Patent Grant
8669377
The application claims the benefit of Taiwan Patent Application No. 100141688, filed on Nov. 15, 2011, in the Taiwan Intellectual Property Office, the disclosures of which are incorporated herein in their entirety by reference.
The present invention relates to a Cinnamomum subavenium extract. In particular, the present invention relates to a supercritical carbon dioxide extract of C. subavenium, and the preparation process and the uses thereof. The C. subavenium supercritical extract is effective in anti-human urothelial carcinoma.
Cinnamomum subavenium belongs to Cinnamomum genus, Lauraceae family, an endemic species grown in the forest at an altitude of 500 to 1000 meters in Taiwan, and also is nominated as Cinnamomum randaiense, obtuseleaf cinnamon bark, Cinnamomum osmophloeum, etc. It is also called as fragrant cinnamon because of its barks and leaves having fragrance. The volatile oil refined from barks of C. subavenium may be the essence material for cosmetics, and the leaf oil refined from leaves thereof may be the raw material for food and cigarettes, or pesticides. In traditional Chinese medicine, C. subavenium also is used to treat a various of diseases, such as stomachache, chest pain, abdominal pain, hernia, diarrhea, rheumatism, nausea, vomiting and so on.
Taiwan patent publication No. 200924788 discloses a pesticide combination, including C. osmophloeum essence oil and other essence oils for food industries and for bacteriostatic activity. That patent application only discloses the formulation made by essence oils containing C. osmophloeum essence oil, whereas it does not disclose the extraction method of C. osmophloeum essence oil and the components therein.
Taiwan patent publication No. 200914036 discloses a skin-used agent for prevent biting midges, including C. osmophloeum, betel nut and other major components. However, it does not disclose the preparation method of C. osmophloeum and the components contained therein.
Since there is not the components in C. subavenium and its preparation method disclosed in the prior art, it is impossible for one skilled in the art to use the components of C. subavenium in the medicines, cosmetics and other fields.
It is therefore attempted by the applicant to deal with the above situation encountered in the prior art.
For extracting the various components from C. subavenium, avoiding the use of organic solvents and reducing energy consumed in the extraction process, C. subavenium is extracted by using supercritical CO2, by the inventors of the present invention, to obtain the plural compounds wherein subamolide A ([(3Z,4R,5R)-3-tetradecylidene-4-hydroxy-5-methoxy-5-methylbutanolide]) is the major component. C. subavenium supercritical CO2 extract can be effectively in inhibiting the growth of urothelial carcinoma and treating cancers. In addition, the combination of alkylating agent (e.g. cisplatin (cis-diamminedichloridoplatinum; “CDDP”)) (or nucleoside analog (e.g. gemcitabine (4-amino-1-(2-deoxy-2,2-difluoro-β-D-erythro-pentofuranosyl)-pyrimidin-2(1H)-on; “Gem”)) with C. subavenium supercritical CO2 extract or the combination of alkylating agent (or nucleoside analog) with subamolide A would synergistically inhibit the growth of urothelial carcinoma. Therefore, alkylating agent and C. subavenium supercritical CO2 extract (or subamolide A) can be prepared as a pharmaceutical composition, and nucleoside analog and C. subavenium supercritical CO2 extract (subamolide A) can be prepared as another one, and both pharmaceutical compositions are used for treating human urothelial carcinoma or cancers relevant to the urinary system.
The present invention provides a preparation method for C. subavenium extract, including steps of: (a) drying C. subavenium plant; (b) pulverizing the plant into plural particles; and (c) extracting the plural particles with supercritical CO2 to obtain the C. subavenium extract including subamolide A.
Preferably, the step (a) further includes step of (a1) drying the stem of C. subavenium plant. The step (c) is performed at a pressure of 150 to 350 bar, a temperature of 45° C. to 55° C., a flow rate of the supercritical CO2 from 4 to 6 L/hr and a packing density of material between 250 g/L and 320 g/L. In some embodiments, the step (c) is further performed at 250 bar, 45° C., the flow rate of 4 L/hr and the packing density of material of 320 g/L.
The present invention further provides a pharmaceutical composition of C. subavenium extract for treating cancer cells and/or growth inhibition of the cancer cells, and the pharmaceutical composition includes: a first component having subamolide A; and a second component being selected from a group consisting of monoterpene, sesquiterpene, sesquiterpene derivative, saturated fatty acid, butanolide, phytosterol, triterpene, phytosterone and a combination thereof.
Preferably, (1) monoterpene includes but not limit to eugenol; (2) sesquiterpine includes but not limit to α-cubebene, α-bergamotene, trans-α-bergamotene, γ-elemene, β-acoradene, α-zingiberene, cis-α-bisabolene, β-bisabolene, β-curcumene, δ-amorphene and trans-α-bisabolene; (3) sequiterpene derivative includes but not limit to cedr-8-ene, α-curcumene, nerolidol, caryophyllene oxide, humulene-1,2-epoxide, cubenol, τ-cadinol, α-cadinol, epi-β-cadinol, epi-β-bisabolol, epi-α-bisabolol, α-bisabalol and 1,10-dihydro nootkatone; (4) saturated fatty acid includes but not limit to ethyl palmitate; (5) butanolide includes but not limit to isolinderanolide B, linderanolide B and secosubamolide; (6) phytosterol includes but not limit to β-sitosterol and γ-sitosterol; (7) triterpene includes but not limit to betulin; and (8) phytosterone includes but not limit to sitostenone.
The present invention further provides a pharmaceutical composition, including: subamolide A with a first effective amount; and an ingredient with a second effective amount and being an alkylating agent or a nucleoside analog.
Preferably, subamolide A is a component of a supercritical CO2 extract of C. subavenium or is a component of a methanol (MeOH) extract thereof. Alkylating agent includes but not limit to cisplatin, carboplatin (cis-diammine(1,1-cyclobutanedicar-boxylato)platinum (II)) and oxaliplatin ([(1R,2R)-cyclohexane-1,2-diamine]-(ethanedioato-O′,O′)platinum (II)), and nucleoside analog includes but not limit to deoxyadenosine analog, deoxycytidine analog, deoxyguanosine analog, deoxythymidine analog, deoxyuridine analog, 6-thiohypoxanthine and fluorouracil. One of the deoxycytidine analog is Gem.
The above objectives and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings.
a) is a diagram showing the percentage of each cell cycle phase after the subamolide A treatment on NTUB1 cells.
b) is a diagram showing the intracellular reactive oxygen species (ROS) M1 ratio after the subamolide A treatment on NTUB1 and SV-HUC-1 cells.
a) is an immunoblotting spectrum showing the cellular protein expressions after the subamolide A treatment on NTUB1 cells.
b) and 5(c) respectively are the diagrams showing the fold of changes of (b) Bax/Bcl-2 ratio and (c) cytochrome c after the subamolide A treatment on NTUB1 cells.
a) and 6(b) are the immunoblotting spectra showing the protein expressions of NTUB1 cells post the subamolide A treatment.
a) and 7(b) respectively are the diagrams showing (a) the cellular viability and (b) the combination index (CI) of the combinational cytotoxic effect of subamolide A with CDDP on NTUB1 cells for 24, 48 and 72 hours. CI is obtained from the median-effect analysis performed by the computer software Calcusyn™.
c) and 7(d) respectively are the diagrams showing (c) the cellular viability and (d) the CI of the combinational cytotoxic effect of subamolide A with Gem on NTUB1 cells for 24, 48 and 72 hours. CI is obtained from the median-effect analysis performed by the computer software Calcusyn™.
a) and 8(b) respectively are the diagrams showing (a) the cellular viability and (b) the CI of the combinational cytotoxic effect of C. subavenium supercritical CO2 extract (containing the major component, subamolide A) with CDDP (or Gem) on NTUB1 cells for 24 hours. CI is obtained from the median-effect analysis performed by the computer software Calcusyn™.
The present invention will now be described more specifically with reference to the following Embodiments. It is to be noted that the following descriptions of preferred Embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
C. subavenium extract (or nominated as C. subavenium MeOH extract) or C. subavenium supercritical CO2 extract in the present invention can be used in inhibiting the growth of urothelial carcinoma and treating cancers. Alternatively, the major component, subamolide A, in C. subavenium extract or C. subavenium supercritical CO2 extract can be prepared as an anticancer pharmaceutical extract. Alternatively, an effective amount of subamolide A and an effective amount of other ingredient also can be prepared as an anticancer pharmaceutical extract.
C. subavenium extract or C. subavenium supercritical CO2 extract is able to synergistically inhibit the growth of urothelial carcinoma with an alkylating agent (an anticancer drug). Alternatively, subamolide A in C. subavenium extract or C. subavenium supercritical CO2 extract is able to synergistically inhibit the growth of urothelial carcinoma with the alkylating agent.
C. subavenium extract or C. subavenium supercritical CO2 extract is able to synergistically inhibit the growth of urothelial carcinoma with a nucleoside analog (an anticancer drug). Alternatively, subamolide A in C. subavenium extract or C. subavenium supercritical CO2 extract is able to synergistically inhibit the growth of urothelial carcinoma with the nucleoside analog.
The terms, e.g. “extract”, “compound”, “alkylating agent”, “analog”, “pharmaceutical composition”, “extract” and so on, herein have an effective amount upon usage, indicating an minimum effective amount for cytotoxicity, an effective amount within a scope or an maximum effective amount.
Alkylating agent includes but not limit to cisplatin, carboplatin and oxaliplatin, nucleoside analog includes but not limit to deoxyadenosine analog, deoxycytidine analog (e.g. Gem), deoxyguanosine analog, deoxythymidine analog, deoxyuridine analog, 6-thiohypoxanthine and fluorouracil, etc.
Experiments and Results:
I. Preparation and Analysis of C. Subavenium Supercritical CO2 Extract
C. subavenium supercritical CO2 extract of the present invention was made by extracting C. subavenium with supercritical CO2. C. subavenium's stems could be adopted as the material for extraction. First, the dried stems of C. subavenium (5 kg) were mechanically or physically pulverized as particles with average diameter of 1˜2 mm. Next, the experiment design was performed by using the orthogonal array of Taguchi methodology. The orthogonal array L9(34) was selected for permutation and combination in the present invention, and 4 control factors were determined. Control factor A was extraction pressure, control factor B was extraction temperature, control factor C was flow rate of supercritical CO2 fluid mass, and control factor D was packing density of material. The parameters for determining each control factor were described as follows. Extraction pressure was 150, 250 and 350 bar respectively, extraction temperature was 45, 50 and 55° C. respectively, flow rate of supercritical CO2 fluid mass was 4, 5 and 6 L/hr respectively, and packing density of material was 250, 285 and 320 g/L respectively.
Next, please refer to apparatus diagram in
C. subavenium supercritical CO2 extract was analyzed to afford 34 components (Table 1) using gas chromatography-mass spectrometry (GC-MS) known by one skilled in the art. Components in Table 1 can be divided as monoterpene (e.g. eugenol), sesquiterpine (e.g. α-cubebene, α-bergamotene, trans-α-bergamotene, γ-elemene, β-acoradene, α-zingiberene, cis-α-bisabolene, β-bisabolene, β-curcumene, δ-amorphene and trans-α-bisabolene), sequiterpene derivative (e.g. cedr-8-ene, α-curcumene, nerolidol, caryophyllene oxide, humulene-1,2-epoxide, cubenol, τ-cadinol, α-cadinol, epi-β-cadinol, epi-β-bisabolol, epi-α-bisabolol, α-bisabalol and 1,10-dihydro nootkatone), saturated fatty acid (e.g. ethyl palmitate), butanolide (e.g. isolinderanolide B, linderanolide B, secosubamolide and subamolide A), phytosterol (e.g. β-sitosterol and γ-sitosterol), triterpene (e.g. betulin) and phytosterone (e.g. sitostenone).
Since C. subavenium supercritical CO2 extract are extracted from C. subavenium, the types of components, and the species and amounts of compounds are influenced by factors such as climate, temperature, moisture, rainwater, cultivation environment, soil, harvest and so on. Therefore, the components and ratios of C. subavenium supercritical CO2 extracts are diverse with the batches. One skilled in the art can arbitrarily prepare the pharmaceutical extract including subamolide A and/or other ingredients in accordance with the types and ingredient of these components based on the present invention.
aKovats index relative to n-alkanes (C10-C40) on a DB-5MS column.
bReference kovats index on a DB-5MS column.
cRelative percentage calculated by integrated peak area in Thermo Xcalibur ™ data analysis program.
dIdentification based on comparison of the mass spectrum, co-injected with standard & Kovats index on a DB-5MS column in reference.
It could be known from Table 1 that subamolide A ([(3Z,4R,5R)-3-tetradecylidene-4-hydroxy-5-methoxy-5-methylbutanolide; formula I; 35.1% of percentage) was the major component in C. subavenium supercritical CO2 extract.
The experimental design in the present invention was processed using the orthogonal array L9(34) of Taguchi methodology to establish the best operational condition with extraction pressure of 150 bar, extraction temperature of 45° C., flow rate of supercritical CO2 fluid mass of 4 L/hr and packing density of material of 250 g/L. It could be known from the signal to noise ratio (SN ratio) that the major factors to influence the yield of C. subavenium supercritical CO2 extract sequentially were flow rate of supercritical CO2 fluid mass, extraction temperature, packing density of material and extraction pressure, and the best predicted operational condition were extraction pressure of 250 bar, extraction temperature of 45° C., flow rate of supercritical CO2 fluid mass of 4 L/hr and packing density of material of 320 g/L. Yields obtained from the above two operational conditions were 7.7% and 7.8% respectively, indicating that the operational conditions established from two experimental methods were similar, and confirming that the difference between the experimental result and the predicted SN ratio was less than 5. That is, the selected experimental condition was established, and the yield of supercritical extraction was 5.39% to be better than yield of organic solvent extraction of 1.54%.
Cytotoxicity assay was performed by using C. subavenium supercritical CO2 extract obtained according to the preparation method mentioned above. Please refer to
II. Preparation of C. Subavenium Extract
Additionally, subamolide A (purity>90%) was isolated from the stems of C. subavenium. Briefly, the air-dried stems were extracted with MeOH at room temperature. The MeOH extract, obtained by concentration under reduced pressure, was suspended in H2O and then partitioned with chloroform (CHCl3) to yield fractions soluble in CHCl3 and H2O. The CHCl3 soluble fraction was chromatographed over silica gel using n-hexane-ethyl acetate (EtOAc)-MeOH mixtures as eluents and separated into five fractions. Fraction 2 was re-subjected to silica gel column chromatography and purified by preparative thin layer chromatography using n-hexane-EtOAc to yield subamolide A. Subamolide A was dissolved in dimethyl sulfoxide (DMSO) and stored at −20° C.
III. Statistical Analysis
Data of the following experimental results were expressed as means±SD. Statistical analyses were performed using the Bonferroni t-test method after ANOVA for multigroup comparison and the Student's t-test method for two group comparison, with *P<0.05, **P<0.01 and ***P<0.001 were considered to be statistically significant.
IV. Cytotoxic Effect of Subamolide A on Various Cell Lines
Cellular cytotoxicity of tested compounds was performed by using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Briefly, the cells were plated with a density of 1×103 cells/well in 96-well plates and incubated at 37° C. overnight before drug treatment. Cells were then cultured in the absence or presence of various concentrations (0.3, 1, 3, 6, 9, 12, 15 and 20 μM) of subamolide A at 37° C. for 72 hours. Subsequently, 50 μL of MTT (2 mg/mL in PBS) was added to each well and allowed to react for another 4 hours. Following centrifugation at 1000×g for 10 minutes, media were removed and 150 μL DMSO were added to each well. The proportions of surviving cells were determined by absorbance spectrometry at 540 nm using a microplate reader. The cell viability was expressed as the survival ratio to the un-treated control. The IC50 values of each group were calculated by the median inhibitory analysis and presented as means±SD. The combinational effects of two compounds were analyzed by median-effect analysis. To evaluate the combined effects in growth inhibition, a corresponding combination index (CI) was adopted for the measurement. The combination index of <1, =1, or >1 denotes synergic, additive, or antagonistic effect, respectively.
Please refer to
V. Subamolide A Induces Apoptosis in NTUB1 Cells
Please refer to
VI. Quantitative Analysis of Intracellular Reactive Oxygen Species (ROS)
Production of ROS was analyzed by flow cytometry. Briefly, cells were plated in 6-well plates, and dichlorofluorescein diacetate (DCFH-DA, 10 μl) were added to the treated cells 30 minutes prior to harvest. The cells were collected by trypsinization and washed with PBS. The green fluorescence of intracellular DCF (2′,7′-dichlorofluorescein) was then analyzed by FACScan™ flow cytometer with a 525-nm band pass filter. The ROS production efficiency (M1 ratio) was calculated as “[counts of treated sample in M1−counts of control in M1]/counts of control in M1×100”.
ROS causes a wide range of adaptive cellular responses ranging from transient growth arrest to permanent growth arrest, apoptosis, or necrosis, depending on the amount of ROS. Here, the effect of subamolide A on the intracellular ROS level in NTUB1 and SV-HUC-1 cells were evaluated.
Please refer to
VII. Mitochondrial Apoptotic Pathway by Mitochondrial Membrane Potential (MMP; Δψm) measurement
MMP levels were measured by the lipophilic cation JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′,-tetraethylbenzimidazolylcarbocyanine iodide) fluorescent dye. Briefly, cells were plated and treated as the aforementioned conditions. JC-1 (1 μM) was added to the treated cells 30 minutes prior to harvest. The cells were collected by trypsinization and washed with PBS. The red (aggregated JC1; R1 region) and green (monomeric JC1; R2 region) fluorescence signals were analyzed immediately by FACScan™ flow cytometer and Cell Quest™ software.
To determine whether subamolide A induces cytotoxicity by triggering the mitochondrial apoptotic pathway, the changes of mitochondrial membrane potential (Δψm) in subamolide A-treated NTUB1 cells were measured. It was that subamolide A converted JC-1 from aggregate form (red fluorescence; R1) to monomer form (green fluorescence; R2) indicated the disruption of mitochondrial function at 10 μM of subamolide A treatment (3.52% versus 52.04%).
In addition, please refer to
VIII. Effect of Subamolide A-Induced Apoptosis in NTUB1 Cells
Please refer to the immunoblotting spectrum in
IV. Combinational Cytotoxicity of Subamolide A with Cisplatin or Gemcitabine on NTUB1 Cells
Cisplatin (CDDP) and gemcitabine (Gem) are commercialized chemotherapeutic agents, in which the IC50 values of CDDP and Gem to NTUB1 cells (a seeding number of 1×103 cells/well at beginning) are 3 μM and 8 nM at 72 hours (data not shown). Please refer to
Since subamolide A is the major component of the C. subavenium supercritical CO2 extract, a more excellent inhibition effect would be obtained by one skilled in the art by co-treating the C. subavenium supercritical CO2 extract containing subamolide A with CDDP (or Gem) on the urothelial carcinoma cells.
The aforementioned assay was made by combining several concentrations of subamolide A with those of CDDP (or Gem) for analyzing the synergistic effect. The concentrations of subamolide A and CDDP (or Gem) can be arbitrarily adjusted to be administered on NTUB1 cells or other cell lines or otherwise be administered on the animals with the appropriate physiological conditions (e.g. body weight, age, sex, disease, etc.) and appropriate experimental conditions by one skilled in the art. For instance, in a cellular experiment with NTUB1 cells at a density of 1×103 cells/well, the concentration of subamolide A was adjusted as lower than 1 μM, within 1 to 10 μM, or higher than 10 μM, the concentration of CDDP was adjusted as lower than 1 μM, within 1 to 3 μM, or higher than 3 μM, and the concentration of Gem was adjusted as lower than 2 μM, within 2 to 8 μM, or higher than 8 μM. In the animal mode, (1) the appropriate amounts of subamolide A and CDDP (or Gem), (2) the appropriate amounts of the C. subavenium MeOH extract (containing an appropriate amount of subamolide A) and CDDP (or Gem), or (3) the appropriate amounts of the C. subavenium supercritical CO2 extract (containing an appropriate amount of subamolide A) and CDDP (or Gem) were administered in accordance with animals' conditions, e.g. body weight, age, sex, disease, etc. Furthermore, the appropriate amounts of subamolide A (in the C. subavenium MeOH extract or the C. subavenium supercritical CO2 extract) and CDDP (or Gem) administered on the human being could be calculated/reduced by one skilled in the art. The pharmaceutical composition including the appropriate amounts of subamolide A and CDDP (or Gem) could be administered on the animal cells and animals, which include human beings, rodents, other mammals and so on.
In addition, please refer to
While the invention has been described in terms of what is presently considered to be the most practical and preferred Embodiments, it is to be understood that the invention needs not be limited to the disclosed Embodiments. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
| Number | Date | Country | Kind |
|---|---|---|---|
| 100141688 A | Nov 2011 | TW | national |
| Number | Name | Date | Kind |
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| 7326734 | Zi et al. | Feb 2008 | B2 |
| 20090247480 | Tidmarsh | Oct 2009 | A1 |
| 20110212194 | Wang et al. | Sep 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 200914036 | Apr 2009 | TW |
| 200924788 | Jun 2009 | TW |
| 0072861 | Dec 2000 | WO |
| Entry |
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| Zubrick, The Organic Chem Lab Survivial Manaual, 1984, p. 111-127, 193-195. |
| Liu et al., “Subamolide A, a component isolated from Cinnamomum subavenium, induces apoptosis mediated by mitochondria-dependent, p53 and ERK1/2 pathways in human urothelial carcinoma cell line NTUB1,” Journal of Ethnopharmacology (2011) 137:503-511. |
| Chen et al., “Cytotoxic Constituents of the Stems of Cinnamomum subavenium,” J. Nat. Prod. 2007, 70, 103-106. |
| Shen et al., “Isolinderanolide B, a Butanolide Extracted from the Stems of Cinnamomum subavenium, Inhibits Proliferation of T24 Human Bladder Cancer Cells by Blocking Cell Cycle Progression and Inducing Apoptosis,” Integr Cancer Ther 2011 10:350. |
| Number | Date | Country | |
|---|---|---|---|
| 20130122110 A1 | May 2013 | US |
