Radix fici simplicissimae compound extract as well as preparation method and application thereof

Abstract

The invention discloses a radix fici simplicissimae compound extract as well as a preparation method and application thereof, and belongs to the field of biological medicines. The medicinal plant compound extract derived from the radix fici simplicissimae is successfully prepared and is proved to have a good lung cancer resisting effect, and proliferation, migration, invasion and apoptosis of lung cancer cells and angiogenesis can be inhibited by adjusting related pathways so as to be used for tumor treatment. The radix fici simplicissimae compound extract disclosed by the invention has the advantages of tumor growth resistance, good biocompatibility and the like, and can be used as a novel high-efficiency and low-toxicity medicine for tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of biological anticancer drugs, and in particular to a compound extract of beech root, a preparation method and an application thereof. Background Art

[0002] Malignant tumors pose a serious threat to life and health. Early-stage clinical tumor treatments are mainly based on surgery, radiotherapy, and hyperthermia, which are effective but have significant side effects and a narrow scope of application. For mid- to late-stage and metastatic unresectable tumors, as well as adjuvant therapy after resection, drug intervention is the most important approach. From traditional cytotoxic drugs and hormonal drugs to precise molecular targeted drugs and immune checkpoint inhibitors, drug therapy for tumors has entered a new stage. However, to date, the therapeutic efficacy of these chemical and biological drugs on tumors is still very limited. Discovering new anticancer drugs from traditional Chinese medicine, using them for the sole treatment of tumors or integrating them with Western medicine to improve anticancer efficacy, reduce toxic side effects, and improve patients' quality of life, is an important development direction and drug research and development trend in tumor treatment.

[0003] Extracts from medicinal plants have been shown to possess significant therapeutic benefits and are expected to develop into a new class of highly effective, low-toxic pharmaceutical preparations. Compared with Western medications, these agents offer significant advantages, including reduced side effects, improved quality of life for cancer patients, and prolonged survival. This makes them valuable clinically for use in areas such as oncology, inflammation, and immunotherapy, and holds enormous promise for the development of innovative pharmaceutical preparations.

[0004] Beech root is the dried root of Actinidia chinensis (Actinidia chinensis), a large deciduous vine of the Actinidia family. It is a traditional medicinal herb used by the She ethnic group and a popular folk herbal remedy. It is a representative remedy in Jin medicine for treating tumors and other diseases. It primarily grows in tall grassy thickets or scrubland on mountainous terrain at altitudes between 250 and 1,000 meters. It has detoxifying, swelling-reducing, and heat-clearing properties.

[0005] In recent years, research on the She medicine Phoebe quinata root has mainly focused on the effects of single extracts in preventing gastric cancer, blood cancer, and melanoma, while there is little research on compound products. Summary of the Invention

[0006] In response to the existing technology, the present invention provides a compound extract of beech root and a preparation method and application thereof.

[0007] The present invention provides a method for preparing a compound extract of Phragmites australis root, the preparation method comprising the following steps:

[0008] 1) grinding the beech root and sieving to obtain beech root powder;

[0009] 2) soaking the beech root powder obtained in step 1) in double-distilled water overnight, and then boiling under reduced pressure reflux to obtain a beech root crude aqueous extract slurry; filtering out the residue in the slurry, collecting the juice, and concentrating the juice to obtain a beech root crude aqueous extract concentrate; adding anhydrous ethanol to the concentrate, allowing it to stand, and collecting the supernatant; and rotary evaporating the supernatant until it is free of alcohol, thereby obtaining a beech root aqueous extract concentrate A prepared by the reduced pressure reflux method;

[0010] 3) soaking the beech root powder obtained in step 1) in double-distilled water overnight, adding cellulase, extracting under ultrasound, and then boiling under reduced pressure reflux to obtain a beech root crude aqueous extract slurry; filtering out the residue in the slurry and collecting the juice, concentrating the juice to obtain a concentrate of the beech root crude aqueous extract; adding anhydrous ethanol to the concentrate, allowing it to stand, and collecting the supernatant; and rotary evaporating the supernatant until there is no alcohol taste, thereby obtaining a beech root aqueous extract extract B prepared by an ultrasound-enzyme-assisted reduced pressure reflux method;

[0011] 4) compounding the beech root water extract extract A obtained in step 2) and the beech root water extract extract B obtained in step 3) to obtain a beech root compound extract extract;

[0012] 5) The beech root compound extract obtained in step 4) is placed on a magnetic stirrer for stirring, and freeze-dried to obtain a beech root compound extract.

[0013] According to a preferred embodiment of the present invention, in step 1), the beech root is crushed and then passed through a 50-100 mesh sieve to obtain beech root powder.

[0014] According to a preferred embodiment of the present invention, in step 2) and step 3), the number of times of boiling under reduced pressure reflux is 1-10 times, and the boiling state (slight boiling) in each boiling under reduced pressure reflux is maintained for 0.5-5 hours.

[0015] According to a preferred embodiment of the present invention, in step 2) and step 3), the method for concentrating the juice is rotary evaporation; the volume after concentration is 1 / 5-1 / 3 of the original volume.

[0016] According to a preferred embodiment of the present invention, in step 2) and step 3), the amount of anhydrous ethanol or double distilled water added to the concentrate is 2-6 times the volume of the concentrate; the standing time is 8-24 hours, and the standing temperature is 0-4°C.

[0017] According to a preferred embodiment of the present invention, in step 2) and step 3), the supernatant is collected by vacuum filtration.

[0018] According to a preferred embodiment of the present invention, in step 3), the amount of cellulase added is 0.5%-3% of the mass of the beech root powder; the ultrasonic extraction time is 10-30 min, and the ultrasonic extraction temperature is 20-55°C.

[0019] According to a preferred embodiment of the present invention, in step 4), the beech root water extract extract A prepared by the reduced pressure reflux method and the beech root water extract extract B prepared by the ultrasound-enzyme assisted reduced pressure reflux method are mixed in a mass ratio of 1:0.5-1:10 to obtain the beech root composite extract extract. Preferably, the mass ratio of the beech root water extract extract A and the beech root water extract extract B is 1:1-1:2.

[0020] According to a preferred embodiment of the present invention, in step 5), the magnetic stirring temperature is 20-35° C., and the stirring time is 6-24 h; the freeze-drying conditions are 2-4° C., and the drying time is 18-24 h.

[0021] The present invention also provides a compound extract of Phragmites australis root prepared by the above method.

[0022] The present invention also provides a use of a compound extract of beech root prepared by the above method in the preparation of an anti-lung cancer drug. Preferably, the concentration of the compound extract of beech root in the anti-lung cancer drug is greater than 100 μg / mL, preferably 100-400 μg / mL; preferably, the mass ratio of beech root aqueous extract extract A to beech root aqueous extract extract B in the compound extract of beech root is 1:1-1:2.

[0023] The present invention provides a method for preparing a compound extract of beech root. The provided beech root extract is cheap, easy to obtain, safe and low-toxic, and can overcome the problems of high cost, poor efficacy, easy drug resistance, large toxic side effects and adverse reactions of tumor treatment drugs. The present invention provides for the first time the specific application and mechanism analysis of the compound extract of beech root in lung cancer. The compound extract of beech root described in the present invention has the advantages of simple preparation method, anti-tumor growth, good biocompatibility, etc., and can be used as a new type of high-efficiency and low-toxic drug for tumor treatment. The present invention will provide new reference significance and value for the further development of various medicinal plant extracts and their application in tumor treatment. The present invention found that compared with a single beech root extract, it has a significant effect in anti-lung cancer activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be further described below in conjunction with the accompanying drawings:

[0025] Figure 1 The effects of a single agent (water extract 1# and alcohol extract 2# prepared by traditional reduced pressure reflux, and water extract 3# prepared by ultrasound-enzyme assisted reduced pressure reflux) on the proliferation of A549 tumor cells at different concentrations in one embodiment of the present invention.

[0026] Figure 2 The effect of different ratios of the compound extracts 1# and 3# (4#) at different concentrations on the proliferation of A549 tumor cells in an example of the present invention is shown;

[0027] Figure 3 This is the effect of different concentrations of the compound extract 4# of the root of Phragmites australis on the proliferation of LO-2 cells in one embodiment of the present invention;

[0028] Figure 4 This is the effect of 400 μg / mL of the compound extract 4# of the root of Phragmites australis on the migration level of A549 cells in one embodiment of the present invention;

[0029] Figure 5 This is the effect of 400 μg / mL of the compound extract 4# of the root of Phragmites australis on the invasion level of A549 cells in one embodiment of the present invention;

[0030] Figure 6 This is the effect of 400 μg / mL of the compound extract 4# of the root of Phragmites australis on the apoptosis level of A549 cells in one embodiment of the present invention;

[0031] Figure 7 This is the inhibition of subcutaneous lung cancer transplant tumor size in mice by the compound extract 4# of the root of Phagmus quinata in one embodiment of the present invention;

[0032] Figure 8 This is a Western blot analysis of the effect of the compound extract 4# of the root of Phagmus quinata on TGF-β / Smad, PI3K / AKT / FOXO and PI3K / AKT / mTOR pathway proteins in subcutaneous lung cancer transplanted tumors in mice in one embodiment of the present invention;

[0033] Figure 9 This is an immunofluorescence staining image of angiogenesis-related proteins VEGF and CD31 in subcutaneous lung cancer transplanted tumors in mice using the compound extract 4# of the root of Ficus microcarpa in one embodiment of the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described and illustrated below in conjunction with specific embodiments. The embodiments are merely illustrative of the present disclosure and do not limit its scope. The technical features of the various embodiments of the present invention may be combined accordingly, provided that there is no conflict between them.

[0035] The human lung cancer cells (A549) and normal human liver cells (LO-2) used in this example were cultured in complete culture medium containing 10% fetal bovine serum, penicillin (50 U / mL) and streptotoxin (50 U / mL) at 37°C in a 5% CO2 environment.

[0036] Example 1: Method for preparing single beech root extract by traditional reduced pressure reflux method

[0037] First, two groups of beech root powder were soaked in double-distilled water and anhydrous ethanol overnight, respectively, and then boiled three times by traditional reduced pressure reflux and kept in a slightly boiling state for 1 hour to obtain crude water extract and alcohol extract slurries of beech root; then, they were filtered using filter meshes to collect the juices, and the two juices were concentrated by rotary evaporation to obtain concentrates of crude water extract and crude alcohol extract of beech root; then, 3 times the volume of anhydrous ethanol was added to the obtained crude water extract concentrate and precipitated for 12 hours, and 3 times the volume of double-distilled water was added to the obtained crude alcohol extract concentrate and precipitated for 12 hours, and the supernatants were collected respectively; finally, the corresponding supernatants were respectively evaporated by rotary evaporation until there was no alcohol taste, to obtain extracts of beech root water extract and alcohol extract, and freeze-dried at 4°C for 20 hours to obtain beech root water extract 1# and beech root alcohol extract 2# prepared by traditional reduced pressure reflux method, respectively.

[0038] Example 2: Preparation of a single water extract from the root of Phragmites australis by ultrasound-enzyme-assisted reduced pressure reflux method

[0039] First, the beech root powder was soaked in double-distilled water overnight, and then cellulase was added according to 2% of the mass of the beech root powder. The extract was extracted under ultrasound at 25°C for 20 minutes, and then the extract was boiled under reduced pressure reflux for 3 times, and each time the reduced pressure reflux was boiled to a slightly boiling state and maintained for 1 hour to obtain a crude water extract slurry of the beech root; the juice was then filtered and collected using a filter, and the juice was concentrated by rotary evaporation to obtain a concentrate of the crude water extract of the beech root; then 3 times the volume of anhydrous ethanol was added to the obtained crude water extract concentrate and precipitated for 12 hours, and the supernatant was collected; finally, the obtained supernatant was rotary evaporated until there was no alcohol taste to obtain an extract of the beech root water extract, and freeze-dried at 4°C for 20 hours to obtain beech root water extract 3# prepared by ultrasound-enzyme assisted reduced pressure reflux method.

[0040] Example 3: Preparation method of compound extract of Phegnum aviculare root

[0041] Based on implementation cases 1 and 2, the extracts of the beech root water extract 1# prepared by the traditional reduced pressure reflux method and the beech root water extract 3# prepared by the ultrasound-enzyme assisted reduced pressure reflux method were mixed evenly in mass ratios of 1:0.5, 1:1 and 1:2, respectively, placed on a magnetic stirrer, stirred at 25°C for 12 h, and vacuum-frozen at 4°C for 20 h to obtain three beech root compound extracts 4# with different ratios.

[0042] Example 4: Specific inhibition of survival rate of tumor model by single and combined extracts of Prunus mume

[0043] Cell proliferation and survival were detected using a CCK-8 assay. A549 and LO-2 cells were seeded onto 96-well plates at a density of 5,000 cells / well and incubated overnight in a 37°C, 5% CO2 incubator. After cell attachment, the medium was changed and the cells were treated with 25, 50, 100, 200, and 400 μg / mL of 1# water extract of beech root, 2# alcohol extract of beech root, 3# water extract of beech root, and 4# compound of beech root in different proportions (the total concentration of the compound beech root extract was 25, 50, 100, 200, and 400 μg / mL, respectively, consistent with the control groups). Blank and control wells were also set. After administration, the cells were incubated in the incubator for 24 hours. Then, 10 μL of CCK-8 solution diluted with fresh serum-free culture medium was added. After incubation for another 1 hour, the absorbance at 450 nm was measured using a microplate reader.

[0044] like Figure 1 As shown in the figure, compared with the extract 2# of the root of the Chinese jujube, the viability of A549 cells was dose-dependent with the extracts 1# and 3# of the root of the Chinese jujube, with the maximum tumor inhibition rates reaching 63.52% and 66.0%, respectively. This indicates that the extracts 1# and 3# of the root of the Chinese jujube effectively inhibited the growth and survival of A549 tumor cells and had a strong anti-tumor effect. In addition, as Figure 2 As shown, under the premise of consistent total concentration, compared with the beech root extracts 1# and 3#, the beech root compound 4# after compounding in a mass ratio of 1:1 and 1:2 has better anti-lung cancer effect, with the maximum tumor inhibition rate reaching 76.40% and 77.90%, respectively. There is no significant difference between the two, but compared with the anti-lung cancer effect of single beech root extract, both are significantly improved, increasing by 12.88% and 11.90%, respectively. This shows that the anti-lung cancer effect of the beech root extract 1# and 3# compound is better than that of the single extract 1# and 3#, and the anti-lung cancer effect shows a trend of first increasing and then stabilizing with the increase of the total compound ratio of 3#. Therefore, a 1:1 compound ratio was selected for the subsequent anti-lung cancer pharmacodynamic evaluation study of the compound system. In addition, as Figure 3 As shown, the compound extract 4# of the root of Psoralea corylifolia (compounded according to the mass ratio of 1# and 3# of 1:1) had low toxicity to L0-2 cells, indicating that it had high biosafety.

[0045] Example 5: The ability of the compound extract 4# (1:1 compound) from the root of Psoralea corylifolia to specifically inhibit migration, invasion and promote apoptosis in tumor models

[0046] A549 cells were plated in a 24-well plate at a density of 5 × 10 4Cells were seeded at a rate of 100 μL / well in a 24-well plate, and 400 μL of cell suspension was added to each well. A blank culture medium group and a 400 μg / mL beech root extract group were set up. The plates were incubated in a 37°C, 5% CO2 incubator for 24 hours. The plates were then washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 5 minutes, and washed twice with PBS. The invasion of cells in each group was observed under a light microscope, photographed, and analyzed using Image J 1.8.0 software.

[0047] 80 μL of Matrigel gel was added to each well of a 24-well plate and incubated in a cell culture incubator for 30 minutes until solidified. A549 cells were seeded at 5 × 10⁴ cells / well in a 24-well plate, and 400 μL of the cell suspension was added to each well. A blank culture medium group and a 400 μg / mL beech root extract group were set up. The culture plates were incubated in a 37°C, 5% CO₂ incubator for 24 hours. The plates were then washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 5 minutes, and washed twice with PBS. The invasion of cells in each group was observed under a light microscope, photographed, and analyzed using Image J 1.8.0 software.

[0048] Take a 6-well plate and plate A549 cells at a density of 1×10 5 Cells were seeded at a rate of 100 μg / well in a 6-well plate, and 2 mL of cell suspension was added to each well. The plates were incubated overnight at 37°C with 5% CO2. The next day, the old culture medium was discarded, and 400 μg / mL of a complex extract of Psoralea corylifolia root was added to the treated group, while blank culture medium was added to the blank group. The cells were then incubated for another 24 hours. The number of apoptotic cells in the different groups was qualitatively and quantitatively analyzed using an apoptosis kit.

[0049] like Figure 3 、 4 As shown in Figures 5 and 5, in the blank control group, the migration rate and cell invasion number of A549 cells were higher, and the cell apoptosis was lower, while the compound extract of the root of Psoralea corylifolia at a concentration of 400 μg / mL could significantly inhibit the migration, invasion and apoptosis of A549 cells, exerting an anti-tumor effect.

[0050] Case Study 6: Mechanism of Inhibition of Tumor Cells by Beech Root Extract 4# and Expression Verification of Major TGF-β / Smad, PI3K / AKT / FOXO, and PI3K / AKT / mTOR Pathway Proteins

[0051] Take a 6-well plate and plate A549 cells at a density of 2×10 5 / well were seeded in 6-well plates, and 2 mL of cell suspension was added to each well. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for routine culture overnight. The next day, the original culture medium was discarded, and 400 μg / mL of culture medium containing the compound extract of the root of beech was added to each well of the treatment group, and the cells were cultured in the incubator for another 24 hours. Total RNA was then isolated, purified, quality controlled, and total protein was extracted. Western blot experiments were used to qualitatively and quantitatively analyze cells in different groups, and Image J1.8.0 image processing software was used to analyze the optical density values ​​of the bands to determine the effects of the compound extract of the root of beech on the expression of TGF-β / Smad, PI3K / AKT / FOXO and PI3K / AKT / mTOR pathway proteins in tumor cells. Figure 6 As shown in the results, the compound extract of the root of Ficus microcarpa mainly intervened in the TGF-β / Smad, PI3K / AKT / FOXO and PI3K / AKT / mTOR pathways, and downregulated the expression of related proteins, indicating that the pathway was inhibited.

[0052] Case Study 7: Evaluation of the Anti-tumor and Anti-angiogenic Effects of Beech Root Extract 4# in a Subcutaneous Lung Cancer Mouse Model

[0053] The experimental animals were 6-8 week old male BALB / c nude mice, inoculated with 0.1 mL of 1×10 8 / mL A549 cell suspension was used to establish a subcutaneous transplantation lung cancer model. When the average tumor volume reached 50-100mm 3 Tumor-bearing nude mice were randomly divided into three experimental groups, with 6 mice in each group. Every two days, one group was given PBS, one group was given 10 mg / kg paclitaxel (PTX) positive drug, and the other group was given 600 mg / kg of the compound extract of the root of the Chinese wolfberry tree, solution 4. The body weight and tumor volume of the mice were monitored every other day. On the 21st day, the mice were killed and the tumors of each group were collected. The results are shown in Figure 2. Figure 7 As shown in the figure, the tumors in the control group grew rapidly within 21 days, while the paclitaxel (PTX) and the compound extract of the root of beech (SMTG) group 4 significantly delayed the growth trend of the tumor. Western blot experiments were used to determine the expression of TGF-β / Smad, PI3K / AKT / FOXO and PI3K / AKT / mTOR pathway proteins. Tunel and immunofluorescence staining were used to analyze the expression of VEGF and CD31, proteins related to apoptosis and angiogenesis in tumor tissues, respectively. Image J1.8.0 was used for image processing. Figure 8 、 9As shown in the results, compared with the control group, the expression of proteins in various pathways and the expression of VEGF and CD31 were lower, and the positive rate of cell apoptosis was higher, which confirmed that the compound extract 3# of the root of pheasant can intervene in the TGF-β / Smad, PI3K / AKT / FOXO and PI3K / AKT / mTOR pathways in vivo to achieve tumor treatment, and realize the function of inhibiting lung cancer growth and promoting apoptosis.

[0054] The above-described embodiments merely illustrate several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. Persons skilled in the art will readily appreciate that variations and modifications may be made without departing from the scope of the present invention, all of which fall within the scope of protection of the present invention.

Claims

1. A method for preparing a compound extract of beech root, characterized in that: The preparation method comprises the following steps: 1) crushing the beech root and sieving it to obtain beech root powder; 2) soaking the beech root powder obtained in step 1) in double distilled water overnight, and then boiling under reduced pressure reflux to obtain a beech root crude water extract slurry; filtering out the residue in the slurry and collecting the juice, and concentrating the juice to obtain a beech root crude water extract concentrate; adding anhydrous ethanol to the concentrate, letting it stand, and collecting the supernatant; rotary evaporating the supernatant until there is no alcohol taste, to obtain a beech root water extract extract A prepared by the reduced pressure reflux method; 3) soaking the beech root powder obtained in step 1) in double distilled water overnight, adding cellulase, extracting under ultrasound, and then boiling under reduced pressure reflux to obtain a beech root crude water extract slurry; filtering out the residue in the slurry and collecting the juice, concentrating the juice to obtain a concentrate of the beech root crude water extract; adding anhydrous ethanol to the concentrate, standing it, and collecting the supernatant; rotary evaporating the supernatant until there is no alcohol taste, to obtain a beech root water extract extract B prepared by an ultrasound-enzyme-assisted reduced pressure reflux method; 4) compounding the beech root water extract extract A obtained in step 2) and the beech root water extract extract B obtained in step 3) to obtain a beech root composite extract extract; 5) The compound extract of the root of beech obtained in step 4) is placed on a magnetic stirrer for stirring, and freeze-dried to obtain a compound extract of the root of beech.

2. The preparation method according to claim 1, characterized in that: In step 1), the beech root is crushed and passed through a 50-100 mesh sieve to obtain beech root powder.

3. The preparation method according to claim 1, characterized in that: In step 2) and step 3), the number of times of reduced pressure reflux boiling is 1-10 times, and the boiling state is maintained for 0.5-5h in each reduced pressure reflux boiling.

4. The preparation method according to claim 1, characterized in that: In step 2) and step 3), the method for concentrating the juice is rotary evaporation; the volume after concentration is 1 / 5-1 / 3 of the original volume.

5. The preparation method according to claim 1, characterized in that: In step 2) and step 3), the amount of anhydrous ethanol added to the concentrated solution is 2-6 times the volume of the concentrated solution; the standing time is 8-24 hours, and the standing temperature is 0-4° C.; and the supernatant is collected by vacuum filtration.

6. The preparation method according to claim 1, characterized in that: In step 3), the amount of cellulase added is 0.5%-3% of the mass of the beech root powder; the ultrasonic extraction time is 10-30 minutes, and the ultrasonic extraction temperature is 20-55°C.

7. The preparation method according to claim 1, characterized in that: In step 4), the beech root water extract extract A prepared by the reduced pressure reflux method and the beech root water extract extract B prepared by the ultrasound-enzyme assisted reduced pressure reflux method are mixed uniformly in a mass ratio of 1:1-1:2 to obtain the beech root composite extract extract.

8. The preparation method according to claim 1, characterized in that: In step 5), the magnetic stirring temperature is 20-35° C., and the stirring time is 6-24 h; the freeze-drying conditions are 2-4° C., and the drying time is 18-24 h.

9. The compound extract of the root of Physalis philadelphica prepared by the method according to any one of claims 1 to 8.

10. Use of the compound extract of the root of Physalis philadelphica according to claim 9 in the preparation of anti-lung cancer drugs.

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