Patent Application
20200290943
The present invention relates to an isolation method of chlorosesamone or chlorosesamone-containing fraction from sesame plants, and to the composition of chlorosesamone-containing fraction , and to an application of the fraction.
According to the top ten causes of death announced by the Ministry of Health and Welfare of Taiwan in 2017, the most common cause of death is malignant tumor and results in 48,037 deaths which is approximately equal to 28.0% of total deaths, indicating a high percentage. Furthermore, the top five cancers are lung cancer, liver cancer, colorectal cancer, breast cancer, and oral cancer, forming a threat to people in Taiwan. Therefore, cancers therapy remains highly important.
Currently, the clinical treatments of cancer mainly include immunotherapy, monoclonal antibody therapy, targeted therapy, radiation therapy, cancer surgery, and chemotherapy. However, an early detection of cancer is not easy, and tumor metastasis usually has occurred when patients noticed discomfort. In addition, the applicability of lumpectomy is limited, and most anticancer drugs not only affect the growth of normal cell, but also have strong sides effects including vomiting, limb weakness, hair loss, decreased white blood cells, and anemia, which seriously affect the patient's life quality and psychological status. Thus, the efficacy of anticancer drugs is still poor.
In recent years, the researches of Chinese herbal medicine indicate that many natural foods have anticancer effects, and thus the extraction of Chinese medicine ingredients with anticancer potentials is also an important direction for cancer therapy.
Seeds of the Pedaliaceae family are oil-rich seeds and edible, so that the sesame plants are planted globally. Among these, seeds of Sesamum indicum is one of the representative plants. Based on documents of traditional Chinese medicine, sesame seeds have benefits of body strength, liver and kidney function, internal organs nourishing, eyesight improvement, etc. In addition, the Chinese medicine has shown that sesamin purified from sesame seeds has anticancer effect on human liver and breast cancer cell. Moreover, myristic acid contained in the sesame seeds also has anticancer property. However, there is still no related researches to investigate other non-edible parts of the sesame plants for medical effect.
The present invention discloses an isolation procedure of the Chlorosesamone or Chlorosesamone-containing fraction from roots of Sesamum indicum. In an embodiment, roots of sesame plants belonged to genus Sesamum is used for experiment, and Chlorosesamone or Chlorosesamone-containing fraction, D2 fraction are extracted from sesame roots and further apply to anticancer treatment in breast, liver, blood, and lung cancer cells.
In accordance with the present invention, an isolation method of chlorosesamone-containing fraction from roots of sesame plants includes an extraction step and a separation step. In the extraction step, roots of sesame plants are extracted at room temperature to obtain a crude extract. In the separation step, a separation is performed to obtain fractions of D1 and D2 after an ethyl acetate layer is removed from the crude extract of extraction step, wherein D2 fraction contains chlorosesamone.
By the aforementioned method, D2 fraction can be obtained and exhibits a growth inhibition effect to cancer cells.
Further, the extraction step is performed by using alcohol, n-hexane, ethyl acetate, acetone, organic solvent, or a supercritical extraction method, wherein the extraction is carried out by using roots of genus Sesamum plants plus an organic solvent with a weight ratio of 1:2 at room temperature. Further, the separation step uses a Diaion HP20 column, which is eluted with H2O/MeOH (weight ratio 40/60); 100% MeOH; 100% acetone; H2O/acetone (weight ratio 30/70) for the separation.
Additionally, the isolation method of the present invention to obtain chlorosesamone from roots of genus Sesamum plants includes an extraction step, a separation step and a purification step. The extraction step and the separation step are the same as those described above, so that they will not be respected. After previous two steps completed, the purification step is carried out. In the purification manipulation, D2 fraction is further purified by a silica gel column (n-hexane/benzene) at room temperature.
Further, the solvent n-hexane/ethyl acetate with a ratio of 1:1 is used for the recrystallization in a water bath with 60 degrees Centigrade to obtain chlorosesamone.
Further, chlorosesamone after the crystallization is remelted by 100% dimethyl sulfoxide (DMSO), and store at −20 degrees Centigrade.
The present invention further provides a medicine application for cancer treatment and the medicine contains chlorosesamone or chlorosesamone-containing fraction , which is extracted from roots of a sesame plants. In addition, the medicine form includes powder, tablet, liquid, plaster, drink or spray.
In the data provided by this embodiment, D2 fraction has a significant effect on inhibiting growth of breast, liver, blood and lung cancer cell. In an embodiment, the 50% inhibition concentration of D2 fraction ranges from 38.2 to 181.9 μg/ml, indicating that D2 fraction has a good anticancer activity. In a preferred embodiment, the treatment time in cells is 24 hours.
To make it easier for our examiner to understand the objective of the invention, its structure, innovative features, and performance, we use a preferred embodiment together with the attached drawings for the detailed description of the invention.
Roots of sesame plants in family Pedaliaceae is used in an embodiment for the illustration of this invention, wherein roots are washed and then extracted, wherein the whole plant, a slice or a powder of the plant is used. In
At room temperature, an organic solvent such as alcohol, n-hexane, ethyl acetate or acetone, or a supercritical extraction method is used to obtain a crude extract, wherein roots of genus Sesamum and the organic solvent with a weight ratio of 1:2 are used for the extraction. After the ethyl acetate layer is obtained, a Diaion HP20 column, which is eluted with H2O/MeOH (weight ratio 40/60); 100% MeOH; 100% acetone; H2O/acetone (weight ratio 30/70)), is used for the separation to obtain fractions of D1 and D2.
With reference to
In addition, the liquid containing D2 fraction or chlorosesamone is concentrated, dried, recrystallized, and then remelted by 100% DMSO, and finally store at −20 degrees Centigrade.
Further, D2 fraction or chlorosesamone can be combined with other compositions to form a powder, a tablet, a liquid, a plaster, a drink or a spray, or mixed with other compositions to be made as a medicine for consuming, injection, coating, or taking. In addition, the Pedaliaceace plants can include the genus Sesamum and the genus Trapella.
The following experiments further describe the embodiments of the present invention used for the anticancer treatment of the breast, liver, blood and lung cancer respectively.
In the first embodiment, MDA-MB-231, a human breast cancer cell and 4T1, a mouse breast cancer cell, Huh-7 and HepG2, a human liver cancer cell and BNL-1-MEA, a mouse liver cancer cell, HL60, a human blood cancer cell and WEHI-3, a mouse blood cancer cell , and A549, a human lung cancer cell and LLC1, a mouse lung cancer cell are used for the experiments. A 96-well plate is used for cell culture. In the test, cells are treated with 0, 25, 50, 100, 200, 400 μg/mL of D2 fraction for 24 hours, and then subjected to cell viability assay (or MTT assay). In cell viability assay, cells are washed with 1× PBS once and treated with 0.5 mg/mL of MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After incubation at 37 degrees Centigrade for 1˜2 hours MTT is removed and cells are washed with 1× PBS once, followed by dimethyl sulfoxide (DMSO) to dissolve the cells. Finally optical density (OD) of 570 nm is detected and medium only is used as a 100% cell viability.
The results of above embodiment show that treatment of D2 fraction for 24 hours can inhibit the growth of MDA-MB-231 and 4T1 breast cancer cell, Huh-7, HepG2, and BNL-1-MEA liver cancer cell, HL60 and WEHI-3 blood cancer cell, and A549 and LLC1 lung cancer cell significantly. The 50% inhibition concentration of D2 fraction in different cells ranges from 38.2 to 181.9 μg/ml. Therefore, D2 fraction can be developed as anticancer medicine.
The results of above embodiment show that the chlorosesamone can inhibit the growth of 4T1 mouse breast cancer cell and HepG2 human liver cancer cell dramatically. This 50% inhibition concentration of chlorosesamone ranges from 0.8 to 1.3 μg/ml. Taken together, chlorosesamone is the major anticancer compound in D2 fraction.
| Number | Date | Country | Kind |
|---|---|---|---|
| 108108952 | Mar 2019 | TW | national |
