TW201019947A - Plant derived compounds and compound formulae containing the same for the treatment of cervical cancer - Google Patents

Abstract

A compound formula for reducing the activity of a cervical cancer cell in a subject is provided. The compound formula includes at least one compound selected from the group consisting of baicalein, baicalin, berberine hydrochloride, and derivatives thereof, and at least one compound selected from the group consisting of isopsoralen, triptolide, quercetin, gossypol-acetic acid, and derivatives thereof.

Description

201019947 VI. Description of the Invention: [Technical Field] The present invention relates to a plant-derived compound for treating cervical cancer; in particular, a compound comprising the above compound. [Prior Art]

Cervical cancer ranks second in Taiwan's female cancer deaths, killing more than a thousand lives each year. It has been confirmed that human papilloma virus (HPV) infection is one of the necessary causes of cervical cancer. HPV vaccines have been developed in the United States and European countries to effectively prevent a small number of human papillomavirus strains, which have been shown to be associated with 70% of cervical cancer lesions. However, Taiwan's local R&D units have not made significant progress in the prevention and treatment of cervical cancer. Human papillomavirus is a double-stranded DNA virus without an outer membrane, and its appearance is a symmetric icosahedron. The genome of human papillomavirus is surrounded by the protein shell of 72 protein capsomer compositions. The HPV genome is highly susceptible to mutation in the host. Currently, approximately 250 HPV strains have been identified; in general, the structure of human papillomavirus often varies with region, lifestyle, ethnicity, and route of infection. As noted above, HPV vaccines are only targeted at certain high-risk strains; therefore, women must be on a regular basis even if they have been vaccinated with HPV; ^ uterus = smear. In addition, the 'HPV vaccine should be injected before infection, so the main target of the vaccine is women who have not had sex. After infection with human papillomavirus, the virus may induce mutations in the cell surface. At present, the (four) sub-cancers include cancer resection, radiation therapy and/or chemotherapy. ‘, ^ 3 201019947 In view of this, there is an urgent need to propose a treatment method for cervical cancer. SUMMARY OF THE INVENTION Accordingly, one aspect of the present invention provides a composition for reducing the activity of cervical cancer cells. According to a specific embodiment of the present invention, the composition comprises at least one of the following compounds: isopsoralen, triptolide, baicalein, gallic acid, quercetin, gossypol acetate, baicalin, hydrochloric acid Berberine and its derivatives, and in an amount sufficient to reduce the activity of cervical cancer cells in a subject. Another aspect of the present invention provides a composition for reducing the viral activity of human papillomavirus in a virus-infected cell or a virus-infected subject. According to a particular embodiment of the invention, the above composition comprises at least one of the following compounds: flavin, gallic acid and its derivatives, and is present in an amount sufficient to reduce viral activity in a virus-infected cell or virus-infected subject. Yet another aspect of the invention provides a method of reducing the activity of cervical cancer cells. According to a specific embodiment of the present invention, the method comprises at least administering to the cervical cancer cell a composition comprising at least a therapeutically effective amount of isopsoralen, triptolide, safrole, gallic acid, suede Ordinary, cotton aphid acetic acid, yellow bitter, hydrochloric acid small test or its derivatives. Still another aspect of the present invention provides a method for reducing viral activity of a human papillomavirus in a subject infected with a virus-infected cell or virus. 201019947, in accordance with an embodiment of the present invention, the method comprising at least administering to the subject a virus-infected cell or a virus-infected subject a composition comprising at least a therapeutically effective amount of baicalein, gallic acid or a derivative thereof. The above and other features, aspects and advantages of embodiments of the present invention will become more apparent from the aspects of the appended claims. The above summary and the following detailed description are merely illustrative, and are intended to provide a further detailed description of the claimed subject matter. ❿ [Embodiment] Among the currently identified human papillomavirus lines, 15 are classified as high risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, respectively). 58, 59, 68, 73 and 82). More specifically, about 70% of cervical cancer cases are associated with HPV 16 and HPV 18. Although the above lines are classified as “high risk” types, infection with high-risk HPV lines does not necessarily lead to cervical cancer. On the other hand, even viruses that may be of high risk type (26, 53 and 66) and low risk types (6, 11, 40, 42, 43, 44, 54, 61, 7〇, 72, 81 and CP6108) Infection can also cause cervical cancer. Traditional medicine and Chinese herbal medicine have been used for thousands of years. Due to advances in modern science and medicine, it has been confirmed that Chinese herbal medicine can effectively prevent, treat and/or alleviate various diseases and discomforts. In addition, Chinese herbal medicines are known to have fewer side effects on the human body. Recently, both the Eastern and Western medical professions have proposed traditional medicine to match common cancer treatments such as chemotherapy, or to alleviate their side effects. • The inventors explored a variety of botanical Chinese herbal medicines to find Chinese herbal ingredients that can treat cervical cancer and/or human papillomavirus, including psoralen (ΡίΌΓύτ/ββ cory/zyb/z.flr), Leigong Vine ( wilfordii Hook. F.), Jingu {Scutellaria baicalensis Georgi), HX Cornus officinalis, Blot I { Sophore flavescents j".), Cotton seeds and Yellow Lotus (Cop along Frimc/〇. Extraction The above-mentioned botanical Chinese herbal medicine, and the active ingredient thereof is purified by high performance liquid chromatography (HPLC), and the purity of each active substance obtained is 97% or more. The following Table 1 lists the used substances. The name and structure of the plant and its active compounds. Table 1 The active compound of the specific plant. Its structural formula number Plant-derived active compound / knot formula A psoralen isopsoralen. Human A^. ChH6〇3 B Leigong Teng Leigong Rattan lactone CH3 δΗ C2〇H24〇6 C Astragalus scutellariae Cl5Hl〇〇5 6 201019947

OH Ο

D Hawthorn Gallic acid

(H0)3C6H2C02H E Bitter quercetin

C15H10O7 F Cotton Seed Gossypol Acetate C3〇H3〇08 · C2H4〇2

G Astragalus Baicalin C2lHi8〇ll 201019947

According to one aspect of the present invention, an in vitro test was conducted to investigate the effect of the above compounds for reducing the activity of cervical cancer cells (HeLa cells). According to another aspect of the present invention, an in vitro test is conducted to investigate the effect of the above compound for reducing the viral activity of human papillomavirus in a virus-infected cell and/or a virus-infected subject. Noun Definitions Terms used in this specification generally have the meanings that are common in the art, whether under the context of the present invention or any other context. Certain terms are used in the specification to enable those skilled in the art to understand the invention. Synonyms for certain nouns are provided in the specification. However, the reference to one or more synonyms does not imply the exclusion of other synonyms. In the present specification, the use of the exemplified descriptions, including any examples of the nouns described herein, are merely illustrative and should not be construed as limiting the scope and meaning of the invention or any exemplary term. The invention is not limited to the specific embodiments described in the specification. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. When there is a conflict between the two, this specification includes the noun definition part and will be avoided. "Treatment" covers the provision or treatment of any disease in humans. The so-called treatment involves inhibiting the disease, preventing the development of the disease, slowing down the disease, for example by degrading it, or recovering or repairing the loss, loss, or defect; or stimulating an inefficient physiological process. The above nouns include obtaining an ideal pharmacological and/or physiological effect covering the treatment of any pathological condition or condition in humans. The above effects may be prophylactic, i.e., capable of completely or partially preventing the condition and or associated symptoms; or may be therapeutically, i.e., capable of partially or completely curing the condition and/or the adverse effects caused by the condition. Accordingly, the present invention provides for the treatment and prevention. It contains (1) preventing the disease or recurrence of a subject who may have a disease but has not yet developed symptoms; (2) inhibiting the disease, such as preventing the development of the disease; (3) stopping or eventually stopping the disease or at least the associated symptoms, and The patient is no longer suffering from the disease or its symptoms, such as degrading the condition or its symptoms, for example by replying or repairing the function of loss, loss, or defect; or stimulating an inefficient physiological process; and (4) mitigating, easing Or to ameliorate a condition or a related condition, wherein the term "improvement" broadly refers to reducing the strength of at least one parameter, such as the cellular activity of a cancer cell or a virus-infected cell, which may be cell proliferation or cellular metabolic activity. > "Effective dose" means that the amount of the compound is sufficient to produce a desired effect on the subject being treated. For example, an effective dose of a composition according to a specific embodiment of the present invention, in the embodiment of the present invention, should be sufficient to reduce the activity of cervical cancer cells in a subject. According to another specific embodiment of the present invention, the effective dose combination = should be used to reduce the viral activity of the human papilloma virus in the squamous infection of the infected or virgin infection. The precise dosage will vary with the purpose of the treatment, and the skilled artisan can utilize known techniques to determine such dosages. It is also known in the related art that it may be necessary to adjust the dosage for systemic or topical administration, annual weight, general health, gender, eating habits, time of administration, interaction of music, and severity, and this is known. The skilled artisan can determine this dosage via routine experimentation. And "pharmaceutically acceptable carrier" means a non-toxic solid, semi-solid or liquid filler, a riding agent, a packaging material, a formulation auxiliary #], and a type of excipient. The pharmaceutically acceptable ketone is not toxic to the user at the dose and concentration and can be compatible with the pelvic ingredients of the formulation. 〃 Du materials and methods

*Biomaterials and chemicals

HeLa cell line was from Dr. Shen Xinxin from the Institute of Biomedical Research, Institute of Industrial Technology. The cells are adherent cells, and DMEM (Dulbecco, sm〇dified Eagle, s medium) is used to proliferate and maintain HeLa cells. The medium is supplemented with 〇% fetal bovine serum (FBS), and 1.5 g/L of hydrogen carbonate. Sodium (NaHc〇3), lmM sodium pyruvate, and O.lmM non-essential amino acid. Human umbilical vein endothelial cells (HUVEC) were purchased from the Hsinchu Food Industry Research Institute. The Medium 1" medium was used to proliferate and maintain huve cells, and 10% FBS, hepatic anticoagulant and EGFp were added to the medium. 201019947 The human 293FT cell line is from the American company invitr〇gen Corporation. 293FT cells were propagated and maintained in DMEM, and 10% fetal bovine serum (FBS), 0.1 mM non-essential amino acid, and 500 ug/ml of geneticin antibiotic (G418) were added to the medium. The plastids pl6sheLL and pCIneoEGFP were from Dr. Ronald Schiller of the National Cancer Institute. The DPBS-Mg buffer contained 100 ml of DPBS, 475 μM of 2M magnesium chloride (MgCl2), and 1 ml of 100 Χ antibiotic stock solution. 0 DMEM, Medium 199 medium, geneticin antibiotic, non-essential amino acid, lipofectamine 2000, cell culture reagents Opti-ΜΕΜ-Ι and DPBS were purchased from American company Gibco Invetrogen. The FBS is purchased from the Israeli company Biological Industries Ltd. Hepatic antithrombin, EGFP, and MTT, Brij-58, 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazoliu-m bromide were purchased from American company Sigma-Aldrich Corp. 〇

Doxorubicin HC1 is a commercially available chemotherapeutic drug that can be used to treat cancer, purchased from Sigma-Aldrich Co.

Corning® 96-well plate (Cat. No. Cat. 3603) was purchased from the American company Corning Inc. ♦ Cytotoxicity Assay (MTT Analysis) MTT assay was used to evaluate cells of the compounds of the present invention against HeLa cells and HUVEC cells. toxicity. HUVEC cells were used to study the cytotoxicity of the above compounds against normal human cells. Further, the chemotherapeutic drug doxorubicin HC1 was used as a control group to investigate the effectiveness of the compound according to the specific examples of the present invention. Briefly, the MTT assay consists of at least three steps: (A) Implant the cells into 96-well plates 3 hours prior to treatment, with HeLa cells at a concentration of 1×104 cells per well, and HUVEC cells at a concentration of 1×104 cells per well. 2X104 cells; (B) The cells are then treated with different concentrations of test samples and cultured for a predetermined period of time (24, 48 and 72 hours); (C) after the predetermined period of time, the test samples are removed and MTμΙ of MTT reagent (1 mg/ml) was added to the wells; (D) 96-well plates were incubated for 5 hours at 37 ° C with 5% carbon dioxide; (E) Remove MTT reagent 'and in each well Add ι〇〇μι DMSO; (F) Use an ELISA analyzer to read the optical density (OD) of each well at 560 nm. Each test was repeated 3 times and the results presented in the table of this specification ❹ were the average of 3 replicates. The 〇D. values of the control group and the experimental group were recorded. Cell viability was calculated in each sample as follows: Cell viability (%) = 〇.D. Experimental group / O.Dm * 1 〇〇〇 /. Thereafter, the cell viability was plotted against the compound concentration, and the GraFit. data analysis software (Erithacus Software Ltd.) was used to calculate the IC5 enthalpy of each compound. • Preparation of HPV16 pseudovirus expressing green fluorescent protein (GFP). Papillomavirus protein shell 12 modified with 2 3 9 F τ cells and codons 12 201019947 Gene L1 and L2, plastid: pl6shell and GFP reporter plastid pCIneo-GFP was co-transfected 'to observe the infection state of the cells. Prepare and obtain HPV 16 pseudovirus that can express GFP according to the technical manual provided by the National Cancer Institute. Please refer to http://home.ccr.cancer.gov/lco/production.asp for the above technical manual. In the example, the viral power of the HPV 16 pseudovirus expressing GFP used was about 4*1〇8 infectious units per ml.

♦ Screening of anti-infective compounds HeLa cells were seeded in a % well plate at a density of 6 x 103 cells per well. Different volumes of 2X HPV 16 pseudovirus were added after 24 hours. After 48 hours, the ELISA analyzer was used to read the fluorescence intensity of the sample, and the j-graph shows the results. Figure 2 shows the signal-to-noise ratio in each HPV 16 pseudovirus volume. When selecting a screening platform, the infection rate is about 20%. Therefore, a 96-well plate containing approximately 0.25 μM of HPV 16 pseudovirus per well was used as a platform for screening anti-infective compounds. At this dose, the signal noise ratio is about 2 〇_2 5 7. The screening method is as follows: The density of the cells will be (Α) about 6Χ103 fine HeLa cells per well into the 96-well plate 24 hours before the treatment; B) Cells were treated with different concentrations of test samples to prepare cells for infection with HPV 16 pseudovirus. (:卞^ '25 hours; Cultivate Village 13 201019947 - \C) Use the ELISA analyzer to read the fluorescence intensity of the sample (Excitation : 485 nm, Emission : 535 nm) and perform according to the method described above. MTT analysis. Red algae gum was used as a positive control drug. Each test was repeated 2 times, and the results listed in the present invention are the average of 2 replicates. Cell viability and IC5Q values were obtained using the methods described above. Results Reference cytotoxicity of the active compound A-oxime to HeLa cells According to one aspect of the present invention, a series of experiments were conducted to determine the cytotoxicity of each compound against HeLa cells. The active compounds listed in Table 1 and the positive control compound, Doxorubin HC1, were administered to HeLa cells and HUVEC cells at various concentrations. In the relevant experiments, HeLa cells in the non-active compounds A-Η and Doxorubin HC1 (addition concentration of xanth/mi) were used as negative control. The survival rates of HeLa cells and HUVEC cells at 24, φ 48 and 72 hours after treatment were obtained according to the methods described in the above Materials and Methods section. Tables 2 and 3 list the results of the MTT analysis. In the present specification, 'Compound A to sputum are isopsoralen, triptolide, baicalein, gallic acid, quercetin, gossypol acetate, berberine and berberine hydrochloride, respectively. Table 2 Survival rates of active compounds and HeLa cells at different concentrations for a predetermined period of time ----- (% of untreated blank control group), _ - Compound A: isopsoralen 0 pg/ml 0.125 μ ^ιηΐ 1·25 tig/nil 12.5 μβ/ml 125 pg/ml 14 201019947

24 hours 100 91 88 70 18 48 hours 100 99 75 30 11 72 hours 100 98 69 10 7 Compound B: triptolide 0 pg/ml 0.001 pg/ml 0·01 pg/ml 0.1 μ^πιΐ 1 pg/ml 24 Hour 100 98 89 33 26 48 hours 100 94 37 14 12 72 hours 100 81 15 9 9 Compound C: baicalein 0 μ^/τηΐ 0.125 μ^ιηΐ 1_25 pg/ml 12.5 μξ/ηά 125 μ^ιηΐ 24 hours 100 104 126 109 134 48 hours 100 107 110 73 68 72 hours 100 99 106 66 29 Compound D: gallic acid 0 pg/ml 0.125 pg/ml 1.25 μβ/πιΐ 12·5 pg/ml 125 pg/ml 24 hours 100 85 84 90 17 48 hours 100 96 91 82 11 72 hours 100 101 103 99 8 Compound E: quercetin 0 μ^ιηΐ 0.1 μ^πχΐ 1 μ§/ϊη\ 10 pg/ml 100 pg/ml 24 hours 100 93 96 83 — 48 hours 100 102 102 51 32 72 hours 100 95 91 33 12 Compound F: gossypol acetate 0 μ^ηιΐ 0_1 pg/ml 1 pg/ml 10 μ§/ηύ 100 pg/ml 24 hours 100 101 97 80 17 48 hours 100 103 105 52 13 72 hours 100 98 98 40 10 Compound G: baicalin 0 pg/ml 0.1 μ^ιηΐ 1 μ§/πι1 10 pg/ml 100 pg/ml 24 hours 100 10 4 103 109 68 48 hours 100 94 96 98 50 15 201019947 72 hours 100 97 97 97 37 Compound Η: berberine hydrochloride 0 μ^ιηΐ 0.1 μ^ιηΐ 1 pg/ml 10 pg/ml 100 pg/ml 24 hours 100 89 87 82 28 48 hours 100 98 84 71 17 72 hours 100 98 91 62 10 Control : Doxorubin HC1 0 pg/ml 0.00058 pg/rnl 0.0058 pg/ml 0.058 pg/ml 0.58 pg/ml 24 hours 100 96 95 84 46 48 Hours 100 100 92 81 23 72 hours 100 97 89 72 11 As can be seen from Table 2, the cell viability of HeLa cells treated with active compound AH was lower than the cell viability in the negative control group after different predetermined times. In contrast to Doxorubicin HC1, the active compounds proposed in the examples of the present invention, such as isopsoralen (Compound A), triptolide (Compound B), gallic acid (Compound D), under otherwise identical conditions ), gossypol acetate (compound F), and berberine hydrochloride (compound H) are more cytotoxic to HeLa cells. For example, in the group treated with 1 〇〇 gg/mi of triptolide, the survival rates of HeLa cells at 24, 48, and 72 hours were 20% and 13 °, respectively. And 9%. On the other hand, to 0.58

The Hg/ml Doxorubicin HC1 treated HeLa cells had 46%, 23%, and 11% survival at 24, 48, and 72 hours, respectively. Table 3 Survival of different concentrations of active compound and HUVEC cells at predetermined times (% of untreated blank control group) 16 201019947

Compound A: Isopsoralen 0 pg/ml 0.125 μ^/ναΐ 1.25 pg/ml 12.5 pg/ml 125 pg/ml 24 hours 100 103 103 100 67 48 hours 100 104 96 83 31 72 hours 100 92 87 80 19 Compound B: triptolide 0 pg/ml 0.001 pg/ml 0.01 pg/ml 0.1 pg/ml 1 pg/ml 24 hours 100 93 94 59 71 48 hours 100 102 111 36 35 72 hours 100 108 114 23 23 Compound C: Baicalin 0 pg/ml — 1.25 pg/ml 12.5 pg/ml 125 pg/ml 24 hours 100 — 109 88 199 48 hours 100 — 94 75 78 72 hours 100 — 83 69 57 Compound E: quercetin 0 μ§/ Ηη\ 0.1 pg/ml 1 μ^ιηΐ 10 μ^ιηΐ 100 μ^πιΐ 24 hours 100 104 108 101 120 48 hours 100 102 95 87 51 72 hours 100 98 94 80 26 Compound F: Gossypol vinegar 4 ester 0 pg/ Ml 0.1 pg/ml 1 pg/ml 10 μ^ιηΐ 100 pg/ml 24 hours 100 93 88 80 20 48 hours 100 104 104 85 17 72 hours 100 103 98 71 17 Compound G: baicalin 0 pg/ml -- 1.25 Μ〇/τη\ 12.5 μ§/ηύ 125 pg/ml 24 hours 100 — 98 97 81 48 hours 100 — 96 101 76 72 hours 100 -- 88 75 56 Compound Η: berberine hydrochloride 17 201 019947

24 hours 48 hours 72 hours

100 1 Hg/ml 10 pg/ml 100 pg/ml — 89 76 23 81 64 16 77 51 15 ~Control : Doxorubin HC1 0.145 pg/ml 0.290 μ^ιηΐ 0.580 pg/ml 111 100 90 77 56 19 68 38 14 Table 2 and 矣1 human tear, 3 can be found, at a specific concentration, the above activation: Xi compound has higher selectivity for HeLa cells, and prisoner is more suitable to use the mucus to reduce the cervix in the subject Cancer cell activity] In the case of triptolide (Compound B), the cell survival rate of huvec cells and HeLa cells in triptolide at ο·οι pg/ml was 114% at 72 °C after treatment. With 15%. Active Compound A-Η Inhibits ICS0 Value of HeLa Cells The data in Tables 2 and 3 are plotted as a line graph to illustrate the relationship between compound concentration and cell viability. Figure 3_u is a line graph showing the relationship between the survival rate of HeLa cells and the degree of compound used in each experimental group and control group. The GraFit data analysis software was used to calculate the ic50 value of each compound inhibiting HeLa and HUVEC cells. The results are shown in Table 4. The IC50 of each compound to HUVEC cells and the ratio of IC5〇 to HeLa cells are also listed in Table 4. 201019947 Active compound and control compound inhibit IC50 of HeLa cells and HUVEC cells at predetermined time

Compound A: Isopsoralen 24 hours IC50 (pg/ml) 48 hours IC50 (pg/ml) 72 hours IC50 (μ^ιηΐ) HeLa 27 5 2 HUYEC >125 57 38 HUVEC/HeLa ratio &gt ;4.6 11.4 19 Compound B: Triptolide 24 hours IC50 (ng/ml) 48 hours IC50 (ng/ml) 72 hours IC50 (ng/ml) HeLa 72.08 7.24 2.87 HUVEC >1000 151 84 HUVEC/HeLa Ratio >13.9 20.9 29.3 Compound C: xanthine 24 hours IC50 (pg/ml) 48 hours IC50 (pg/ml) 72 hours IC50 (pg/ml) HeLa >125 >125 37 HUYEC >125 >125 >125 HUVEC/HeLa ratio — — >3.4 Compound D: IC50 for pentaic acid for 24 hours (pg/ml) IC50 for 48 hours (pg/ml) IC50 for 72 hours (pg/ml) HeLa 37 26 45 Compound E: quercetin 24 hours IC50 (pg/ml) 48 hours IC50 (pg/ml) 72 hours IC50 (gg/ml) HeLa >100 20 6 HUVEC >100 104 36 HUYEC/ HeLa ratio — 5.2 6 Compound F: gossypol acetate 19 201019947 24 hours IC50 (pg/ml) 48 hours IC50 (pg/ml) 72 hours IC50 (pg/ml) HeLa 29 12 8 HUVEC 30 33 23 HUVEC /HeLa ratio 1.0 2.8 2.9 Compound G: IC50 (pg/ml) for 24 hours IC50 (μ^ιηΐ) IC50 (pg/ml) for 72 hours HeLa >125 >125 92 HUVEC >125 >125 218 HUVEC /HeLa ratio — — 2.4 Compound H: berberine hydrochloride 24 hours IC50 (pg/ml) 48 hours IC50 (pg/ml) 72 hours IC50 (pg/ml) HeLa 39 21 15 HUVEC 30 15 9 HUVEC/ HeLa ratio 0.8 0.7 0.6 Control : DoxorubinHCl IC50 for 24 hours (μ_) IC50 for 48 hours (μ_) IC50 for 72 hours (pg/ml) HeLa 0.48 0.20 0.12 HUVEC >0.58 0.299 0.22 HUVEC/HeLa ratio >1.2 1.5 1.8 As can be seen from Table 4, the active compounds A to guanidine were effective in reducing HeLa cell activity. Therefore, according to one aspect of the present invention, a composition for reducing the activity of cervical cancer cells comprises at least one of the following compounds: isopsoralen, tripterygium wilfordii, safrole, gallic acid, quercetin Gossypol acetate, baicalin, berberine hydrochloride and its derivatives, and the content thereof is sufficient to reduce the activity of cervical cancer cells in the subject. 20 201019947 , It is understood by those skilled in the art that the higher the ratio of a compound to the IC5 HU of HUVEC cells and HeLa cells, the higher the selectivity of the compound for HeLa cells, and thus the more suitable for reducing the The active ingredient in the composition of cervical cancer cells active in the subject. Thus, according to one embodiment of the present invention, a composition for reducing the activity of cervical cancer cells comprises at least one of the following compounds: isopsoralen, tripterygium wilfordii, scutellaria, plum|peelin, cotton aphid Acetic acid vinegar, yellow april and its derivatives, and its content is sufficient to reduce the activity of cervical cancer cells in the subject. Reference Method According to another embodiment of the present invention, a compound having a multiplying effect is proposed. The above cytotoxicity assay was carried out using a compound containing two or more active compounds, and the results of the cytotoxicity analysis of the partial compound for HeLa cells are listed in Table 5.

The IC50 compound number of the compound inhibited HeLa cells 48 hours after the treatment. IC5〇 (pg/ml) Ml Compound H: Compound A = 2:1 0.14 M2 Compound C: Compound A = 2:1 0.3 M3 Compound G: Compound A = 2:1 0.2 M4 Compound Η: Compound E = 2:1 0.19 M5 Compound C: Compound E = 2:1 0.72 M6 Compound Η: Compound B = 3:1 0.62 (ng/ml) M7 Compound C: Compound B = 3 :1 1.28 (ng/ml) M8 Compound G: Compound B = 3:1 0.77 (ng/ml) 21 201019947 M9 Compound H: Compound F = 2:1 0.08 M10 Compound C: Compound F = 2:1 0.1 Mil Compound G: Compound F = 2:1 0.13 Comparing Table 4 with Table 5, it can be found that the IC5() required for the compound composition is much smaller than the IC5Q of the individual single compound. That is, the activity of the compound according to the embodiment of the present invention The compounding effect is exhibited between the compounds. Taking compound Ml (IC5〇=0.14) as an example, compound M1 contains compound η (berberine hydrochloride, IC5〇= 2l) and compound Α (iso-psoralen, IC5〇= • n.4) ·' and compound The ic5 M of M1 is 0.14, which is much smaller than the ic50 of the individual constituent compounds. Thus, a specific embodiment of the present invention provides a compound comprising at least one of the following compounds: baicalein, baicalin, berberine hydrochloride and derivatives thereof; and at least one of the following compounds: isopsoralen, triptolide , quercetin, gossypol acetate and its derivatives. Screening of Spring Anti-Infective Compounds ❹ According to another aspect of the present invention, a series of experiments were conducted to confirm whether active compound A to sputum can inhibit HPV 16 pseudovirus infection of HeLa cells. In the relevant experiments, the active compounds listed in Table 1 and the positive control compound, a red algae, were added to the above screening platform at different concentrations. Figures 13 through 15 show some of the results of the relevant experiments, where the bar graph represents the amount of HPV 16 pseudovirus in the sample, and the results of the MTT analysis are represented by a line graph. Figure 15 shows the test results of red algae; red, algae inhibits the HPV 16 pseudovirus with a jc50 of about 〇.7 pg/ml, which is the same as the literature (see 'Carrageenan Is a Potent Inhibitor 22 201019947 of Papillomavirus Infection, PloS Pathogens. 2006; 2:617) 0 The test results show that baicalin (compound c) and gallic acid (compound D) can inhibit HPV16 pseudovirus infection of HeLa cells at appropriate concentrations. It does not cause significant cytotoxicity to HeLa cells. From the data shown in Fig. 13 and Fig. 14, it can be calculated that baicale and gallic acid inhibit the HPV 16 pseudovirus by 1 (:5〇 value is about 8.2 and about 8.9 pg/ml, respectively. Meanwhile, κ芩素The survival rate of jjeLa cells treated with gallic acid is higher than about 80%. DETAILED DESCRIPTION OF THE INVENTION The results contained in the present invention are set forth below. In one aspect of the present invention, it is proposed to reduce the neutrons of the subject. Cervical = live = compound. The above composition contains at least the following compounds. Heterophyllin, Tripterygium wilfordii, yellow saponin, no acid, quercetin 'cotton __ purpose, yellow material, hydrochloric acid == ::r target cervical cancer 2nd cervical cancer = mentality, the subject to be treated may be a patient according to a specific embodiment of the invention, the upper substance 'containing a single active compound. μ is The early composition of the root complex (4) 'The above composition can be a compound of the compound group exhibits a multiply effect. The compound is replenished with a small amount of U - the following compounds: yellow material, yellow acid small 23 201019947 And its derivatives; and at least one of the following compounds: isopsoralen, tripterygium § 1. Quercetin, vinegar, and its derivatives. From further embodiments according to the present invention, the above composition further comprises at least a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, =S spin sugar, glycerin, physiological saline, ethanol, and the above group. On the medicine ^^: she can face additional (four) agents, such as wetting agent or $Ub^J pH or adjuvant 'these extra When the agent can strengthen the Φ Φ + |, other materials such as an antioxidant, a (four) agent, a viscosity stabilizer, and the like may be added. In the present invention, another detachment is caused by a cell or a virus infection. The combination of the above composition to the viral activity of the human papillomavirus is a gallic acid and is derived from the following compounds: - yellow avidin, virus infected in a test subject and sufficient to reduce viral infection Cell or god, acceptable for the treatment of j virus activity. According to the principle of the present invention and according to the invention - the object can be a human suffering from cervical cancer. For high-risk human mastoid = application of the human milk to be treated Virus can be a virus Human mastoid. For example, 'high-risk human papillae (HPV 18) 〇 mother 6 (HPV 16) or human papillomavirus 18 according to the invention, which contains only a few examples of the above-mentioned composition Another compound according to the present invention may be a single compound, which contains at least two kinds, and the above composition may be each active-compound in the compound composition. In the compound composition, the compound may contain at least two substances. The addition effect can be exhibited. For example, up to > 'one of the following compounds: baicalein, baicalin, 24 201019947 berberine hydrochloride and its derivatives; and at least one of the following compounds: isopsoralen, tripterygium s Purpose, quercetin, cotton vinegar and its derivatives. According to still another embodiment of the present invention, the above composition further comprises at least one pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers are as described above. In still another aspect of the invention, a method for reducing cervical cancer cell activity is provided. The above method comprises at least administering to the cervical cancer cells a therapeutically effective amount of a composition comprising at least one of the following compounds: isopsoralen, triptolide, flavin, gallic acid, quercetin, cotton Phenol acetate, baicalin, berberine hydrochloride and its derivatives. In accordance with the principles and spirit of the present invention, a subject who is treatable may be a human suffering from cervical cancer. According to a particular embodiment of the invention, the composition for administration may be a single composition comprising only one of the above active compounds. According to another embodiment of the invention, the composition for administration may be a combination composition comprising at least two of the above active compounds. In the above combination, each active compound in the composition may exhibit a multiplication effect. For example, the above compound may comprise at least one of the following compounds: baicalein, baicalin, berberine hydrochloride and its derivatives; and at least one of the following compounds: isopsoralen, triptolide, quercetin, gossypol Acetate and Its Derivatives 0 According to another embodiment of the invention, the above composition further comprises at least one pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers are as described above. In the form of a pharmaceutical dosage form, the composition of the present invention can be administered in the form of a salt acceptable for its pharmaceutically 25 201019947, or it can be used alone or in combination with other pharmaceutically active compounds. Used in combination. The target composition can be synthesized according to the possible mode of administration. In another aspect of the present invention, a method for reducing viral activity of a human papillomavirus in a virus-infected cell or a virus-infected subject is provided, the method comprising at least a virus-infected cell or a virus-infected cell-subject A composition comprising a therapeutically effective amount is administered, the composition comprising at least three of the following compounds: baicalein, gallic acid and derivatives thereof. According to the principles and spirit of the present invention, a subject who is treatable can be a human having cervical cancer. According to a specific embodiment of the present invention, the human papillomavirus to be treated may be a high-risk human papillomavirus. For example, the high-risk human virus can be human papillomavirus 16 (HPV 16) or human mastoid 1 (HPV18).帑 18 According to one embodiment of the invention, the composition for administration may be a single composition comprising only one of the above active compounds. According to another embodiment of the invention, the composition for administration may be a combination of 3 and at least two of the above active compounds. Each active compound in the composition in the upper block composition can exhibit a multiplication effect. For example, the above compound may comprise at least one of the following compounds: baicalein, lysine, berberine hydrochloride and its derivatives; and at least one of the following compounds: psoralen, triptolide, quercetin, cotton Phenol acetate is similar to its derivative 0. In the form of a pharmaceutical amount, the composition of the present invention can be administered in the form of its pharmaceutically acceptable salt, or the composition of the compound can be used alone, or Other pharmaceutically active compounds are used in a suitable combination or combination. The target composition can be synthesized according to the possible mode of administration. A person skilled in the art can easily contemplate various modifications and variations of the present invention without departing from the scope or spirit of the invention. The scope of the present invention is defined by the scope of the appended claims, unless otherwise claimed. Φ [Simplified description of the drawings] Fig. 1 is a line diagram showing the relationship between the volume of HPV 16 pseudovirus and the relative fluorescence intensity in an experimental example of the present invention; Fig. 2 is a line diagram showing the first diagram The relationship between the HPV 16 pseudovirus volume and the signal noise ratio in the experimental example; Fig. 3 is a line graph showing the isopsoralen (compound) after 24, 48 and 72 hours of treatment in an experimental example of the present invention. A) Relationship between concentration and HeLa cell survival rate; ❹ Figure 4 is a line graph showing the concentration of triptolide (Compound B) and HeLa after 24, 48 and 72 hours of treatment in an experimental example of the present invention. Relationship between cell viability; Fig. 5 is a line graph showing the relationship between the concentration of baicalein (Compound C) and the survival rate of HeLa cells after 24, 48 and 72 hours of treatment in an experimental example of the present invention; Figure 6 is a line graph showing the relationship between the concentration of gallic acid (Compound D) and the survival rate of HeLa ' cells after 24, 48 and 72 hours of treatment in an experimental example of the present invention; 27 201019947 7th The figure is a line drawing showing an experimental example of the present invention, after being processed 24, 48 and 72 After the time, the relationship between the concentration of quercetin (compound E) and the survival rate of HeLa cells; Fig. 8 is a line diagram showing the experimental example of the invention: after 24, 48 and 72 hours of treatment, gossypol acetic acid Relationship between ester (compound F) concentration and HeLa cell survival rate; Figure 9 is a line graph showing the concentration of baicalin (compound G) after 24, 48 and 72 hours of treatment in an experimental example of the present invention. Relationship between HeLa cell survival rate; Fig. 10 is a line graph showing the ratio of berberine hydrochloride (compound oxime) to HeLa cell survival rate after 24, 48 and 72 hours of treatment in an experimental example of the present invention. Figure 11 is a line graph showing the relationship between the concentration of 'Doxorubin HC1 (control group) and the survival rate of HeLa cells after 24, 48 and 72 hours of treatment in the control group of the present invention; Fig. 12 is a broken line Figure 'further clarifies the relationship between the concentration of triptolide (Compound B) and the survival rate of HeLa cells after 24, 48 and 72 hours of treatment in the experimental example of the present invention; Figure 13 is a diagram showing the experimental example of the present invention. 'Bakein (Compound C) after 48 hours of treatment Relationship between degree and HeLa cell survival rate (shown by a line graph); and the relationship between the concentration of baicalein (compound c) and the HPV 16 pseudovirus infection rate (shown in bar graph) 'Figure 14 is shown in the present invention In the experimental example, the relationship between the concentration of gallic acid (Compound D) and the survival rate of HeLa cells after 48 hours of treatment (shown in a line graph); and the concentration of gallic acid (Compound D) with HPV 16 28 201019947

Relationship between pseudovirus infection rates (shown in bar graph); and Figure 15 shows the concentration of red algae (control) and HeLa cell survival after 48 hours of treatment in the control group of the present invention. Relationship (shown on a line graph); and the relationship between the concentration of red algae (control) and the HPV 16 pseudovirus infection rate. 29

Claims (1)

201019947 VII. Patent application scope: 1. A compound for reducing the activity of cervical cancer cells in a subject, the compound comprising at least: at least one selected from the group consisting of baicalein, baicalin, berberine hydrochloride and derivatives thereof a compound; and at least one compound selected from the group consisting of psoralen, triptolide, quercetin, gossypol acetate and derivatives thereof. 2. A compound as claimed in claim 1 wherein the subject is a human. 3. The compound of claim 1, further comprising at least one pharmaceutically acceptable carrier. 4. A compound for reducing the viral activity of human papillomavirus in a virus-infected cell or a virus-infected subject, the compound comprising at least: φ selected from baicalein, baicalin, berberine hydrochloride and derivatives thereof At least one compound; and at least one compound selected from the group consisting of psoralen, triptolide, quercetin, gossypol acetate and derivatives thereof. 5. The compound of claim 4, wherein the human papillomavirus is a high-risk human papillomavirus. 6. The compound according to claim 5, wherein the high wind 30 201019947 human HPV is human papillomavirus 16 . 7. The compound of claim 5, wherein the high risk human papillomavirus is human papillomavirus 18 . 8. The compound of claim 4, wherein the subject is a human. 9. The compound of claim 4, further comprising at least one pharmaceutically acceptable carrier. 10. A method for reducing the activity of a cervical cancer cell, the method comprising administering at least one therapeutically effective amount of a compound to the cervical cancer cell, wherein the compound comprises at least: baicalein, baicalin, hydrochloric acid At least one compound selected from the group consisting of berberine and a derivative thereof; and at least one compound selected from the group consisting of psoralen, triptolide, quercetin, cotton acetate and derivatives thereof. 11. The method of claim 10, wherein the subject is a human. 12. The method of claim 10, wherein the compound further comprises at least one pharmaceutically acceptable carrier. 31 201019947 13. A method for reducing the viral activity of a virus-infected cell or a virus-infected papilloma virus in a subject, the method comprising at least administering a therapeutically effective amount of one of the compounds to the virus-infected cell or the virus Infected cell subject, wherein the compound comprises at least: at least one compound selected from the group consisting of baicalein, baicalin, berberine hydrochloride and derivatives thereof; and psoralen, tripterygium vinegar, quercetin, cotton At least one compound selected from the group consisting of acetic acid vinegar and its derivatives. The method of claim 13, wherein the human papillomavirus is a high-risk human papillomavirus. 15. The method of claim 14, wherein the high risk human papillomavirus is human papillomavirus 16 . 16. The method of claim 14, wherein the high wind A dangerous human papillomavirus is human papillomavirus 18 . 17. The method of claim 13, wherein the subject is a human. 18. The method of claim 13, wherein the compound further comprises at least one pharmaceutically acceptable carrier. 32