TW201519896A - A herbal extract and a usage of manufacturing a lung cancer drug thereof - Google Patents

Abstract

A herbal extract for manufacturing a lung cancer drug is disclosed. The herbal extract comprises Antrodia cinnamomea and Cordyceps militaris in a weight ratio of 1: 5 to 5: 1. The herbal extract is manufactured by a method which comprises: mixing Antrodia cinnamomea and Cordyceps militaris in a weight ratio of 1: 5 to 5: 1 to obtain a mixture, extracting the mixture with a solvent in a weight-volume percentage of 50% at 40-90 DEG C to obtain a herbal extracting liquid and condensing the herbal extracting liquid to obtain the herbal extract.

Description

Chinese herbal medicine extract and use thereof for preparing lung cancer medicine

The present invention relates to a Chinese herbal medicine extract, in particular to an extract which inhibits the growth of lung cancer cells, and to the use of the Chinese herbal medicine extract for the preparation of a lung cancer medicament.

According to statistics from the Department of Health, cancer is the top ten cause of death, with lung cancer being the number one death. It is known that radiotherapy, chemotherapy and surgical resection are the main treatments for western medicine. However, due to the ineffective surgical results, radiotherapy and chemotherapy can excessively damage normal cells near the tumor, leading to side effects and treatment. There will still be a possibility of tumor metastasis.

Because traditional Western medicine lung cancer therapy has side effects that make patients extremely uncomfortable, although Chinese medicine is not as effective as Western medicine, it is less harmful to the human body and is therefore gradually accepted by patients. Among the precious Chinese herbal medicines in Taiwan, Antredia cinnamomea belongs to a fungus whose carcass is bell-shaped or plate-shaped and grows only on the high-altitude evergreen broad-leaved burdock tree ( Cinnamomum kanehirae ). Phytochemical studies have shown that Antrodia camphorata is rich in Triterpenes, superoxide dismutase, steroids and polysaccharides that inhibit the growth of tumor cells.

However, in the traditional Chinese medicine classification, Antrodia camphorata is cold and has strong anti-free radical ability. Short-term use can help people with inflammatory symptoms to eliminate excessive free radicals, which can relieve the discomfort caused by inflammation. However, free radicals are related to the blood circulation dynamics and the expansion of blood vessels. If they are taken for a long time, excessive free radicals in the body will cause poor blood circulation, leading to end. Insufficient blood flow to the tip microvessels is the so-called "body cold qi deficiency" phenomenon of Chinese medicine. Therefore, Antrodia camphorata can reduce the side effects of patients who have just undergone chemotherapy or radiation therapy, but at this time, the patient's immunity is quite weak. If the cow's anthocyanin is used excessively, it will cause more damage.

Cordyceps militaris , also known as Cordyceps militaris , is golden and orange-yellow and is a precious medicinal fungus. Cordyceps militaris is a kind of warm medicinal material, rich in Cordycepin, protein, superoxide dismutase and amino acids and trace elements required by the human body. Cordyceps sinensis has the functions of relieving asthma, relieving cough and tonifying kidney. It is also called the three major tonics in ginseng and antler. Cordycepin also proven to be activated by melanoma (mouse melanoma) mouse lung cancer cells on the adenosine receptor A ₃ (adenosine receptors A3) inhibit cell proliferation effect.

In view of this, based on the problem of "the side effects of the Western medicine lung cancer therapy" and the "cold traits of the burdock", it is extremely necessary to provide a modified Liaozhizhi formula to prepare a lung cancer drug to solve the above problems.

The main object of the present invention is to provide a Chinese herbal medicine extract which can be used as a lung cancer drug and assists in the treatment of western medicine lung cancer therapy to alleviate the side effects of radiation therapy and chemotherapy.

The second object of the present invention is to provide a Chinese herbal medicine extract, which improves the medicinal properties of Antrodia camphorata and reduces the side effects caused by long-term use of debilitating body.

A second object of the invention is to provide a Chinese herbal medicine extract for use in the preparation of a medicament for lung cancer, which uses the herbal extract as an active ingredient to inhibit the growth of lung cancer cells.

In order to achieve the foregoing object, the technical means and the efficiencies achievable by the technical method include: a Chinese herbal medicine extract comprising Astragalus and Cordyceps sinensis in a weight ratio of 1:5 to 5:1. Wherein, the preparation method of the Chinese herbal medicine extract comprises: weighing by 1: Mixing Antrodia camphorata and Cordyceps militaris in a ratio of 5 to 5:1 to obtain a mixture; mixing the mixture with a solvent in a mixing ratio of 50% by weight and extracting at 40 ° C to 90 ° C to obtain a mixture a Chinese herbal extract; and concentrating the Chinese herbal extract to obtain the herbal extract.

A Chinese herbal medicine extract of the present invention, wherein the weight ratio is preferably 5:1 of Antrodia camphorata and Cordyceps militaris.

A Chinese herbal extract of the present invention, wherein the solvent is preferably 95% ethanol.

A Chinese herbal medicine extract of the present invention, wherein the heating is preferably carried out at 50 ° C in a water-tight manner.

A Chinese herbal medicine extract of the present invention, wherein the extraction time of the Chinese herbal medicine extract is preferably 8 hours.

The use of a Chinese herbal extract of the present invention for the preparation of a lung cancer drug is carried out by administering the Chinese herbal extract to a desired individual to inhibit the growth of lung cancer cells.

The use of a Chinese herbal extract of the present invention for the preparation of a medicament for lung cancer, wherein the herbal extract is preferably administered orally to a subject in need thereof.

The use of a Chinese herbal extract of the present invention for the preparation of a medicament for lung cancer, wherein preferably 0.1 to 0.6 g of the herbal extract is administered per kg of the body weight of the individual.

The herbal extract of the invention is rich in active ingredients such as triterpenoids, polysaccharides and cordycepin, can effectively reduce the survival rate of lung cancer cells, and can effectively inhibit the growth of lung tumors in living, so as to achieve the effect of poisoning lung cancer cells. .

The herbal extract of the invention can effectively inhibit the growth of lung cancer cells, and can combine the traditional western medicine lung cancer therapy to reduce the dose of radiation therapy or chemotherapy, and achieve the effect of reducing the side effects caused by the conventional western medicine lung cancer therapy.

The herbal extract of the present invention has a unilateral comparison with Antrodia camphorata or Cordyceps militaris. The lung cancer cell inhibiting effect is better, and the cold trait of A. angustifolia is improved through the combination with Cordyceps militaris, thereby reducing the side effects caused by long-term use of Antrodia camphorata alone.

The use of a Chinese herbal medicine extract for preparing a lung cancer medicine is effective for inhibiting proliferation of lung cancer cells by an active ingredient rich in triterpenoids, polysaccharides and cordycepin, and has the effect of preventing tumor from proliferating or spreading. .

Figure 1A shows the analysis of the six major indicators of the triterpenoids of the extract of Antrodia camphorata in Group A1 of this test.

Figure 1B shows the analysis of the six major indicators of the triterpenoids of A. angustifolia and Cordyceps militaris in Group A2 of this test.

Figure 1C shows the analysis of the six major indicators of the triterpenoids of A. striata and Cordyceps militaris in Group A3 of this test.

The 1D figure is the analysis chart of the six major indicators of the triterpenoids of the A4 and the Cordyceps militaris extracts in Group A4 of this test.

Figure 1E shows the analysis of the six major indicators of the triterpenoids of A. angustifolia and Cordyceps militaris in Group A5 of this test.

The 1F figure is the analysis chart of the six major indicators of the triterpenoids of the A6 and the Cordyceps extracts in the A6 group of the test.

The 1G figure is the analysis chart of the six major indicators of the triterpenoids of Cordyceps militaris extract in group A7 of this test.

Figure 2 is a graph showing the cell viability of human lung cancer cells treated with herbal extracts from each group of the test.

Figure 3A shows the appearance of a tumor (arrow circle) after continuous implantation of RO water for 30 days after implantation of a mouse lung cancer cell.

Figure 3B shows the tumor appearance of human lung cancer cells implanted in mice after continuous implantation of the herbal extract of the preferred embodiment for 30 days at a dose of 0.1 g/kg/day after the mouse is implanted into human lung cancer cells. (dotted circle).

Figure 3C shows the tumor appearance of human lung cancer cells implanted in mice after continuous implantation of the herbal extract of the preferred embodiment for 30 days at a dose of 0.3 g/kg/day after implantation of the mouse into human lung cancer cells. (dotted circle).

The 3D image shows that after the mouse is implanted into human lung cancer cells, the herbal extract of the preferred embodiment is continuously administered at a dose of 0.6 g/kg/day for 30 days, and the tumor appearance of the human lung cancer cells implanted into the mouse is shown. (dotted circle).

The above and other objects, features and advantages of the present invention will become more <RTIgt; The extract comprises Astragalus and Cordyceps sinensis in a weight ratio of 1:5 to 5:1. Antrodia camphorata can be further divided into fruit bodies and mycelium. Among them, the preferred ones can be selected from the natural body of A. angustifolia. The amount of triterpenoids contained in the body of A. angustifolia is higher than that of mycelium; In the case of Cordyceps militaris, the fruiting bodies are selected, and the content of active ingredients such as adenosine and cordycepin in the Cordyceps militaris fruit body is higher than that of the mycelium.

Preferably, the herbal extract in the preferred embodiment can be obtained by the following methods, but not limited thereto. The preparation method of the herbal extract in the preferred embodiment comprises: mixing an appropriate amount of Antrodia camphorata and Cordyceps militaris to obtain a mixture; extracting the mixture with a solvent to obtain a Chinese herbal extract; and concentrating the Chinese herbal extract, That is, the herbal extract of the preferred embodiment can be obtained.

Specifically, the mixture is obtained by mixing Antrodia camphorata and Cordyceps militaris in a ratio of 1:5 to 5:1 to obtain the mixture, preferably 5:1 by weight of Antrodia camphorata and Cordyceps militaris. Preferably, the Antrodia camphorata and the Cordyceps militaris can be broken into powder particles and sieved (screen number 30). Particle screening is performed to increase the mixing efficiency and subsequent extraction efficiency.

The mixture is mixed with the solvent in a proportion of 50% by weight of the solvent, wherein the solvent is preferably 95% ethanol, and is extracted by heating at 40 ° C to 90 ° C for 8 hours to obtain the Chinese herbal extract. Wherein the heating method is preferably 50 ° C water heating. In addition, the above extraction may be repeated several times, so that the active ingredient rich in Antrodia camphorata and Cordyceps militaris can be completely dissolved in the solvent, which is widely used by those having ordinary knowledge in the art, and will not be described herein.

The concentrate of the Chinese herbal extract is preferably vacuum filtered using a No. 3 filter paper, and at least half of the solvent is removed by concentration under reduced pressure, and lyophilized at -60 ° C to obtain the herbal extract having a water content of less than 2%. The concentrate of the Chinese herbal extract is more concentrated, so that only a small amount of the Chinese herbal extract can be used to achieve the best effect.

In the preferred embodiment, the herbal extract contains a plurality of active ingredients such as triterpenoids, cordycepin and polysaccharide, so that the herbal extract has a poisoning effect against lung cancer cells, inhibits proliferation of lung cancer cells, and prevents tumor expansion.

In order to confirm that the herbal extracts of the preferred embodiment have triterpenoids, cordycepins and polysaccharides, and the Chinese herbal extract can inhibit the lung cancer cells, the following test is carried out: in this test, the first step is as follows. The weight ratio of the herbal medicines shown in the table was mixed to a total amount of 500 g to obtain a mixture of each group; the mixture was continuously mixed with 95% ethanol in a proportion by weight of 50% by weight, and extracted by heating at 50 ° C for 8 hours. Then, it was vacuum filtered through a No. 3 filter paper, concentrated under reduced pressure to remove at least half of the solvent and freeze-dried at -60 ° C to obtain a herbal extract of each group.

(A) Yield

The test was carried out with each group of herbal extracts as shown in Table 1, and the yield calculation formula was (Chinese herbal extract weight/total weight 500 g) × 100%; the results are shown in Table 2. The highest in group A2 (9.12±0.3%).

(B) Triterpenoid content detection

Continue to weigh 0.2 grams of each group of herbal extracts into a spiral test tube, add 5 milligrams The methanol was stirred, ultrasonically shaken for 15 minutes, centrifuged at 3000 rpm for 10 minutes, and 5 ml of the supernatant was placed in a clean test tube, and then heated to dryness in a 100 ° C water bath.

Then, a Purospher STAR (Merk) RP-18e (5 μm) 250 mm × 4 mm column was used for analysis. Acetonitrile (ACN) and 0.085% phosphoric acid solution were mixed in a volume of 47:53 as a mobile phase at a flow rate of 1 ml/min. The absorbance at a wavelength of 254 nm was detected for analysis, and the results are shown in Figures 1A to 1G, respectively.

The results of total triterpenoid analysis are shown in Table 3, in which the total triterpenoid content of the A1 group is the highest (32.45±1.12%); the total triterpenoid content of the A2 group is the second (28.77±1.08%); The amount of triterpenes was the lowest (18.26 ± 1.23%).

The burdock triterpenoids have six major components, namely anthin K, antcin C, zhankuic acid C, dehydrosulphurenic acid, The results of analysis of each group are shown in Table 4 for the results of analysis of each group of zhankuic acid A and dehydroeburicoic acid. Please also refer to Figures 1A, 1B, 1C and 1E respectively, which are Groups A1, A2, A3 and A5, respectively. Six major triterpenoid indicators were measured, of which the percentage of the six major triterpenoids in the A1 group was the highest (68.78%), and the second was the A2 group (37.61%) (please refer to Table 4). In Groups A4, A6 and A7, it is not possible to obtain the content of the six major triterpenoid indicators through the instrument (please refer to Figures 1D, 1F and 1G respectively).

(C) Cordycepin content test

In this test, samples of Groups A1 to A7 were used, followed by C ₁₈ (5 μm) 150 mm × 4.6 m column analysis. The methanol phase with a volume ratio of 40:60 was used as the mobile phase at a flow rate of 1 ml/min. The absorbance at 260 nm was used for subsequent analysis.

The content of cordycepin was as shown in Table 5. Among them, the content of cordycepin in group A7 was the highest (8.25±0.29%); the content of cordycepin in group A6 was the highest (6.57±0.24%); the group A1 could not be detected. The amount of cordycepin.

(D) Detection of polysaccharide content

This test uses galactose as a standard and uses its concentration and absorbance to make a standard curve. Continue to configure the Chinese herbal medicine extract solution, take 1 ml of each group of herbal extract solution, add 1 ml of 5% phenolic solution, and then add 5 ml of concentrated sulfuric acid to obtain the mixed solution of each group, so that the mixed solution of each group is cooled for 30 minutes, then shake They were uniformly mixed, and their absorbance was measured at 490 nm using a spectrophotometer (Hitachi U1800 Spectrometer), and the concentration was calculated by a standard curve.

The content of the polysaccharide was as shown in Table 6, wherein the highest amount of polysaccharide (61.33±2.09%) was higher in Group A2, higher than that in Group A1 (60.09±1.56%); and the polysaccharide contained in Group A7. The lowest amount is 13.09 ± 1.37%.

Based on the above test results, the herbal extracts in the preferred embodiment are rich in triterpenoids, cordycepin and polysaccharides, and have a poisoning effect on lung cancer cells.

In order to prove that the herbal extracts in the preferred embodiment have better lung cancer cell inhibition effects than the unilateral ones, the following experiments are continued:

(E)Effects of Chinese herbal extracts on the survival rate of human lung cancer cell lines

The test used human lung cancer cells (A549 cells) purchased from the Food Industry Development Institute, which were cultured in 10% fetal bovine serum (FBS, purchased from Biological Industries, Kibbutz beit haemek), 2 mmol/L L- L-glutamine (purchased from HyClone, USA), 1 x non-essential amino acids (purchased from HyClone, USA), 100 μg/ml streptomycin, and 100 U/ml penicillin (purified from HyClone, USA) The culture solution (F-12K) was placed in an incubator at 37 ° C, 5% carbon dioxide and 95% humidity, and the fresh culture solution was changed every two days.

When the lung cancer cell line is subcultured, the culture solution and the cells can be centrifuged at 1000 rpm for 5 minutes, the supernatant is removed, a new culture solution is added, and the number of cells is maintained in a 10 cm culture dish. 1 × 10 ⁵ to 1 × 10 ⁶ cells/ml.

When the test is carried out, the 10 cm culture dish of the lung cancer cell line which has been 8 to 9 minutes old can be taken out, the discolored culture medium is removed, the cells are washed by adding 8 ml of PBS buffer solution, and trypsin/ethylenediamine acetic acid is added. Trypsin/EDTA), after 1 to 3 minutes of reaction, gently shake the culture dish to detach the cells from the wall, add the warmed culture medium, gently disperse the cells, and then place the cell-containing culture solution into the centrifuge tube. Inside, the cells were evenly dispersed, and 20 μl of the lung cancer cells were taken out, and 20 μl of 0.04% trypan blue (Trypan Blue) was added for staining, placed in a cell counter, and the number of viable cells was counted under a microscope, and the cell survival rate was not less than 85% of the trials are available.

The concentration of the lung cancer cells was adjusted to 1 × 10 ⁵ cells/ml with a serum-containing culture solution, and 100 μl of the lung cancer cells were seeded in a 96-well plate so that each well contained 1 × 10 ⁴ cells, and at 37 Incubate overnight at °C in an incubator containing 5% carbon dioxide.

After 24 hours of incubation, the samples as shown in Table 7 (4 concentrations of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, and 0.125 mg/ml, respectively) were added, and after mixing, at 37 ° C, The 5% carbon dioxide incubator was incubated overnight for 24 hours.

After 24 hours, the culture solution was removed, rinsed with PBS buffer solution, and then 100 μl of fresh culture medium containing CCK-8 was added, placed at 37 ° C, reacted for 2 hours in a 5% carbon dioxide incubator, and shaken for 5 minutes. The absorbance of each slot at a wavelength of 450 nm is detected. The cell survival rate conversion formula of the test results was "absorbance value of each experimental group" / "absorbance value of control group without any drug treatment" × 100%.

Please refer to Figure 2, in the E4 group at a concentration of 1mg / ml, the survival rate of the lung cancer cells is only 11.56 ± 1.18%, the inhibition effect is significantly higher than the E3 group (18.24 ± 2.16%); In the E9 group, the inhibitory effect was the worst at the concentration of 1 mg/ml, and the cell survival rate was as high as 95.26±3.14%. From this, it can be seen that the E4 group of the preferred embodiment has an effect of preferably inhibiting tumor growth.

The Chinese herbal medicine extract of the preferred embodiment has a poisonous killing function for lung cancer cells, and the Chinese herbal extract having a weight ratio of 5:1 to Antrodia camphorata and Cordyceps militaris is the best.

(F) Evaluation of the efficacy of Chinese herbal extracts on tumor suppression in tumor mice

This trial used Balb/C male mice purchased from the Animal Center of the National Cheng Kung University School of Medicine. The mice were all over 8 weeks old and weighed between 20 and 25 grams. The mice were housed in the SPF Animal Center of the University of Stanley, and maintained in an animal room at room temperature of 25 ± 1 °C for 12 hours each. Moreover, mice were free to eat and drink; feed (purchased from LabDiet, Inc., USA) and drinking water were supplied by the Animal Center of the University of Science and Technology.

The human lung cancer cell line (such as the A549 cell strain described above, purchased from the Food Industry Development Research Institute) was diluted with physiological saline to a concentration of 5×10 ⁶ cells/ml, and the lung cancer cell strain was subjected to the subgingival area of the mouse. Subcutaneous injection.

Next, the herbal extract of the preferred embodiment (the weight ratio of Antrodia camphorata to Cordyceps militaris to 5:1, which is the aforementioned Group A2) was orally administered to the mouse, and RO water was used as the control group. Two doses per day (3 doses of 0.1, 0.3, and 0.6 g/kg/day, respectively) were administered for 30 consecutive days, and tumor area was recorded weekly by x-ray irradiation. After the tumor mice were sacrificed, the body weight of the mice was weighed, and the tumor, spleen and thymus were removed, and the tumor inhibition rate, spleen coefficient and thymus coefficient were calculated. The blood of the mouse was also taken through a fully automatic blood analyzer (sysmex). F-800, Japan) Assessment of liver and kidney function: blood glutamic acid transaminase (GOT), serum acetone transaminase (GPT), blood urea nitrogen (BUN) and creatinine Variety.

Referring to Figures 3A, 3B, 3C and 3D, in the preferred embodiment, the herbal extract can effectively reduce the tumor volume and growth rate compared to the RO water group, and with the Chinese herbal extract As the dose increases, the inhibitory effect of tumor growth is also relatively increased.

Continued to refer to Table 8, there is no significant difference in body weight, thymus and spleen weight of lung cancer mice with different doses of herbal extracts, indicating that the Chinese herbal extract does not damage other non-tumor areas.

Please refer to Table 9, the liver function index GOT and GPT values in tumor mice will decrease as the dosage of herbal extracts in the preferred embodiment increases, while the renal function index BUN and creatinine No significant difference, showing the Chinese herbal extract in the experimental dose range It has the ability to protect the liver in the body, but it does not cause kidney damage.

In summary, the herbal extract of the present invention is rich in active ingredients such as triterpenoids, polysaccharides and cordycepin, and can effectively reduce the survival rate of lung cancer cells, and can effectively inhibit the growth of lung tumors in living bodies, so as to achieve lung cancer killing. The efficacy of cells.

Furthermore, the herbal extract of the present invention can effectively inhibit the growth of lung cancer cells, and can combine the conventional western medicine lung cancer therapy to reduce the dose of radiation therapy or chemotherapy, thereby reducing the side effects caused by the conventional western medicine lung cancer therapy.

In addition, the herbal extract of the present invention has a better lung cancer cell inhibiting effect than the burdock or the northern cordyceps unilateral, and improves the cold trait of the Antrodia camphorata through the combination with the Cordyceps militaris, thereby reducing the side effects caused by long-term use of Antrodia camphorata alone. The effect.

In addition, the use of the Chinese herbal medicine extract of the present invention for preparing a lung cancer medicine is effective for inhibiting the proliferation of lung cancer cells by the active ingredients rich in triterpenoids, polysaccharides and cordycepin, and has the tumor unable to proliferate or spread. The effect.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

Claims (8)

The invention relates to a Chinese herbal medicine extract, which comprises an Antrodia camphorata and a Cordyceps militaris in a weight ratio of 1:5 to 5:1, wherein the preparation method of the Chinese herbal medicine extract comprises: mixing the Antrodia camphorata in a ratio of 1:5 to 5:1 by weight And Cordyceps sinensis to obtain a mixture; mixing the mixture with a solvent in a mixing ratio of 50% by weight, and heating at 40 ° C to 90 ° C for extraction to obtain a Chinese herbal extract; and extracting the Chinese herbal medicine The liquid is concentrated to obtain the herbal extract. The herbal extract according to claim 1, wherein the extract comprises Astragalus and Cordyceps militaris in a weight ratio of 5:1. The herbal extract according to claim 1, wherein the solvent is 95% ethanol. The herbal extract according to claim 1, wherein the heating is heated at 50 ° C. The herbal extract according to claim 1, wherein the extraction time of the Chinese herbal extract is 8 hours. The use of a Chinese herbal medicine extract for preparing a lung cancer medicine, wherein the Chinese herbal medicine extract is administered to a desired individual to inhibit the growth of lung cancer cells, wherein the Chinese herbal medicine extract comprises a weight ratio of 1:5 to 5:1. Antrodia camphorata and Cordyceps militaris, and the Chinese herbal medicine extract is prepared by mixing Antrodia camphorata and Cordyceps militaris in a ratio of 1:5 to 5:1 by weight to obtain a mixture; and mixing the mixture with a solvent, The mixing ratio is 50% by weight and is extracted by heating at 40 ° C to 90 ° C to obtain a Chinese herbal extract; The Chinese herbal extract is concentrated to obtain the herbal extract. The use of the herbal extract according to claim 6 of the patent application for the preparation of a lung cancer drug, wherein the herbal extract is administered orally to a desired individual. The use of the herbal extract according to claim 6 of the patent application for the preparation of a lung cancer medicament, wherein 0.1 to 0.6 g of the herbal extract is administered per kg of the desired individual body weight.