TW200816926A - A black tea extract and uses thereof - Google Patents

Abstract

The present invention provides an ammonium hydroxide extract of black tea, characterized by producing three specific peaks at mass/charge ratio of 204.5, 226.3 and 368.6, respectively, when the ammonium hydroxide extract is subjected to electron spray ionization (ESI) mass spectrometry with a negative ESI state, a scanning range of 150-2000 mass/charge ratio, and a flow rate of 20 μl/min for the injection and LC pumps. The uses of the ammonium hydroxide extract of black tea of the invention in preventing sun-burn, inhibiting tumor growth and cancer metastasis are also provided.

Description

200816926 IX. Description of the invention: [Technical field of the invention] The present invention relates to a black tea extract, in particular to a gas-water stroke of black tea, which has the functions of preventing skin bruising, preparing for inhibiting growth of malignant tumors, and inhibiting cancer cells. The effect of the transfer. [Prior Art] Black tea is a popular beverage. During the process of making black tea, the polyphenols in the tea will undergo oxidative polymerization by polyphenol oxidase, resulting in bisflavanol and theaflavin. , the formation of thearubigin and other oligomers (oligomers). (Wei et al,

Scavenging of hydrogen peroxide and inhibition of ultraviolet light-induced oxidative DBA damage by aqueous estracts from green and black teas. Free Radio Biol Med. 1999, Jun; 26(11-12): 1437_35; Lambert et al., Inhibition of carcinogenesis by polyphenols : evidence from laboratory investigations. Am J Clin Nutr. 2005 Jan; 81(1 Suppl): 284S-291S, Review). In the aqueous extract of black tea, the solid of μ2〇/〇 is the theaflavin which provides the color and taste of black tea, and the water extract of most black tea is thearubigin, which has a wide range of molecular weight but characteristic of nature. Not yet certified. Previous studies have found that black tea extracts have anti-oxidant, anti-carcinogenic and anti-mutation effects (Lambert et al., 2005), and it is generally assumed that these effects are derived from polyphenols in black tea, such as theaflavins and thearubigins. In the fermentation process of tea leaves, theaflavins and thearubigin are products of oxidation and condensation reactions. In recent years 5 200816926, several methods for extracting biologically active substances from black tea have been developed. The most commonly used solvents in the initial extraction step are boiling water and ethanol, followed by a series of organic solvents to obtain polyphenols or tea pigments. In 2002, Hung et al. reported that black tea pigment can be obtained by ammonia water extraction and subsequent organic solvents, and further studies have found that these pigments have an improved effect on magnetic resonance imaging (Teng I) and hydrazine. Liver damage caused by (Hydrazine) has protective effects. Hung et al. believe that the extracted black tea pigment is meianin (Hung et al.' Gastrointestinal enhancement of MRI with melanin

Drawn from tea leaves (Thea sinesis Linn.). J

Ethnopharmacol. 2002 Jan; 79(1): 75_9; Hung et al.

Protection of tea melanin on hydrazine-induced liver injury.

Life Sci· 2003 Jan 17; 72(9): 1061-71). Black tea extracts having the effects of inhibiting the growth of malignant tumors and inhibiting the metastasis of cancer cells have not been reported in the foregoing studies and reports. [Summary of the Invention] The present invention is a m纟X tea extract obtained by extracting fine ammonia water, and the characteristics of the red money water extract are determined by electrospray ionization mass spectrometry (ESI_MS) as negative electrode=sprinkle free state. The scan range is from 15G to · f charge ratio, and when the flow rate of the injection pump and the LC pump are 2 〇 microliters/min, the mass/charge ratio is about 2〇4·5, 226 3 and 368 respectively. 6 of the three special values. On the other hand, the present invention provides the use of the black tea extract, and the composition thereof for preventing sunburn, 200816926, and the use of the ===:: scavenging free radicals. In one aspect, the present invention provides the black tea extract for use in antioxidants and compositions thereof for use in antioxidants.

Further, in another aspect, the present invention provides the use of the black tea extract for inhibiting cancer, cell metastasis, and a pharmaceutical composition for preventing metastasis of cancer cells. [Embodiment] The present invention provides a black tea extract obtained by extracting ammonia water, which is characterized in that the eosin residual ammonia aqueous extract is subjected to electrospray ionization mass spectrometry (ESI_MS) under the condition of a negative electric current, a spray free state, and a scan. In the mass/charge ratio ranging from 15〇 to 2500, and when the flow rate of the injection ▲ floc and LC pump is 20 μl/min, the mass/charge ratio is generated, which is about 204.5, 226·3 and 368.6. Specific peaks. According to an embodiment of the present invention, the method for preparing the black tea ammonia water extract comprises: immersing black tea leaves in Μ0% ammonia water and filtering to obtain filtered 'night, ✓ chen, monk the filtrate and performing acid titration (pH is in the range) Inside), the solution obtained by titration is then centrifuged, and the supernatant is removed to obtain a precipitate. The precipitate is then placed at -VC to -8 (TC is allowed to freeze and after the thawing of the precipitate is subsequently removed) to remove the red residue aqueous ammonia extract. 7 200816926 According to another embodiment of the invention, after the supernatant 'You can also carry out the second extraction, === solution to remove the M0% ammonia water and then concentrate it, then carry out the acid drop ^t substance in the solution), and then put it to the Luyi I value in the 1-4 range After the beads, remove the supernatant. Finally, the knot will be 4 ° and then the sink will be a black tea ammonia extract. W ^ 仃 仃 freeze drying to take in a specific embodiment of the invention,

V has the first-time and second extraction steps, according to the shell, "to be taken, and finally, to (4) the object is cooled; the extract is continued. Therefore, the extraction step 2 of the invention: #黑茶的氨水The extraction method is not limited to a specific extraction number. According to the above extraction method of the present invention, according to the material analysis, the 4^2 water-slipping state is scanned from 150 to 25 〇; =^^ The flow rate of the second and LC pumps is 2 〇 microliters per minute. The second generation is 204'5^ 2263 # 368·6 m The peak value of the face is analyzed. Other black tea extracts = the clothes of the shirt (four), the red money of the present invention The water extract does have a physical property of 4 inches, which is different from the main active ingredient melanin of the black tea ammonia water extract described in the prior art. The black tea ammonia water extract provided by the present invention is novel. Further 'via spectroscopic spectrometer Spectrophotometer analyzes the absorbance of the black tea ammonia water extract of the present invention, and finds that the black tea ammonia water extract of the present invention can absorb light having a wavelength range of 200-400 nm, which covers the wavelength of ultraviolet light, and confirms that the black tea ammonia water extract of the present invention has ultraviolet light absorption. It 8 200816926 effect, can be used for sun protection. In addition, in the test for scavenging free radicals and reducing power, it was also confirmed that the black tea ammonia aqueous extract of the present invention has the ability to scavenge free radicals and reducing power. According to the present invention, the present invention provides a The use and composition for preventing skin sunburn, which comprises the use of the black tea ammonia aqueous extract of the present invention as an active ingredient, /, a drop-off or a cosmetically acceptable carrier, because most skin sunburns are long-term exposures. The ultraviolet light exposure in the sunlight is caused, and the aqueous extract of the red tea of the present invention can effectively absorb the ultraviolet light in the sunlight, and can effectively prevent the skin from being sunburned. It can also be used as a cosmetic composition or a pharmaceutical composition, etc. According to the present invention In a preferred embodiment, the method for preparing the skin-inhibiting composition is the active ingredient of the black tea ammonia aqueous extract of the present invention, and the medical or cosmetically acceptable carrier, including water, emulsion, cream, ^Poor or a combination of them. And because the purpose of this group of money is to prevent = sunburn, the composition can be made into different forms, including sprays, ", lotion, pore cream or mask method, in order to facilitate the need for users to use the 'money frequency or dose can be adjusted according to the user's actual needs.,, mouth & the present invention to prevent skin sunburn The composition may optionally include makeup: an adjuvant or additive for hanging, such as an emulsifier, a tackifier, an interfacial active chelating agent, an antibacterial agent, a preservative, a coloring agent, a fragrance, etc. The professional knowledge, experience and needs of the company are selected from the appropriate substances and the amount of the additives is determined. Further, the composition for preventing sunburn of the present invention may further contain other active agents to provide additional skin. Benefits. Common active agents include, but are not limited to, Moisturizer II 9 200816926 Humectants, antioxidants, sunscreens, anti-aging agents, whitening, and emollients. Month Months and provide a free radical scavenging material and pharmaceutical composition. ^中=乐组成物 The extract of the black tea ammonia water of the present invention is used as an active ingredient: a medically acceptable excipient is mixed. The medically acceptable excipients include binding agents, fillers, and stabilizers. Further, the present invention also provides an anti-oxidation use and a doctor's advice. == The extract of the broad-band tea ammonia aqueous extract is used as an active ingredient; The medical agent includes a binder, a money filling, and a stabilizer. Then again! The invention also provides a use for inhibiting the growth of malignant tumors and a pharmaceutical active ingredient, which is mixed with a medically acceptable agent, and includes an excipient, a filler and a stabilizer. With scorpion cancer. The use of the basal cell, the basal cell, and the use of the present invention - a preferred embodiment of the present invention, the black tea ammonia of the present invention, can be transferred to the rectal cancer cells and the mobility of the needle Invasive ability. Silk cancer cells = tree _ real _, the hair of the "medicine composition system,", wood milk water draw is active age, (four) academically woven shirt w-shaped agent mixed 200816926, which is medically acceptable The excipient includes a binder, a filler and a stabilizer. Similarly, the pharmaceutical composition can be prepared into a drip, a syrup, a powder, a tablet, a capsule, a spray or a patch to facilitate the user's taking. However, the pharmaceutical composition of the present invention is not limited to the above several forms, and anyone skilled in the art can clearly understand that other modes of administration of the above composition are within the scope of the present invention. The pharmaceutical composition of the present invention can be based on those skilled in the art. The appropriate amount of the composition of the present invention can be used according to its own expertise, experience and needs. The frequency and dosage can be determined according to the severity of the symptoms, or according to the instructions or recommendations of a physician or the like. The present invention is specific but not limited The examples are described in more detail. Example 1: Preparation and identification of black tea ammonia water extract 1.1 Preparation of ammonia water extract The black tea leaves are boiled in hot water for 10 minutes, and boiled for cooking. The volume of the red tea leaves was soaked in 2% ammonia water at room temperature and stirred overnight at room temperature. The next day, the filtration was carried out, and the filtrate was treated under vacuum at a vacuum concentration machine at 50 ° C to make the total volume before concentration. 1/2. The obtained concentrate was titrated to ρΗ2·5 with 5N hydrochloric acid, and allowed to stand at room temperature for 4 hours, and then centrifuged at 10,000 g for 10 minutes, and the supernatant was removed to the -20 ° C refrigerator. Freeze. After thawing, remove the supernatant, dissolve the precipitate in 2% ammonia water before the acid titration, carry out the second extraction, and treat the remaining total volume at 5 CTC with a vacuum concentrator with a rotation speed of 90 rpm. 1/2 to 1/3. Acid titration to π 200816926 ρΗ2·5, and allowed to stand at room temperature for more than 4 hours, then transferred to a -20 ° C refrigerator to completely beaded. Remove the supernatant liquid. Add the 2% ammonia water in the volume before the second acid titration to dissolve it. Concentrate again, repeat the first and second extraction methods, and finally place the precipitate at _80 °C. Hours, in a freeze dryer (dura_DRY; FTS Systems, Inc. San Francisco, USA) Drying. The obtained dried product is the black tea ammonia water extract of the present invention. 1. 2 Electrospray ionization mass spectrometry (ESI-MS) The dried black tea ammonia water extract obtained in the foregoing Example 1.1 is electrically treated with other forms of extracts. A comparison of the analysis by spray free mass spectrometry, the extracted extracts include an aqueous extract prepared by a conventional method, an ethyl acetate extract and a 70% ethanol extract, wherein the aqueous ammonia extract and the aqueous extract are 1 Torr. The (mM) aqueous ammonia solution was redissolved into a solution of concentration μg/μl (μ§/μ1), while the other two extracts maintained the fluid state without further processing. The soil was continuously injected into the sample in a 250 μΐ syringe. The τ-type valve is used to connect the injection, and the pipeline of the pump and the LC pump is sent to the electric spray free source, and the current velocity of the above-mentioned soil is 20 μΐ/min (minutes). The average spectrum was obtained by completely scanning each of the first few minutes at a mass/charge ratio range of 150 to 25 Torr in the free state of the negative electrode. As shown in Figure 1 (8), the mass/charge ratios of the black tea ammonia extracts in the present invention are three specific values of lang, 226 3 and 368 6 respectively, and can be found in the mass spectrometric analysis of other black tea extracts. The mass/charge ratios of water extraction were (10)·8, 336.6 and _, respectively (the mass/charge ratios exhibited by the 12th 200816926 萃取 酉 酉 extract were 348 8 , 44 〇 6 and 883.0 respectively (Fig. i (10), and The ethanol extract is different from the side, the coffee and the rush ($1_. According to the mass spectrometry analysis above), it can effectively distinguish the water extract from the water, the acetic acid extract and the ethyl acetate. Unless otherwise specified, the black tea extract used in the following examples is the black tea ammonia water extract of the present invention prepared according to the present embodiment. Example 2: Ultraviolet absorption analysis of the black tea ammonia water extract of the present invention 2·ι mouse anti-UV test 8.1〇 weeks old retire cultivar (ICR) mice (purchased from the National Animal Center) After anesthesia with Avertin (15 ml/kg), the back hair was scraped off, as in the first 2 is divided into three areas, painted with water (area b), containing 丨% black tea extract A cream made from the method (area d) and a blank cream without extract (area c). Depending on the application dose (2 mg/cm2 and 2〇mg/cm2) and different UV wavelengths (UV) Light A (UV_A): 320 to 400 nm, UV_B: 290 to 320 nm, UV-C: 200 to 290 nm) The above-mentioned small gas is divided into 6 groups of at least 2 in each group, except for the part to be irradiated. The rest

After being covered with a covering, it was irradiated in a UV-crosslinker (produced by Stratalinker). UV-A irradiation time is 60 minutes (equivalent to 144, 〇〇〇 joules) ' IJV-B irradiation time is 30 minutes (equivalent to 72,000 joules) and UV-C 5 irradiation time is minutes (equivalent to 12,000 joules), and Continue to observe changes in mice. 13 200816926 Four days later, the mice were anesthetized, and the lower back of the page was coated with different skin sounds, soaked in 10% neutral fumarin, and embedded in paraffin, sectioned with Hematoxylin & Eosin (HE)) staining. As shown in Fig. 3, the skin of the mouse without any treatment (ie, area a in Fig. 2) (Fig. 3 (8)) or only water (Fig. 3 (b)) is irradiated with UV-B. After that, the epidermal cell layer increased significantly. Compared to the skin of mice that have not been treated with any treatment or only with water, application of mouse skin at a dose of 20 mg (containing 1% black tea ammonia extract cream) / cm 2 (skin area) can completely inhibit UV Skin hyperplasia caused by -B (Fig. 3 (d)), and skin whitening alone can only partially inhibit skin hyperplasia caused by ultraviolet rays (Fig. 3 (4)). Further, as shown in Fig. 4 and Fig. 5, the skin of the mouse treated with the black tea ammonia aqueous extract of the present invention can also significantly block the skin hyperplasia caused by 1^^8 or 11 (^). The black tea ammonia water extract of the invention can effectively protect the skin from skin damage caused by UV-A, UV-B and UV-C, and achieve the function of protecting the skin. 2.2 Absorbance value test dissolves black tea extract into secondary water After 20 gg/mi solution, the absorbance of the black tea ammonia aqueous extract of the present invention was measured by a spectrometer (BECKMAN DU9640 spectrophotometer) at a wavelength range of 200 to 600 nm and a scan rate of 1200 nm/min, and the ultraviolet light-visible absorption spectrum was recorded. 0 14 200816926

As shown in Fig. 6, the absorbance value of the black tea ammonia water extract of the present invention is distributed in the range of wavelengths of 200 to 400 nm, and the range covers υν·Α, uv_B and UVC hybrids, thereby confirming the tree makeup Wei The water extract can absorb the ultraviolet light to protect the skin. 2. 3 scavenging free radical test - 0.05% of f broken 1 of the present invention, black tea ammonia aqueous extract solution, 5 wins of country P (IU) of vitamin E, 01 mg / nil of vitamin C, 2 mg / ml BHT • and 0 05% synthetic melanin (melanin) was mixed with 4〇〇μ1 of 〇1M Tm-HC1 (ρΗ7·4), and then added 5〇〇(4) of 礼$礼u_diphenyl-2-picrylhydryl (DPPH) Mixing was allowed to stand at room temperature for 2 minutes and then detected at an optical density (OD) of 517 nm. The Benximing black tea ammonia water extract solution reacts with the 〇??^^ reagent, and the result is as shown in Fig. 7, the bribe is a miscellaneous group, and the light absorption value is eliminated. RESULTS: The black tea ammonia water extract of the present invention and the m photo of the known free radicals show that the ammonia aqueous extract of the present invention has the ability to scavenge free radicals. The effect of the black tea ammonia water extract of the present invention on scavenging free radicals is better than that of synthetic melanin. . 2.4 Reducing power test 250 μl of 0.05% of the black tea ammonia extract of the present invention, 5 kj of vitamin Ε, 0·1 mg/mi of vitamin C and 2 mg/ml of hydrazine respectively with 25 〇 of pH 6.6 PBS and 250 μM 1% K3Fe(CN)6 mixed, placed in 5〇t: water/slot 20 knives. After the action, transfer to 250 μΐ of tcA on ice, 15 200816926 and mix evenly and centrifuge at 6, _ for 1 〇. The supernatant was taken at 45 〇 μΐ plus an equal volume of water and 15 μl of 〇.1% FeCl3_ ′ was allowed to stand at room temperature for 5 minutes, and measured at an optical density of 700 nm. The main principle of the reducing power test is to determine the amount of Prussian blue (ferric iron cyanide, (4) Qing] 3) after the reaction, as a high and low index of reducing power. As shown in Fig. 8, the black tea ammonia aqueous extract solution, the synthesized melanin and the known reducing power BHT, vitamin E and vitamin φ were compared with each other, and the black tea ammonia aqueous extract and the melanin, BHT, and the present invention were shown. Vitamin E has the same reducing power as vitamin C. Example 3 · Analysis of cancer cell growth and metastasis 3.1 Cell culture and growth analysis The human rectal cancer cell lines Hct η 6 and SW480 used in the laboratory and the HeLa line of the cervical cancer cell line were purchased from the food industry development research. The Center for Biological Resource Conservation and Research (Hsinchu, Taiwan), the skin cancer cell line A431 • and the basal cell carcinoma cell line (BCC) (Jee et al., 0ncogene 20(2)· 198-208, 2001) by Guo Dr. Bright (National Taiwan University Medicine - Hospital, Department of Toxicology). All cell lines were cultured at 37 in DMEM (Dulbecco's modified Eagle's medium) medium (Biowest) containing 10% fetal bovine serum (produced by Bi〇west). € Tissue incubator with 5% carbon dioxide. First, the cells were seeded at a density of 5×l (a density of 1 day in a six-well plate, followed by a false (m〇ek) treatment (not treated with the black tea ammonia aqueous extract of the present invention) or at different concentrations (25 16 200816926 gg/ml and The cells were treated with the black tea ammonia aqueous extract of the present invention at 125 pg/m. After 48 hours, the vertebral blue discharge test was performed (t ring n-exdusi (10). The number of viable cells was counted using a hemocytometer. As shown in Fig. 9, the rectum The cancer cell lines HCT116 and sw4(10) were inhibited by 30-40% and 50_60/, respectively, after being treated with μ gg/ml and 125 pg/ml of the black tea ammonia aqueous extract of the present invention for two days.结果. The results show that the black tea ammonia water extract of the present invention can significantly inhibit the growth of rectal cancer cells in vitro. In addition, the black tea ammonia water extract of the present invention can also inhibit the cervical cancer cell line He e L a , The growth of the skin cancer cell line A431 and the basal cell carcinoma cell line proves that the red residual ammonia water extract of the present invention has the effect of inhibiting growth of different cancer cells. 3.2 Flow number cytometry analysis to investigate whether the cell growth slowdown is increased or not Related to cell death, cells were fixed in ice-cold 70% ethanol for 3 min, followed by 2 〇〇 from §/1111 10 RNAse A (Pr〇mega produced) and 5 〇yg/ml propidium iodide (propiciium) Io (d) e) (produced by Sigma) The cells were treated at 37 ° C for 30 minutes, and the stained cells were analyzed by flow cytometry (BD Bi〇science). Endonucleases activated by the caspase pathway Cleavage of chromosomal DNA symbolizes apoptosis, and the breakdown of apoptotic cells causes the total amount of DNA to decrease to a lower amount than that of cells during the cell growth (G1) period. As a result, the DNA of apoptotic cells is lower than the DNA profile. The peak amount of cellular DNA during the growth period. As shown in Fig. 1, the rectal cancer cell lines HCT116 and SW48〇 cells treated with 25|^/1111 of the black tea ammonia aqueous extract of the present invention 17 200816926 accounted for the cell population in the sub G1 period, respectively. 44% and 53%. Conversely, only 2% and 5% of human G1 0 phase cells were detected in the pseudo-treated HCT116 and SW480 cells. These results show that the black tea ammonia extract of the present invention can significantly cause the rectum Cancer cell death. 3. 3 cancer cell migration test ( Migration assay) - The cancer cells are seeded in 6-well plates and cultured in DMEM medium supplemented with 〇% fetal bovine serum until the cancer cells are overgrown (eonf|uence), and the cancer is scraped with a sterile 25 μm pipette tip. The cell monolayer was used to create a cleaned zone, and the cells were moved to the cleaned area within 48 hours under a microscope. The image was taken every six hours while scratching the monolayer of the cancer cell and after the scraping, and the measured migration rate was the moving distance (micron time (hours). As shown in Fig. 11, the invention was 25 pg/ml The rectal cancer cell lines HCT116 and SW480 cells treated with black tea ammonia extract reduced the migration rate by 53% and 64%, respectively, compared with the pseudo-treated φ HCT116 and SW480 cells, and the 125 mg/ml black tea ammonia water extract of the present invention. The treated HCT116 and SW480 cells reduced the migration rate by 89% and 94%, respectively. Therefore, treatment of rectal cancer cells with the black tea ammonia aqueous extract of the present invention can significantly reduce the migration ability of rectal cancer cells. 3.4 Artificial basal layer invasion test ( Matrigel invasion assay) The cell invasion assay is in the BioCoatTM MatrigelTM Invasion Chamber (Becton-Dickinson? 18 200816926)

Bedford, ΜΑ) is analyzed according to the steps provided by the manufacturer. The cells (2xl〇5) in 500 μl of DMEM medium were added to each well and placed in a tissue culture incubator containing 5% carbon dioxide at 37 ° C for any time to invade the artificial basal layer for 48 hours. The non-invasive cells on the upper surface of the trough membrane were removed, and the invading cells on the lower surface of the trough membrane were fixed with 100% methanol, followed by cell staining with 〇·5% crystal violet solution (Sigma). After washing twice with distilled water, the tank was air dried. The number of invading cells in the visual field was calculated under a phase contrast microscope, and the mean standard deviation of the number of cells in five different fields of view under the microscope was calculated. As shown in Fig. 12, after treatment of rectal cancer cell lines HCT116 and SW480 cells with 25 Hg/ml of the black tea ammonia aqueous extract of the present invention, the number of invasive cells was reduced by 85% and 97%, respectively, and 125 pg/ The treatment of HCT116 and SW480 cells by the ammonia aqueous extract of the present invention of mi of the present invention reduced the number of invasive cells by 97% and 99%, respectively. These results show that the black tea ammonia aqueous extract of the present invention can also effectively prevent the invasion of cancer cells. [Simple description of the diagram] Figure 1 is a graph of the analysis of different black tea extracts by electrospray ionization mass spectrometry. (a) is an extract of black tea ammonia water of the present invention; (b) is an aqueous extract; (c) is an ethyl acetate extract; and (d) is an ethanol extract. Figure 2 is a macro image (macr〇sc〇pic jmage) showing the skin of a far-parent breed (ICR) mouse after shaving the hair. The skin is marked with a, b, c, d. Different processing areas. 19 200816926 Fig. 3 shows the magnification of the skin tissue cut in the back skin of the outbred mice and then treated with IJV-B. (a) 400 skins without any treatment; (e) skin with a blank cream; (b) skin with a water-ammonia extract cream. Horse smear containing 1% black tea Figure 4 shows the skin tissue sections obtained by UVC irradiation on the back of the overcrossed mice. Talk about: Magnification after different treatments. (8) Skin that has not been treated to _ times skin; (8) Skin that is applied with a blank cream; Water of the water-washing cream. ',', smear 3 1% black tea ammonia Figure 5 shows the magnification of the far-off product. Skin/Slice 'This image has double skin. (8) For the skin that has not been treated, (8) For the analysis of the black water ammonia extract of the present invention, the sample of the black tea ammonia water extract of the present invention is used to remove the tea ammonia water extract and other known reducing powers. The black tea ammonia water extract of the present invention and others have been Knowing the length of the sample = the data of the present invention, the pure water extract inhibits the growth of cancer cells 20 200816926 The figure shows the effect of analyzing the black tea ammonia aqueous extract of the present invention on the death of rectal cancer cells by flow rate cytometry. Fig. 11 is a bar graph for analyzing the ability of the aqueous extract of black tea of the present invention to inhibit the migration of rectal cancer cells. Fig. 12 is a bar graph for analyzing the invasive ability of the black tea ammonia aqueous extract of the present invention to inhibit rectal cancer cells.

twenty one

Claims (1)

200816926 X. Applying for the patent garden: & ★ Kind of red money water extract' is derived from the scent of water, which is the black tea ammonia extract money _f score _ class condition is 贞 电 嗜 ^ ^ The state of the 'scanning range is within the charge ratio of 15G to ·, and the flow rate of the injection pump LC pump is 2 〇 microliters / minute, resulting in three specific peaks of mass / charge, applying .5, 2263 and 368.6 . ] 2'If the application surface _ the first reward of the black tea ammonia water extract, the black tea ammonia water extract is prepared by the method comprising the following steps: Μ (8) the red glutinous rice immersed in the ammonia water and still transfer the transition liquid; (b) The filtrate is concentrated and subjected to acid titration; and (e) an aqueous ammonia extract is obtained by obtaining a precipitate. 'The preparation method 3_, as claimed in the application of the formula, the black tea ammonia water extract method described in the second item further includes after step (c): (0 completely freezes the sink; and (ii) thaw the precipitate 4. The upper layer liquid is removed. 4. The black tea ammonia water extract according to claim 2, wherein in the step (9), the filtrate is concentrated by a vacuum decompressor at 5 〇〇c. The composition of the black tea ammonia water extract according to the above-mentioned patent application, which further comprises a medically acceptable carrier. 6. The composition of claim 5, wherein the carrier comprises Water, lotion, cream or cream. 3 7. If the application of m® is the domain of the 5th, the injection and the medical treatment can be used to receive the excipients. The composition of claim 7, wherein the jujube comprises a combination, a filler or/and a stabilizer. 9. A composition for preventing skin damage, comprising the black tea ammonia water as described in the scope of the patent application. Extraction _ miscellaneous ingredients 鄕 cut to accept the carrier. -1 〇 _ as claimed in the scope of the ninth item To prevent skin sunburn composition ^ Among them, the agent includes water, lotion, cream and cream. 11.11·Please please fine _ 9 willow (four) stop skin _ domain, which can be made into a solid agent, milk T, lotion , cream or mask. 12.1 A free radical pharmaceutical composition comprising the black tea aqueous ammonia extract as described in the scope of the application of the patent application as an active ingredient and a commercially acceptable excipient. An oxidized pharmaceutical composition comprising a black tea aqueous ammonia extract as described in the scope of application of the patent application as a heterogeneous component and a pharmaceutically acceptable excipient. The application of the patent scope of the black tea ammonia water extract described in the second paragraph is an active ingredient and a medically acceptable agent. The pharmaceutical composition for inhibiting metastasis of cancer cells, which comprises the content of claim 1 of the patent scope. The black tea aqueous ammonia extract is an active ingredient and a scientologically acceptable excipient. The pharmaceutical composition according to claim 12, 13, 14, or 15, wherein the excipient includes a binder and a filler. Or / and stabilizers. The pharmaceutical composition according to Item 12, 13, 14 or 15 may be prepared as a drip, a syrup, a powder, a medicinal tablet, a capsule, a spray or a patch according to paragraph 23, 2008. A pharmaceutical composition for inhibiting the growth of a malignant tumor, wherein the malignant tumor includes a rectal cancer, a cervical cancer, a skin cancer, or a basal cell carcinoma. 19. The pharmaceutical composition for inhibiting metastasis of cancer cells according to claim 15 of the patent application, wherein Cancer cells include rectal cancer cells. 2. A black tea aqueous extract is used for the preparation of a composition for preventing sunburn. The black tea aqueous extract is a black tea gas-water extract as described in the patent application (3). A kind, * ammonia water stroke for the preparation of a free radical-decomposing pharmaceutical composition. The feng shui extract is a black tea gas-water extract as described in the application of the patent scope. The preparation of a pharmaceutical composition for inhibiting the growth of a malignant tumor is as described in claim 1 of the patent application, wherein the black tea aqueous extract is used for the use of the black tea ammonia water extract. • A kind of black tea ammonia water extract for use in wearing, wherein the black tea ammonia is used as a pharmaceutical composition for cancer cell transfer, and the black tea ammonia water extract is used. The use of the invention is as described in claim 1, wherein the malignant tumor includes rectal cancer, cervical cancer, skin cancer, or basal cell carcinoma. 26. The use of claim 24, wherein the cancer cells comprise rectal cancer cells. 25