.742 minutes. The use of claim 1, wherein the medicament further comprises an anti-tumor agent. The use of claim 2, wherein the anti-tumor agent is paclitaxel. The use of claim 1 wherein the medicament is formulated in a pharmaceutically acceptable carrier. The use of claim 1, wherein the medicament is administered to the patient by injection, orally or topically. A method of preparing a scented leaf juice comprising the steps of: (a) harvesting leaves of Plectranthus amboinicus ; (b) washing the leaves obtained in step (a) with distilled water; (c) removing water from the leaves; d) grinding, stirring, agitating, cutting or chopping the leaves obtained from the step (c) to obtain a leaf juice; wherein the hand-scented leaf juice is used for the manufacture of a medicament for treating hepatocellular carcinoma and/or melanoma, wherein 50 microliters of juice-based hand geranyl inner diameter (ID) of 4.6 mm and a length (L) of 15 cm ZORBAX ^TM C18 column for high performance liquid chromatography (HPLC), using acetonitrile and 0.1% TFA mixture The elution was carried out in 30 minutes for separation, wherein the concentration of acetonitrile in the mixture was a linear gradient of 0-60%, the flow rate was 1 ml/min, and a spectrum was obtained at 214 nm, which included a residence time of 1.756, respectively. , peaks of 2.537, 7.118, 7.851, 9.715, 10.278, 10.864, 11.212, 12.287, 12.799, 13.178, 13.413, 14.027, 14.794, 16.253 and 18.742 minutes. The method of claim 6, wherein the method for preparing leaf juice further comprises the step (d2) of centrifuging the leaf juice obtained from the step (d) to remove tissue fibers. The method of claim 6, wherein the method for preparing leaf juice further comprises a leaf juice sterilization step. The method of claim 8, wherein the leaf juice is sterilized by filtration. The method of claim 6, wherein the method for preparing leaf juice further comprises a leaf juice concentration step.