TWI400079B - Antimicrobial activity of Pennisetum active substance, preparation method and application thereof - Google Patents

Description

Pennisetum active substance with anticancer activity, preparation method and application thereof

The present invention relates to the technical field of an active substance having anticancer activity and a preparation method thereof, and particularly to an extractor prepared from a natural herb.

Pennisetum purpureum ( Pennisetum alopecuroides (L.) Spreng, also known as Chinese Pennisetum) is a perennial Gramineae plant with high yield and can be one of the main herbaceous materials of herbivores. Upright, hairless, flattened on both sides, waxy, plant height 100~210 cm, leaf leathery, 30~60 cm long, 5~8 mm wide, strip-shaped, often involute. Leaf sheath flat pressure ridge, brush-like. Panicle-like panicles, cylindrical, 10–15 cm long, small spikelets with 2 small flowers, ca. 5 mm long, spindle and branch densely pilose, calyx-like branchlets 1–1.5 mm long, often purple Can be used to clear the lungs and relieve cough, cooling blood and eyesight.

Cancer, a disease caused by the abnormality of the cell growth and proliferation mechanism. In addition to uncontrolled growth, cancer cells also locally invade the surrounding normal tissues (invasion) and even transfer to other parts of the body (distal metastases) via the circulatory system or lymphatic system. There are many types of cancer, and the severity of the condition depends on where the cancer is located and the extent of malignant growth, and whether distant metastases occur. Most cancers require different treatments depending on their type, location, and stage of development, usually treated with surgery, chemotherapy, or radiation therapy or a combination thereof. If the cancer is left untreated, usually the end result will result in death. However, there are limitations and side effects of surgery, chemotherapy or radiation therapy.

In recent years, many have been derived from plant extract components and their derivatives, such as vinblastine (vincristinem and vinblastine), camptothecin, taxol and its derivatives. Paclitaxel and docetaxel have been widely used in clinical chemotherapy for malignant tumors. This shows that the research of plant ingredients as a drug development is still a study with both theory and effectiveness.

In view of this, the inventor of the present invention extracts an active substance from a natural herb and analyzes whether it has anticancer activity, and hopes to provide a natural anticancer active substance for use by a general user or a cancer patient. To improve, control, treat or prevent the onset or deterioration of cancer. Therefore, this article "the active substance of Pennisetum with anticancer activity, preparation method and application thereof"

All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be common to those of ordinary skill in the art.

An object of the present invention is to provide an active substance of Pennisetum having anticancer activity, which has an effect of inhibiting proliferation of cancer cells and achieving anti-hepatocarcinogenesis.

A second object of the present invention is to provide a method for preparing an active substance of Pennisetum having anticancer activity, which is obtained by extracting Pennisetum as a material to obtain the active substance of Pennisetum having anticancer activity.

Another object of the present invention is to provide an anti-cancer activity of the active substance of Pennisetum, which is used for preparing an anti-cancer composition, a drug or the like, which comprises food, drink and health food. It is easy to use in daily life, such as additives, medical compositions, etc., for general users or patients, and can be easily taken for long-term use for daily health care, cancer prevention, or disease control.

The active substance of Pennisetum which can achieve the above object is produced by the following steps: Step 1: Treating Pennisetum with acid, alkali and solvent respectively; Step 2: Processing the product after Step 1 Cell disruption, filtration, column After chromatography, ZE5 extract, ZE50 extract, and ZE501 extract can be separately prepared, and these extracts are active substances of Pennisetum having anticancer activity.

The preparation of the Pennisetum active substance obtained as described above was subjected to cytotoxicity analysis of various cancer cells to evaluate the anticancer activity of the active substance of the Pennisetum. The following examples demonstrate that the Pennisetum active substance provided by the present invention has a selective inhibitory effect on cancer cells, especially a lung cancer cell and a breast cancer cell, and has a significant inhibitory effect (or poisoning effect) on normal cells. No significant cytotoxicity.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. The drugs and biological materials used in the present invention are commercially available and are readily available. The following are only examples of available pipelines.

Example 1 Preparation of Pennisetum Active Substance

Referring to FIG. 1 , in this embodiment, the commercially available Pennisetum is used as raw material (for example, it can be purchased from Chulu Ranch), and the 2 kg of Pennisetum raw materials are soaked in 70% ethanol and divided into three groups. Two groups of them were subjected to acid treatment and alkali treatment. The three groups (after acid treatment, after alkali treatment and after ethanol treatment) were separately subjected to cell disruption step, solid-liquid separation, column chromatography and low-temperature concentration. Finally, three extracts were obtained, respectively named as ZE5. Extract, ZE50 extract, ZE501 extract.

The material of the pennisetum can be further subjected to coarse grinding to prepare a powder, a coarse abrasive, and the like for various subsequent processing materials, and then subjected to acid, alkali or solvent treatment. This raw material coarse grinding step only affects the efficiency of subsequent processing.

The "acid treatment" is carried out by acid hydrolysis of 30% to 50% concentrated hydrochloric acid (HCl).

Wherein the "alkali treatment" refers to placing the material of Pennisetum in an alkaline solution for about 24 hours; wherein the alkaline solution can adjust the environment to pH value by ammonia water (NH ₄ OH) (pH) is 8.5, making it alkaline.

The "cell disruption treatment" is performed by ultrasonication (Sonicaton) or by microwave-assisted extraction (Microwave assistant extraction) at 80 ° C for 2 to 3 hours for cell disruption treatment.

The "solid-liquid separation" includes, but is not limited to, centrifugation, natural sedimentation, filtration, and the like, and the solid-liquid separation method to which the present invention is applied.

The "column chromatography" is performed by column chromatography on an HP-20 column or an alumina (Al ₂ O ₃ ) column, and using alcohol as a flow wash to collect 30% to 50% of the alcohol stream. By washing the fraction, ZE5 extract, ZE50 extract, and ZE501 extract can be separately prepared.

The ZE501 extract prepared as described above is a product which can be commercialized and is named "Zeurogen".

Example 2 Analysis of anticancer activity 2.1 Materials and methods

In this example, the Stanforhodamine B assay was used to evaluate the toxicity of the active substance of Pennisetum to the following cancer cells. The cancer cell comprises: human lung carcinoma cell line (A549), human breast adenocarcinoma cell line (MDA-MB-231), human hepatocellular carcinoma cell line (Hep), human colon Cancer cell DLD-1 (human colorectal Adenocarcinoma cell line). In addition, human normal skin fibroblast CCD 966 (human skin fibroblast) was used as a normal cell as a control group.

2.2 Cell culture and grouping

The above cell lines were cultured in a medium (containing DMEM (Dulbecco's Modified Eagle Medium, Hyclone SH30022.02), 10% FBS (Fetal bovine serum), 1×Penicillin/Streptomycin) at 37 ° C, 5% CO ₂ atmosphere. In the culture, when the cells were cultured to a number of 1 × 10 ⁶ or more, they were randomly assigned to 96-well culture dishes (5000 cells/well). After one day of culture, different concentrations (4, 2, 1, 0.5, 0.25 μg/μL) and species of Pennisetum extract (prepared in Example 1) were added for two days for SRB analysis.

2.3 SRB analysis (Sulforhodamine B assay)

The cells were fixed with 50% TCA, and a quantity of Sulforhodamine B was placed in each well for coloration. Finally, the amount of protein in the surviving cells was analyzed by spectrophotometer in 565 nm spectral absorption value, thereby measuring the cell survival number.

2.4 Experimental results ZE5 extract

Please refer to Figure 2A, Figure 2B and Table 1. Figure 2A shows the cells of Pennisetum extract ZE5 for human lung cancer cell A549, human breast cancer cell line MDA-MB-231 and human normal skin fibroblast CCD 966. Survival analysis; Figure 2B shows cell viability analysis of Heterophyllum extract ZE5 for human hepatoma cells Hep G2, human colon cancer cell DLD-1 and human normal skin fibroblast CCD 966; ZE5 is toxic to human lung cancer cell A549 and human breast cancer cell line MDA-MB-231, and its IC50 (drug concentration for inhibiting 50% cell growth) is 0.42 μg/μL and 0.36 μg/μL, respectively; however, these concentrations are for humans. positive Normal skin fibroblast CCD 966, human hepatoma cell Hep G2 or human colon colon cancer cell DLD-1 have no obvious cytotoxicity. This means that the Pennisetum extract ZE5 provided by the present invention has a selective poisoning effect on cancer cells (especially lung cancer cells and breast cancer cells) and does not harm normal cells. Figure 5A shows the cell distribution of human breast cancer cell line MDA-MB-231 without the treatment of Pennisetum extract ZE5, and Figure 5B shows the cell distribution of human breast cancer cell line MDA-MB-231 treated with Pennisetum extract ZE5. The cell viability of human breast cancer cell line MDA-MB-231 (Fig. 5B) was significantly decreased after treatment with Pennisetum extract ZE5 (0.5 μg/μL).

ZE50 extract

Please refer to Figure 3 and Table 1. Figure 3 shows the extract of Pennisetum ZE50 for human lung cancer cell A549, human breast cancer cell line MDA-MB-231, human liver cancer cell Hep G2, human colorectal cancer cell DLD-1, Cell viability analysis of human normal skin fibroblast CCD 966; the ZE50 of Pennisetum extract 3.650 for human lung cancer cell A549, human breast cancer cell line MDA-MB-231, IC50 was 3.6 μg/μL and 3.5 μg/μL, respectively; These concentrations (≒4.0 μg/μL) still have the same degree of cytotoxicity for human normal skin fibroblast CCD 966, human hepatoma cell Hep G2 or human colon colon cancer cell DLD-1. This means that the different extraction methods or extracts provided by the present invention have different poisoning effects and abilities for the selective poisoning effect of the same kind of cancer cells and normal cells.

ZE501 Extract

Please refer to Figure 4A for treatment of human lung cancer cell A549 without Pennisetum extract ZE501. Cell distribution map, while Figure 4B shows the cell distribution after treatment with extract ZE501 (0.5 μg/μL); Figure 6A shows the distribution of human normal skin fibroblast CCD 966 without the treatment of Pennisetum extract ZE501 Fig. 6B shows the cell distribution after treatment with extract ZE501 (0.5 μg/μL). Figure 7 shows the cell viability of human lung cancer cell line A549 and human normal skin fibroblast CCD 966 at different concentrations of Pennisetum extract ZE501. The konjac extract (0.5 μg/μL) was clearly observed. After treatment, the cell viability of human lung cancer cell A549 (Fig. 4B) and human breast cancer cell line MDA-MB-231 (Fig. 5B) decreased significantly; however, the cell viability of human normal skin fibroblast CCD 966 was not Significant impact (comparison of Figure 6A and Figure 6B).

Example 3 Analysis of extract components Analysis methods and results

Referring to Table 2, the composition of the ZE501 extract was analyzed by a conventional method. For example, the protein content was determined by the Biuret test; the saponin content was determined by the Vanillin-Perchloric acid test; the alkaloids content was determined by the Bromocresol Green test; the phenols were determined by the Folin-Ciocalteu colorimetric method. (Phenolic) content; determined by the Christ-Müllers test Flavonoids content. The results showed that the ZE501 extract consisted mainly of saponin and flavonoids.

The active substance, preparation method and application thereof of the Pennisetum with anticancer activity provided by the invention have the following advantages when compared with other traditional anticancer drugs, therapies and processes:

1. The active substance of Pennisetum having anticancer activity of the present invention can effectively inhibit the growth of lung cancer cells and breast cancer cells and has no obvious toxicity to normal cells.

2. The anti-cancer activity of the Pennisetum active substance of the present invention, which is extracted from the natural herb "wolftail grass", has a wide source of raw materials, is easy to obtain, and can be prepared with the simple process of the present invention. Active substance of cancer.

The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

In summary, the technical features disclosed in this case have fully complied with the statutory invention patent requirements of novelty and progress. If you apply in accordance with the law, you are requested to approve the application for this invention patent. Inspired by the invention, to the sense of virtue.

Figure 1 is a flow chart for preparing the active substance of Pennisetum according to the present invention.

Figure 2A shows the cell viability of human lung cancer cell line A549, human breast cancer cell line MDA-MB-231 and human normal skin fibroblast CCD 966 at different concentrations of Pennisetum extract ZE5; Figure 2B shows different concentrations of Pennisetum Analysis of cell viability of extract ZE5 against human hepatoma cells Hep G2, human colon colon cancer cell DLD-1 and human normal skin fibroblast CCD 966.

Figure 3 shows different concentrations of Pennisetum extract ZE50 for human lung cancer cells A549, human breast cancer cells MDA-MB-231, human liver cancer cells Hep G2, human colon cancer cells DLD-1, human normal skin fibroblasts CCD 966 Cell viability analysis.

Figure 4A shows the cell distribution of human lung cancer cell A549 without the treatment of Pennisetum extract ZE501; Figure 4B shows the cell distribution of human lung cancer cell A549 treated with Pennisetum extract ZE501.

Figure 5A shows the cell distribution of human breast cancer cell line MDA-MB-231 without the treatment of Pennisetum extract ZE501; Figure 5B shows the cell distribution of human breast cancer cell line MDA-MB-231 treated with Pennisetum extract ZE501 Figure.

Figure 6A shows the cell distribution of human normal skin fibroblast CCD 966 without the treatment of Pennisetum extract ZE501; Figure 6B shows the cell distribution of human normal skin fibroblast CCD 966 treated with Pennisetum extract ZE501 Figure.

Figure 7 shows different concentrations of Pennisetum extract ZE501 for human lung cancer cells A549, human positive Cell viability analysis of normal skin fibroblast CCD 966.

Claims (5)

An active substance of Pennisetum toxic to lung cancer cells, which is prepared by the following steps: Step 1: Treating Pennisetum with 70% ethanol to obtain an ethanol extract; Step 2: taking step one The ethanol extract is subjected to alkali treatment to obtain an alkali extract; Step 3: the step 2 alkali extract is sequentially subjected to cell disruption treatment, solid-liquid separation, column chromatography, and low-temperature concentration step, The ZE501 extract is obtained, and the extract is an active substance of Pennisetum having anti-lung cancer cell activity. The active substance of Pennisetum having a lung cancer cell as claimed in claim 1, wherein the alkali treatment in the second step refers to treating the pennisetum in an alkaline solution for treating the alkaline solution. It can be adjusted to a pH of 8.5 by aqueous ammonia (NH ₄ OH). The cellulite active substance having the poisonous killing lung cancer cell according to the first aspect of the patent application, wherein the cell disruption treatment in the third step is ultrasonic wave oscillation treatment or microwave assist extraction method (80). Extract at °C for 2~3 hours. The pennisetum active substance having a lung cancer cell as claimed in claim 1, wherein the column chromatography is performed on a HP-20 column or an alumina (Al ₂ O ₃ ) column. Analyze, using alcohol as a running lotion, collect 30% to 50% alcohol washes. A pharmaceutical composition comprising the Pennisetum active substance having a lung cancer cell as described in claim 1 of the patent application.