TW201008572A - Anti-lung cancer Glossogyne tenuifolia substance and method of preparing the same - Google Patents

Abstract

This invention relates to an anti-lung cancer Glossogyne tenuifolia substance and a method of preparing the same. The preparation method comprises solvent extraction of Glossogyne tenuifolia, followed by separation method like column chromatography, high performance chromatography, and the likes. As such, compound and composition capable of preventing or inhibiting growth of lung cancer cells can be prepared. In addition, thins invention also discloses an application method of the anti-lung cancer substance.

Description

201008572 . IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a medicinal herb, a remedy for a lung cancer, and a method for preparing the same, and more particularly, It is derived from the anti-lung cancer bio-aerobic substance in Glossogyne tenui folia, which can be used to inhibit or prevent lung cancer by solvent extraction, column chromatography and high-efficiency column chromatography. Cell growth. [Prior Art] ©#CANCER is a cancer with low cure rate and recurrence after cure. The treatment methods include: surgical therapy, radiation therapy and chemotherapy, among which chemotherapy is still i, supplemented with hands Mixed application of # and radiation therapy to prevent metastasis and recurrence of cancer to improve long-term survival. Therefore, the development of 'new anti-lung cancer compounds will help improve the effectiveness of chemotherapy. Cancer cell death patterns include two major modes: cell necrosis (necr〇sis) and apoptosis (apoptosis). "Cell necrosis" is a trauma caused by external or external environment, such as ischemia caused by ischemia, hypoxia or complement attack, foreign toxins, etc. 'This machine is often accompanied by an inflammatory reaction and causes Multiple cells in the tissue are killed at the same time. (Van Cruchten, S. and Van Den, BW Morphological and biochemical aspects of apoptosis, oncosis and necrosis. Anat. Histol. Embryol. 2002, 31, 214-223) "Apoptosis" is also known as the "Cell Project" "Scheduled death" (programmed ce 11 death), due to DNA damage, can not be repaired 201008572. The process of causing cells to die (Lowe, SW and Lin, AW Apoptosis in cancer. Carcinogenesis. 2000, 21, 485-495. FiT chenkov, AA Apoptosis modulators. Biomed. Khim. 2003, 49, 333-359), and also an inevitable replacement process for normal tissues inside the organism; in vivo, Apoptosis often plays an important role in maintaining a constant number of homeostasis. Many studies have shown that a defect in the apoptotic pathway is one of the important factors leading to cancer occurrence (Tsujimoto, Y., Cossman, J., Jaffe, E. and Croce, CM Involvement of the be 1-2 gene in human follicular lymphoma. Science 1985, 228, 1440-1443; * Hengartner M. 0. The biochemistry o Nature: toxic cytokines I cancer targets. Hematol. Oncol. Clin. North Am. 2002, 16, 1255- 1 267; Ferreira, CG, Epping, M., Kruyt, FA, et-a 1. Apoptosis: target of cancer therapy. Clin. Cancer Res. 2002;8:2024 -2034); Therefore, how to effectively guide cancer cells into the apoptotic pathway is one of the important molecular mechanisms of anticancer drugs. Glossogyne tenuifolia is also known as "wind blessing" , Alias "Shansongzi", "South Xiangru" "Iron fishing rod" "Golden key" 6 201008572 . Waiting for 'a genus Compositae, perennial perennial grass, dampness, swelling, promoting blood circulation, treating colds, lung heat, Acute tonsillitis, efficacy. The plant has antipyretic, detoxification, fever, abdominal heat dysentery, hot edema bronchitis, urethritis and liver protection, and other domestic and foreign research reports on the analysis of grass by Xiangzi in the fragrant grass: Lin Zhehui et al. Acid, luteolin,

1114〇1111-711 This 0 is the heart and 1[(:1 and other chemical components (Lin Zhehui, Zhou Zhengren, Liu Guogui: Research on the ingredients of Xiangru. The first series of TCM literature abstracts in Taiwan] 193 (1975)); Lin Ronggui's research It is confirmed that the extract of fragrant grass can treat hepatitis with chemical injury (Lin Ronggui: Investigation and study of medicinal plant resources in Wuhu County. Master's thesis of Pharmacy Department of China Medical College (1997)); Cai Jiahong's research results show that fragrant grass The extract has a significant inhibitory effect on the mutation-induced mutation effect of Salm〇neHa • typhimurium T1 535 NQN0 (Cai Jiahong: Ten kinds of Taiwan's anti-mutagenic research) · Master's thesis, Department of Food Science, National Pingtung University of Science and Technology (2000)). According to a study by Wu et al., the ingredients obtained from the separation of fragrant grass are oleanolic acid ' luteolin ' luteolin-7-glucoside , β -sitosterol ' stigmasterol , stearic - acid and palmitic acid , etc . The anti-inflammatory and anti-hepatitis B surface antigen function of Xiangru was studied, and it was found that the fragrant grass can inhibit the surface antigen expression of hepatitis B (Wu MJ, Wan g L, Ding HY, Weng CY and Yen JH. ( 2004 ) G1ossogyne tenu i folia acts to inhibit inflammatory mediator production in a macrophage cell line by downregulating LPS-7 201008572 induced NF-kappa BJ Biomed. Sci. 11(2): 186-199) ; At the same time, the crude extract of the fragrant grass has strong antioxidant capacity (Wll MJ, Huang CL, Lian TW, Kou MC and Wang L. (2005a). Anti oxidant activity of G1 ossogyne tenui folia J. Agric. Food Chem. 53, 6305-6312 ; Hsu HF, Houng JY,

Chang CL, Wu CC, Chang FR and Wu YC. (2005)

Antioxidant Activity, Cytotoxicity and DNA Information of G1ossogyne teniufolia. J. Agric. Food Chem., 53: 6117-6125; Yang, J.H., Tsai, S.Y., Han, C.M., Shih, C. C., Mau, J. L., 2006.

Antioxidant properties of Glossogyne tenuifolia. 'Am. J. Chin. Med. 34, 707-720); in addition, the ethanol extract of fragrant ruthenium has an anti-inflammatory effect by inhibiting the transcription of i NOS to inhibit nitric oxide synthase The expression, in turn, inhibits the biosynthesis of nitric oxide, which is 〇16311〇14 3(^(1 and 1111:6〇1111-7-莒11^03 1 (16 is the indicator component of Xiangru anti-inflammatory (Wu MJ, Weng CY, Ding HY and Wu PJ (2005b)

Anti-inflammatory and antiviral effects of Glossogyne tenuifolia. Life Sci. 76(10): 1135-1146) • In addition, the flavonoids Luteolin and 11^6〇1丨11-7-glucoside in the fragrant grass are also It has been shown to have antioxidant effects and is toxic to cancer cell lines such as MDA-MB_231, MCF-7 (breast cancer), Hep G2, Hep 3B (liver cancer) and A549 (lung cancer), especially for Hep G2 liver cancer cells. Most notable (Hsu HF, Houng JY, Chang CL, Wu CC, Chang FR and Wu YC. (2005) Antioxidant 8 201008572 • Activity, Cytotoxicity and DNA Information of

Glossogyne teniufolia. J. Agric. Food Chem., 53: 6117-6125). The inventor of this case has been committed to the research and development of related fields for many years, and has a considerable understanding of related technologies. In view of the previous research, it has been confirmed that the substances in Xiang.4 grass have poisoning effect on cancer cells such as liver cancer and lung cancer, but their effects The machine has not been discussed and implemented in detail. It is the inventor's research in the spirit of excellence. After several years of thinking and exploration, and after many times of close testing and discussion, the present invention has finally been completed. SUMMARY OF THE INVENTION The main object of the present invention is to disclose Dehydr〇c〇stus and Glossogin, two biologically active substances derived from the scented grass which inhibit the growth of lung cancer cells, and a method for preparing the same At the same time, the application method of the anti-lung cancer substance disclosed by the present invention is provided. According to the above object, the method for preparing the anti-lung cancer substance and the cockroach of the genus of the genus of the present invention is in accordance with the needs of industrial application, thereby establishing a method for separating and purifying the lung cancer-resistant substance of the fragrant grass, and carefully discussing the operating conditions thereof. • In the end, the manufacturing process for industrial scale production will be included, and the six kings will include: ' (1) Extracting the fragrant grass with water or organic solvent to obtain a volume--a service such as a grass stem (2) The fragrant solvent obtained in the step (1) is extracted and layered by using different materials to obtain the extract of the fragrant grass extract. The extract of the fragrant grass obtained in the step (2) is used as the total layer B of the crushed rubber. Separate to obtain an effective distinction of the fragrant grass; 9 (3) 201008572 . (4) Separate and purify the effective fraction of the fragrant grass obtained by the preparative high-performance liquid chromatography column. The three anti-lung cancer active compounds disclosed in the invention. The method for applying the anti-lung cancer biological active substance of the fragrant grass comprises: (1) using a crude extract containing the anti-lung cancer substance disclosed by the present invention as a human body for preventing the production of lung cancer cells; (2) The use of the extract containing the anti-lung cancer substance of the scented grass of the present invention, the use of hydrazine as a human body to prevent the production of lung cancer cells; (3) the use of an effective distinguishing substance containing the anti-lung cancer substance of the fragrant grass disclosed in the present invention as a human prevention Application of lung cancer cell production; * (4) using the method disclosed in the present invention to prepare various pure substances as a human body for preventing the production of lung cancer cells; (5) using a crude extract containing the lung cancer-resistant substance of the fragrant grass disclosed in the present invention As an application for inhibiting the growth of human lung cancer cells; (6) using an extract containing the anti-lung cancer substance disclosed by the present invention, ® as an application for inhibiting the growth of human lung cancer cells; (7) using the present invention The effective differentiation of the scented grass against lung cancer substances - the use of 'skin' as a method for inhibiting the growth of human lung cancer cells; (8) using the method disclosed in the present invention to obtain various pure substances as a human body Application of the growth of cancer cells. In order to have a clear understanding of the purpose, efficacy and structural features of the present invention, the following preferred embodiments are described in conjunction with the drawings. 201008572. [Embodiment] The fragrant grass of the present invention is resistant to lung cancer. The substance and its preparation method mainly reveal the separation and purification of Dehydrocostus lactone and G1 ossogi η from the fragrant ruthenium and the separation methods thereof, and reveal the application methods of these anti-lung cancer substances to prove that the two substances can inhibit lung cancer Cell growth; see the first figure 'which lists the chemical structure of the substance and reveals the chemical formula of the substance as follows: (1) Dehydrocostus lactone : 6-Hydroxy-guaia- © 4(15),10(14) , lK13)-trien-12-oic acid lactone. As shown in the first figure (A). (2) Glossogin · 1 -Acetoxy-4-〇-isova1yry1eugeno1. ' As shown in the first figure (B). • The term “MTT analysis” as used in this manual refers to an anticancer drug screening process constructed by the National Cancer Institute's Cancer Treatment Department's Developmental Therapy Program in the 1980s (A1 ley MC et ▲ a 1. , Feasibility of drug screening with panels of ❹ human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48: 589-601, 1988; * Scud i ero DA et a 1. , Evaluation of a soluble tetrazolium/ Formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res. 48: 4827-4833, 1988;

Vi st i ca DT et a 1., Tetrazo1ium-based assays for cellular viability: a critical examination of 201008572 selected parameters affecting formazan. Cancer Res. 51: 2515-2520, 1991; Monks A. et al., Feasibility of a high -flux anti cancer drug screen using a diverse panel of cultured human tumor cell lines. J. Nat. Cancer Inst. 83: 757-766, 1991). In this analysis method, a substance having an anticancer effect (in the present invention, an anticancer substance derived from the separation of fragrant grass) is tested for its ability to inhibit the growth of a lung cancer cell line. The analytical principle is 3-[4,5-dimethylthiazol-2-❹yl]-2,5-diphenyltetrazolium bromide (MTT). The yellow water-soluble solid can be dehydrogenase in the intracellular granule (mi tochondr ia). (dehydrogenase) metabolism, the tetrazolium ring 'tetrazolium ring' is cut and reduced to purple insoluble sputum foraza (formaza η). Because the mitochondrial enzyme is active, the measured absorbance is directly proportional to the number of viable cells, so the amount of hyperthyroidism can be used to assess cell viability. The term "1C" value in the context of the present specification means a dose which causes 50% of lung cancer cells to die when an anticancer substance is administered. When ▲ IC5. The lower the value, the lower the dose of the anticancer sample, and the death of 50% of the cancer cells, which means that the ability to inhibit the growth of cancer cells is better. The following is a detailed description of various embodiments of the present invention as follows: Example 1, the scented column chromatography effectively distinguishes the effect of the product on inhibiting the growth of lung cancer cells: The whole plant fragrant grass is powdered into a powder by a fine crusher. 3 kg was placed in 2〇12 201008572. The liter was immersed in 95% ethanol for one day, and the gauze was passed through and then extracted with 9 π liters of 95% ethanol three times. After filtration, the sputum, & used a public 茱 ethanol extract _ pressure concentrated to remove the ethanol solution, lyophilized I to reduce the "hard: / East dry sample sample a total of 776. 67 grams. Take 400 grams of lyophilized The dried product was dissolved in 丨5 male 95% ethanol solution, and 1.5 liters of acetic acid B and _ liter 3 were added for stratification, which was evenly mixed by sowing. After standing to dissolve the solution, the mixture was collected and 7 ^ Ψ was collected. , the ethyl ester layer, this step is repeated 5 times, 5 times of the obtained ethyl acetate layer liquid Hua ~ ~ 仃 decompression concentrated and cold drying, can get 71. 9 grams of product, this product meets the ρ name Fragrant acetic acid acetic acid extract. Take the ethyl acetate extract of 61 军 军 61 61 61 61 61 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 The tube length is 760 mm), the mobile phase is n-hexane and ethyl acetate. The ratio is gradually increased from 90: 10, 80-20 to 1% acetic acid ethyl acetate and finally washed with 100% methanol. 5升升。 The mobile phase is used 1. 5 liters.

On the other hand, a liquid separator is used to collect the liquid flowing out of the chromatography column, and a collection tube is exchanged at a fixed time. During the column separation operation, 'using a thin film chromatography aluminum sheet (MERCK Co.) with different ratios of n-hexane and ethyl acetate developing solution and ethyl acetate and methanol developing solution as the mobile phase, the sample composition in each collecting tube The composition was developed at different positions according to the polarity of the developing liquid, and the position and size of the stain were judged by ultraviolet light at 254 nm and 365 nm for qualitative analysis. At the same time, a sample of the collection tube containing three substances, such as Mokko lactone, Dehydrocostus lactone and Glossogin, was combined, and after concentration under reduced pressure and freeze-dried, a product of 1. 5 5 g was obtained, and the product was named as "Zhezi Grass effectively differentiated. Matter 13 201008572 (effeetive fraction)". Among them, 'Mokko lactone, dehydrocostus lactone and glossogin contain 25.24%, 11.05% and 18.56%, respectively. The A549 lung cancer cell line was cultured in Dulbecco's modified Eagle's medium (DMEM-F12) medium containing 10% fetal bovine serum, 1% penici11in/streptomycin and 1.5 g/1 sodium bicarbonate, and placed at 37 ° C, 5 Incubate in an incubator of % C〇2. 3 xl03 A549 cells were cultured in 96-well culture plates, and each well was added with different concentrations of eucalyptus ethyl acetate extract or fragrant grass virgin, and 10% fetal bovine serum, 1% penicillin /streptomycin and 1.5 g/l sodium bicarbonate in DMEM- 'F12 medium, cultured at 37 ° C, 5% C (h in an incubator. After cultured for 7 hours, remove the original culture solution and add 丨〇 〇μ丨DMEM-F12 medium containing 丄mg/ml MTT was incubated for 1 hour, the culture solution was removed, and then dimethyl sulfoxide (DMMS0) was added and stirred until the precipitate was dissolved to obtain an immunoassay analyzer ( ELISA reader) The absorbance was measured at 550 nm. The results of MTT analysis showed that the ethyl acetate extract of 'Rhodoside' had no significant effect on the growth inhibition of A549 lung cancer cells, while the effective differentiation of the fragrant grasses inhibited the growth of A549 cells. The effect is excellent (as shown in the second figure), its IC5 value reaches 18.23 pg/ml. Example 1, Xiangyou grass column chromatography, Xiangzhuang grass Han contraction differentiation product inhibits the growth of lung cancer cells: 0.95 5 grams dry The effective distinction of the fragrant grass is placed in a packed column of 15〇14 201008572. gram Si-60 Shiyuejiao (40-63 μΐη, Merck Co.) in a chromatography column (inner diameter 45 mm 'tube length 60 0 mm), flowing The phases were n-hexane and ethyl acetate, the ratio was gradually increased from 90:10, 80-20 to ethyl acetate, and the mobile phase was used for each ratio of 200 ml. On the other hand, the liquid collection collector was used for collection. The liquid flowing out of the column is replaced by a collection tube at a fixed time. During the column separation operation, the sample composition in each collection tube is analyzed by a film chromatography method and will contain Mokko iactone, Dehydrocost us. The collection of three substances, such as lac tone and Glossogi η, is combined with the sample. After concentration under reduced pressure and lyophilization, 46.5 mg of the product is obtained. This product is named "Fruit Grass Concentrate". Among them, Dehydrocostus The two substances, lactone and Glossogin, contained '33.03% and 16.45%, respectively. According to the results of MTT inhibition of lung cancer cell growth test in Example 2, the concentration of this fragrant grass stalk was inhibited by A549 lung cancer cells. The effective distinguishing substance is better (as shown in the third figure), its IC5 value reaches ❹ 11.56pg/ml. Example 2, the anti-cancer component of zizi grass inhibits lung access ^ + > : Approximately 44 mg of fragrant grass concentrated fractions were purified by preparative high-performance liquid chromatography to prepare anti-cancer components. The column used for the analysis was a STAR Si column (RT). -250, Hibar Fertigsaule, 5 Class, 1〇_ χ 250 mm) 'The mobile phase is n-hexane and ethyl acetate, Α μ, more or, ^ ratio is 90:10, volume flow is 1 m 1 /m 1 η. 7 mg of yellow oil (6-hydroxy-guaia- through this purification operation, can be obtained l4 dehydrocostus lactone: 15 201008572 4 (15), 10 (14), ll (13)-trien-12-oic acid 1 Actone): [a]28.2 = -16.67°; IR (CHC13) vmax 1764, 1639 cm-1; EIMS: m/z 231 (M+H)+, 1H NMR (400 MHz) δ 1.42 (1H, dddd, J = 13.2, 11. 5, 10.2, 5.6 Hz, H-8), 1.87 (1H, ddd, J = 13.2, 4 .7, 2. 7 Hz, H-2), 1.95 (1H, ddd, J = 13. 2, 7.6, 1. 7 Hz, H-2), 2. 16 (1H, dddd, J = 12.2, 10 .4, 5.6, 5. 6 Hz, H-9), 2.24 (1H, dddd, J = 13.2, 8. 2, 5. 8, 4. 4 Hz, H-8), 2.87 (1H, dd, J = 9.3, 3. C 1 Hz, H- 5), 2.82-2.95 (2H, m , Hl, H-7), 3. 97 (1H, t, J = 18.4 Hz, H-6), 4.81 (1H, s, H- 15a), 4. 90 (1H, s, H-15b), 5.06 (1H, br d, J = 2.0

Hz, H-14a), 5.27 (1H, br d, J = 2.0 Hz, H-14b), 5.49 (1H, d, J = 3.2 Hz, H-12a), 6.22 (1H, d, J = 3.6 Hz , H-12b); 13C NMR (100 MHz) δ 30.28 (C-2), 30.91 (C-8), 32.57 (C-3), 36.25 (C-9), 45.10 (C-7), 47.58 ( Cl), 52.01 (C-5), 85.23 (C-6), 109.60 (C-f 14), 112.61 (C-15), 120.19 (C-12), 139.71 (C-ll), •149.21 (C -10), 151.22 (04), 1 70.24 (C-13); -mo 1 ecu 1ar: C15H1 802. Also available is 7.3 mg of yellow oil in the form of £1〇880£丨11 (1'- acetoxy-4-O-isovalyryleugenol): [α] 2 7.6 = -34.5. (CHC13); IR (CHC13) vmax 1743, 1587 cm-1; EIMS: m/z 305.8 (MH)+, 1H NMR (400 MHz) δ 1.06 (6H, d, J = 6_8 Hz, H-4" , H-5"), 2.11 (3H, s, H-5'), 2_25 16 201008572 (1H, m, H-3"), 2.45 (2H, d, J = 7·2 Hz 'H-2") , 3.82(3H, s, H_7), 5.26(lH,d,J = i〇.4Hz, H-3' a), 5.31 (1H,d, J = 17.2 Hz, H-3,b),5 99 (1H ddd, J = 17.2, 10.8, 5.6 Hz, H-2'), 6_25 (1H, d, J = 5.6 Hz, H-l'), 6.94 (1H, dd, J = i2_4, 2.0 Hz H- 3), 6.941 (1H, t, J = 17.6 Hz, H-2), 6.99 (1H, d J = 8.4 Hz, H-6), 13C NMR (100 MHz) δ 21.24 (C-5,) 22.33 ( C-4' ', C-5"), 25.90 (C-3"), 43 〇 4 (c_ H 2,), 55.79 (C-7), 75.70 (C-l'), 111.43 (C-2 ), 116.99 (C-3,), 119.58 (C-3), 122.79 (C-6), 135.94 (C-2,), 137.50 (Cl), 139.59 (C-4), i511〇 (c_5), -1 69.90 (C-4'), 171.02 (Cl,,); molecular: • C17H2205. The fourth figure is based on the third example. TT inhibits lung cancer cell growth test results, Dehydrocos tus lactone For the other two substances Gloss〇gin A 5 4 9 the effect of suppressing the cancer cells are excellent, estimated I c 5. The values are 6. 6.27 and 48.49 pg/ml, respectively. - Example 4, fragrant grass column chromatography effectively distinguishes the product from inhibiting the growth of lung cancer cells - long-acting effect: A549 cells are effectively treated with fragrant grass for 24-48 hours, washed with phosphate buffer, and placed in 75 % ethanol was placed at 4 ° C overnight. Subsequently, the cells were washed twice with phosphate buffer, and the cells were suspended in phosphate buffer containing 40 pg/ml propidium iodide and 0.1 mg/ml RNase A, and treated at 37 ° C for 30 minutes. Finally, flow cytometry (flow) 17 201008572 • cytometer) to observe. As shown in the eighth figure, after the A549 cells are effectively treated by 15 column column chromatography, some of the cells will remain in the sub-Gl phase and no longer grow and multiply, so the fragrant grass has a distinction. The product causes lung cancer cells to die by apoptotic pathway. Moreover, the ratio of the number of A549 cells arrested in the sub-G1 phase increases with the treatment time. The dose of the product which effectively distinguishes the applied fragrant grass increases and increases (as shown in the figure). Example 5: Dehydrocostus lactone inhibitory effect of lung cancer cell growth: A549 cells were treated with Dehydrocostus lactone for 24 to 48'. The method described in Example 4 was used to observe the long period of A549 cells. influences. As shown in Figure IX, after treatment with - Dehydrocostus lactone, the growth cycle of some cells will be arrested in the sub-Gl phase and no longer grow and multiply. Therefore, the effective fractionation of the fragrant grass will lead to cell cycle of lung cancer cells. Death through the apoptotic pathway. Moreover, the ratio of the number of A549 cells stagnation in the sub-G1 phase also increases as the treatment time increases Ο 曰 and the dose of the effective sage is effectively increased (as shown in Fig. 2). - Example 6, the active ingredient of the geranium glossogin inhibits the growth of lung cancer cells: A549 cells are treated with Glossogin for 24 to 48 hours, and then the method described in Example 4 is used to observe the growth cycle of A 5 4 9 cells. Shadow. As shown in the tenth figure, after the treatment of GL〇SSOgin, the growth cycle of a cell will be stagnant in the sub-Gl phase and no longer grow and reproduce. The ring part 18 is labeled as above 201008572. • Therefore, the effective differentiation of the product by the scented grass can cause the lung cancer cells to die and die. Moreover, the increase in the number of A549 cells stagnant in the sub-G1 phase was associated with an increase in the effective differentiation of the applied fragrant grasses (as shown in Figure 7). π~Λ Ding Dan is also your ή and practicality. At the same time, it has been found in the technology of such techniques at home and abroad. It has not been found that the structure with the same approximation exists first, and should be named "progressive" "Combined with industrial use": and the patent requirements of «©" are submitted in accordance with the law. However, the above description is only a preferred embodiment of the present invention, and the application of the present specification and the scope of the patent application are all other equivalents, and are included in the invention. Will also follow the dose on the progress of the literature together with the "new creative deduction" method of the end of the knot 3 0 201008572. [Simplified schematic] The first series shows the chemical formula of Dehydrocostus lactone and Glossogin. The second series shows the inhibition ratio of the effective differentiation of the fragrant grass to the growth of A549 lung cancer cells. The third series shows the inhibition ratio of the Fragrant Grass Concentrate on the growth of A549 lung cancer cells. The fourth panel shows the inhibition ratio of two anti-cancer compounds of sage, A 5 49 lung cancer cell growth ® . The fifth series shows the ratio of cells treated with different doses of fragrant grass column chromatography to distinguish the A549 lung cancer cells from sub-G1 after 24 and 48 hours. The sixth series shows that A549 is treated with different doses of Dehydrocostus lactone. The ratio of cells in lung cancer cells that were arrested in the sub-G1 phase after 24 and 48 hours. The seventh series shows the ratio of cells in A 5 49 lung cancer cells treated with different doses of G1 〇 s s 〇 g i η, which persisted in the s u b - G1 phase after 24 and 48 hours. • The eighth series shows that A549 cells are effectively distinguished by the column chromatography of fragrant grasses. The ninth panel shows the results of treatment of A549 cells with Dehydrocostus lactone. The tenth series shows the results of treatment with Glossogin on A549 cells. [Main component symbol description] None 20

Claims (2)

201008572 . X. Patent application scope: 1. A fragrant grass anti-lung cancer substance, which contains the following ingredients: Dehydrocostus lactone: 6-Hydroxy-guaia-4(15),10(14),11(13)-trien-12- 〇ic acid lactone ; Glossogin : 1 ^ce^〇xy~4-〇-is〇valyryleugenol ° 2.According to In the patent H, the incense of the grass is the anti-lung cancer substance, wherein β, the Xiangzhi grass anti-lung cancer substance can be used alone. 3. According to the towel, please note that the fragrant grass of the patent scope is the anti-lung cancer substance, wherein the scented anti-lung cancer substance can be used in combination. 4. According to the patent application scope of claim 1, the fragrant 11 grass anti-lung cancer substance, wherein the fragrant grass anti-lung cancer substance can be used as a composition. 5_ - A method for preparing a succulent herb against lung cancer, comprising: (a) extracting the fragrant grass with water or an organic solvent to obtain a crude extract of fragrant grass; (b) obtaining the fragrant scent of (a) The crude extract of the grass is extracted by other different organic solvents to obtain the extract of the fragrant grass; - (4) the extract of the fragrant grass obtained in the step (8) is subjected to the separation of the stalks of the stalks to obtain the active ingredients of the fragrant grasses. Distinguishment; Knife (d) 7 Step (c) Known that the active ingredient of the fragrant grass is purified and separated using a preparative type of liquid chromatography column. '俾(4) 仏咖... lactone and Glossogin and other two anti-lung cancer Active substance. 6. The method for extracting the crude extract of the scented scented grass according to the method of claim 5, wherein the step (a) is used for extracting the crude extract of the scented scented grass. 21 201008572. The solvent is ethanol and a sub-alcohol. 7. According to the fragrant method described in claim 5, the step (b)# a. Preparation of grass anti-lung cancer substance 诹 The solvent used for the extraction and preparation of fragrant machine is ethyl acetate. According to the patent application of the Tutu flute c method, the preparation of the (4) grass anti-lung cancer substance described in the step (c) is used to produce the fragrant grass with #^" The column packing is Si, Shishijiao. Heart division 9 · According to the patent application gluten κ = ❹ J range item 5 according to the method of the zizi grass anti-lung cancer method, wherein the fishing equipment of Zhou Cong 4 Unsuccessful (C) used to make the active component of the Fraxin grass substance, the mobile phase of the layer is n-hexane and ethyl acetate. 10. According to the application of the patent of Mingqing Patent Mingtuzhe scope 5, the grass is anti-lung cancer The chromatographic separation step for preparing the substance f and/or the step (C) for producing the active ingredient of the fragrant grass comprises one or more chromatographic separation operations. 11. According to the patent claim 5 The method for preparing an anti-lung cancer substance, wherein the high-performance liquid chromatography column for producing the anti-cancer substance of the sage grass in the step (d) is a normal phase rubber tube column. The door is according to the fifth aspect of the patent application. Method for preparing anti-lung cancer substance of fragrant grasses, wherein step (d) is used High performance liquid chromatography flow mushroom grass anticancer substances phase of n-hexane and ethyl acetate. 22