TWI235664B - Extract from the leaves of Toona sinensis Roem., and the preparation process and uses thereof - Google Patents

Abstract

Disclosed are extracts from the leaves of Toona sinensis Roem. prepared by extracting the leaves of Toona sinensis Roem. using water and an alcohol in sequence. Also disclosed are processes for preparing extracts from the leaves of Toona sinensis Roem., and uses of such extracts in the manufacture of medicaments for use in the treatment of ovarian cancer and/or bladder cancer.

Description

1235664 发明 Description of the invention: [Technical Field of the Invention Λ] Field of the Invention The present invention relates to the extract 5 and its preparation method derived from the leaves of Toona sinensis It is used for the treatment of ovarian and / or bladder cancer. T litr ^ Background of the invention Toon (Latin Toona sinensis Roem or Cedrela 10 sinsensis, English name Chinese mahogany cedar or Chinese Toona) is a species belonging to the family Polygonaceae (Meliaceae) is a perennial deciduous tree plant with bark brown and young leaves are edible and can be harvested throughout the year. Toon is native to southeast China, southwest to north China, and is now grown worldwide (Jennifer M Edmonds and Martin Staniforth, TOONA 15 SINENSIS {Meliaceae), Curtis's Botanical magazine, 15 (3) 5 186-193, 1998; Xiao-Dong Luo et al., Fitoterapia, 71? 492-496, 2000; Jong-Cheol Park et al.? Kor. J. Pharmacogn, 27 (3), 219-223, 1996). Toona sinensis has become a tree species with extremely high economic value because the whole plant can be used. According to literature reports, each of Toona sinensis Plant parts (seeds, root bark, bark, leaf axis, leaves) have health or healing functions (Edmonds JM & Staniforth M, 1998, supra; Park JC α / · 5 1996, supra). In terms of food, 'Xian Juan Xuan Oil is very suitable because of its colorless and scented aroma. 1235664 Cooperates as a food oil. The buds and leaves of Toona sinensis are rich in nutrients such as carotene, amino acids and vitamins, and are very popular. The old leaves of Toona sinensis can be used as high nutritional value feed for livestock. In the medicinal aspect, according to the literature (Edmonds JM & Staniforth 5 M, 1998, supra; Xiao-Dong Luo α /., Supra; Park JC W α /., 1996, supra), the bark, root bark and seeds of Toona sinensis are used to treat neuralgia, hemostasis, cold expelling, analgesic, stomach, duodenal ulcer, gonorrhea, menstrual disorders, roundworm, typhoid suppression , Anti-amoebia, rheumatoid arthralgia and anti-cancer (Yu Mingming and Zhang Zedang, Jounal of Anhui 10 University Natural Science Edition, No. 4, 91-94, 1990; Liu Yuezhen and Li Yuping, He North Forestry Science and Technology, No. 4, 51-52, December 1997). According to the literature, Toona sinensis leaves have anti-inflammatory, detoxifying, insecticidal, curative effects on enteritis, dysentery, anthracnose, scabies, lacquer sores, scabies, white baldness, and medicine for improving physical fitness. In addition, according to legend, after drinking the water extract of Toona sinensis leaves, it was found that the symptoms of hypertension and branuria could be improved. Recently, Xu Shengguang et al. Studied aqueous extracts of Toona sinensis leaves, and found that the aqueous extract of Toona sinensis leaves can reduce blood glucose in diabetic rats induced by Alloxan (Wang PH et al., Toona Sinensis increase GLUT4 glucose 20 transporter protein in adipose tissue from Alloxan-induced diabetic rats, Annual Conference of Biomedical Science, p. 198, 2001), and in another study, it was found that the aqueous extract of Toona sinensis leaves inhibited human lung adenocarcinoma A549 (human lung adenocarcinoma cells A549) proliferation effect (i / WC / n'w C / mwg 1235664 al · (2002), American Journal of Chinese Medicine, Vol · 30,

Nos · 2 & 3, 307-314. In other aspects, 'Toon leaves can be used as a dye or glaze. In addition, Toon's wood is solid, detailed, not warped, cracked, and wet. Therefore, Toon's wood is often used as a material for high-end furniture, shipbuilding, bridges, etc., and also used in the manufacture of badminton rackets, soldiers Racket, tennis racket and musical instruments. Toon is also an afforestation tree species that prevents earth and rock flow when applied to afforestation. In addition, toon wood oil can be extracted from the wood of toon, and the oil 10 can be used as a flavoring agent for cigars. To the best of the applicant's knowledge, there is no literature or pre-patent case that has stated or implied that "Toon leaves or extracts derived from toon leaves have the activity of inhibiting the growth of ovarian cancer cells, and therefore can be used for preparation For the treatment of ovarian cancer. " [Summary of the Invention] SUMMARY OF THE INVENTION Therefore, in a first aspect, the present invention provides an extract obtained from toon leaf, which is prepared by a method comprising the following steps: Prepared by a method 20 comprising the following steps: (1) extracting toon leaves with water under heating to obtain a first extract with water extraction, and (2) extracting step with an alcohol (1) The obtained water-extracted first extract is obtained as an alcohol-extracted second extract. 1235664 In a second aspect, the present invention provides a method for preparing an extract of Toona sinensis leaves, which comprises the following steps: () extracting the leaves of Citrus curd with water under heating to obtain an aqueous extract solution (b) drying the aqueous extraction solution obtained from step (a) to obtain a dried first extract; (c) dissolving the first extract obtained from step (b) in an alcohol solvent To form an alcohol extraction solution; and (d) removing the alcohol solvent from the alcohol extraction solution obtained in step (c) to dryness to obtain a dried second extract. The extract of Toona sinensis leaves according to the present invention has been shown to be effective in inhibiting the growth of ovarian cancer cells (especially SK0V3 and PA_υ. Therefore, in a third aspect, the present invention provides a pharmaceutical composition comprising The above mentioned and other objects, features, and advantages of the present invention are measured in accordance with the above-mentioned and other tonic leaf extracts from the growth of nested cancer cells. After attaching the attached drawings, it will become obvious' in the drawings: the drawings briefly illustrate FIG. 1—a water-extracted product derived from toon leaves (that is, the “Toon” obtained in Example 021 described later) 5_4 ,,) an HPLC profile; Figure 2 is an alcohol-extracted voluminous leaf extract (that is, obtained in Example 1 of the text) obtained according to a preferred embodiment of the method of the present case. "Toon 5-2 ,," one of the HPLC elution diagrams;! 235664 Figure 3 shows the cytotoxicity of 5 toon leaf extracts obtained by different purification treatments on bladder cancer cell line T24 Effectiveness (cytotoxicity effect), which H20, 50% EtOH, and 99.5% EtOH, which were used as the extraction solvents for these toon leaf extracts, were used as a control group 5; Figure 4 shows toon leaf extracts obtained according to one of the preferred embodiments of the method in this case ("Toon 5-2") for the growth inhibitory effect of different cancer cell lines, where the control group is the experimental group without the extract of Toona sinensis; Figure 5 shows the Toona sinensis obtained according to a preferred specific example of the method of the present case Effect of 10-leaf extract ("Toona sinensis 5-2") on the cell morphology of ovarian cancer cell line SKOV3 at different concentrations (1 mg / ml, 100 pg / ml), of which the control group was taken without leaves Fig. 6 shows the Toona sinensis leaf extract ("Toona sinensis 5-2") obtained according to one of the preferred embodiments of the method at different concentrations (1 mg / m, 10 pg / ml). 15 For Cell morphological effects of ovarian cancer cell line PA-1, in which the control group was the experimental group without the extract of Toona sinensis leaves; and Figure 7 shows that nude mice with tumors grown by ovarian cancer cells were administered at low doses and high levels. The dose is based on one of the preferred examples of the method in this case. The tumor volume changed after the toona sinensis leaf extract ("Toon 5-2") was obtained, and the tumor volume was measured once a week for a total of 7 weeks. L EMBODIMENT 3 Detailed description of the preferred embodiment (Detailed description of the invention) One of the previous studies led by Dr. Xu Shengguang (i / WC / z / w ei al · (2002), American Journal of Chinese Medicine, Vol. 30, 10 1235664 2 ά l 307-W4) One of the aqueous extracts of Chinese toon leaf is based on

Hui-Chiu Chang et al. (1998), Am J. Chin, Med, Vol. 26: Phantom 40 was prepared by the method disclosed in which 100 g of Toona sinensis leaves were added to 1000 mL of The solution was boiled and heated in water until 1,005 mL of the solution remained, and then centrifuged at 1,000 xg for 20 minutes to obtain a supernatant solution for the experiment of this research. The above-mentioned aqueous extract of Toona sinensis leaves has been found through experiments to effectively block the expression of human lung cancer cell A549 by inhibiting the expression of cyclin D1 (cyelin m) and cyclin E (cyclin E). 10 cell cycle progression (cell cycle). Shen Qingren further tried to extract Toona sinensis leaves by various processing methods, and knew that an Toona sinensis leaf extract was prepared by a method including the following steps: (1) Toona sinensis leaves were extracted with water under heating. Then, a first extract 15 obtained by water extraction is obtained, and (2) the first extract extracted by water in step (丨) is extracted with an alcohol to obtain a second extract extracted by alcohol. In Yu, the present invention also provides a method for preparing toona sinensis leaf extract, which includes the following steps: (a) extracting toona sinensis leaves with water under heating to obtain an aqueous extract solution; (b ) Drying the aqueous extraction solution obtained from step (a) to obtain a dried first extract; (the first extract obtained from step (b) is dissolved in an alcohol solvent to form 1235664 a An alcohol extraction solution; and (d) removing the alcohol solvent in the alcohol extraction solution obtained in step (C) to dryness to obtain a dried second extract. Preferably, in step (a) of the method of the present case ), 5 of the aqueous extraction solution is obtained by boiling and concentrating the water with toon leaves added to an appropriate amount, and then filtering. The filtering treatment can be performed using gauze, cotton or a filter with a selected mesh size. In a preferred embodiment of the present invention, the filtering treatment is performed with gauze and cotton. In another preferred embodiment of the present invention, the 忒 filtering treatment is performed with a 70 mesh screen. -10。 Preferably, this Steps (b) to (d) of the method are performed under low temperature conditions so that the destruction of the active components contained in the leaves of Toona sinensis can be reduced to a minimum. Preferably, step (13) of the method of the present case The drying process performed in the process is selected from the group consisting of: cold; east drying (lyophiiizati), I5 low temperature spray-drying, low temperature evaporation (e-), and One of these combinations. I In a preferred embodiment of the present invention, the drying treatment performed in step 是 is cold; the east drying treatment. Preferably, step (b) of the method of the present case may include the following processing steps: ⑴ Centrifuging the aqueous extraction solution obtained in step ⑷ to obtain a 20% solution and a sink; and ⑼ subjecting the upper solution obtained in step ii to a drying treatment. Preferably, step ii is borrowed It is performed by centrifuging the aqueous extraction solution obtained from step (b) at a low temperature (for example, 4.0 ° C. at a nominal rpm.) Preferably, the drying treatment for step ⑼ is selected from the group consisting of- 12 1235664 Group: cold bead drying treatment (lyophil ization), low-temperature spray-drying, low-temperature evaporation, and a combination thereof. In a preferred embodiment of the present invention, the drying process performed in step ⑴ is a freeze-drying process 5 In addition, the precipitate obtained in step 可以 can also be dried in a similar manner to produce a dry product. Preferably, the alcohol solvent removal performed in step (d) is carried out by a member selected from the group consisting of The treatments in the group were performed: evaporation, lyophilization, spray-drying, and a combination of 10 and one of these. In a preferred embodiment of the present invention, step (d) is performed by a freeze-drying process. Preferably, in step (d) of the method of the present case, before the alcohol solvent is removed, the alcohol extraction solution obtained from step (c) may be filtered or centrifuged to remove insoluble substances that may be contained therein. . More preferably, in step (d), the alcohol extraction solution obtained in step (c) is first centrifuged at 3000 rpm at 4 ° C for 12 minutes, and then the alcohol solvent is removed. The alcohol suitable for step (c) of the method of the present case may be, for example, ethanol, methanol, propanol, isopropanol, n-butanol, isobutanol, or a combination thereof. In a preferred embodiment of the present invention, the alcohol used in step (c) 20 of the method of the present case is ethanol. As shown in Example 1 below, the first extract obtained from step (b) of the method of the present case and the second extract obtained from step (d) of the method of the present case were sampled for HPLC analysis, and It was confirmed that the peaks of the main compounds of the two extracts were indeed different. 13 1235664 Shen Qiaoren also tried to further extract the second extract obtained in step ⑷ of the method of this case, including: (·); the second extract was dissolved in 50/0 ethanol, and then centrifuged, and To the clarified liquid portion and a precipitate portion; and then the upper clarified liquid portion and the precipitated portion are respectively subjected to cold beam drying to obtain a dried third extract derived from the clarified liquid portion and i The fourth extract of the substance portion; (ii) dissolving the fourth extract obtained in the above step ⑴ in 25% ethanol, and then centrifuging to obtain a supernatant liquid portion and a precipitate portion; and Cold-drying the upper clear liquid portion and the sinking object portion respectively to obtain a dried fifth extract derived from the upper clear liquid portion and a sixth extract derived from the sink liquid portion; 15 π obtained in ~ ⑻ ™ read reverse osmotic water (R0water), and then centrifuge to get the-upper liquid part and-sediment; then the upper liquid part and the sink part were separately Cold and dry, and get-dried from the The first reverse osmosis water extract of the upper liquid portion and the second reverse osmosis water extract derived from the precipitate portion; and ㈣ dissolving the second reverse osmosis water extract obtained in the above step 溶于 in the reverse osmosis rigid water Then, it is centrifuged to obtain an upper clear solution part and a precipitate part; then the upper clear solution part M and the precipitate part are respectively subjected to cold beam drying to obtain—the dried part originated from the upper clear solution part. The third reverse osmosis water extract and c 14 1235664 a fourth reverse osmosis water extract derived from the precipitate portion, wherein in steps (i), (Π), (iii) and (iv) above, The separation of the clear solution part from the precipitate part was achieved by centrifugation at 3000 rpm at 4 ° C for 12 minutes. 5 In order to investigate the biological activities of the various toona sinensis leaf extracts obtained above, the applicant used selected toona sinensis leaf extracts to treat several types of cancer cells derived from women's urogynecological system to observe the toona sinensis leaf extracts. Whether the substance is cytotoxic. At the beginning, the applicant used bladder cancer cell line T24 (bladder cancer 10 cell line T24) for preliminary anti-cancer cell activity screening, and found that the second extract obtained according to the method of this case has the best inhibition of cancer cell growth And the first extract obtained by step (b) of the method of the present case is second. Therefore, the applicant further used the second 15 extract obtained according to the method of the present case to treat two ovarian cancer cell lines SKOV3 and PA-1, two cervical cancer cell lines HeLa and HeLaS3, and an endometrium. Cancer (endometrial cancer) cell line RL95-2 'and the second extract was found to have selective cytotoxicity to ovarian cancer cells. Therefore, the present invention also contemplates the use of the Toona sinensis leaf extract in the preparation of a pharmaceutical composition for treating ovarian cancer. Thus, the present invention provides a pharmaceutical composition comprising: (a) a therapeutically effective amount of the second extract prepared by the method as described above in this case; and (b) a pharmaceutically acceptable amount Vehicle. 15 1235664 The pharmaceutical composition containing toon leaf extract prepared according to the method of this case can be used to treat an ovarian cancer or bladder cancer. Therefore, for an individual with cancer or bladder cancer, the toon leaf extract prepared according to the method of the present case can be administered to the individual. The present invention also provides a pharmaceutical composition which can be used for treating bladder cancer, comprising: (a) a therapeutically effective amount of an toon leaf extract selected from the following ... (I) an toon leaf extracted by water and The resulting extract, and (II) an extract of 10 from Sinensis leaves in sequence via water and an alcohol, and (b) a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier, this term refers to carriers well-known in the art that are suitable for pharmaceutical manufacturing, and include, but are not limited to, water, physiological saline, glycerol, organic solvents, Stabilizers, chelating agents, preservatives 15, emulsifiers, suspending agents, diluents, gels, liposomes, etc. The pharmaceutical composition according to the present invention can be prepared using techniques well known to those skilled in the art. Into a form suitable for parenteral, oral or topical administration. 'This includes, but is not limited to, injections, solutions, capsules, dispersions. Suspension, etc. 20, etc. In order to manufacture an oral solid product, an excipient and, if necessary, a binder, a disintegrant, a lubricant, a colorant, a flavoring agent And / or the like can be mixed with the toon leaf extract according to the present invention. The resulting mixture can then be formed into lozenges, coated by a method known per se 16 1235664 method Tablets, granules, powders, capsules These additives can be those commonly used in today's technical fields, including excipients: lactose, sucrose, sodium chloride, glucose, Dianfen, carbonic acid # 5, kaolin, microcrystalline Cellulose 5 (micro-crystalline cellulose) and silicic acid; binders: water, ethanol, propanol, sucrose solution, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl fiber Cellulose, hydroxypropyl starch, methyl cellulose, ethyl cellulose, shellac, calcium phosphate and polyvinylpyrrolidone; disintegrants: dry starch, algae 10 sodium alginate ), Powdered agar; fat, sodium bicarbonate, carbonate 1 bow, sodium lauryl sulfate, monoglycerol stearate and lactose; lubricant: purified talc, hard Stearate salts, borax and polyethylene glycol; and bridges: strict sugar, bitter orange peel, citric acid ) Tartaric acid. To make an oral liquid product, a flavoring agent, a buffering agent, a stabilizer, and the like may be mixed with the toon leaf extract according to the present invention. The resulting mixture may then be borrowed It is formed into a solution for oral administration, a syrup, an elixir or the like by 20 methods known per se in this art. In this example, the flavoring agent may be the same as that mentioned above. Examples of the buffering agent are sodium citrate, and examples of the stabilizer are tragacanth, gum arabic, and gelatin. To make an injectable product, a pH regulator, a buffering agent, an ampoulant 17 1235664, an isotonicity, and the like may be mixed with the toon leaf extract according to the present invention. The resulting mixture can then be formed into a subcutaneous, intramuscular, or intravenous injection by a method known per se in the art. Examples of the pH regulator and buffer include sodium citrate, sodium acetate, and sodium sulfate. Exemplary stabilizers include sodium pyrosulfite, ethylenediaminetetraacetic acid (EDTA), thioglycollic acid, and thiolactic acid. Examples of isotonicity include sodium chloride and glucose. Preferably, the pharmaceutical composition according to the present invention is manufactured in a form suitable for injection, such as powder injection, lyophilization product for injection, and injection injection. , Oily injection, liposome injection and other forms. As used herein, the term "therapeutically effective amount" means that when a pharmaceutical composition containing 15 toon leaf extracts according to the present invention is administered to an individual in need of the composition for treatment, one is sufficient to provide the desired effect. The amount of therapeutic effect that does not cause undesired serious harm to non-standard tissues or organs. The therapeutically effective amount will vary depending on various factors including, for example, the type of disorder, the weight, age, physical condition and response of the individual to be treated, the route of administration of the drug, and the like. The therapeutically effective amount can be determined by those skilled in the art. The dosage and number of administrations of the pharmaceutical composition according to the present invention will vary depending on the severity of the disease to be treated, the route of administration, and the weight, age, physical condition and response of the individual to be treated. 1 18 1235664 In general, the daily dosage of the pharmaceutical composition according to the present invention is usually 0.67 mg / Kg body weight to 6.7 mg / Kg body weight, which is in the form of a single dose or divided into several doses, and can be used without Enteral, oral or topical administration. Preferably, the pharmaceutical composition according to the present invention can be injected via intraperitoneal injection5, continuous intravenous injection, topical arterial single injection, and direct local tumor injection ( topical tumor direct injection). In a preferred embodiment, the pharmaceutical composition according to the present invention can be administered by intraperitoneal injection or local tumor direct injection. The pharmaceutical composition according to the present invention can be administered daily. Preferably, it can be administered continuously for 5 days and then rested for 2 days for a period of 43 days or until remission occurs. The pharmaceutical composition according to the present invention may be administered alone or in combination with other treatment methods or drugs for treating ovarian cancer. These treatments include chemotherapy and external beam radiation therapy, and these treatments include, but are not limited to, paclitaxel, cisplatin, carboplatin, Cyclophosphamide and doxorubicin 〇 The present invention will be further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not It should be construed as a limitation on the practice of the invention. 19 1235664 Example I. Preparation of toon leaf extract The toon leaf system used in this example was collected from an toon tree planted in Tuku Town, Yunlin County, Taiwan, Taiwan. Extraction procedure: Man 5 took young toon leaves and washed them with water, and then used 4 L of RO water per kg of toon leaves, mixed an appropriate amount of reverse osmosis water with toon leaves, and boiled them for 30 minutes. minute. After that, the toon leaves were taken out, and the extract was concentrated under low heat, and then filtered through a 70 mesh filter. The obtained filtrate can be directly lyophilized using a Virtis machine to obtain a crude extract of Toona sinensis (called Toona 5_4) (also called "TSL-CE"). Generally speaking, processing 100 g of Toona sinensis leaves in this way can obtain about 5-6 g of lyophilized powder on average. In addition, the filtrate obtained by the above filtering treatment can be subjected to a centrifugal treatment before freeze-drying: centrifugation at 3000 rpm (Beckman 15 AvantiTM J-30I) at 4 ° C for 12 minutes to obtain a supernatant liquid portion And a sanctuary part containing insoluble matter. The supernatant liquid was then freeze-dried using a Virtis machine to obtain a water extract as a lyophilized powder, which was called "TSL-1". The "Toon 5-4" and "TSL-1" obtained above are both "the first stroke-extracted items of 20" in the present invention, and for convenience of illustration, the applicant uses "TSL -1 "to perform the following further extraction procedure.

Extraction procedure B The "TsLq", extract (50 g) obtained in the above extraction procedure A was dissolved in 99.5% ethanol (400 ml) for alcohol extraction. The alcohol solution formed was 4 20 1235664. (: below Centrifuge at 3000 rpm (Beckman Avanti ™ J-30I) for 12 minutes to obtain a supernatant solution portion and a precipitate portion. The supernatant solution portion was then freeze-dried using a Virtis machine to obtain a frozen solution. The further purified alcohol 5 extract of dry powder, which is called "Toon 5-2, '(also known as" TSL-2 "). 100 g of" TSL-1 "extract can obtain about 1 g of" Toon 5-2 "lyophilized powder.

Extraction procedure C The part of the precipitate obtained in the above extraction procedure B was freeze-dried in a Virtis machine and then dissolved in 50% ethanol. The resulting 50% alcohol solution was centrifuged at 3000 rpm (Beckman AvantiTM J-30I) at 4 ° C for 12 minutes to obtain an upper liquid portion and a precipitate portion. The upper liquid portion was connected and cold-beam dried using a Virtis machine to obtain an extract in the form of a bundle of dry powder, which was called "Toon 5. · 5" (also called "TSL-3").

15 Extraction sequence D The portion of the precipitate obtained in the above extraction procedure C was freeze-dried in a Virtis machine and then dissolved in 25% ethanol. The resulting 25% ethanol solution was centrifuged at 3000 rpm (Beckman AvantiTM J-30I) at 12 ° C for 12 minutes to obtain an upper liquid portion and a precipitate portion. Then, a 20 Virtis machine was used to freeze-dry the supernatant and the precipitate, respectively, to obtain two lyophilized powders called "TSL-4" and "TSL_4P" extraction products, respectively.

Extraction procedure E The extraction product "TSL-4P" obtained in the above extraction procedure D was dissolved in water of 〇21 1235664, and the resulting aqueous solution was centrifuged at 3000 rpm (Beckman Avanti ™ J-30I) at 4 ° C. It took 12 minutes to obtain an upper clear solution part and a sediment part. Next, the Virtis machine was used to freeze-dry the supernatant and the precipitate, respectively, and two lyophilized powders were obtained, which are called "TS-H20-1" (also called "TSL-5 ") And" TSL-5P ".

Extraction procedure F The extraction product "TSL-5P" obtained in the above extraction procedure E was dissolved in RO water, and the resulting aqueous solution was centrifuged at 3000 rpm (Beckman 10 AvantiTM J-30I) at 4 ° C for 12 minutes. To obtain a clear solution portion and a precipitate portion. Next, the Virtis machine was used to freeze-dry the upper liquid part and the precipitate part respectively, and the two freeze-dried powders were respectively called "Toon 5-4R" (also known as "TS-H20-2" Or "TSL-6") and "TSL-7". 15 Example 2. HPLC analysis of Toona sinensis leaf extract To understand the distribution of the main components of the Toona sinensis leaf extract obtained according to the method of the present case, the “Toona sinensis 5_4” obtained by the extraction procedure A and extraction procedure B of Example 1 and "Toona 5-2" was used for analysis by high pressure liquid chromatography (HPLC). 20 Experimental operation method: In this example, the HPLC analysis instrument was Hitachi L-7100 pump, L-7420 uv / vis detector, D- 2410 degaser; analysis software is D-7000 HPLC System Manager; analysis column is Mightysil RP-18 GP 250-4.6 (5μηι); analysis conditions: gradient solvent A: methanol; B: water; 0 ~ 10 22 1235664 minutes 20% ~ 50% A; 10 ~ 20 minutes 50% ~ 70% A; 20 ~ 40 minutes 70% A; flow rate 1 ml / min; wavelength 254 nm. Results: Fig. 1 and Fig. 2 show the HPLC elution diagrams of "Toon 5-4" and "Toon 5-2", respectively. Comparing Fig. 1 and Fig. 2 shows that under the same separation conditions, "Toon" 5-4 "and" Toon 5-2 "contain different main compounds. "Toon 5-4" and "Toon 5-2" have the same compound peaks at the retention time points of 5, 6 and 8 minutes, and most of the compounds contained in "Toon 5_2" are Appeared in the 3 ~ 4 peaks ran out of the surface, but "Toon 5-4" has another 2 ~ 3 peak signals at the retention time points of 10 25 and 26 minutes. Example 3 · Toon leaf extract XTT cell proliferation assay To screen extracts of Toona sinensis leaves that have the activity of inhibiting the growth of cancer cells, this example uses XTT cell proliferation analysis (XTT cell proliferation 15 analysis), and was collected from American type culture The Center [American Type Culture Collection (ATCC), PO Box 1549, Manassas, VA 20108 USA] bladder cancer cell line T24) was used to test 5 toon leaf extracts prepared in Example 1, Including "Toon 5-4", "Toon 5-2", "Toon 5-5", "Toon 5-5R", and "Toon 20 Toon 5_4R". Experimental operation method: 5 obtained in Example 1 "Toona sinensis 5-4 ,, "toon 5-2 ',' 5-5 toon", "Toon 5-5R" and "toon 5-4R" is used to perform XTT cell proliferation assay (Roche Molecular 23 1235664

Biochemicals). The test concentrations of each toon leaf extract were 1, 10, and 100 pg / mL, and H20, 50% EtOH, and 99.5% EtOH were used as control groups. Bladder cancer cells T24 were cultured in 10% fetal calf serum (FCS), 5 10,000 units / mL penicillin, 10 mg / mL streptomycin, and 0.025 mg / mL amphotericin B. (Amphotericin B) in DMEM-F12 medium. XTT cell proliferation analysis (Roche) was performed by inoculating cells at a concentration of 5x103 cells / 100 μΐ / well into each well of a culture plate 10 of a 96 well, and placing the culture plate in a 5% The C02 incubator lasted 24 hours. After that, the culture medium was replaced with toona sinensis extracts with different concentrations (100 pg / mL, 10 pg / mL and 1 pg / mL), and each toona sinensis extract was dissolved in PBS. The culture was continued for 72 hours. . After that, remove the culture medium and add 100 μΐ of fresh medium and 50 μΐ of XTT mixed reagent (xTT reagent: electronic coupling reagent = 50: 1) which has been pre-adjusted, and place the culture plate in the incubator for 4 After that, the absorbance values (OD = OD492-ODD690) were measured by ELISA reader at the wavelengths of 492 nm and 690 nm, and the concentration of the drug that inhibited the growth of cancer cells by 50% (IC5G) was calculated, that is, the experiment The absorbance value of the group was one-half of the cell concentration of that of the control group. Results: FIG. 3 shows five toon leaf extracts “Toon 5-4”, “Toon 5-2”, “Toon 5-5”, “Toon 5-5R” and “Toon 5-5R” prepared in Example 1. The results of XTT cell proliferation analysis of Toona 5_4R ", the lower the absorbance value, the lower the number of 24 1235664 cells that survived. It is clear from Fig. 3 that "Toon 5-2" is the most cytotoxic to the bladder cancer cell line T24, and "Toon 5-4" is the second. In addition, "Toon 5-2" was dose-dependent 'on the cytotoxicity of bladder cancer cell line T24, and the calculated IC50 was 71.3 pg / mL. 5 Example 4: Effect of Toona sinensis leaf extract on the cell cycle of bladder cancer cell line T24 This example further explores the effect of "Toon 5-2" on the cell cycle of bladder cancer cell line T24. The unsynchronized bladder cancer cell line T24 was treated with different doses of "Xiang Juan 5-2" for 24 hours, and then analyzed with a fluorescent activated cell sorter [Fluorescence-activated cell sorter ( FACS) analysis] to assess cell cycle changes. Experimental method: Cancer cells were inoculated into a 6 cm culture plate at a concentration of 2xl05 cells / plate and cultured for 24 hours, and then the medium was changed to "Toon 5-2" containing 10 15 or 100 pg / mL. And continue incubation for 24 hours. After that, trypsin was used to treat the culture plate and the collected cells were neutralized with the medium, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed and 300 μΐ of PBS was added to evenly disperse the cells. Slowly 700 μΐ of 99.5% alcohol was added to fix the cells. After standing at 4 ° C for 20 to 30 minutes, centrifuge again to remove the supernatant, and add PBS containing 0.1% tnton and 0.05% RNase. After incubation at 37 ° C for 1 hr, centrifugation was performed to remove the supernatant, and PBS containing Propidlum iodide (Propidlum iodide) at 40 pg / ml was added. After the light-shielding effect was performed at 4 ° C for several minutes, it was washed through a sieve with a 60 μηι Mesh Filter, and then analyzed by a flow cytometer. 25 1235664 Results: See Table 1 below, the applicant found that the bladder cancer cell line T24 at the G2 stage increased after treatment with "Toon 5.2", especially when treated with 100 // g / mL. Since the combination therapy using monogrammed drugs that are in cells at 5 stages of different cell cycles is the standard approach for cancer treatment, the results shown in Table 1 provide clinical application of toon leaf extract according to the present invention. Useful information. Table 1 Percentage of cells in various cell cycle stages after 24 hours of treatment with "Toon 5-2" G1 S G2 Control group 45 14.1 40.9 100 // g / mL 8.6 27.9 63.5 10 // g / mL 33.7 13.6 52.8 1 β g / mL 43.9 13.2 42.9 Example 5. Toxin-cancer leaf cancer cell test (| > | 10 anti-cancer cell test) In this example, “Toon 5-2” was used to treat several species. Cancer cells derived from women's urogynecological system, including two ovarian cancer cell lines SKOV3 and PA-1, two cervical cancer cell lines HeLa and HeLaS3, and an endometrium 15 Cancer (endometrial cancer) cell line RL95-2 to observe whether "Toon 5-2" is cytotoxic to these cancer cells. Source and culture of cancer cell lines: In this example, the SKOV3, PA-: 1, HeLa, HeLaS3, and RL95-2 used were purchased from ATCC, and these cancer cell lines were all cultured at 20 with 10% FCS, 10,000 units / mL penicillin, 10 mg / mL 26 1235664 streptomycin, 0.025 mg / mL amphotericin B in DMEM-F12 medium. Experimental operation method: The cancer cells were seeded at a concentration of 15 × 105 cells / plate in a 10 cm culture 5 plate and cultured overnight. After that, the medium was changed to a medium containing 1 mg / mL "Toon 5-2" ("Toon 5-2" was first dissolved in PBS for concentration adjustment), and the culture was continued for 24 or 48 hours. Thereafter, trypsin (0.05% trypsin / 0.02% EDTA in PBS) was used to treat the culture plate, and the collected cells were placed in microtubes. Place 10 tubes of microtests on an ice bath, suspend the cells in suspension (0.05% trypsin / 0.02% EDTA in PBS), and trypan blue at a ratio of 1: 1 To mix uniformly, and then count the number of surviving cells with a hemacytometer under a microscope (Nikon TS100). Results: 15 To further confirm whether "Toona 5-2" has cytotoxicity to cancer cells derived from women's urogynecological system, ovarian cancer cells were treated with "Toona 5_2" at a concentration of 1 mg / mL. SKOV3 and PA-B cervical cancer cell lines HeLa and HeLaS3 and endometrial cancer cell line RL95_2 lasted for 24 hours or 48 hours, and 20 were compared with the control group without "Toon 5-2", the results obtained It is shown in FIG. 4, where the ordinate represents the cell survival rate (%) compared to the control group. It is clear from Fig. 4 that at an effective concentration of 1 mg / mL, for ovarian cancer cell lines SKOV3 and PA-1, "Toon 5-2" has shown a strong growth inhibitory effect under a 24-hour treatment time ( Can almost completely inhibit the growth of these two types of ovarian cancer cells. For the endometrial cancer cell line RL95-2 and the cervical cancer HeLaS3, "Toon 5-2," the cancer cell growth inhibitory effect shown in the 48-hour treatment time is not high. For cervical cancer cells HeLa strain "Toon 5-2" exhibited 60% of cancer cell growth inhibitory effect under a 48-hour treatment time. Table 2 below shows the IC% values of "Toon 5_2" for each of the five cancer cell lines tested. The ICm value is calculated based on the fact that 50% of the cells will survive 48 hours after dosing. The total is taken as the drug concentration of 100%). As shown in "2", the IC50 values of "Xiang Juan 5_2" for Indocarcinoma cell line H) SKOV3 and PAU are 28, respectively, called Lan and 10 Chan. These data show that Toona 5_2 "has the potential to be made into an effective anti-ovarian cancer drug. Table 2

Cancer cell line IC $ 〇SKOV3 28 pg / mL PA-1 10 gg / mL HeLa 1 mg / mL HeLa S3 > 1 mg / mL RL95-2 ^ > 1 mg / mL Example 6 Cell Morphology and Fine Cell Cycle of Indo-Cancer Cancer Cells This example further studies the changes in cell morphology and cell cycle produced by Indo-cancer cells after receiving the effect of the voluminous leaf extract of the present invention. Experimental operation method: According to the method described in the experimental operation method of Example 5, India 20 nested cancer cell line SKOV3 and pupae were cultured in a culture plate, and were respectively i 28 1235664 mg / mL, 100 pg / mL and 10 pg / mL "Toona sinensis 5_2", after treatment, observe the change in cell morphology under a microscope. In addition, referring to the method described in the experimental operation method of Example 4, the Indian nested cancer cell line SKOV3 was cultured on a culture plate And processed with "Toon 5-2" at ^ 005 Kg / mL and 10 Kg / mL, respectively, and then performed facs analysis. Results: Figure 5 and Figure 6 respectively show "Toon 5-2" in the The morphological effects caused by the ovarian cancer cell lines SKOV3 and PA-1 at the doses used. As shown in Figure 5 and Figure 6 (at a magnification of 100X), the treatment was performed at a dose of} mg / mL for 24 hours. Later, "Toon 5-2" caused a large number of apoptosis and cell floating. However, after being treated at 100/1111 for 24 hours at a dosage of 10 pg / mL, "Toon 5_2" , Still can cause significant cell death of ovarian cancer cell lines SK0V3 and PA · 1 individually. Perish. 15 In addition, the ovarian cancer cell line SK0V3 was treated with different doses of "Toon 5 · 2" for 24 hours, and the cell cycle distribution of the ovarian cancer cell line SK0V3 was observed by FACS analysis. The results are shown Table 3 below. Table 3 Percentage of ovarian cancer cells at the cell cycle stage after 24 hours of treatment with "Toon 5-2" (mean ± SD) G1 S G2 / M Control group 59.0 ± 0.2 27.3 ± 0.9 13.8 ± 〇.7 10 gg / mL 53.7 ± 1.3 32.1 ± 1.4 14.2 ± M 100 pg / mL 54.2 ± 1.6 20.9 ± 0.9 25.0 ± 2.5 29 1235664 Male nude mice (Foxnlnu / Foxnlnu) were inoculated with 2x10 SK0V3 cells / 01 mL PBS (24_29 g, 5-6 weeks old, n = 5) After subcutaneous development of a tumor of at least 0.3 mm (about one week), intraperitoneal injection of Toona sinensis 5-2 is performed to observe the treatment effect. 5 The preparation of Toona 5_2 was as follows: Toona 5-2 was dissolved in PBS and then treated, and formulated into 67.25 mg / mL and 672.5 mg / mL, and then toon 5-2 was given according to the weight of each nude mouse (dose: 0.6725 pg / g body weight or 6.725 pg / g body weight). Results: 10 Male nude mice with significant tumor mass developed by subcutaneous injection of ovarian cancer cell line SKOV3 (2xl05 cells) were injected intraperitoneally with 0.67 pg / g body weight (low dose) or 6.7 gg / g body weight (high dose). 'Toon 5-2' was injected for 5 days per week for a total of 7 weeks. From the anatomical examination, it was found that the test group administered with a high dose (6.7 tons / g body weight) of the extract of Cinnamomum japonicus 15 showed that the tumors in the naked body had shrunk to a nearly disappearing degree, while the low dose was administered. (.67 pg / g body weight) The test group of Toona sinensis leaves also showed that the tumors in the nude mice had shrunk. As for the control group injected with phosphate buffered saline (PBS), tumors in nude mice showed continuous growth. Compared with the control group treated with PBS, the intraperitoneal injection treatment of "Toon 5-2" 20 significantly inhibited the growth of tumor mass in a dose-dependent relationship. Figure 7 shows the change in tumor mass in nude mice after 7 weeks of observation. In particular, in nude mice injected with a high dose of "Toon 5-2", the original tumor mass in the body almost disappeared. In addition, see Table 5, 1235664 After intraperitoneal injection of "Toon 5-2" for 7 weeks, it did not cause any significant toxicity to the bone marrow, kidney, or liver of the test nude mice. Table 5 Mean + SD Deviation Control group Low dose high dose WBC (10 * 3 / μ1) 3.32 ± 0.88 3.143 ± 0 · 67 4.562 ± 1.14 RBC (10 * 6 / μ1) 9.34 ± 0.17 9.2110.41 9 · 755 ± 0.19 HGB (g / dl) 12.45 ± 0.20 12.5 ± 0.42 13.525 ± 0.32 HCT (%) 41.5 ± 0.76 41.266 ± 1.83 44.85 ± 0.91 MCV (fl) 44.45 ± 0.65 44 · 833 ± 0 · 50 46 ± 0 · 79 MCH (pg) 13.33 ± 0.14 13.610.15 13 · 875 ± 0.13 MCHC (%) 30 · 05 ± 0 · 61 30 · 333 ± 0.39 30 · 15 ± 0 · 33 BUN (mg / dL) 29.3 ± 2.3 28 · 0 ± 2.0 25 · 0 ± 4 · 0 Creatinine (mg / dL) 0.43 ± 0.11 0.47 ± 0 · 04 0.47 ± 0.16 AST (IU / L) 101.3 ± 23.3 111 · 7 ± 24.3 150.0 ± 30.7 ALT (IU / L) The experimental results above 29.8 ± 17.7 48.0 ± 20.0 60.7 ± 51.4 clearly show that the Toona sinensis extract 5 according to the present invention has a high potential to develop into an anti-ovarian cancer drug. All documents cited in this specification are incorporated in this case as a reference. In case of conflict, the detailed description of the case, including the definition, will prevail. Although the invention has been described with reference to the specific examples described above, it will be apparent that many modifications and changes can be made without departing from the scope and spirit of the invention. Therefore, it is intended that the present invention is limited only by those indicated in the scope of patent applications attached to the document. [Brief description of the figure] Figure 1 is an HPLC elution profile of a product extracted from water of Toona sinensis ("Toona sinensis 5_4" obtained in Example 15 1 hereinafter); 2 is an HPLC elution chart of one of the toon leaf extracts obtained through alcohol extraction 32 1235664 (that is, "toon 5_2," obtained in Example 1 below) obtained according to one of the preferred methods of the present case; 3 shows the 5 cytotoxicity effects of five toon leaf extracts obtained by different purification treatments on bladder cancer cell line T24, which are used as such toon leaves H2O, 500 / oEtOH and 99 · 5% EtOH of the extraction solvent of the extract were used as a control group; FIG. 4 shows the toon leaf extract (ie "Toon 5-2") obtained according to a preferred embodiment of the method of the present case. ) 10 effects on the growth inhibition effect of different cancer cell lines, wherein the control group is the experimental group without the toon leaf extract; FIG. 5 shows the toon leaf extract (that is, the " Toon 5-2 ,,) at different concentrations 1 mg / ml, 100 pg / ml) on the cell morphology of ovarian cancer cell line SKOV3, in which the control group is the experimental group without the extract of Toona sinensis; 15 FIG. 6 shows a preferred specific example according to the method of this case. The effect of the toon leaf extract ("Toona sinensis 5-2,") on the cell morphology of ovarian cancer cell line PA-1 at different concentrations (1 mg / ml, 10 pg / ml). Experimental group of toon leaf extract; and FIG. 7 shows that nude mice bearing tumors grown by ovarian cancer cells were administered 20 drugs at low and high doses according to one of the preferred embodiments of the method of the present invention Changes in tumor volume after the extract (that is, "Toon 5-2"), in which the tumor volume is measured once a week for a total of 7 weeks. [The main elements of the round form represent the symbol table] (none) 33

Claims (1)

I2 ^ S6S4 .V, g revision month said to make up for the problem _ _ _: pick up the patent scope 0 9212 5 5 2 3 patent application for the revision of the patent scope date of revision: July 1993 1. An toon leaf extract Is obtained by a method comprising the following steps: (1) extracting toon leaves with water under heating to obtain a first extract with water extraction, and (2) extracting with an alcohol The water-extracted first extract obtained in step (1) is used to obtain an alcohol-extracted second extract. 10 2. A toon leaf extract, which is prepared by a method comprising the following steps: (a) extracting toon leaf with water under heating to obtain an aqueous extract solution; 15 20 (b) drying the aqueous extraction solution obtained in step (a) to obtain a dried first extract; (c) dissolving the first extract obtained in step (b) in an alcohol solvent To form an alcohol extraction solution; and (d) removing the alcohol solvent in the alcohol extraction solution obtained in step (c) to dryness to obtain a dried second extract. 3. The toona sinensis leaf extract as claimed in the scope of patent application No. 1 or 2 can be used to prepare a medicine for treating ovarian cancer or bladder cancer. 4. A method for preparing an toon leaf extract, comprising the following steps: (a) extracting toon leaf with water under heating to obtain an aqueous extract solution; 33 1235664 (b) Drying from the aqueous extraction solution of step (a) to obtain a dried first extract; (c) dissolving the first extract from step (b) in an alcohol solvent to form an alcohol extraction solution And 5 (d) removing the alcohol solvent in the alcohol extraction solution obtained in step (c) to dryness to obtain a dried second extract. 5. The method according to item 4 of the scope of patent application, wherein in step (a), the aqueous extraction solution is obtained by boiling and concentrating water added with toon leaves to an appropriate amount, and then filtering. 10 6. The method according to the scope of patent application, wherein the filtering treatment is performed by using an appliance selected from the group consisting of gauze, cotton, a filter with a selected mesh size, and a combination thereof . 7. The method according to item 4 of the patent application, wherein the drying treatment performed in step (b) is selected from the group consisting of freeze-drying treatment, low-temperature spray 15 spray-drying, Low temperature evaporation, and one of these combinations. 8. The method according to item 7 of the scope of patent application, wherein the drying treatment performed in step (b) is a freeze-drying treatment. 9. The method according to item 4 of the scope of patent application, wherein step (b) includes the following processing steps 20 steps: (i) centrifuging the aqueous extraction solution obtained from step (a) to obtain a supernatant and a precipitate And (ii) subjecting the upper liquid obtained in step (i) to a drying treatment. 10. The method according to item 9 of the scope of patent application, wherein the drying place performed in step (ii) 34 1235664 is selected from the group consisting of freeze drying treatment, low temperature spray drying treatment, low temperature evaporation treatment, and One of these combinations. 11. The method according to item 4 of the patent application, wherein the alcohol used in step (c) is selected from the group consisting of ethanol, methanol, propanol, isopropanol, 5 n-butanol, Isobutanol, and one of these combinations. 12. The method of claim 11 in which the alcohol used in step (c) is ethanol. 13. The method according to item 4 of the scope of patent application, wherein the removal of the alcohol solvent in step (d) is performed by a process selected from the group consisting of freeze drying, drying process, evaporation process, Spray drying treatment, and one of these combinations. 14. The method as claimed in claim 13 wherein step (d) is performed by freeze-drying. 15.-A pharmaceutical composition for treating a cancer selected from ovarian and bladder cancer, comprising: 15 (a)-a therapeutically effective amount of one of the toon leaf extracts selected from: (i) a Toon leaf extract obtained by extracting toon leaf via water; (ii) Toon leaf extract as applied for patent application item 1 or 2; or (iii) By applying patent application items 4 to 14 Toon leaf extract prepared according to any one of the methods 20; and (b) a pharmaceutically acceptable carrier. 16. A pharmaceutical composition for inhibiting the growth of a cancer cell selected from the group consisting of ovarian cancer cells and bladder cancer cells, comprising: (a) a therapeutically effective amount of one of the toon leaf extracts selected from: 35 1235664 (i) Toona sinensis leaf extract obtained by extracting toona sinensis leaves with water; (ii) Toona sinensis leaf extract as claimed in item 1 or 2 of the scope of patent application; or (iii) by scope of patent application The toon leaf extract prepared by the method 5 of any one of items 4 to 14; and (b) a pharmaceutically acceptable carrier. 36