Patent Application
20220175729
This non-provisional application claims priority of Taiwan Invention Patent Application No. 109143029, filed on Dec. 7, 2020, the contents thereof are incorporated by reference herein.
The present invention is directed to pharmaceutical use of a chlorophyll derivative for cancer therapy, and more particularly to pharmaceutical use of chlorophyllide for cancer therapy.
Plants are the foundation of traditional medicines. A number of plant extracts possess anti-cancer properties, including Annona muricata L., Carica papaya, Colocasia gigantea, Annona squamosa Linn, Murraya koenigii L., Olea europaea L., Pandanus amaryllifolius Roxb., Chenopodium quinoa, Toona sinensis, Myristica fragrans, Thermopsis rhombifolia, and Cannabis sativa. The potential anti-cancer activities of these plants are associated with various bioactive compounds, including chlorophyll, pheophorbide, alkaloid, terpenoid, polysaccharide, lactone, flavonoid, carotenoid, glycoside, and cannabidiol. Beside the possibility of anti-cancer functions, compounds in plant extract demonstrate to exert function of anti-oxidation, anti-inflammation and attenuate side effects induced by chemotherapeutics. Additionally, those bioactive factors, especially chlorophyll and its derivatives, demonstrate potential for the treatment of cancer.
Chlorophyll, the most abundant pigment on earth, is present at high levels in green leafy plants, algae, and cyanobacteria. The catabolic derivatives of chlorophyll are chlorophyllide (chlide), pheophytin, pheophorbide, and phytol. Studies demonstrate that chlorophyll can reduce the growth and proliferation of MCF-7 breast carcinoma cells. Chlorophyll is also reported to promote cell differentiation, and to induce cell cycle arrest and apoptosis in HCT116 colon cancer cells. Chlorophyllide a/b and pheophorbide a/b are reported to reduce hydrogen peroxide-induced strand breaks and oxidative damage, and aflatoxin B 1-DNA adduct formation in hepatoma cells. Chlorophyllide is shown to decrease the levels of hepatitis B virus without affecting cell viability and viral gene products in tetracycline-inducible HBV-expressing HepDE19 cells. In human lymphoid leukemia molt 4B cells, pheophorbide a and phytol are able to induce programmed cell death. Phytol can also reduce inflammation by inhibiting neutrophil migration, reducing the levels of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and oxidative stress. Pheophorbide a, in photodynamic therapy, is found to increase the levels of cytosolic cytochrome c, and is also tested against human pancreatic cancer cells (Panc-1, Capan-1, and HA-hpc2), hepatocellular carcinoma cells (Hep 3B), uterine sarcoma cells, human uterine carcinoma cells, and Jurkat leukemia cells. Pheophorbide is also shown to decrease the levels of procaspase-3 and -9 in Hep3B, Hep G2, and human uterine sarcoma MES-SA cells.
Extensive studies are performed with chlorophyllin (chllin, Cu-chl). Chlorophyllin, a semisynthetic, Cu-coupled, and water-soluble derivative of chlorophyll, is shown to significantly decrease the growth of mutagen-induced cancer cells. In vitro and in vivo studies are suggested that chlorophyllin possesses anti-genotoxic functions against compounds present in cooked meat, including N-nitroso compound and fungal toxin, aflatoxin B1 (AFB1), and dibenzo[d,e,f,p]chrysene (DBC). The regulation of cancer growth by chlorophyllin seems to involve the deactivation of key signal transduction pathways, including the nuclear factor kappa B, Wnt/b-catenin, phosphatidylinositol-3-kinase/Akt, and expressed E-cadherin and alkaline phosphatase pathways.
The amount of chlorophyll degraded globally each year is estimated to exceed 1000 million tons, and this is mostly derived from agriculture and food processing waste. Except for the edible parts of vegetables and fruits, most chlorophyll from low-value agricultural waste can only be degraded naturally. By using low-value agricultural waste as sources to collect chlorophyll, the cost of extraction can be reduced and maximum value of agriculture waste can be reached. Therefore, agricultural waste is potentially useful in the biomedical industry as a high-value nutraceutical and pharmaceutical material.
The present invention is made based on the discovery that a product obtained by treating a plant leaf extract with chlorophyllase exhibits the activity of inhibiting the cancer cell survival, wherein its active ingredient at least includes chlorophyllide.
The present invention is made based on the discovery that the combination of doxorubicin and a product obtained by treating a plant leaf extract with chlorophyllase create a synergistic effect on the activity of inhibiting the cancer cell survival, wherein its active ingredient at least includes chlorophyllide.
Therefore, an embodiment of the present invention provides a method for treating cancer, the method including the step of: administering a therapeutically effective dose of chlorophyllide to a subject in need thereof.
Preferably, the cancer is breast cancer, liver cancer, colon adenocarcinoma, glioblastoma, lung cancer, buccal cancer, stomach cancer, colorectal cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain cancer, prostate cancer, ovarian cancer, cervical cancer, intestinal cancer, or bladder cancer.
Preferably, the cancer is drug-resistant cancer.
Preferably, the cancer is anthracycline-resistant cancer.
Preferably, the anthracycline-resistant cancer is doxorubicin-resistant cancer, daunorubicin-resistant cancer, arubicin-resistant cancer, epirubicin-resistant cancer, idarubicin-resistant cancer, valrubicin-resistant cancer, or mitoxantrone-resistant cancer.
Preferably, the cancer is triple-negative breast cancer.
Another embodiment of the present invention provides a method for treating cancer, the method including the step of: administering a product obtained by treating a plant leaf extract with chlorophyllase to a subject in need thereof, wherein the product comprises a therapeutically effective dose of chlorophyllide.
Preferably, the product is produced by the following steps of: providing plant leaves; performing extraction on the plant leaves with a solvent to obtain a crude extract; and treating the crude extract with chlorophyllase to obtain the product.
Preferably, the solvent is ethanol or hexane.
Preferably, the cancer is breast cancer, liver cancer, colon adenocarcinoma, glioblastoma, lung cancer, buccal cancer, stomach cancer, colorectal cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain cancer, prostate cancer, ovarian cancer, cervical cancer, intestinal cancer, or bladder cancer.
Preferably, the cancer is drug-resistant cancer.
Preferably, the cancer is anthracycline-resistant cancer.
Preferably, the anthracycline-resistant cancer is doxorubicin-resistant cancer, daunorubicin-resistant cancer, arubicin-resistant cancer, epirubicin-resistant cancer, idarubicin-resistant cancer, valrubicin-resistant cancer, or mitoxantrone-resistant cancer.
Preferably, the cancer is triple-negative breast cancer.
Another embodiment of the present invention provides a pharmaceutical composition, the composition including: a therapeutically effective dose of chlorophyllide and a therapeutically effective dose of anthracycline.
Preferably, the anthracycline is doxorubicin, daunorubicin, arubicin, epirubicin, idarubicin, valrubicin, or mitoxantrone.
Yet another embodiment of the present invention provides a pharmaceutical composition, the composition including: a product obtained by treating a plant leaf extract with chlorophyllase and a therapeutically effective dose of anthracycline, wherein the product comprises a therapeutically effective dose of chlorophyllide.
Preferably, the anthracycline is doxorubicin, daunorubicin, arubicin, epirubicin, idarubicin, valrubicin, or mitoxantrone.
Preferably, the product is produced by the following steps of: providing plant leaves; performing extraction on the plant leaves with a solvent to obtain a crude extract; and treating the crude extract with chlorophyllase to obtain the product.
Preferably, the solvent is ethanol or hexane.
Yet another embodiment of the present invention provides a method for treating cancer, the method including the step of: administering any of the foregoing compositions to a subject in need thereof.
Preferably, the therapeutically effective dose of chlorophyllide is from 12.5 to 100 μg/mL, and the therapeutically effective dose of anthracycline is from 0.625 to 20 μg/mL.
Preferably, the cancer is breast cancer, liver cancer, colon adenocarcinoma, glioblastoma, lung cancer, buccal cancer, stomach cancer, colorectal cancer, nasopharyngeal cancer, skin cancer, kidney cancer, brain cancer, prostate cancer, ovarian cancer, cervical cancer, intestinal cancer, or bladder cancer.
Preferably, the cancer is drug-resistant cancer.
Preferably, the cancer is anthracycline-resistant cancer.
Preferably, the anthracycline-resistant cancer is doxorubicin-resistant cancer, daunorubicin-resistant cancer, arubicin-resistant cancer, epirubicin-resistant cancer, idarubicin-resistant cancer, valrubicin-resistant cancer, or mitoxantrone-resistant cancer.
Preferably, the cancer is triple-negative breast cancer.
The detailed description and preferred embodiments of the invention will be set forth in the following content, and provided for people skilled in the art to understand the characteristics of the invention.
The compound chlorophyllide used in the content is represented by a general formula (I)
in which Me is a Mg atom, and R is a CH3 group or a CHO group. Generally, while R is a CH3 group, the compound is called “chlorophyllide a”; while R is a CHO group, the compound is called “chlorophyllide b”.
The leaves of guava, sweet potato, banana, Chinese toona, logan, wax apple, mango, caimito, and cocoa were used to extract chlorophyll. 10 g (wet weight) of leaves were washed, dried, and ground into powder with a pestle and mortar. Leaf mixtures were then frozen in liquid nitrogen and stored at −80° C. in a deep freezer. Chlorophyll was extracted by immersing leaves in ethanol solvent (or hexane solvent) for 48 h. Ethanol crude extracts (or hexane crude extracts) from leaves were centrifuged at 1500 g for 5 min and keep at −20° C. for further experiments. To measure the concentrations of chlorophyll a/b, the crude extracts were passed through a 0.22-μm filter and the absorbance was measured at 649 and 665 nm, which were the major absorption peaks of chlorophyll a and b, respectively. The estimated concentrations of chlorophyll a and b in crude extracts were calculated according to the following equation:
chlorophyll a concentration (n/mL)=13.7×A665−5.76×A649;
chlorophyll b concentration (n/mL)=25.8×A665−7.6×A649.
The chlorophyll a/b concentrations in crude extracts calculated with the empirical equation were multiplied by the volume of the solvent that resulted in the relative chlorophyll mass values in the given samples. When the dry and wet weights of the plant species are known, the content of chlorophyll a/b and the mass of crude extracts relative to the mass of the dry plant can be calculated and expressed as mg/gDW.
Chlamydomonas reinhardtii chlorophyllase was produced as described previously (Molecules. 2015 Feb. 24; 20(3):3744-57; Biotechnol Appl Biochem. 2016 May; 63(3):371-7). Recombinant Chlamydomonas reinhardtii chlorophyllase was expressed, purified, and then lyophilized. The reaction mixture contained 0.5 mg of recombinant chlorophyllase, 650 μL of the reaction buffer (100 mM sodium phosphate, pH 7.4, and 0.24% Triton X-100), and 0.1 ml of crude extracts from leaves (100 mM chlorophyll). The reaction mixture was incubated at 37° C. for 30 min in a shaking water bath. The enzymatic reaction was stopped by adding 4 mL of ethanol, 6 mL of hexane, and 1 mL of 10 mM KOH, respectively. The reaction mixture was vortexed vigorously and centrifuged at 4000 rpm for 10 min to separate the two phases. The upper layer contained the untreated chlorophyll a/b; the bottom layer was chlorophyllase-treated crude extracts comprising chlorophyllide a/b. The chlorophyllase-treated crude extracts containing chlorophyllide a/b mixtures were then concentrated and the solvent was removed by evaporation under reduced pressure at 40° C. on a rotary evaporator. The concentrated crude extracts were processed by lyophilization, weighed, and stored at −80° C. for further experiments.
Chlorophyll was extracted from leaves of 9 plant species, including guava, sweet potato, banana, Chinese toona, logan, wax apple, mango, caimito, and cocoa. Ethanol crude extracts were treated with chlorophyllase to generate chlorophyllide, and then lyophilized in order to measure the weight. The results are listed in Table 1. Significantly, the most chlorophyll a level was observed in Chinese toona (9.8 mg/gDW), followed by mango (8.407 mg/gDW). The lowest chlorophyll a levels were present in banana (2.921 mg/gDW) and sweet potato (3.481 mg/gDW). For chlorophyll b, Chinese toona possessed the highest content (5.419 mg/gDW), followed by cocoa (4.485 mg/gDW) and mango (2.599 mg/gDW). The lowest levels of chlorophyll b were found in sweet potato (0.996 mg/gDW), banana (1.031 mg/gDW), and caimito (1.493 mg/gDW). Of the species analyzed, leaves of cocoa and caimito contained the highest level of ethanol crude extracts, at 412.65 and 397.62 mg/gDW, respectively. The lowest weight of ethanol crude extracts was obtained from sweet potato (43.175 mg/gDW), banana (47.76 mg/gDW), and wax apple (94.29 mg/gDW).
Ipomoea batatas
Syzygium
samarangense
Psidium guajava
Musa paradisiaca
Toona sinensis
Dimocarpus
longan
Mangifera indica
Pouteria Caimito
Theobroma cacao
To analyze chlorophyll and chlorophyllide, the mixtures containing chlorophyllase-treated crude extracts were analyzed by using HPLC as described previously. Chlorophyllide was detected at a wavelength of 667 nm and identified by absorption spectra, peak ratios, and co-migration with authentic standards.
The HPLC separation system was applied to determine the amount of chlorophyll a/b and chlorophyllide a/b in crude extracts. Since the provision of commercial standards was limited, it was not possible to identify all peaks in all crude extracts by HPLC. Herein, the standards used in this study, including chlorophyll a, chlorophyll b, chlorophyllide a, and chlorophyllide b were selected based on our previous studies. HPLC results were obtained using mobile phases consisting of ethyl acetate/methanol/H2O2=44:50:6. Samples were quantified using photodiode array detection in the region 200-400 nm based on the retention times and UV spectra compared with the standards.
Five eukaryotic cell lines were used to assess cytotoxicity in in vitro assays: human fibroblast cells (NIH/3T3), human breast cancer cell lines (MCF7 and MDA-MB-231), hepatocellular carcinoma cells (Hep G2), colorectal adenocarcinoma cells (Caco2), and glioblastoma cells (U-118 MG) were purchased from the American Type Culture Collection (ATCC) (Manassas, USA). Cells were cultured were in DMEM (Dulbecco's modified eagle medium) supplemented with 10% fetal bovine serum (FBS), Eagle's Minimum Essential Medium (EMEM), with 10% FBS and 0.01 mg/mL insulin, Leibovitz's L-15 Medium (L15) with 10% FBS, EMEM with 10% FBS, EMEM with 20% FBS, and DMEM with 10% FBS, respectively. The cells were maintained at 37° C. under a humidified atmosphere of 5% CO2, except for MDA-MB-231. The cells were treated with increasing concentrations of chlorophyllide in ethanol extracts (50, 80, 100, 150, and 200 μg/mL), cultured in an incubator at 37° C. for 48 hr, and the cellular morphology was observed. Following incubation, the cells were observed under an inverted microscope.
Cell viability was examined by the ability of the cells to cleave the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (Sigma Chem., St. Louis, Mo.) by the mitochondrial enzyme succinate dehydrogenase following a previously described procedure (Cold Spring Harbor protocols 2018, 2018(6): pdb prot095505). Cells were incubated at the temperature used to acclimatize cell lines. The background absorbance of the culture medium was subtracted from the measured absorbance. Cells (5×104/well) were stimulated with different doses of chlorophyllide (50, 80, 100, 150, and 200 μg/mL). At the end of the incubation period, 24 hr post stimulation, 20 μL of the MTT solution was added per well. After treatment for 24 hr, supernatants were removed from the wells and 1% MTT solution was added to each well. The plates were incubated for 4 hr at 37° C. and the optical density was determined at 595 nm using a multi-well spectrophotometer (Multiskan, Thermo Fisher Scientific, Waltham, Mass.). All measurements made in the 96-well plates were performed using five technical replicates. In addition, cell viability was examined microscopically for the presence of cytopathic effect (CPE). The half-maximal inhibitory concentration (IC50) was defined as the concentration required to inhibit cell viability by 50%. The IC50 value and the standard error of the mean (SEM) were calculated using a non-linear regression curve contained in the SigmaPlot™ statistical software. A calculated selectivity index (SI) evaluated the relationship between cytotoxicity of cancer cells and normal cells. The SI was calculated from the IC50 of normal NIH-3T3 versus cancer cells. The cytotoxicity effect was considered to have high selectivity for cancer cells if the SI exceeded 2. Values in Tables 2 and 3 were evaluated by linear regression analysis, and correlation coefficients between chlorophyll/chlorophyllide content and cytotoxic activity were calculated by Pearson's correlation coefficient. The values were between +1 (black color) and -1 (red color). The absolute value of correlation coefficient ranges 0.7-0.99, 0.4-0.69, 0.1-0.39 and 0.01-0.09, which was defined as highly, moderately, modestly and weakly correlations.
The cytotoxic effect of 9 chlorophyllase-treated ethanol crude extracts from plants against human fibroblast cells (NIH/3T3), human breast cancer cell lines (MC7 and MDA-MB-231), hepatocellular carcinoma cells (Hep G2), colorectal adenocarcinoma cells (Caco2), and glioblastoma cells (U-118 MG) were determined by MTT assay at a concentration range of 50-200 μg/mL. As shown in
Based on the dose-response curve, the IC50 of each extract was calculated, and these are summarized in Tables 2 and 3. MCF7 cells were more sensitive to chlorophyllide in ethanol extracts of wax apple and banana with an IC50 of 88.87 and 104.41 μg/mL, respectively. MDA-MD-231 cells were most sensitive to sweet potato and wax apple, with IC50 values of 82.9 and 97.83 μg/mL, respectively. In Hep G2 cell lines, sweet potato had the lowest IC50 at 63.73 μg/mL, while those of other plants were nearly 200 μg/mL. In Caco2 cells, the IC50 value of sweet potato was 80.73 μg/mL. U-118 MG cells, which represent the most sensitive of the tested cell lines, were responsive to sweet potato, wax apple, banana, and guava, with IC50 values of 43.17, 52.64, 119.59, and 133.55 μg/mL, respectively (P<0.01).
Selectivity index (SI) is defined as the ratio between the IC50 of each plant extract in cancerous and normal NIH/3T3 cells. An SI exceeding 2 was considered to indicate high selectivity. The SI values were calculated to verify the therapeutic potential of plant extracts. Banana had the highest SI value at 4.6, 4.02, 2.57, and 2.5 in MCF7, U-118 MG, MDA-MB-231, and Hep G2 cell lines, respectively. Wax apple and guava had the highest selectivity, with SI values of 2.75 and 2.37, respectively, in U-118 MG cell lines. Toona showed high selectivity towards MDA-MB-231 cell lines with an SI of 2.12. Among the extracts tested, sweet potato exhibited promising cytotoxicity with the lowest IC50 values (43.17-82.9 μg/mL) in U-118 MG, Hep G2, Caco2, and MDA-MB-231 cells. However, the highest SI found for sweet potato was 1.915, in U-118 MG cell lines.
Values in Tables 2 and 3 were evaluated by linear regression analysis, and correlation coefficient was calculated by Pearson's correlation coefficient and shown in Table 4 and
To confirm that chlorophyllide in ethanol extracts has an important effect on cell viability, the cytotoxicity of chlorophyll and chlorophyllide in sweet potato leaf ethanol extracts and of chlorophyllin against MCF7, MDA-MD-231, Hep G2, Caco2, and U-118 MG cell lines were compared. Chlorophyll, chlorophyllide, and chlorophyllin were analyzed in an MTT assay at concentrations between 0 and 200 μg/mL. As shown in
The DPPH assay was used to evaluate the free radical-scavenging of chlorophyllide. Briefly, DPPH (8 mg) was dissolved in methanol (100 mL) to obtain a stock solution of 80 μg/mL. Then, 2.95 mL of the working solution was mixed with 50 μL of sample. After incubation in a dark at room temperature for 20 min, the absorbance was measured at 517 nm. The DPPH scavenging effect (%) was determined using the following formula:
where Ac was the absorbance of the blank control, Ai was the absorbance in the presence of the samples, and Aj was the absorbance of the samples alone. Vitamin B2 was used as a reference standard compound. The EC50 value, which is the concentration that can inhibit 50% of DPPH free radicals, was obtained by extrapolation from regression analysis.
The anti-oxidant capacities of chlorophyll and chlorophyllide from sweet potato leaf ethanol extracts and chlorophyllin were compared by DPPH assay.
The colorimetric MTT viability assay was performed as above, except for the chemicals for stimulation. As shown in
The colorimetric MTT viability assay was performed with the treatment of 0.625 μg/mL doxorubicin in combination of the treatment of the chlorophyllase-treated hexane crude extract from sweet potato containing various concentrations of chlorophyllide. As shown in
The colorimetric MTT viability assay was performed with the treatment of the chlorophyllase-treated hexane crude extract from sweet potato containing 100 μg/mL chlorophyllide in combination of the treatment of various concentrations of doxorubicin. As shown in
The gene expression profiles of the MCF7 breast cancer cells treated with chlorophyllide or MDA-MB-231 breast cancer cells treated with chlorophyllide and the corresponding untreated cells were analyzed by using next generation sequencing (NGS).
The Gene Ontology analysis was performed to classify the foregoing 2 differentially expressed gene groups.
The KEGG pathway enrichment analysis was also performed to classify the foregoing 2 differentially expressed gene groups. In the KEGG pathway enrichment analysis, all of the foregoing differentially expressed genes were classified according to different KEGG pathways including “metabolism”, “genetic information processing”, “environmental information processing”, “cellular processes”, “organismal systems”, and “human diseases”.
As shown in
As shown in
While the invention has been described in connection with what is considered the most practical and preferred embodiments, it is understood that this invention is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.
| Number | Date | Country | Kind |
|---|---|---|---|
| 109143029 | Dec 2020 | TW | national |
