Anticancer agent containing tea fruit extract

Abstract

To solve the problem in which: while a molecular targeting agent is highly effective in the treatment of cancer, no improvement has yet been observed in survival rates for advanced cancers with metastasis; therefore, it is a critical challenge to develop an antitumor drug effective against metastatic cancer.SOLUTION: An anticancer agent containing a squeezed tea fruit residue extract as an active ingredient is capable of suppressing the proliferation of cancer cells and also capable of suppressing cell migration, thereby effectively suppressing cancer progression.SELECTED DRAWING: Figure 5

Description

[Technical field]

[0001] The present invention relates to an anticancer agent which contains an extract of tea seeds and has the effect of inhibiting the proliferation and migration of cancer cells. [Background technology]

[0002] Cancer is the leading cause of death in Japan, and the number of deaths continues to increase year by year. With the development of molecular targeted drugs, the mortality rate from these cancers has improved significantly. However, molecular targeted drugs often have strong side effects, and their use imposes a burden on patients.

[0003] In recent years, natural materials such as medicinal plants have been attracting attention as useful resources for developing antitumor drugs because they are safe and have few side effects. Various phytochemical substances with antitumor effects have been discovered and are now being used clinically as antitumor drugs.

[0004] Patent Document 1 states that the oil extracted from tea seeds contains ingredients such as amino acids, catechins, caffeine, vitamin C, β-carotene, vitamin E, and oleic acid, and can therefore be effectively ingested to have benefits such as preventing carcinogenesis, enhancing immune function, preventing aging, and preventing arteriosclerosis. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP2004-89146A Summary of the Invention [Problem to be solved by the invention]

[0006] Although molecular targeted agents are highly effective, the survival rate of advanced cancers with metastasis has not yet improved. Therefore, it is an important task to develop effective antitumor drugs for metastatic cancer. [Means for solving the problem]

[0007] The present invention has been made to solve the above-mentioned problems, and provides an anticancer agent which is a material derived from a natural product and which inhibits, in particular, the migration and invasion of cancer cells.

[0008] Specifically, the anti-cancer agent according to the present invention is characterized in that it contains an extract of the residue of pressed tea seeds as an active ingredient. Effect of the Invention

[0009] The anticancer agent according to the present invention inhibits the proliferation, migration and invasion of cancer cells, and is therefore expected to effectively inhibit the metastasis of cancer. In addition, the anticancer agent according to the present invention is derived from natural sources, and is almost free of side effects. Therefore, by taking the agent when cancer is discovered early, as well as during the prognosis period of cancer treatment, it is possible to inhibit the progression of cancer and postpone the time until surgery is required. Furthermore, by inhibiting the metastasis of cancer even in cases where the cancer is inoperable, it is possible to make the cancer inoperable by combining the agent with other treatment methods. [Brief description of the drawings]

[0010] [Figure 1] 1 is a graph showing the growth inhibitory effect of tea fruit extract, which is an active ingredient of the anticancer agent of the present invention, on gastric cancer cells (KATO-III). (a) shows the result 24 hours after the start of culture, and (b) shows the result 48 hours after the start of culture. [Diagram 2] 1 is a graph showing the growth inhibitory effect of tea fruit extract, which is an active ingredient of the anticancer agent of the present invention, on colon cancer cells (SW480). (a) shows the result 24 hours after the start of culture, and (b) shows the result 48 hours after the start of culture. [Diagram 3] The results of a Boyden chamber test on cell migration of gastric cancer cells (KATO-III) treated with tea seed extract, an active ingredient of the anticancer drug of the present invention. (a) is an example photograph and a table of results for the blank, and (b) is an example photograph and a table of results for the tea seed extract. [Figure 4]The results of a Boyden chamber test were used to examine the cell migration of colon cancer cells (SW480) by tea seed extract, which is an active ingredient of the anticancer agent of the present invention. (a) is an example photograph and a table of results for the blank, and (b) is an example photograph and a table of results for the tea seed extract. [Diagram 5] 1 shows the results of a scratch test of gastric cancer cells (KATO-III) using tea fruit extract, which is an active ingredient in the anticancer agent of the present invention. [Figure 6] 1 shows the results of a scratch test of colon cancer cells (SW480) using tea seed extract, which is an active ingredient in the anticancer agent of the present invention. [Figure 7] 1 is a graph showing a part of the results of mass spectrometry of tea fruit extract, which is an active ingredient of the anticancer agent of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The anticancer agent according to the present invention will be described below with reference to examples and drawings. Note that the following description is an example of one embodiment of the present invention and an example, and the present invention is not limited to the following description. The following description can be modified within the scope of the present invention. In addition, embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. In addition, all of the documents described in this specification are incorporated herein as references. In this specification, when "A to B" is described in relation to a numerical range, the description intends "A or more and B or less."

[0012] The anti-cancer agent according to the present invention contains as its active ingredient a substance obtained by extraction with acetic acid and methanol from the residue left after squeezing tea seeds.

[0013] Tea fruits are the fruits of the tea plant (Camellia sinensis), an evergreen tree of the Theaceae family. There are no particular restrictions on the type of tea fruit, but the fruit of the "Yabukita" variety, which is the most widely cultivated in Japan, can be used. Tea fruits harvested between October and January are used.

[0014] The anti-cancer agent of the present invention uses the residue obtained by squeezing the tea berries. The squeezing method is not particularly limited, but generally involves drying the berries in the sun, cracking the outer shell, and squeezing the fruit inside. The squeezed oil from the tea berries is then used in various ways. In the present invention, the squeezed residue is used.

[0015] The residue left after squeezing is called "squeezing residue." Although it is called squeezing residue, it is a very hard solid. Therefore, the squeezing residue is first crushed. It is preferable to crush it finely, but it may be crushed into small pieces by hitting it with a hammer or the like. The crushed squeezing residue is called "crushed residue."

[0016] The ground residue is dissolved in a polar solvent with a neutral to acidic pH (pH 1-7) to obtain an extract. For example, a mixture of water and alcohol can be used as the polar solvent, and lower alcohols are preferably used as the alcohol, with ethanol being preferred. Of course, other alcohols can also be used. This is because even if a toxic solvent is used during extraction, it can be removed by purification later. Also, acetic acid can be used preferably to make the pH more acidic.

[0017] The ratio of water to alcohol and the amount of acid added are preferably such that the acid is 0.1% by mass to 1.0% by mass to the volume of the mixed liquid, and the alcohol is 5% by volume to 80% by volume of the mixed liquid, and the mixed liquid is highly polar and has the ability to dissolve hydrophobic groups. When extracting components from the pulverized residue, the temperature of the mixed liquid is 0°C to a temperature up to the boiling point of the mixed liquid, preferably 15°C to 60°C, more preferably 20°C to 40°C. Note that dissolution and extraction may be accompanied by physical vibration or stirring, such as a magnetic stirrer or ultrasonic waves. In addition, when extracting at a temperature near the boiling point of the mixed liquid, an appropriate reflux device may be used.

[0018] After extraction, the mixture is filtered to remove solids. The resulting filtrate is called "tea seed extract." This tea seed extract is the tea seed extract of the present invention.

[0019] The anti-cancer agent according to the present invention has this tea fruit extract as an active ingredient. The anti-cancer agent may be used as a liquid medicine in the form of a tea fruit extract, or may be used in the form of a powder solidified by a method such as freeze-drying. The medium containing the polar solvent used in the extraction process from the tea fruit may be removed by purification when the anti-cancer agent is prepared.

[0020] In addition, the anticancer agent according to the present invention may contain carriers, diluents, excipients, or combinations of two or more thereof that are commonly used in biological preparations. For example, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components may be used.

[0021] If necessary, other common additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added. In addition, diluents, dispersants, surfactants, binders, lubricants, and lubricants may be added to the composition to prepare an injectable preparation such as an aqueous solution, suspension, emulsion, etc., or a pill, capsule, granule, or tablet. Of course, the composition may be formulated in a suitable manner depending on each disease or on the ingredients to be mixed.

[0022] In addition, the anticancer agent according to the present invention can be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally or locally as an injection preparation) or orally depending on the intended method. The dosage may be adjusted depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity.

[0023] The liquid preparation for oral administration of the composition of the present invention includes suspensions, liquid preparations for internal use, emulsions, syrups, etc. In addition to these, various excipients, so-called wetting agents, sweeteners, flavoring agents, preservatives, etc. may be included in addition to water and liquid paraffin, which are commonly used simple diluents.

[0024] Preparations for parenteral administration include aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. The anticancer agent according to the present invention can be administered by any device that can transfer the active ingredient, tea fruit extract, to target cells. Preferred administration methods and preparations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drip injections, etc.

[0025] Injectables can be prepared using aqueous solvents such as physiological saline and Ringer's solution, or non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.).

[0026] Pharmaceutical carriers may be included, such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium metabisulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to prevent the growth of microorganisms (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0027] The anticancer agent according to the present invention may further contain a pharma- ceutical acceptable additive, which may include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carbonauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. EXAMPLES

[0028] <Tea seed extract> The tea cherries used were picked and harvested during the tea leaf harvest season. They were dried in the sun immediately after harvest. The outer shells of the tea cherries were cracked by drying in the sun. The pulp inside the shells was removed and further dried in a dryer. The drying temperature was about 55°C, and the drying time was about 12 hours. The pulp was then pressed to extract the pressed oil. The pulp from which the pressed oil was extracted is the pressing residue.

[0029] The pressed residue was pulverized with a hammer to produce a pulverized residue, the size of which ranged from a powdery state of less than 0.5 mm to approximately 3 mm.

[0030] 100 mg of the ground residue was added to 1 mL of a mixture of water and methanol with added acetic acid and suspended uniformly. The acetic acid content of the mixture was 0.2 mass to volume %, and the methanol content of the water and methanol mixture was 25 volume %. The mixture with the ground residue suspended was placed in an ultrasonic water bath for 10 minutes to extract the soluble components of the tea seeds (tea seed extract).

[0031] After the ultrasonic water bath, insoluble matter was precipitated by centrifugation, and 400 μL of the supernatant was filtered using a 0.45 μm membrane filter to obtain a tea bean extract (liquid) containing a tea bean extract.

[0032] The obtained tea seed extract (liquid) was diluted 100, 200, 400, 800, 1600, 3200, and 6400 times using a medium containing 10% FBS to prepare 1 mL of tea seed extract samples (hereinafter simply referred to as "samples") with the following concentrations: 1000 μg / mL, 500 μg / mL, 250 μg / mL, 125 μg / mL, 62.5 μg / mL, 31.25 μg / mL, and 15.625 μg / mL.

[0033] The 10% FBS-containing medium was prepared by adding 55 mL of FBS (Fetal Bovine Serum) (manufactured by CCP, model: CCP-FBS-BR-500) to 500 mL of RPMI-1640 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0034] In addition, to eliminate bias in the results due to the cytotoxicity of the solvent itself in the tea seed extract (liquid), the 0.2% acetic acid content and 25% methanol content in each sample were adjusted to a constant value. Specifically, 20 μL of 0.2% acetic acid-containing 25% methanol was added to 1 mL of control, and 10 μL was added to 1 mL of medium for dilution of samples with a concentration of 500 μg / mL or less.

[0035] <Cultured cancer cells> Cultured colon cancer cells SW480 and cultured gastric cancer cells KATO-III were purchased from American Type Culture Collection, Inc. All cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum under a 5% CO2 environment.

[0036] <Cell proliferation assay> Cells were plated in a 96-well plate at 5 × 10 3 The next day, the culture medium was replaced with a medium containing tea fruit extract (liquid) (62.5 μg / mL to 1000 μg / mL) or the extract solvent, and cell proliferation was measured after 24 and 48 hours using a WST-8 cell counting reagent (Wako Pure Chemical Industries, Ltd.).

[0037] <Cell migration and invasion assay> In vitro cell migration assays were performed using the Boyden chamber method. Cells were plated at 1 × 10 5 The cells were dispensed at 1 cell per insert and incubated at 37℃ in 5% CO2. Tea fruit extract (liquid) (1000μg / mL) or medium containing the extract solvent was added to the lower layer as a chemical attractant. The number of cells that passed through the insert was counted after 26 hours for SW480 and 24 hours for KATO-III. All assays were performed in triplicate, and the number of cells present in 5 randomly photographed fields was counted in a blinded experiment.

[0038] To confirm the effect on migration, a scratch assay was performed. SW480 or KATO-III cells were cultured at 3 × 10 5 The cells were dispensed into a 24-well plate at 100 cells / well, incubated at 37°C, and cultured to become confluent. The center was scratched using a 200-μL micropipette tip, and the cells were washed with PBS to remove detached cells. The medium was then replaced with tea fruit extract (liquid) (1000 μg / mL) or extract solvent-containing medium, and the scratched area was photographed 0 and 6.5 hours later using an EVOS FLoid Cell Imaging Station.

[0039] <Statistical analysis> All data are presented as mean ± standard error. Data were analyzed using Dunnett's test, and P values ​​< 0.05 were considered significant. Analyses were performed using GraphPad Prism version 8.0 (GraphPad Software).

[0040] <Result> [Effect of tea seed extract on cancer cell proliferation] Figures 1 and 2 show the effect of tea fruit extract on the proliferation of gastric cancer cells (KATO-III) and colon cancer cells (SW480). In each figure, (a) shows the results 24 hours after the start of culture, and (b) shows the results 48 hours after. In each graph, the vertical axis shows the absorbance at 450 nm, and the horizontal axis shows the sample concentration (μg / mL). In each graph, "*" indicates a significance level of 0.05 (P<0.05), "**" indicates 0.01 (P<0.01), "***" indicates 0.001 (P<0.001), and "****" indicates 0.0001 (P<0.0001).

[0041] As a result of the WST-8 assay, referring to FIG. 1, the addition of tea fruit extract significantly inhibited the proliferation of KATO-III cells in a concentration-dependent manner after 24 hours (250 μg / mL; P<0.05, 500 μg / mL; P<0.001, 1000 μg / mL; P<0.0001) and 48 hours (62.5 μg / mL; P<0.0001, 125 μg / mL; P<0.0001, 250 μg / mL; P<0.0001, 500 μg / mL; P<0.0001, 1000 μg / mL; P<0.0001).

[0042] Also, referring to Figure 2, the proliferation ability of SW480 cells was also significantly inhibited in a concentration-dependent manner after 24 hours (62.5 μg / mL; P<0.05, 125 μg / mL; P<0.01, 250 μg / mL; P<0.0001, 500 μg / mL; P<0.0001, 1000 μg / mL; P<0.0001) and 48 hours (125 μg / mL; P<0.0001, 250 μg / mL; P<0.0001, 500 μg / mL; P<0.0001, 1000 μg / mL; P<0.0001).

[0043] [Effect of tea seed extract on cancer cell migration] Furthermore, the effect on cell migration was examined using the Boyden chamber method. The results are shown in Figures 3 and 4. Figure 3 shows the results for KATO-III. Figure 3(a) shows the blank result, and Figure 3(b) shows the result in the case of tea seed extract (liquid) (1000 μg / mL). Figure 4 shows the results for SW480. Figure 4(a) shows the blank result, and Figure 4(b) shows the result in the case of tea seed extract (liquid) (1000 μg / mL).

[0044] Each figure consists of five photographs and one table. The five photographs (a) and (b) are photographs of cells that passed through the insert in the cases of Blank 1 and Sample 1, respectively. Note that Blank is the case where an extractant and a chemical attractant were added to the lower layer, and Sample is the case where tea fruit extract (liquid) and a chemical attractant were added. Blanks 1 to 3 and Samples 1 to 3 show the results for each experimental run.

[0045] The table also shows the number of cells that passed through the insert for each sample, with the average shown as "average" at the end of the table.

[0046] Referring to FIG. 3, the results for KATO-III were that in the blank, an average of about 120 cells passed through the insert (FIG. 3(a)), whereas in the case where tea fruit extract was added (FIG. 3(b)), about 1.3 cells passed through the insert.

[0047] Referring to FIG. 4, the results for SW480 showed that in the blank, an average of about 109 cells passed through the insert (FIG. 4(a)), whereas in the case where tea fruit extract was added (FIG. 4(b)), about 0.4 cells passed through the insert.

[0048] Thus, the effect of tea fruit extract (liquid) (1000 μg / mL) on cell migration ability was significantly suppressed in both KATO-III and SW480 (P<0.0001).

[0049] Furthermore, a scratch assay was performed to confirm the inhibitory effect of tea seed extract on cell migration. The results are shown in Figures 5 and 6. Figures 5 and 6 show the results for KATO-III and SW480, respectively. Each figure shows photographs for the control and tea seed extract at the start of the scratch assay (0 hr) and after 6.5 hours (6.5 hr). The distance between the vertical lines in each figure is the scratch width Sw. The scratch width Sw at 0 hr is represented as "Sw0" and the scratch width Sw after 6.5 hours is represented as "Sw65".

[0050] Referring to Figure 5, in the case of the KATO-III, the scratch width Sw65 6.5 hours after the start of the test was narrower than the scratch width Sw0 at the start of the test (0 hr) for the control. On the other hand, when the tea seed extract was used, the scratch width Sw65 6.5 hours after the start of the test was almost the same as the scratch width Sw0 at the start of the test (0 hr).

[0051] 6, similar results were obtained for SW480, that is, for the control, the scratch width Sw65 6.5 hr after the start of the test was narrower than the scratch width Sw0 at the start of the test (0 hr). On the other hand, when the tea seed extract was used, the scratch width Sw65 6.5 hr after the start of the test was almost the same as the scratch width Sw0 at the start of the test (0 hr).

[0052] The above results show that the addition of tea fruit extract (liquid) (1000 μg / mL) almost completely inhibited the cell migration ability of both KATO-III and SW480 (P<0.0001).

[0053] <Analysis of Tea Seed Extract> 100 mg of the ground residue described in the <Tea seed extract> section was weighed out and uniformly suspended in 1 mL of 25% methanol containing 0.2% acetic acid. This suspension was placed in an ultrasonic water bath for 10 minutes, after which the insoluble matter was precipitated by centrifugation, and 200 μL of the supernatant was filtered using a 0.45 μm membrane filter. The resulting filtrate was diluted 10-fold with 10% methanol containing 0.1% formic acid to prepare an analytical sample (10 mg / mL). Thermo Exactive was used for the analysis. The analytical conditions are shown in Table 1.

[0054] [Table 1]

[0055] The measurement results are shown in Figure 7. The top result is the LC with UV detection (280 nm), and the results below are all LC-MS results. Looking at the LC with UV detection (280 nm), it can be determined that there are three main peaks: 13.14 minutes, 18.43 minutes, and a relatively large peak after 18.43 minutes.

[0056] The second row is the MS of total ions (100-1000). The value "NL:5.69E8" written to the right of the chromatogram can be used as a rough estimate of the MS detection intensity.

[0057] From the third row onwards, the chromatograms are shown of the MS where the main peaks were selected in order of the earliest elution time. Although catechin in the fourth row was not considered to be the main peak, the molecular weight of the catechins was specified and displayed. The catechins have an NL of 8.10E5, which is about 0.14%, which is the ratio of NL:8.10E5 to the total ion NL of 5.69E8. Looking at this detection time (14.69 minutes) with the UV in the first row, no peaks are seen in that vicinity.

[0058] Considering that the λmax of catechin in an acidic state is 270-280 nm, it was not detected in the chromatogram at 280 nm. Therefore, even if catechins were present, they were in very small amounts. Therefore, it can be said that the ability of tea fruit extract to inhibit cancer cell proliferation and migration described above is not due to catechins.

[0059] As described above, the tea fruit extract (liquid) that is the active ingredient of the anticancer agent according to the present invention contains almost no catechins, but it has been shown to exert the function of inhibiting the proliferation and migration of cancer cells. [Industrial Applicability]

[0060] The anticancer agent containing the tea fruit extract of the present invention as an active ingredient can inhibit the proliferation and migration of cancer cells, and can be suitably used in the early stages of cancer detection and in the prognosis of cancer treatment.

Claims

1. An anti-cancer drug whose active ingredient is an extract from the residue of pressed tea seeds.

2. 2. The anticancer agent according to claim 1, wherein the tea seeds are "Yabukita" of Camellia sinensis.

3. The anticancer agent according to claim 1 or 2, wherein the extract is an extract using a polar solvent having a pH of 1 to 7.

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