WO2016049811A1 - Antitumour composition rich in triterpenoids and preparation method thereof - Google Patents

Abstract

Disclosed is an antitumour composition rich in triterpenoids comprising 10-15 parts by weight of Antrodia fruiting body extracts, 10-25 parts by weight of Antrodia mycelium extracts and 60-80 parts by weight of Ganoderma lucidum fruiting body extracts. The composition contains 10-17% by weight of a triterpenoid compound. The method for preparing the composition comprises mixing the fat-soluble extracts and water-soluble extracts of the two fungi in a proper proportion to obtain the composition. The composition can reduce the tumour marker content, and can reduce the economic burden of cancer patients.

Description

 Antitumor composition rich in triterpene and preparation method thereof Technical field

 The invention relates to the field of anti-tumor compositions, in particular to an anti-tumor composition rich in triterpenoids, a preparation method and application thereof.

Background technique

In today's world, tumors are another major killer of human deaths after cardiovascular disease, and the incidence of cancer is increasing year by year. At present, the diagnosis and treatment of tumors are mainly concentrated on conventional methods such as surgery, chemotherapy, radiotherapy, etc., but due to the resection of the primary lesions, the wounds are likely to cause safety hazards, and the chemotherapy method has large side effects, and the radiotherapy has damage to the body. These can cause or aggravate side effects such as non-infectious inflammatory reactions in the body. Therefore, these conventional methods for treating tumors are limited.

In-depth research found that traditional Chinese herbal medicines and botanicals and other natural products have significant effects in the treatment of cancer. For example, the invention patent entitled "Anti-tumor activity of Ganoderma lucidum fruit body extract and preparation method thereof" discloses that Ganoderma lucidum contains various anti-tumor active ingredients. However, the anti-tumor effect of Ganoderma lucidum is not high, and patients need to take a large amount of Ganoderma lucidum to inhibit the growth of tumors. This not only causes the patient to have a large economic burden during use, but also causes excessive damage to the body. Due to its unique growth environment, Nigella sinensis has accumulated a large number of anti-cancer ingredients - triterpenoids. The anti-cancer effect of the same quality of Antrodia camphorata is far better than that of Ganoderma lucidum, but the price of Antrodia camphorata is very expensive, which is more than a dozen of the same quality Ganoderma lucidum. Even dozens of times.

technical problem

In view of the above deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a lower cost triterpene-rich antitumor composition.

Technical solution

The solution adopted by the present invention to solve the above technical problems is to provide a triterpene-rich antitumor composition comprising 10-15 parts by weight of an extract of Antrodia camphorata fruit body, 10-25 parts by weight of an extract of Antrodia camphorata mycelium and 60 to 80 parts by weight of the extract of Ganoderma lucidum fruit body; the weight percentage of the triterpenoid compound in the composition is 10 to 17%.

It is also an object of the present invention to provide a method for preparing the above-described triterpene-rich antitumor composition, comprising:

Step A: preparing the extract of the body extract of Antrodia camphorata, the mycelium extract of Antrodia camphorata, and the extract of Ganoderma lucidum fruit body;

Step B: Weigh 10 to 15 parts by weight of the extract of the body extract of A. angustifolia, 10 to 25 parts by weight of the mycelium extract of Antrodia camphorata, and 60 to 80 parts by weight of the extract of the Ganoderma lucidum fruit body are uniformly mixed.

Further, the preparation of the extract of Antrodia camphorata fruit body, the extract of Astragalus membranaceus mycelium and the extract of Ganoderma lucidum fruit body respectively comprises the step A1: placing the raw material of A. angustifolia fruit body or Astragalus membranaceus mycelium or Ganoderma lucidum fruit body The extract is collected by reflux extraction or percolation in 95% ethanol; the extract is recovered into ethanol, and then eluted with ethyl acetate on a diatom column, and the eluate is collected and ethyl acetate is recovered to obtain the raw material. Ethanol extract.

Further, the ratio of the liquid to liquid of 95% ethanol and the raw material in the reflux extraction is 6-8 L/kg; the ratio of the mixture of 95% ethanol and the raw material in the percolation extraction is 20 L/kg.

Further, the step A1 further comprises: adding the ethanol extract of the raw material to a saturated aqueous solution of cyclodextrin, ultrasonically mixing to obtain a suspension of the cyclodextrin inclusion, and statically drying and pulverizing to obtain water extraction of the raw material. Things.

Further, the ultrasonic mixing temperature was 30 ° C and the ultrasonic mixing time was 40 min.

Further, the standing was placed under refrigeration at 0-5 ° C for 24 h; the drying temperature was 60 ° C.

Further, the preparation of the extract of A. angustifolia, the extract of the mycelium of A. angustifolia and the extract of the Ganoderma lucidum fruit body further comprises the step A2: extracting the raw materials extracted through the step A1, respectively, and extracting with water, and then adding 95% ethanol to alcohol content of 75%, after complete precipitation, the supernatant was taken for filtration and steaming, and the resultant was freeze-dried to obtain an aqueous extract of each raw material.

Beneficial effect

The invention provides a triterpene-rich antitumor composition by subjecting the mycelium, fruit body and ganoderma lucidum fruit body of Antrodia camphorata to alcohol extraction and water extraction respectively and mixing in an appropriate ratio, and has remarkable antitumor activity. The anti-tumor composition of the invention can be used for preventing and treating tumors, and the therapeutic effect is close to the expensive Antrodia camphorata, which improves the anti-tumor efficiency of the unit value composition, reduces the economic burden of the tumor patients, and avoids excessive consumption of the ganoderma lucidum. Excess residues caused by drugs can cause damage to the body.

DRAWINGS

Figure 1 is a graph showing the results of testing the effects of different compositions of Experimental Example 1 on tumor cells of the present invention;

Figure 2 is a graph showing the results of testing the effects of different compositions of Experimental Example 2 on T cell transformation and spleen cell antibody production in tumor-bearing mice;

Fig. 3 is a graph showing the results of testing the effects of different compositions of Experimental Example 2 on the levels of cellular immune factors in the serum of tumor-bearing mice.

Embodiments of the invention

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The composition of Antrodia camphorata and Ganoderma lucidum used in the present embodiment is an extract of Antrodia camphorata fruit body, Antrodia camphorata mycelium and Ganoderma lucidum fruiting body. The triterpenoids were extracted from the three raw materials with 95% ethanol, and concentrated, and then embedded with β-cyclodextrin. The alcohol-treated drug residue is then subjected to water extraction, concentrated, dried, and mixed with the alcohol extract contained in the cyclodextrin. Weigh 10 to 15 parts by weight of the extract of Antrodia camphorata fruit body, 10 to 25 parts by weight of the extract of Antrodia camphorata mycelium and 60 to 80 parts by weight of the extract of the Ganoderma lucidum fruit body. The total weight of the active ingredient in the obtained extract is 10-17%, wherein the total triterpenoid content from Antrodia camphorata is 8-12%, which is derived from the total weight loss of Ganoderma lucidum. At 2-5%.

The specific preparation method is as follows:

(1) Firstly crush a certain quality of the body of A. sinensis through a 20 mesh sieve and place it in 95% ethanol (the ratio of material to liquid is 6~8). L/kg) reflux extraction 3 times, each time 2h; or 95% ethanol (liquid to liquid ratio of 20 L/kg) percolation extraction. The extract obtained by reflux extraction or osmosis extraction was recovered into ethanol, and then applied to a diatom column for elution, and the diatomaceous earth used was 1-1.5 times of the fungal raw material. Then, elute with ethyl acetate (feed ratio is 6~8) L/kg), the eluate was collected, and ethyl acetate was recovered to prepare an alcohol extract of the starting material.

(2) The β-cyclodextrin was dissolved in distilled water at 30 ° C to prepare a saturated solution. The extract prepared in the step A1 was dissolved in a small amount of ethanol, and then added dropwise to a continuously stirred saturated aqueous solution of β-cyclodextrin, and the triterpenoid was encapsulated by ultrasonication at 30 ° C for 40 minutes. The obtained suspension of the inclusions was stored at 0-5 ° C for 24 h, and after suction filtration, dried at 60 ° C, and finely obtained to obtain a cyclodextrin-encapsulated alcohol extract.

(3) Powdered medicinal slag extracted with the above 95% ethanol, water (stock to liquid ratio of 8~12) L/kg) decoction extraction twice, each extraction for 2h, the extract is concentrated to a relative density of 1.5-2.0 Kg/L, then 95% ethanol is added to the alcohol content of about 75%. After complete precipitation, filtration and rotary evaporation are carried out, and the resultant is freeze-dried to obtain an aqueous extract of the raw material.

(4) The alcohol extract and the water extract of the combined raw materials are uniformly mixed and dried until the moisture content is less than 4%, that is, the extract of the body extract of Antrodia camphorata.

(5) Same as above, the mycelium extract of Antrodia camphorata and the fruit body of Ganoderma lucidum and the Ganoderma lucidum fruit body were respectively obtained according to the above method.

Weighing 10 to 15 parts by weight of the extract of the body extract of A. angustifolia, 10 to 25 parts by weight of the mycelium extract of A. niger, and 60 to 80 parts by weight of the extract of the Ganoderma lucidum fruit body are uniformly mixed by a multi-dimensional mixer, that is, Anthraquinone and Ganoderma lucidum pharmaceutical composition. The total weight of the active ingredient in the composition is 10-17%, wherein the total weight of the total triterpenoids is 8-12%, and the total weight of the ganoderma lucidum is 2-5%.

Example 1

The preparation procedure was the same as described above, except that the composition of the composition was changed to 12 parts by weight of the extract of Antrodia camphorata fruit body, 20 parts by weight of the extract of Astragalus membranaceus mycelium and 68 parts by weight of the Ganoderma lucidum fruit body.

Example 2

The preparation steps are the same as described above, and only the composition is changed to become a single-flavored extract of Ganoderma lucidum fruiting body.

Example 3

The preparation procedure was the same as described above, except that the composition of the composition was changed to 14 parts by weight of the extract of Antrodia camphorata fruit body and 18 parts by weight of the extract of Astragalus membranaceus mycelium.

Experimental Example 1: Effect of composition on tumor

A total of 40 Balb/c-nu nude mice with a uniform size and weight of (20±2) g and good condition were randomly divided into 5 groups, 8 in each group. Under sterile conditions, log liver cancer (HepG2) cells were diluted with PBS to a cell suspension of 1×10 ⁷ /mL, and inoculated in the SPF clean room in the right forelimb of the right forelimb of the nude mice, 0.1 mL/only, establish a load. Tumor nude mouse model.

They were randomly divided into five groups: model group, composition group, Ganoderma lucidum group, Antrodia group and cyclophosphamide group. The composition group, the Ganoderma lucidum group and the Antrodia camphorata group were respectively administered with the drugs prepared in Examples 1, 2 and 3, and the doses were all 300 mg/kg/day. The model group was given the same amount of normal saline daily. The cyclophosphamide (CTX) group was intraperitoneally injected with cyclophosphamide at a dose of 30 mg/kg/day. After continuous treatment for 4 weeks as described above, all nude mice were sacrificed by cervical dislocation, tumors were removed, tumor weight was recorded, and tumor inhibition rate was calculated. The results of the effects of the above five different extracts on tumor cells are shown in Figure 1. (Note: compared with the model group: *P<0.05, **P<0.01; compared with the composition group: ##P<0.01.)

Experimental Example 2: Effect of antitumor composition on immune system

40 clean-grade Balb/c-nu nude mice were selected, about 2 weeks old, with uniform size and body weight (20±2) g. The logarithmic liver cancer (HepG2) cells were diluted with PBS to a cell suspension of 1×10 ⁷ cells/mL, and inoculated into the right forelimb of the right forelimb of the nude mice in a SPF clean room, 0.1 mL/cell, to establish a tumor-bearing nude mouse model. . They were randomly divided into 5 groups, 8 in each group, which were: model group, composition group, Ganoderma lucidum group, Antrodia group and cyclophosphamide group. In addition, 8 blank control groups were also set up, and tumor cells were not inoculated.

Among them, the composition group, the Ganoderma lucidum group and the Antrodia camphorata group were respectively administered with the drugs prepared in the respective Examples 1, 2, and 3, and the doses were all 300 mg/kg/day. The model group and the blank control group were intragastrically administered with the same amount of normal saline daily. The cyclophosphamide (CTX) group was intraperitoneally injected with cyclophosphamide at a dose of 30 mg/kg/day. The treatment was continued for 4 weeks as described above, and the blood was collected from the eyelids on the last day, and the serum was separated; the spleen was aseptically taken to prepare a spleen cell suspension. Detection of T cell production and transformation ability, spleen cell antibody production ability and serum cellular immune factors (interleukin IL-2, IL-12, tumor necrosis factor TNF-) using MTT assay α The content changes, and the test results are shown in Figures 2 and 3. (Note: *P<0.05, **P<0.01 compared with the blank control group; #P<0.05, ##P<0.01 compared with the model group; aP<0.05, aa compared with the composition group P < 0.01. )

The above experimental results show that the tumor inhibition rate of the same dose of the triterpene-rich antitumor composition of the present invention is lower than that of the Antrodia camphorata group, but is significantly higher than that of the single-flavored Ganoderma lucidum group, which is about 4 times. From the aspects of improving immune cell viability and immune molecule level to improve the anti-tumor immune function of tumor-bearing mice, the anti-cancer effect of the anti-tumor combination extract of the present invention is similar to that of the Antrodia camphorata group in a dose-dependent manner. It can be seen that the antitumor effect of the triterpenoids in the composition is related to the improvement of the immune function of the body. The anti-cancer effect of Antrodia camphorata extract is most obvious, but its price is extremely expensive due to the scarcity of Antrodia camphorata resources. The anti-cancer effect of the extract of single-flavored Ganoderma lucidum at the same dosage is not obvious, and it has a good anti-cancer effect only when taken in large doses. However, taking large doses of Ganoderma lucidum is harmful to the human body. Therefore, some Ganoderma lucidum is replaced by Antrodia camphorata, and the extract is prepared and then prepared into a composition according to an appropriate ratio, thereby greatly reducing the production cost while ensuring anti-tumor efficiency.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims (8)

A medicinal fungus composition rich in triterpenoids, comprising 10-15 parts by weight of an extract of Antrodia camphorata fruit body, 10-25 parts by weight of an extract of Antrodia camphorata mycelium and 60-80 parts by weight of Ganoderma lucidum The fruit body extract; the triterpenoid compound in the composition has a weight percentage of 10 to 17%. A method for preparing a triterpene-rich antitumor composition, comprising: Step A: preparing the extract of the body extract of Antrodia camphorata, the mycelium extract of Antrodia camphorata, and the extract of Ganoderma lucidum fruit body; Step B: Weigh 10 to 15 parts by weight of the extract of the body extract of A. angustifolia, 10 to 25 parts by weight of the mycelium extract of Antrodia camphorata, and 60 to 80 parts by weight of the extract of the Ganoderma lucidum fruit body are uniformly mixed. The method for preparing an anti-tumor composition according to claim 2, wherein the preparation of the extract of the Antrodia camphorata fruit body, the extract of the Antrodia camphorata mycelium and the extract of the Ganoderma lucidum fruit body comprises the step A1: The raw material of Antrodia camphorata fruit body or Antrodia camphorata mycelium or Ganoderma lucidum fruit body is placed in 95% ethanol for reflux extraction or osmotic extraction, and the extract is collected; the extract is recovered into ethanol and then eluted with EtOAc on a diatom column. The eluate was collected and ethyl acetate was recovered to obtain an ethanol extract of the starting material. The method for preparing an antitumor composition according to claim 3, wherein a ratio of 95% ethanol to the raw material in the reflux extraction is 6-8 L/kg; the ratio of the mixture of 95% ethanol and the raw material in the percolation extraction is 20 L/kg. The method for preparing an antitumor composition according to claim 3, wherein the step A1 further comprises: adding an ethanol extract of the raw material to a saturated aqueous solution of cyclodextrin, and ultrasonically mixing to obtain a cyclodextrin inclusion. The suspension of the material was allowed to stand on a static dry pulverization to obtain an aqueous extract of the raw material. The method for preparing an antitumor composition according to claim 5, wherein the ultrasonic mixing temperature is 30 ° C, and the ultrasonic mixing time is 40 Min. The method for producing an antitumor composition according to claim 5, wherein the standing is refrigerated at 0 to 5 ° C for 24 hours; and the drying temperature is 60 ° C. The method for preparing an anti-tumor composition according to claim 3, wherein the preparation of the extract of the Antrodia camphorata fruit body, the extract of the Antrodia camphorata mycelium and the extract of the Ganoderma lucidum fruit body further comprises the step A2: The raw materials extracted by the step A1 are respectively extracted by boiling with water, and then 95% ethanol is added to the alcohol content of 75%. After the whole precipitation is completed, the supernatant is taken for filtration and steaming, and the obtained product is freeze-dried to obtain each raw material. Water extract.