TWI238852B - A new extraction method and application of antrodia camphorata to reduce the damage from chemotherapy for cancer and enhance the erythropoiesis in bone marrow - Google Patents

Abstract

We found a new extraction method and application of Antrodia camphorata in promoting and protecting the Erythropoiesis after the chemotherapy of cyclophosphamide. We extracted the Antrodia camphorata with the following procedure: 3 gram powder dissolved in water with 121 DEG C for 10-30 minutes, then centrifuge to discard the precipitates, add the same volume of 95% ethanol, then shaking for 8 hours, centrifuge again with 4000 rpm for 10 minutes, discard the precipitates, keep the supernatant do the following experiments. In the in vivo experiment, we found the peripheral blood cell number increase from 2.7000 ± 1.0801x10<3>/ul to 4.6250 ± 4.2161x10<3>/ul after the cyclophosphamide injection in the water and Antrodia camphorata treatment, respectively. In the in vitro experiment, we scarified the mice with cyclophosphamide injection after 6 days feeding with Antrodia camphorata, took out of mice bone marrow cells and cultivated to count the colony numbers, the number increase from 23.7500 ± 5.9090 to 42.0000 ± 4.9666 in the positive and negative control, respectively.

Description

1238852

[Technical field to which the invention belongs] Cancer adjuvant therapy, medicinal fungi, functional health foods, etc., blood cell proliferation. [Previous technology] Antrodia cinnamomea is a species unique to Taiwan, so no research report on antrodia camphorata has been found in other countries around the world. In terms of solid fruiting body cultivation, many domestic fungal cultivation experts have attempted solid state cultivation of Antrodia camphorata fruit without success; currently only the domestic Weixiang Biotech Company successfully cultivates solid fruiting body Antrodia camphorata. Weixiang also applied for this technology to patent the solid culture method of Chapter 4 of the cultivation technology and its use, the obtained solid culture, and its products and uses. This research raw material is obtained from the powder of Antrodia camphorata fruit produced by Weixiang Company. In Weixiang's patent CN1379082A, it is mentioned that the Antrodia camphorata fruiting body obtained by this production method has three kinds of ingredients consistent with that of wild Antrodia camphorata. This cannot be achieved by general liquid cultivation. In terms of applications, Weixiang applied for patents for liver protection, treatment or inhibition of cancer cell growth, inhibition of peroxidation, anti-free radical or anti-lipid peroxidation, and skin reconstruction functions in the same patent. Antrodia angustifolia is generally circulated with functions such as calming the nerves, removing qi and qi, promoting blood circulation and removing stasis, detoxification and swelling, and restless pain. So far, it has not been found that Antrodia camphorata has the function of promoting the proliferation of white blood cells. Therefore, the present invention still uses an extraction method. The obtained extract can improve the human hematopoietic function. Antrodia camphorata has identified the structure of the chemical structure. There are 17 new 'α-methylergostane type compounds, including antcin A, anticin B, anticin C, antcin E, antcin F, methyl antcinate'G and methyl antcinate H, discovered by Cheng Yihua, et al., Of the Department of Chemistry of Normal University. Yang Shu Zhankuic acid A, Zhankuic acid B, Zhankuic acid C, Zhankuic acid D, and Zhankuic acid E discovered by Qi et al. Among them, antcin A, anticin B, Zhankuic acid A, and Zhankuic acid C murine leukemia) cytotoxin activity, anticholinergic and antiserotonin activity; six new lanostane type compounds, including 15 a -acetyldehydro- sulphurenic acid found by Yang Shuqi et al., dehydroeburicoic acid dehydrosulphurenic acid and other three, of which dehydroeburicoic acid has been proven to be resistant Inflammatory effects, there are four other l, 3-benzodioxloe compounds, including antrocin, 4,7-dimethoxy-5-methyl-1,3-benzodioxole and [2,2 ', 5,5,- tetramethoxy -3,4,3 ', 4'-bimethyl enedioxy-6,6'-dimethyl-biphenyl and the like. 1238852 [Content] The extraction method of Antrodia camphorata in the present invention is to take 3g Antrodia camphorata powder in 40 ml H £), apply 121 kg at 1 kgw / cm2 for 10-30 minutes at 121 ° C, and centrifuge at 4000 rpm for 10 min. Take the supernatant and add an equal volume of 100% alcohol, so that the final alcohol concentration is 50%, shake at room temperature for 10 hours, centrifuge at 4000 rpm for 10 min, collect the supernatant, and then freeze-dry Dry and add water to reconstitute the extracts with different concentrations of 1-10 mg / ml. And after cyclophosphamide (cancer treatment agent) was administered to mice, the extract of Antrodia camphorata extracted by this special process was taken, and the effect on peripheral blood cells was observed. 1. Negative control group 2.7000 soil 1.0801 X 10 view, experiment Group 4.6250 ± 4.2161 X KP / d. It was confirmed that this extract can play a role in the body to slow the damage of hematopoietic function by chemotherapy and promote the proliferation of white blood cells. 2. Six days after the administration, the bone marrow cells of the mice were separated and subjected to a colony counting experiment. The results of the colony counting experiment were negative in the control group 23.7500 ± 5.9090 and positive control group 42.0000 ± 4.9666. This experiment proves that the extract obtained from the original Antrodia camphorata extraction method can be used as an adjuvant treatment for cancer, in order to slow down the problem of hematopoietic function caused by chemotherapy, and promote the proliferation of white blood cells. [Embodiment] 3 g of Antrodia camphorata powder was dissolved in 40 ml of sterile water, and the pressure was applied at 121 ° C and 1 kgw / cm2 for 10-30 minutes. The supernatant was centrifuged and then extracted with 30-90% alcohol and removed by centrifugation. Precipitate, and the supernatant was diluted at different times for in vivo and in vitro experiments in mice. I. Research materials and methods: * 1. Experimental animals: BALB / c (BALB / cByJ) mice, which were purchased from The Jackson Laboratory, Bar Harbor, ME ·, USA, and were obtained from the Animal Center of National Cheng Kung University School of Medicine Feed and breed with standard feed and drinking water. The experiment used male rats aged 8-12 weeks. 2. Isolation of mouse bone marrow cells: Aseptically, take the two thigh bones of the mouse, carefully remove the muscles, use a 3mL syringe with a 27G needle, take 3mL of HBSS, rinse the bone marrow cells, and centrifuge to 10mL 20 % FBS IMDM resuspenked, slowly add IMDM medium containing cells to the Ficoll solution, centrifuge and layer the Ficoll solution at 1500 rpm, take the middle cell layer at the solution interface, and resuspend with 10 mL 20% FBS IMDM After washing, centrifugation at 1300 rpm, take about 106 cells / ml in IMDM medium (2 ng GM-CSF / per ml) with 5% FBS, and culture in 96 well plate. After 24 hours, add Antrodia camphorata extract for 96 hours. After the MTT test. 3. Colony counting experiment: After isolating mouse bone marrow cells in the above method, adjust the cell concentration to 10 cells / ml. Take 0.9 ml and add 2.4 ml of STEMLINE ™ Methylcellulose medium without Ϊ238852 growth factors, mouse (Sigma S4062) , And the medium contains 10ng / ml GM-CSF, put it in a culture plate for 7-10 days, and then take it out to calculate the number of colony. 4. Effect of cyclophosphamide treatment on peripheral blood cells: BALB / c mice were injected intraperitoneally with a dose of cyclophosphamide 160mg / kg (SigmaC0768), while Antrodia camphorata extract was fed, and the white blood cells of peripheral blood were observed. Whether the number is affected. 5. Collection of blood samples: 5.75-inch pasteur narrow-necked glass tubes that have been sterilized are used for blood collection. Blood is collected from the eye socket and anticoagulant is added. About 0.2 mL of blood is added to 30 ul of EDTA (72 mg / ml). In a test tube of), the mixture was evenly counted with a blood sorting counter (ERMA particle counter) (PC608, ERMA Inc., Tokyo, Japan) to obtain the amount of white blood cells in the blood. 6. Antrodia cinnamomea extraction method: take 3g antrodia cinnamomea powder in 40 ml H £), sterilize at high temperature and autoclave, centrifuge at 4000 rpm for 10 min, add the supernatant and add equal volume of 100% alcohol to make alcohol The final concentration is 50%, shaking at room temperature for 8 hours, centrifuging at 4000 rpm for 10 min, collecting the supernatant, drying it by freeze-drying, and reconstituting it with water to obtain extracts with different concentrations. 7. Test substance administration route: a stainless steel feeding needle was put on a 1.0 mL sterile plastic syringe daily, and 0.2-0.4 mL was fed by tube irrigation. The control group was fed with gadolinium and the experimental group was fed with Antrodia camphorata. Stainless steel feeding needles are usually immersed in 95% alcohol, and then wet with H2O before use. 8. Biostatistical analysis: The experimental results are expressed by the mean standard deviation of the mean, and the SigmaPlot software is used to draw the chart, and the Student T test is used to determine whether the control group and the experimental group have reached a significant difference in biostatistics. For example, 7 値 is less than 0.05, which represents a significant difference. 2. Experimental Design 1. Water extract from Antrodia camphorata was precipitated with different alcohol concentrations. The supernatant solution was tested in vitro. 3 g of Antrodia camphorata powder was dissolved in 40ml of alcohol or aqueous solution. After high temperature and high pressure extraction, 30% , 50%, 75%, and 90% alcohol were precipitated. After drying, the concentrations were adjusted to 0.25 g of an extract of Antrodia camphorata powder source μ h20, and the supernatant solution (Table 1) (Figure 1) And added to the cells separately. It was found to have the highest myeloproliferative effect in the 50% alcohol-extracted supernatant. ♦ 1238852 Table I. Antrodia camphorata water pumped and precipitated at different alcohol concentrations. The supernatant solution was subjected to bone marrow hyperplasia MTT test results. Dilution W00X dilution 500 × dilution mox dilution 50X dilution 5X Antrodia camphorata water extract 0.8363 ± 0.1242 ρ = 0.334 0.9203 ± 0.1046 ρ &lt; 0.05 1.0247 ± 0.1421 p &lt; 0.05 1.1390 ± 0.1811 p &lt; 0.05 90% alcohol supernatant 0.8730 + 0.0507 ρ &lt; 0.05 0.05 240 + 0.0695 p &lt; 0.05 0.9987 ± 0.0747 p &lt; 0.05 0.5933 + 0.0539 p &lt; 0.05 75 % Ethanol supernatant 0.9733 ± 0.0570 ρ &lt; 0.05 0.9583 ± 0.0566 p &lt; 0.05 0.9690 ± 0.0185 ρ &lt; 0.05 05 1.0650 ± 0.0811 p &lt; 0.05 50% alcohol supernatant U290 ± 0.0724 p &lt; 0.05 1.2297 ± 0.0795 p &lt; 0.05 1.5377 ± 0.0573 p &lt; 0.05 30% alcohol supernatant 0.9300 ± 0.1029 p &lt; 0.05 1.0540 ± 0.0405 p &lt; 0.05 1.1517 + 0.1157 p &lt; 0.05 0.9123 ± 0.1181 p = 0.060

Positive control (2ng / well): 1.0940 ± 0.0585 Negative control · 0.7698 ± 0.0290 &lt; note &gt; p 値 is compared with Negative control 2 · Changes in peripheral blood cells of mice treated with immunosuppressant and Antrodia cinnamomea: divided into: Three groups, four in each group, were the control group, the negative control group, and the experimental group. The negative control group and the experimental group were treated with cyclophosphamide, 160 mg / kg on day 0. The experimental group was fed with Antrodia camphorata extract at the same time. The negative control group was fed with sterile water until the end of the experiment. Days 0, 3, 5, and 7 Blood was collected from the eye socket, and the number of white blood cells f in the peripheral blood was measured. After the administration of cyclophosphamide immunosuppressant, the white blood cell count was reduced to the minimum on day 5 regardless of whether the Antrodia camphorata extract was fed, but the experimental group with the Antrodia camphorata extract on the 3rd and 5th day Compared with the negative control group, the number of white blood cells was significantly higher than that of the negative control group (Table 2) (Figure 2). phenomenon. Table 2. Changes in the number of peripheral blood cells in mice that were immunosuppressed and fed with Antrodia camphorata. Day 3 Day 5 Day 7 Control group 5.5000 + 0.5568 5.4333 ± 1.0504 7.4333 ± 2.5502 7.7000 ± 0 · 9539 negative Control group (water) 5.866711.9399 2.700011.0801 0.950010.3786 9.5333 ± 5.6766 Experimental group (Antrodia) 6.7333 ± 2.9263 4.6250 ± 4.2161 1.1500 + 0.3697 24.0250 + 35.4014 1238852 3. Mouse bone marrow cell proliferation in vitro colony culture experiment: divided into Three groups, namely the control group, the negative control group, and the experimental group. The negative control group and the experimental group were treated with cyclophosphamide, 160 mg / kg on day 0. The experimental group was fed with Antrodia extract at the same time. The negative control group was fed with sterile water. After six consecutive days of feeding, the bone marrow cells of the femur were sacrificed. A colony counting experiment was performed. The experimental results showed that the quantity of colony fed with Antrodia camphorata water extract was higher than that of colony fed sterile water. It is helpful for maintaining the number of hematopoietic cells in bone marrow cells. Table 3. After 10 days of culturing the bone marrow of the thigh bone of male BALB / c male rats, the amount of colony was calculated. Normal control group (feeding water) Experimental group (feeding Antrodia camphorata) Colony Number 33.OOOOt9.8995 23.7500 ± 5.9090 42.0000 ± 4.9666 &lt; n〇te &gt; A control group (feeding water) and experimental group (feeding Antrodia camphorata) p = 0.03. It was confirmed that Antrodia camphorata powder was extracted through a specific extraction method, 'no matter in vivo or in vitro tests, it can effectively protect the toxocytosis of Cyclophosphamide chemotherapy cells and promote the proliferation of white blood cells. [Schematic description], Figure 1' Zhi: water-extracted liquid with different concentrations of alcohol after being stimulated with supernatant &amp; solution for bone marrow hyperplasia MTT test results

Claims (1)

1238852 Scope of patent application 1. A method for preparing antrodia cinnamomea extract which can improve human hematopoietic function. 3g of antrodia camphorata powder in 40 ml of water is applied at 121 ° C and 1 kgw / cm2 pressure for 10-30 minutes. Centrifuge at 40⑻rpm for 10 min, add equal volume of 100% alcohol to make the final alcohol concentration of 50%, shake at room temperature for 8 hours, and centrifuge at 4000 rpm for 10 min to collect the supernatant. Then freeze-dry it and dry it. Add water to make it into an extract with different concentration of l-10mg / ml.