Patent Application
20170274026
1. Technical Field
The present invention relates generally to active components of petri dish cultured Antrodia cinnamomea, and more particularly to a method of making triterpenoids from petri dish cultured Antrodia cinnamomea, which is effective in anti-cancer.
2. Description of Related Art
Fruiting bodies of Antrodia cinnamomeain dude various components, and some of the active components thereof, such as MMH01 (dehydrosulphurenic acid) and MMH02 (lanostane triterpenoids), are effective in anti-tumor and repressing the growth of cancer cells, see Toxicology in Vitro and International Journal of Gerontology. Petri dish culturing is a conventional way of growing Antrodia cinnamomea. It has many advantages, including fast growing, good anti-cancer effect, and reducing the chopping of Cinnamomum kanehirae.
There are patents disclosing the anti-cancer components of Antrodia cinnamomea, and methods making the anti-cancer components, such as CN102232944B, CN102000047B, CN102232945B, CN102232940B, CN102232943B, CN102232942B, CN102232941B, CN102443613B, CN104177240A, and U.S. Pat. No. 8,546,366. Those patents disclosed that the components in the extract of Antrodia cinnamomea are effective in anti-cancer.
There are patents disclosing the methods of extracting triterpenoids from Antrodia cinnamomea as well. CN102614195A and US2010/0210869A1 disclose a method of extracting ergosterol and lanosterol from fruiting bodies of Antrodia cinnamomea by ethanol and hexane (or ethyl acetate). U.S. Pat. No. 7,994,158 discloses a method, including extracting by water or an organic solvent, such as acetic acid, ethyl acetate, benzene, alcohol, and methylene dichloride, and then absorbing, concentrating, and purifying the extract by silicone and Sephadex. CN101555436B and TWI487531 disclose a method of extracting by ethanol and supercritical carbon dioxide. With the last method, ethanol and supercritical carbon dioxide are sent to a container, in which solid Antrodia cinnamomea is received. However, it is hard to reach a steady condition, or the active components are extracted unstably. Even it is performed by solid-liquid separation and bath extracting, it can't get purified extract, and it has a high cost.
In conclusion, most of the conventional methods of extracting active components from Antrodia cinnamomea use organic solvents to extract, and then perform the steps of concentration, separation, and purification in sequence. They usually have several drawbacks, such as complex processes, residual organic solvent, and toxicity problem. The conventional method of extracting by supercritical liquid directly uses the mixed solvent and carbon dioxide that need a lot of tests and work experiences, and furthermore, it needs the step of purification.
In view of the above, the primary objective of the present invention is to provide a method of making triterpenoids from petri dish cultured Antrodia cinnamomea, which may reach the stable condition and maximum separation with less ethanol and supercritical carbon dioxide in a supercritical status. With various cell tests, it may prove that triterpenoids is effective in anti-cancer. The present invention is a safe, recycling, and power saving method.
The present invention provides a method of making triterpenoids from petri dish cultured Antrodia cinnamomea, including A. providing 1-2 Kg petri dish cultured Antrodia cinnamomea in an extracting container; B. providing a supercritical solvent to the extracting container to obtain an Antrodia cinnamomea extract; after 30-60 minutes, sending the Antrodia cinnamomea extract to a chromatographic column to separate impurities and triterpenoids from the Antrodia cinnamomea extract, and then removing the impurities at a bottom of the chromatographic column, and collecting the triterpenoids containing fraction at a top of the chromatographic column, wherein the supercritical solvent is a saturated mixture of supercritical carbon dioxide and ethanol with a volume ratio of 1:0.1-0.2 (v/v); a pressure of the supercritical solvent is set to 2,000-4,000 psi, a temperature is set to 40-60° C., and a flow rate is set to 3-9 L/hr; and C. testing the triterpenoids containing fraction by cytotoxicity test, cell morphology analysis, cell cycle and apoptosis test, and cell motility test to find an anti-cancer effect of the triterpenoids containing fraction.
The present invention further provides triterpenoids containing fraction, which is effective in anti-cancer, made by the method as described above.
The present invention will be best understood by referring to the following detailed description of some illustrative embodiments in conjunction with the accompanying drawings, in which
The detailed description and technical contents of the present invention will be explained with reference to the accompanying drawings. However, the drawings are for illustration only and cannot be used to limit the present invention.
As shown in
The first step 100 is preparing Antrodia cinnamomea 110, including putting 1-2 Kg petri dish cultured Antrodia cinnamomea in an extracting container. The extracting container is a stainless column with a diameter in a range between 0.036 m and 0.125 m and a height of 0.5 m.
The second step is extracting and separating 120. This step is performed under 2,000-4,000 psi and 40-60° C. A supercritical solvent with a flow rate of 3-9 L/hr is applied to the extracting container for extracting. The supercritical solvent is a saturated mixture of supercritical carbon dioxide and ethanol with a volume ratio of 1:0.1-0.2 (v/v) to obtain an Antrodia cinnamomea extract. After 30-60 minutes, apply the Antrodia cinnamomea extract to a chromatographic column to separate impurities and triterpenoids containing fraction from the Antrodia cinnamomea extract, and then remove the impurities at the bottom of the chromatographic column, and collect the triterpenoid-rich fraction at the top of the chromatographic column.
The chromatographic column is made of stainless, having an interior diameter of 0.036-0.125 m and a height of 1 m. A stainless plate is received in the chromatographic column, which is made of Pro-Pak protruded metal, saddles, rings, structured packing, or knitted packing.
The third step is testing 130, in which the triterpenoids containing fraction is tested by cytotoxicity test, cell morphology analysis, cell cycle and apoptosis test, and cell motility test to evaluate the anti-cancer effect of the triterpenoid containing fraction.
In the cytotoxicity test, HCT116 cells are put into a 96-well culture plate (a culture plate having 96 wells, and each well receives 10,000 HCT116 cells), in which a 200 μL complete medium (McCoy's 5a) is received. The complete medium is replaced by a triterpenoids medium (a medium having the triterpenoid containing fraction at a concentration of 0.25-1.0 mg/mL) in the triterpenoid containing fraction treated group next day. The control group is replaced with 200 μL complete medium only. After 2 days, a ratio of a viable cell count in each well is tested by a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay at an optical density of 570 nm by an ELISA reader.
In cell morphology analysis, HCT116 cells are received in a petri dish, and replaced with the triterpenoids medium (1.0 mg/mL) next day. The petri dish is kept and pictured by an inverted microscope at different times (2, 7, 14, 21, and 28 days).
In cell cycle and apoptosis test, HCT116 cells are treated with the triterpenoids medium (1.0 mg/mL) for a predetermined time, harvested with trypsin-EDTA, and then neutralized with the complete medium to obtain a solution. Next the solution is centrifuged to remove supernatant liquor thereof, washed by phosphate buffered saline, added to 1 mL 70% cool methanol, kept at 4° C. environment for a night, centrifuged again, then washed with 1 mL phosphate buffered saline for suspension, and then added with 50 mg/mL propidiumiodide for a 10 minutes photophobic process. The cell distribution in cell cycle is collected by an excitation beam at 532 nm and an emission beam at 590±40 nm by a flow cytometry.
In cell motility test, HCT116 cells are put in a 6-well culture plate (a culture plate having 6 wells, and 8,000 HCT116 cells are received in each well), and added with a 2 mL complete medium (McCoy's 5a). Next day, a line is drawn on confluent cells by a 200 μL pipette tip, and culture medium is replaced with the triterpenoids medium (0.25-1.0 mg/mL), and pictures of the wound drawn are taken on both Day 0 and Day 1 under a 100× magnification microscope. The control group has a 2 mL complete medium only without any addition, and is kept for 24 hours to take pictures for the measurement of the change in wound width between Day 0 and Day 1. At last, a measurement software (Image J) is applied to measure wound width between 5 opposite points (random) on each picture, and at least 25 data is collected for each conditions to compare averages motility between conditions.
The following description is about the tests of the triterpenoids obtained by the method of the preferred embodiment of the present invention, and the test of the effect of triterpenoids obtained by the preferred embodiment of the present invention on anti-cancer.
First, the measurement triterpenoid content is carried out on the conventional ethanol extract and the triterpenoids containing fraction of the present invention. Ursolic acid is applied as a standard sample, a content of the standard sample is obtained by measuring an optical density (mg/mL) of 548 nm. A measurement of components and contents of the triterpenoids is carried out by a quantity analysis with C18 column of HPLC, and the unit is mg/mL.
As shown in
The tests of effect of the triterpenoid containing fraction on the anti-cancer are described hereafter:
As shown in
Cell cycle reflects the cell's status, cancer cells usually have abnormal division cycles that makes cancer cells grow faster than normal cells, so slowing down the cell cycle of cancer cells may be a way of anti-cancer. As shown in
The test of wound healing is a simple and fast way to examine the cell motility, and also as a hint to tumorigenicity and metastasis of cancer cells. As shown in
In conclusion, the method of the preferred embodiment of the present invention provides saturated supercritical carbon dioxide as the solvent, and uses the chromatography and absorption techniques to massively separate and purify triterpenoids. It could reach the balance with less ethanol and supercritical carbon dioxide under the supercritical condition. The present invention is effective in anti-cancer that has been proved by cytotoxicity test, cell morphology analysis, cell cycle and apoptosis test, and cell motility test. The triterpenoid manufacturing method of the preferred embodiment of the present invention is a simple procedure with no residual solvent problem. It further has some advantages, including low cost, solvent recyclable, safety, and practicable.
It must be pointed out that the embodiments described above are only some preferred embodiments of the present invention. All equivalent structures which employ the concepts disclosed in this specification and the appended claims should fall within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1051019485 | Mar 2016 | TW | national |
