Application of effective fraction of Phytolacca americana fruit fermentation broth extract in preparation of anti-tumor drugs

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to the use of effective parts of the extract of Phytolacca americana fermentation broth in the preparation of anti-tumor drugs. More particularly, the tumor is liver cancer, lung cancer, pancreatic cancer, colon cancer or glioma. The present invention chemically splits the effective parts of the Phytolacca americana fermentation broth for the first time, and conducts pharmacological studies on the anti-tumor activities of different effective parts against different tumors, thereby providing the use of effective parts of the extract of Phytolacca americana fermentation broth in the preparation of anti-tumor drugs, providing new ideas for further developing the application of Phytolacca americana in tumor prevention and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of an effective part of a fermentation broth extract of Phytolacca americana in the preparation of an anti-tumor drug. More particularly, the tumor is liver cancer, lung cancer, pancreatic cancer, colon cancer or glioma. Background Art

[0002] Phytolaccae acinosa Radix is ​​a traditional Chinese medicine. Its perennial root is bitter and cold, and it enters the lung, spleen, kidney, and large intestine meridians. It has the effects of expelling water and reducing swelling, detoxifying and dispersing knots, and diuresis. It is used externally to detoxify and dissipate knots, and is used to treat edema, fullness, carbuncle, etc. In modern clinical practice, it is often used to treat chronic nephritis and ascites due to liver cirrhosis, pericardial effusion, exudative pleurisy, non-infectious pharyngeal edema, bronchitis, functional uterine bleeding, breast hyperplasia, thrombocytopenic purpura, psoriasis, liver ascites tumors, and other diseases.

[0003] The first record of Phytolacca was in Shennong's Herbal Classic, where it was listed as a low-grade herb. It also recorded that "Phytolacca is pungent and flat in taste. It is used to treat water swelling, tumors and paralysis, and to remove carbuncles and swellings, and to kill ghosts and spirits." The "Famous Doctors' Records" of the Han Dynasty said that "Phytolacca is sour in taste. It can treat evil spirits in the chest, edema, paralysis, abdominal distension, and dredge the five internal organs and disperse water vapor." In the "Elbow Prescriptions" of Ge Hong of the Jin Dynasty, it was recorded that "to treat sudden swelling of the whole body and face, one catty of Phytolacca root (scrape off the skin and cut it thinly), boil it until it is rotten, remove the dregs, add one catty of mutton, add onion, soy sauce and salt as you like, and cook it as you like. After the swelling is healed, it is suitable to do this. You can also often pound Phytolacca, mix it with rice, steam it into cakes, and eat it." Hongjing said: Fangjia doesn't use it much, but only to treat edema, cut the raw roots, mix it with carp and cook it into soup. Taoists use it in powder form, and boil it in wine... In fact, the seeds are also a magical medicine. The flower is called Danghua, which is especially good. "Compendium of Materia Medica for Famine Relief" calls it Zhangliu, and its seeds, roots, seedlings, and stems can all be steamed and eaten.

[0004] Phytolaccae acinosa Radix is ​​a perennial plant, and its infructescence is terminal or lateral, cymose, and the annual yield of mature fresh fruit per plant is 5-15kg, which can be continued for many years, with great yield and abundant resources, but its fresh fruit has not been effectively developed and utilized. The inventor has previously developed a method for preparing Phytolaccae acinosa Radix fruit fermentation liquid using fresh fruit of Phytolaccae acinosa Radix with low toxicity as raw material, and the fermentation liquid is used to treat various diseases such as cancer (see CN112190603A). The chemical composition research conducted by the inventor in the early stage shows that the active compounds produced by Phytolaccae acinosa Radix after fermentation include various components such as alkaloids, saponins, flavonoids, polysaccharides, etc. It can be seen that the chemical composition of Phytolaccae acinosa Radix fermentation liquid is very complex, and it is impossible to determine which active ingredients therein are the main active ingredients with anti-tumor effects, and it is even more impossible to determine which specific tumor types these active ingredients may be more effective for.

[0005] Therefore, on this basis, the present invention systematically chemically splits the pokeweed fermentation liquid and conducts pharmacological research on the anti-tumor activity of different effective parts, providing a new idea for further developing the application of pokeweed in tumor prevention and treatment. Summary of the invention

[0006] In view of the unclear problem of the effective anti-tumor parts of the pokeweed fruit fermentation broth extract and in order to improve the anti-tumor effect and targeting of the pokeweed fruit fermentation broth extract, the present invention chemically splits the effective parts of the pokeweed fruit fermentation broth, and conducts pharmacological studies on the anti-tumor activities of different effective parts against different tumors, thereby providing the use of the effective parts of the pokeweed fruit fermentation broth extract in the preparation of anti-tumor drugs.

[0007] Specifically, the present invention is realized through the following technical solutions:

[0008] The invention provides use of an effective part of a pokeweed fruit fermentation broth extract in preparing an anti-tumor drug.

[0009] As an optional mode, in the above-mentioned use, the tumor is liver cancer, lung cancer, pancreatic cancer, colon cancer or glioma.

[0010] As an optional mode, in the above-mentioned use, the method for preparing the pokeweed fruit fermentation liquid comprises the following steps:

[0011] (1) Preparation of fermented yeast liquid: taking ripe and fresh pokeweed fruit, crushing and fermenting, stirring continuously, adding appropriate amount of inorganic salts and white sugar to adjust the culture conditions, and fully fermenting to obtain fermented yeast liquid;

[0012] (2) Preparation of the first fermentation liquid: fresh and ripe pokeweed fruit is taken, washed, drained, and dried in a cool place until 60% to 70% dry, placed in a non-iron fermentation container, 2-3% fermentation yeast solution is added, and the pokeweed fruit is mixed evenly, and aerobic fermentation is performed at 20-30° C. for 30-60 days, and appropriate amounts of white sugar and organic acid are added to adjust the sugar content and acidity, and the fermentation liquid is separated to obtain the first fermentation liquid;

[0013] (3) Preparation of the second fermentation liquid: The first fermentation liquid is placed in a non-iron container, placed in a cold place at 5-25°C, sealed and kept away from light, and fully fermented again for more than 30 days under anaerobic conditions to obtain the second fermentation liquid, i.e., the pokeweed fruit fermentation liquid.

[0014] In the preparation of the fermentation liquid, 0.1-0.5% of ammonium phosphate and 15%-25% of crystallized white sugar are added to adjust the culture conditions; in the preparation of the first fermentation liquid, 15%-25% of white sugar is added to adjust the sugar content, and 0.5%-2.0% of citric acid is added to adjust the acidity.

[0015] The pokeweed fruit fermented liquid contains a mixture of eight bacterial species, namely, Penicillium sublateritium, Golubevia pallescens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozyma guilliermondii, Candida intermedia, Lodderomyces elongisporus and Aspergillus sydowii, wherein the abundance of each bacterial species in the fermented liquid is at least 100 times greater than that of the fresh fruit before fermentation.

[0016] Preferably, the preparation method of the Phytolacca americana fermented liquid can refer to CN112190603A.

[0017] As an optional mode, in the above-mentioned use, the method for preparing the effective part of the Phytolacca americana fruit fermentation broth extract comprises the following steps:

[0018] The fermented liquid of Phytolacca sinensis was ultrasonically extracted with 8 times the amount of 70% methanol for 40 minutes, for a total of 3 times. After extraction, the extracts were combined and concentrated. 80% ethanol was added to the concentrate for alcohol precipitation for 24 hours, for a total of 3 times. After the last filtration, the supernatant was concentrated to obtain mother liquor 1. After 732 type cationic resin was activated, it was rinsed with distilled water to neutrality. After the mother liquor 1 was adjusted to an appropriate concentration, it was loaded and separated. It was first eluted with water until it was colorless, and then eluted with ammonia and ethanol for 4 column volumes to obtain the effective part of the ammonia and ethanol eluent, and then eluted with water. The water eluent was mother liquor 2. After D101 type macroporous resin was activated, it was rinsed with distilled water to neutrality. After the mother liquor 2 was adjusted to an appropriate concentration, it was loaded and separated. The elution order was water-50% ethanol-70% ethanol-95% ethanol. Each solvent was eluted for 4 column volumes, and then replaced with the next solvent to obtain the effective part of the 50% ethanol eluent.

[0019] As an optional mode, in the above-mentioned use, the effective part of the ammonia ethanol eluent is the alkaloid component, and the effective part of the 50% ethanol eluent is the saponin component.

[0020] As an optional mode, in the above-mentioned use, the method for preparing the effective part of the Phytolacca americana fruit fermentation broth extract comprises the following steps:

[0021] The concentrated liquid of the Phytolacca americana fermentation liquid was added with 8 times the volume of 75% methanol and ultrasonically extracted for 3 times, each time for 40 minutes. After the methanol was recovered by a rotary evaporator, the extracts were combined to obtain the total methanol extract of Phytolacca americana. An equal volume of petroleum ether was added to the separatory funnel, and the extraction was carried out three times in a row. After the petroleum ether was recovered from the upper layers, the petroleum ether extraction component of Phytolacca americana was obtained. The total methanol extract was extracted with the ethyl acetate component according to this method to obtain the ethyl acetate extraction component of Phytolacca americana.

[0022] As an optional mode, in the above-mentioned use, preferably, the effective fraction of the ammonia ethanol eluate is used to treat liver cancer or colon cancer, the effective fraction of the 50% ethanol eluate is used to treat lung cancer, pancreatic cancer or colon cancer, and the petroleum ether extraction component and the ethyl acetate extraction component are used to treat glioma.

[0023] As an optional mode, in the above-mentioned use, the drug further comprises one or more chemotherapeutic drugs or molecular targeted drugs commonly used in clinical practice or traditional Chinese medicines with anti-tumor effects.

[0024] As an optional mode, in the above-mentioned use, the chemotherapy drug is selected from one or more of the following: platinums, alkaloids, alkylating agents, antimetabolites or antitumor antibiotics. Preferably, the platinum drug is oxaliplatin for injection, cisplatin for injection, carboplatin for injection, nedaplatin for injection or lobaplatin for injection.

[0025] Preferably, the alkaloid is paclitaxel of natural origin, fully synthesized, semi-synthesized or synthesized by endophytic fungi.

[0026] As an optional method, in the above-mentioned use, the molecular targeted drug is selected from one or more of the following: gefitinib, erlotinib, sorafenib, icotinib, afatinib, dacomitinib, osimertinib, ametinib, cetuximab, imatinib, rituximab, bevacizumab, crizotinib, bosutinib, cabozantinib, dasatinib, everolimus or vemurafenib.

[0027] As an optional mode, in the above-mentioned use, the traditional Chinese medicine with anti-tumor effect is Rhizoma Dioscoreae.

[0028] As an optional mode, in the above-mentioned use, the dosage form of the drug is selected from: tablets, capsules, oral liquid or granules.

[0029] The above dosage forms can be prepared by conventional pharmaceutical preparation methods in the art.

[0030] The percentages of various organic solvents used in the extraction process of the present invention are all volume percentages.

[0031] The beneficial effects of the above technical solution of the present invention are as follows:

[0032] In order to improve the anti-tumor effect and targeting of the extract of Phytolacca americana fermentation broth, the present invention chemically splits the effective parts of the Phytolacca americana fermentation broth for the first time, and conducts pharmacological studies on the anti-tumor activities of different effective parts against different tumors, thereby providing the use of the effective parts of the extract of Phytolacca americana fermentation broth in the preparation of anti-tumor drugs, providing new ideas for further in-depth development of the application of Phytolacca americana in tumor prevention and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 :Effects of alkaloid components on the migration ability of Bel-7402 liver cancer cells (×40). A: blank group; B: low-dose alkaloid group; C: medium-dose alkaloid group; D: high-dose alkaloid group.

[0034] Figure 2 :Effects of different components of Phytolacca oleracea fermented liquid on organ indexes of mice. Among them, K: normal group; A: model group; B: cyclophosphamide group; C: Phytolacca oleracea total fermented liquid group; D: polysaccharide group; E: alkaloid group; F: 50% ethanol group. Compared with the model group, * P<0.05, ** P<0.01, *** P<0.001; compared with the blank group, #### P<0.0001.

[0035] Figure 3 :HE-stained sections of tumor tissues of H22 liver cancer-bearing mice (HE, ×400).

[0036] Figure 4 :Effects of 50% ethanol fraction of Phytolacca japonica fermented liquid on the proliferation of A549 cells. Compared with the model group, *** P<0.001.

[0037] Figure 5 :Effects of 50% ethanol fraction of Phytolacca japonica fermentation liquid on the migration ability of A549 cells. Compared with the model group, *** P<0.001.

[0038] Figure 6 :Effect of 50% ethanol fraction of Phytolacca japonica fermented liquid on the proliferation of pancreatic cancer BXPC3 cells. Compared with the model group, *** P<0.001.

[0039] Figure 7 :Effect of 50% ethanol fraction of Phytolacca japonica fermented liquid on the migration ability of pancreatic cancer BXPC3 cells. Compared with the model group, * P<0.05.

[0040] Figure 8:Effects of Phytolacca americana fermented liquid and its components on the body weight of mice. Compared with the model group, * P<0.05, ** P<0.01, *** P<0.001; compared with the blank group, # P<0.05, ## P<0.01, ### P<0.001.

[0041] Fig. 9 :Effects of Phytolacca americana fermented liquid and its components on the volume and mass changes of subcutaneous transplanted tumors in mice. Compared with the blank group, * P<0.05.

[0042] Fig.10 : Schematic diagram of pancreatic cancer subcutaneous transplanted tumors with Phytolacca americana fermentation broth and its components.

[0043] Fig.11 :Effects of various components of Phytolacca japonica fermentation broth extract on HCT116 cell proliferation. Mean ± SD, n = 6; (A) HCT116 cells were treated with polysaccharide components for 12h, 24h and 48h; (B) HCT116 cells were treated with water components for 12h, 24h and 48h; (C) HCT116 cells were treated with 50% ethanol components for 12h, 24h and 48h; (D) HCT116 cells were treated with 70% ethanol components for 12h, 24h and 48h; (E) HCT116 cells were treated with alkaloid components for 12h, 24h and 48h.

[0044] Fig.12 :Photos of rat colon. A: blank group; B: model group; C: positive drug group; D: pokeweed fermentation liquid group; E: 50% ethanol group; F: alkaloid group.

[0045] Fig.13 :Photos of rat colon tissue sections. A: blank group; B: model group; C: positive drug group; D: pokeweed fermentation liquid group; E: 50% ethanol group; F: alkaloid group.

[0046] Fig.14 : Tumor inhibition rate of each experimental group.

[0047] Fig.15 : Tumor volume increase curves of each experimental group. # Compared with the blank group, P < 0.05, ## Indicates P < 0.01, ### indicates P < 0.001; * indicates P < 0.05 compared with the model group; ** indicates P < 0.01; ***Indicates P < 0.001.

[0048] Fig.16 :Effects of each drug-treated group on organ index and tumor weight. Fig.16 A is the spleen index, Fig.16 B is the thymus index, Fig.16 C is tumor weight. In the horizontal axis, A: model group; B: cyclophosphamide group; C: Phytolacca oleracea fermented liquid group; D: polysaccharide group; E: alkaloid group; F: 50% ethanol group; G: Phytolacca oleracea fermented liquid combined with Rhizoma Dioscoreae group; H: Rhizoma Dioscoreae group. DETAILED DESCRIPTION

[0049] The present invention will be further described with reference to specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0050] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0051] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0052] Statistical analysis was performed using GraphPad Prism 8.0, and all counting data were expressed as mean ± standard deviation. One-way ANOVA was used for comparison among multiple groups, and LSD-t test was used for comparison between two groups. P < 0.05 was considered statistically significant.

[0053] Preparation Example:

[0054] 1. Preparation of Phytolacca japonica fermented liquid:

[0055] The method shown in Example 3 of CN112190603A is adopted, which is specifically as follows:

[0056] 200 kg of fresh and ripe pokeweed fruit were taken, washed, drained, and dried in a cool place until 60% to 70% dry, and then put into a PVC plastic barrel, and eight strains of Penicillium sublateritium, Golubevia pallescens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozyma guilliermondii, Candida intermedia, Lodderomyces elongisporus, and Aspergillus sydowii were added in sequence according to the titer ratio of 3.37:3.31:2.32:2.16:1.52:1.45:1.11:1, and then 2.5 kg of white sugar and 0.6 kg of citric acid were added to adjust the sugar content and acidity of the fermentation liquid. The fermentation environment had a daytime temperature of 25-30°C and a nighttime temperature of 19-27°C. After fermentation for 30 days, the first fermentation liquid was obtained by squeezing and separation with gauze.

[0057] The obtained first fermentation liquid is placed in a clean PVC plastic barrel, sealed and protected from light, anaerobic fermentation is performed, and the temperature is 15-27°C during the day and 8-20°C at night. The fermentation is performed again for 30 days, and the precipitation is fully settled. The precipitation is discarded to obtain the second fermentation liquid, that is, the pokeweed fruit fermentation liquid fermented with 8 strains is obtained. The pokeweed fruit fermentation liquid can be directly prepared into an oral liquid preparation, and can also be used as a raw material.

[0058] 2. Preparation of the effective part of Phytolacca americana fruit fermentation broth extract:

[0059] The pokeweed fermentation liquid prepared in the first part was ultrasonically extracted with 8 times the amount of 70% methanol for 40 minutes, for a total of 3 times. After extraction, the extracts were combined and concentrated, and 80% ethanol was added to the concentrate for alcohol precipitation for 24 hours, for a total of 3 times. After the last filtration, the supernatant was concentrated to obtain mother liquor 1. After activation of 732 type cationic resin, it was rinsed with distilled water to neutrality. After adjusting the mother liquor 1 to an appropriate concentration, it was loaded and separated. It was first eluted with water until colorless, and then eluted with ammonia and ethanol for 4 column volumes to obtain the effective part of the ammonia and ethanol eluent, and then eluted with water. The water eluent was mother liquor 2. After activation of D101 type macroporous resin, it was rinsed with distilled water to neutrality. After adjusting the mother liquor 2 to an appropriate concentration, it was loaded and separated. The elution order was water-50% ethanol-70% ethanol-95% ethanol. Each solvent was eluted for 4 column volumes, and then replaced with the next solvent to obtain the effective part of the 50% ethanol eluent. According to the identification of potassium bismuth iodide experiment, the effective part of the ammonia ethanol eluate is the alkaloid component; according to the qualitative identification of Liebermann-Burchard reaction, the effective part of the 50% ethanol eluate is the saponin component.

[0060] The concentrated solution of the fruit of Phytolacca is added with 8 times the volume of 75% methanol for ultrasonic extraction 3 times, each time for 40 minutes. After the methanol is recovered by rotary evaporator, the extracts are combined to obtain the total methanol extract of Phytolacca, and petroleum ether of the same volume as the total methanol extract is added to the separatory funnel, and the extraction is continued for three times. After the upper layer is combined and the petroleum ether is recovered, the petroleum ether extraction component of Phytolacca is obtained. According to this method, the total methanol extract is extracted with ethyl acetate component and n-butanol component in turn to obtain the ethyl acetate extraction component and n-butanol extraction component of Phytolacca. The lower layer solution after the n-butanol extraction is concentrated, 4 times the amount of 90% ethanol is added, and it is allowed to stand overnight. The precipitated part is the polysaccharide component.

[0061] Effect embodiment:

[0062] Effect Example 1: Study on the Anti-liver Cancer Effect of the Effective Part of Phytolacca americana Fruit Fermentation Extract 1. Effect of the effective fraction (alkaloid component) of the ammonia-ethanol eluate of Phytolacca japonica fermentation liquid on the proliferation of Bel-7402 hepatoma cells

[0063] CCK-8 test was used to evaluate cell viability. 5000 cells were inoculated in each well of a 96-well flat-bottom culture plate. After 24 hours of attachment, the cells were treated with different concentrations of alkaloid components of the fermentation broth of Phytolacca japonica. After 24 hours of culture, CCK-8 reagent was added according to the instructions of the kit. The absorbance (A) was detected by a microplate reader at 450nm. The cell inhibition rate = 100-(A treatment group-A blank group) × 100%. Blank group: No cells were inoculated in the wells, and only the absorbance after adding CCK-8 reagent was measured.

[0064] As shown in Table 1, the results showed that as the mass concentration of alkaloid components increased, the cell survival rate decreased. The IC 50 The concentration is 0.75 g·L -1 So in the subsequent experiments, 0.375 g·L of alkaloid components was selected. -1 , 0.75g·L -1 , 1.5 g·L -1 These three concentrations served as low, medium and high dose groups in subsequent experiments.

[0065] Table 1 Effects of alkaloid components on the proliferation of Bel-7402 hepatoma cells

[0066]

[0067] 2. Effects of alkaloid components on the migration ability of Bel-7402 hepatoma cells

[0068] Bel-7402 hepatoma cells in the logarithmic growth phase were cultured at 5×10 5 Cells / mL were inoculated into 6-well culture plates, 2 mL per well, and placed in a 37°C, 5% CO 2Culture overnight in an incubator. After the cells have grown to 80% adherently, discard the original culture medium and wash twice with PBS. Use a clean 200μL pipette tip to scratch the bottom of the plate vertically, crossing at least 6 horizontal lines in each well. After drawing the lines, gently wash with PBS to remove the scratched cells. The culture medium used in this experiment is RPMI-1640 serum-free culture medium. Take pictures and observe at 0h (T0) and 24h (T1) of culture, and calculate the scratch healing rate using ImageJ software. The experiment was repeated 3 times. Average scratch width = scratch gap area / length, cell migration rate (%) = (average scratch width at T0 - average scratch width at T1) / average scratch width at T0 × 100%.

[0069] The scratch test results are as follows Figure 1 As shown in Table 2, compared with the blank group, the cell migration rates of the low, medium and high doses of alkaloids groups were significantly reduced (P<0.05); compared with the low dose group, the cell migration rates of the medium and high doses of alkaloids groups were significantly reduced (P<0.05); compared with the medium dose group, the cell migration rate of the high dose group was significantly reduced (P<0.05). With the increase of alkaloid concentration, the cell migration rate decreased from 25.90% to 21.30%, 13.50% and 3.71%.

[0070] Table 2 Effects of alkaloid components on the migration ability of Bel-7402 liver cancer cells ( n=3)

[0071]

[0072] Note: Compared with the blank group, 1) P<0.05, 2) P<0.01.

[0073] 3. Effects of alkaloid components on H22 tumor-bearing mice

[0074] (1) Effects of alkaloid components on tumor inhibition rate and organ index in mice

[0075] During the administration of H22 liver cancer-bearing mice, it is necessary to observe and record the mice's eating, drinking, coat color, mental state, and dead animals every day. Starting from the first day of administration, the mice's weight should be weighed and recorded every other day. At the same time, a vernier caliper should be used to measure the long and short diameters of the mouse tumor. After the experiment is over, the tumor volume is calculated according to the formula: Volume = (length × width 2 ) / 2, draw the tumor growth curve.

[0076] like Figure 2As shown in Table 3, compared with the normal group, the spleen index of the model group was significantly increased (P<0.01), and the thymus index was significantly decreased (P<0.01). Compared with the model group, the spleen index of the cyclophosphamide group, the total fermentation broth group, and the alkaloid group was significantly decreased (P<0.01, P<0.05), and the thymus index of the total fermentation broth group and the alkaloid group was significantly increased (P<0.01). Compared with the model group, the tumor weight of the cyclophosphamide group, the total fermentation broth group, the alkaloid group and the 50% ethanol group was significantly reduced (P<0.01). Among them, the tumor inhibition rates of the total fermentation broth group and the alkaloid group were 45.40% and 48.41%, respectively. All the above results showed that the total fermentation broth group and the alkaloid group had stronger anti-tumor effects and less toxicity.

[0077] Table 3 Effects of different components of Phytolacca americana fermented liquid on tumor inhibition rate in mice

[0078]

[0079]

[0080] Note: Compared with the model group, 1) P < 0.05; 2) P<0.01; compared with the cyclophosphamide group, 3) P < 0.05; 4) P<0.01, A: model group; B: cyclophosphamide group; C: Phytolacca americana total fermentation liquid group; D: polysaccharide group; E: alkaloid group; F: 50% ethanol group.

[0081] (2) Observation on the effects of different components of Phytolacca japonica fermented liquid on mouse tumor tissue pathology

[0082] like Figure 3 As shown, HE staining results showed that the tumor cells in the model group were closely arranged, with less hemorrhage and necrosis; compared with the model group, the tumor cells in the cyclophosphamide group had large nuclei, coarse granular chromatin, visible nucleoli, frequent nuclear divisions, and pathological nuclear divisions. The pink state was the tumor necrosis area. Tumor cell rupture and necrosis occurred in the total fermentation liquid group, polysaccharide group, alkaloid group and 50% ethanol group, among which, except for the cyclophosphamide group, the total fermentation liquid group and alkaloid group were the most obvious.

[0083] Effect Example 2: Study on the Anti-lung Cancer Effect of the Effective Part of Phytolacca japonica Fermentation Extract

[0084] 1. Effect of 50% ethanol fraction of Phytolacca japonica fermentation broth on A549 cell proliferation

[0085] CCK-8 test was used to evaluate cell viability. 5000 cells were inoculated in each well of a 96-well flat-bottom culture plate. After 24 hours of attachment, the cells were treated with different concentrations of the fermentation broth components of Phytolacca japonica fruit. After 48 hours of culture, CCK-8 reagent was added according to the instructions of the kit. The absorbance (A) was detected by a microplate reader at 450nm. The cell inhibition rate = 100-(A treatment group-A blank group) × 100%. Blank group: No cells were inoculated in the wells, and only the absorbance after adding CCK-8 reagent was measured.

[0086] like Figure 4 As shown, after the extraction and separation of the fermented liquid of Phytolacca americana, its 50% ethanol component has a significant inhibitory effect on A549 cells, and is concentration-dependent. As the concentration increases, the inhibitory effect on lung cancer cells becomes stronger. When the concentration is 2.5 mg / mL, the inhibition rate is 94.25%.

[0087] 2. Effect of 50% ethanol fraction of Phytolacca japonica fermentation broth on A549 cell migration

[0088] The scratch test was used to detect the effect of drugs on cell migration. 8×10 A549 cells in the logarithmic growth phase were taken per well. 5 The cells were inoculated in a 6-well plate. When the cell adhesion density reached 90%, 3 scratches were made evenly in each well with a 200 μL pipette tip. The cells were washed twice with PBS. Different concentrations of drugs were added to the drug-treated group, and the corresponding volume of sterile double-distilled water was added to the control group. The cells were placed in an incubator and cultured in serum-free medium for 24 hours. The relative distance of scratch healing (%) = (1-24h distance / 0h distance) × 100%. The cell scratch area was analyzed using Image J software.

[0089] like Figure 5 As shown, compared with the scratch healing rate of the control group, after 48 hours of observation, it was found that the scratch healing rate after treatment with the 50% ethanol component of the fermentation liquid extract of Phytolacca americana was significantly reduced, and the difference was statistically significant (P < 0.05).

[0090] Effect Example 3: Study on the Anti-pancreatic Cancer Effect of the Effective Part of Phytolacca japonica Fermentation Extract

[0091] 1. Effect of 50% ethanol fraction of Phytolacca japonica fermentation broth on the proliferation of pancreatic cancer cells BXPC3

[0092] CCK-8 test was used to evaluate cell viability. 5000 cells were inoculated in each well of a 96-well flat-bottom culture plate. After 24 hours of attachment, the cells were treated with different concentrations of the fermentation broth components of Phytolacca japonica fruit. After 48 hours of culture, CCK-8 reagent was added according to the instructions of the kit. The absorbance (A) was detected by a microplate reader at 450nm. The cell inhibition rate = 100-(A treatment group-A blank group) × 100%. Blank group: No cells were inoculated in the wells, and only the absorbance after adding CCK-8 reagent was measured.

[0093] like Figure 6 As shown, after the extraction and separation of the fermented liquid of Phytolacca americana, its 50% ethanol component has a significant inhibitory effect on BXPC3 cells, and is concentration-dependent. As the concentration increases, the inhibitory effect on pancreatic cancer cells becomes stronger.

[0094] 2. Effect of 50% ethanol fraction of Phytolacca japonica fermentation broth on the migration of pancreatic cancer cells BXPC3

[0095] The scratch test was used to detect the effect of drugs on cell migration. 8×10 BXPC3 cells in the logarithmic growth phase were taken per well. 5 Cells were inoculated in a 6-well plate. When the cell adhesion density reached 90%, 3 scratches were made evenly in each well with a 200μL pipette tip. The cells were washed twice with PBS. Different concentrations of drugs were added to the drug-treated group, and the corresponding volume of sterile double-distilled water was added to the control group. The cells were placed in an incubator and cultured in serum-free medium for 24 hours. Relative distance of scratch healing (%) = (1-24h distance / 0h distance) × 100%.

[0096] like Figure 7 As shown, compared with the scratch healing rate of the control group, after 24 hours of observation, it was found that the scratch healing rate after treatment with 50% ethanol component of Phytolacca americana fermentation liquid was significantly reduced, and the difference was statistically significant (P < 0.05).

[0097] 3. Effects of 50% ethanol fraction on Panc02 tumor-bearing mice

[0098] Modeling method: Mouse pancreatic cancer cells Panc02 in logarithmic growth phase were digested with 0.5% trypsin, washed twice by centrifugation with PBS buffer, and the cell concentration was adjusted to 2×10 7 / mL, and the cell suspension was drawn with a 1mL No. 6 sterile needle syringe and inoculated into the right forelimb axilla of the mouse, with 0.1mL inoculated into each mouse. Tumor nodules appeared subcutaneously in the model mice, with a diameter greater than 4mm, indicating that the pancreatic cancer subcutaneous transplanted tumor mouse model was successfully established.

[0099] Dosage and grouping: After 7 days of tumor bearing, C57BL / 6 mice were randomly divided into 4 groups according to the calculation formula of equivalent dose ratio of human and mouse body surface area, including blank group, model group, 5-FU group (20mg / kg / d), SLG (fermented liquid of Phytolacca strychnifolia) group (3g / kg / d), 50%D group (120mg / kg / d), 50%H group (360mg / kg / d), among which the blank group and model group were given pure water according to body surface area conversion. The drug was administered once by gavage every morning, and the 5-FU group was intraperitoneally injected every other day for a total of 21 days.

[0100] (1) Effects of different components of Phytolacca americana fermented liquid on the body weight of mice

[0101] like Figure 8 As shown in the figure, before administration, there was no significant difference in the body weight of mice in each group (P>0.05). After administration, the body weight of the SLG group and the 5-FU group was greater than that before administration (P<0.05). After administration, the body weight of the SLG group increased significantly, and after 12 days of administration, the body weight increased slowly; after administration, the body weight of the 5-FU group increased significantly, but after 12 days of administration, the body weight decreased, which may be due to the side effects of chemotherapy drugs.

[0102] (2) Effects of different components of Phytolacca americana fermented liquid on tumor weight in mice

[0103] After tumor formation, the tumor diameter was measured every 4 days. The longest diameter (a) and the perpendicular diameter (b) were measured using a vernier caliper, and the tumor volume (V) was calculated using the formula V = 0.52 × ab.

[0104] like Fig. 9 As shown, the transplanted tumors of mice in each group grew to the 7th day, and the drug was started when the diameter of the transplanted tumors was about 5mm. There was no statistically significant difference in the tumor volume of mice before drug administration. The results showed that the growth rate of the transplanted tumor volume in the SLG group was significantly lower than that in the model group, but the average volume and growth rate were still greater than those in the 5-FU group. After 21 days of medication, the tumors were peeled off and the average tumor weight of the mice was measured. The average tumor weight of the SLG group was significantly lower than that of the model group, with statistical difference (P < 0.05); the average tumor weight of the 5-FU group was significantly lower than that of the model group, with significant statistical significance (P < 0.01). The average tumor weight of the 50% component of the Phytolacca sinensis fermentation liquid was significantly lower than that of the model group. Fig.10 It is a subcutaneous transplanted tumor of pancreatic cancer containing fermented liquid of Phytolacca strychnifolia and its components.

[0105] Effect Example 4: Study on the Anti-glioma Effect of the Effective Part of Phytolacca americana Fruit Fermentation Liquid Extract

[0106] 1. Material Preparation

[0107] (1) C6 glioma cells

[0108] The glial cell line C6 was cloned from rat glioma induced by N-nitrosomethylurea by Benda et al., and was established after a series of in vitro culture and animal passage alternations. It is an adherent cell.

[0109] (2) Drug preparation

[0110] Weigh 10 mg of petroleum ether, ethyl acetate, and polysaccharide component freeze-dried products, add 1 mL of PBS in sequence, sterilize with a 0.22 μm microporous filter membrane, and prepare a 10 mg / mL stock solution. When CCK8 cells are administered, use Ham's F12-K complete medium to prepare 625 μg / mL, 312.5 μg / mL, and 156.25 μg / mL dilutions of each component.

[0111] 2. Experimental Methods

[0112] (1) Extraction of effective components from Phytolacca japonica fermentation broth

[0113] See Preparative Examples Section 3.

[0114] (2) CCK8 method to determine the effects of the extract components of Phytolacca americana fermentation broth on the proliferation of C6 glioma cells. When C6 cells were in the logarithmic growth phase, the cells were treated and inoculated in a 96-well plate, with 100 μL per well. The culture plate was gently shaken and placed at 37°C and 5% CO. 2 Cultured in an incubator. After 24 hours, each group was medicated and cultured. The experiment was divided into a control group (untreated tumor cells and culture fluid), a drug treatment group (the concentration of each component was 625μg / mL, 312.5μg / mL, 156.25μg / mL), and a zeroing group (equal amount of culture fluid without cells). Each group had 6 replicate wells. After 24 hours of culture, 100μL of 10% CCK8 solution (10μLCCK8+90μL culture fluid) was added in the form of liquid replacement to avoid bubbles. After incubation in a carbon dioxide incubator for 1 hour, the absorbance value at a wavelength of 450nm was measured with an enzyme marker, and the cell survival rate was calculated in Excel according to the formula. The same method was used to measure the effect of the fermentation broth extract of Phytolacca japonica on the survival rate of normal cells BV2.

[0115] 3. Experimental results

[0116] The results of CCK8 experiments showed (Table 4 and Table 5) that the three extracts of Phytolacca sutchuenensis fermentation broth had a certain inhibitory effect on the proliferation of C6 cells, among which the inhibitory effect of the polysaccharide component was weaker, and the inhibitory effect of the petroleum ether component was the strongest. However, the petroleum ether component had a greater effect on the survival rate of microglial BV2 cells. The ethyl acetate component had a better inhibitory effect on C6 cells and was concentration gradient dependent, while the effect on the survival rate of BV2 cells was relatively weak.

[0117] Table 4 Inhibition rate of each extract of Phytolacca japonica fermentation liquid on C6 cells

[0118]

[0119] Table 5 Survival rate of normal BV2 cells acted on by extracts of Phytolacca oleracea fermentation broth

[0120]

[0121]

[0122] Effect Example 5: Study on the Anti-colon Cancer Effect of the Effective Part of Phytolacca japonica Fermentation Extract

[0123] 1. In vitro study on the effects of the fractionated components of Phytolacca japonica fermentation broth on the proliferation of HCT116 colon cancer cells

[0124] (1) Experimental Materials and Methods

[0125] Cell culture: HCT116 cells were inoculated into DMEM complete medium (37°C, 5% CO 2 When the cells grew to 80%, they were digested and passaged with 0.2% EDTA-trypsin.

[0126] Preparation of drug solution: Take the lyophilized powder of each component (prepared in Part 2 of the preparation example), and make it into 100 mg / mL with sterile water before use. After filtering through the membrane, divide it into small packages and store it at -20°C for later use.

[0127] The cell inhibition rate was detected by CCK-8 method: the cells were inoculated into a 96-well plate, with about 100 μL of cell suspension in each well. After culturing in a 37°C incubator for 24 h, culture medium and drug solution were added to prepare different concentrations of drug administration groups (0, 1, 2, 3, 3.5, 4, 5, 7, 8, 10 mg / mL) and cultured for 24 h. A culture medium containing 10% CCK-8 was prepared, 100 μL was added to each well, mixed and placed in an incubator for 1 h. The absorbance A at 450 nm was measured using a microplate reader, and the cell inhibition rate of each group was calculated according to the following formula.

[0128] Cell inhibition rate = 1-(experimental well A-control well A) / (blank well A-control well A)×100%

[0129] (2) Experimental results

[0130] When the HCT116 cells were treated for 12h, 24h and 48h, the 50% ethanol component and the alkaloid component with a mass concentration of more than 3mg / mL had a certain inhibitory effect on proliferation, and the inhibition rate was high. The inhibition rate of HCT116 cells was higher when the treatment time was 24h. Based on the comprehensive experimental results, the 50% ethanol component and the alkaloid component were selected, and the concentration was set below 5mg / mL. The drug action time was 24h as the dosing conditions for subsequent experiments. Fig.11 .

[0131] 2. In vivo study of the fractionated components of Phytolacca japonica fermentation broth on SD rat colon cancer model

[0132] (1) Experimental animals

[0133] 40 SD mice (male, 8 weeks, 180-250 g), rat feed, and bedding were purchased from the Guangdong Medical Laboratory Animal Center. The experimental unit use license number is SCXK (Guangdong) 2017-0125, and the animal license number is SCXK (Guangdong) 2018-0002. All animals were kept under standard laboratory conditions: in a pathogen-free environment, temperature (20°C ± 2°C), humidity (50% ± 10%), and lighting (12 hours / day), with free access to food and water. Animal experiments and operating procedures were approved by the Ethics Committee of Guangdong Pharmaceutical University and followed the Animal Ethics Code.

[0134] (2) Experimental methods

[0135] Preparation of drug solution:

[0136] The pokeweed fermented liquid was freeze-dried and concentrated into powder, and then added with physiological saline to prepare the pokeweed group liquid. Separately, 50% ethanol component and alkaloid component freeze-dried powder were taken and added with physiological saline to prepare 50% ethanol group and alkaloid group liquid.

[0137] Rats were orally administered the drug solution for 14 days, including the blank group and model group with normal saline (0.2 mL / d, ig), the positive drug pentafluoropyrimidine (0.05 g / kg / d, ig), the pokeweed fruit fermentation solution group (3 g / kg / d, ig), the 50% ethanol group (0.56 g / kg / d, ig), and the alkaloid group (0.42 g / kg / d, ig).

[0138] Animal Model:

[0139] A total of 40 rats were divided into 6 groups using a random number table method, including a normal control group, a model group, a group of fermented pokeweed fruit liquid, a group of 50% ethanol fraction, and a group of alkaloids. The rats in the model group, the group of fermented pokeweed fruit liquid, the group of 50% ethanol fraction, and the group of alkaloids were subcutaneously injected with dimethylhydrazine (DMH) 40 mg / kg / d in the first week, once every other day, for a total of 3 injections. In the second week, their drinking water was replaced with 2% dextran sulfate sodium (DSS) solution, and in the third week, they were allowed to drink pure water freely. Two weeks constituted one cycle, for a total of 2 cycles.

[0140] Source:

[0141] The rats were anesthetized by intraperitoneal injection of 8% chloral hydrate, and blood was collected from the abdominal aorta and centrifuged at 4°C and 4000 rpm, and the supernatant was stored at -20°C. The liver, spleen, colon and feces of the rats were collected and stored at -80°C for later use.

[0142] (3) Experimental results

[0143] Depend on Fig.12It can be seen that the colon of each drug group and the positive drug group is longer than that of the model group, and the colon length of the positive drug group is second only to the blank group; in the drug group colon, the colon length of 50% ethanol component> alkaloid component> Phytolacca fruit fermentation liquid component. It can be explained that the 50% ethanol group has the best therapeutic effect, followed by alkaloids.

[0144] Depend on Fig.13 (HE staining sections) It can be seen that the number of abnormal crypt foci (ACF) in the colon of each drug group and the positive drug group is less than that of the model group; in the colon sections of the drug group, the number of ACF is: 50% ethanol component> alkaloid component> pokeweed fruit fermentation liquid component. This shows that the 50% ethanol group has the best therapeutic effect, followed by alkaloids.

[0145] Effect Example 6: Study on the anti-pancreatic cancer effect of Phytolacca japonica fruit fermentation broth extract combined with Rhizoma Dioscoreae

[0146] (1) Main experimental materials

[0147] The extract of Phytolacca americana fermented broth was prepared by the method in Part 1 of the preparation example. Rhizoma Dioscoreae was purchased from Qingping Medicinal Materials Market in Guangzhou City, Guangdong Province. The preparation method of Rhizoma Dioscoreae extract is as follows: weigh 100g of Rhizoma Dioscoreae, add 1L of distilled water and soak for 1h, then decoct for 1h, decoct twice in total, filter out the residue, combine the filtrate, heat and concentrate to a crude drug concentration of 800mg / mL, dilute with distilled water before use, and set aside. Mouse pancreatic cancer cell line Panc02 was purchased from Beina Biotechnology Co., Ltd. (catalog number BNCC338034), and the culture conditions were DMEM medium, 10% FBS, 1% penicillin-streptomycin, humidity of 95%, 5% CO 2 , 37°C. The experimental animals were 32 C57BL / 6 male mice, 4-6 weeks old, weighing (15-17) g, purchased from Guangdong Medical Experimental Animal Center, production license number SYXK (Yue) 2017-0002.

[0148] (2) Experimental methods

[0149] Establishment of pancreatic cancer transplant model:

[0150] Mouse pancreatic cancer cells Panc02 in logarithmic growth phase were digested with 0.5% trypsin, washed twice by centrifugation with PBS buffer, and the cell concentration was adjusted to 2×10 7 / mL, and the cell suspension was drawn with a 1mL No. 6 sterile needle syringe and inoculated into the right forelimb axilla of the mouse, with 0.1mL inoculated into each mouse. Tumor nodules appeared subcutaneously in the model mice, with a diameter greater than 4mm, indicating that the pancreatic cancer subcutaneous transplanted tumor mouse model was successfully established.

[0151] Grouping and dosing:

[0152] After 7 days of tumor bearing, C57BL / 6 mice were randomly divided into 4 groups according to the calculation formula of equivalent dose ratio of human and mouse body surface area, including blank group, model group, 5-FU group (20 mg / kg / d), Phytolacca sinensis fermented liquid group (SLG, 3 g / kg / d), CSL group (105 mg / kg / d), CSL and Phytolacca sinensis fermented liquid combined group (CSL+SLG, 105 mg / kg / d+3 g / kg / ). Among them, CSL and Phytolacca sinensis fermented liquid group were first administered with Phytolacca sinensis, and then with Phytolacca sinensis fermented liquid. The blank group and model group were given pure water according to body surface area conversion. The drug was administered once by gavage every morning, and the 5-FU group was intraperitoneally injected every other day, for a total of 21 days.

[0153] Observation indicators:

[0154] The observed indicators were tumor volume and tumor inhibition rate.

[0155] (3) Experimental results

[0156] Depend on Fig.14 and Fig.15 It can be seen that the tumor volume of mice in the model group continued to increase over time. After drug administration, the growth rate of the tumor volume of mice slowed down. Among them, the tumor volume of the SLG group and the CSL+SLG group was smaller. The tumor inhibition rate showed that the CSL+SLG group had a more obvious tumor inhibition.

[0157] Effect Example 7: Study on the anti-liver cancer effect of Phytolacca japonica fruit fermentation broth extract combined with Rhizoma Dioscoreae

[0158] (1) Main experimental materials

[0159] The extract of Phytolacca oleracea fermented liquid and its effective fraction were prepared by the method in Part 1 of the preparation example. The preparation method of the extract of Rhizoma Dioscoreae is as shown in the effect example 6. H22 mouse hepatoma cells were purchased from Wuhan Pronosai Life Science Co., Ltd. (passaged to the 5th generation). 90 4-6 week old SPF grade ICR mice, male, weighing 18-22 g, were purchased from Guangdong Medical Experimental Animal Center (Guangzhou, China), license number: SCXK (Yue) 2022-0002.

[0160] (2) Experimental methods

[0161] Establishment of H22 liver cancer transplanted tumor mouse model:

[0162] Resuscitate H22 hepatoma cells and adjust the cell density to about 1×10 7Cells / mL, 0.2 mL of H22 liver cancer cell suspension was intraperitoneally injected into ICR mice in good condition as tumor-derived mice. After normal feeding for about 8 days, the mouse abdomen was obviously bulging, and then the mouse was placed on a clean workbench to extract the ascites solution and put it into a centrifuge tube containing saline, and then washed twice with saline, and finally the density of H22 liver cancer cells was adjusted to about 5.0×10 6 Within one hour, 0.2 mL of H22 liver cancer cell suspension was subcutaneously inoculated into the right armpit of ICR mice in good condition to establish an H22 liver cancer cell transplantation mouse model.

[0163] Experimental groups and drug administration:

[0164] H22 liver cancer-bearing mice were raised normally. After obvious tumors grew under the right armpits of the tumor-bearing mice, the tumor volume of each mouse was measured with a vernier caliper. Then, mice with tumor volumes between 70 and 140 mm were selected. 3 The tumor-bearing mice within the range were randomly divided and marked, and the groups and dosages were model group (A), cyclophosphamide group (B, 0.05g / kg), Phytolacca oleracea fermented liquid group (C, 3g / kg / d), polysaccharide group (D, 0.405g / kg / d), alkaloid group (E, 0.375g / kg / d), 50% ethanol group (F, 0.168g / kg / d), Phytolacca oleracea fermented liquid combined with Rhizoma Dioscoreae group (G, 3g / kg / d+3.9g / kg / d) and Rhizoma Dioscoreae group (H, 3.9g / kg / d), and ICR mice in good condition and not inoculated with H22 liver cancer cells were selected as blank control group (Control), with 10 mice in each group. The dosage of Phytolacca oleracea fermented liquid was converted according to the human dosage, and the dosage of each component was calculated according to the separation ratio. The mice were dosed as follows: the blank control group and the model group were gavaged with distilled water; the positive control group was gavaged with cyclophosphamide every other day; the other experimental groups were gavaged with the corresponding drugs according to the dosage every day, and the drugs were administered once a day at a fixed time for a total of 14 days.

[0165] Determination of tumor inhibition rate and organ index in mice:

[0166] After the administration, the mice in each group were anesthetized, blood was collected from the eyeballs and then killed, and then the tumor tissue was quickly peeled off, the thymus, spleen and liver were taken out, and the weights were recorded respectively. The tumor inhibition rate and organ index were calculated according to the formula.

[0167] Tumor inhibition rate (%) = (1-tumor weight of drug administration group / tumor weight of model group) × 100%

[0168] Organ index = organ weight (mg) / body weight (g)

[0169] (3) Experimental results

[0170] Depend on Fig.16 As shown in Table 6, after the combined administration of Phytolacca oleracea fermented liquid and Rhizoma Dioscoreae, the tumor inhibition rate was 51.13%, which was increased compared with the Phytolacca oleracea fermented liquid group. Compared with the model group, the organ function of the Phytolacca oleracea fermented liquid and Rhizoma Dioscoreae combined administration group was restored.

[0171] Table 6 Effect of combined administration of Phytolacca japonica fermented liquid and Rhizoma Dioscoreae on tumor inhibition rate in mice

[0172]

[0173] Note: The adult dosage of Rhizoma Dioscoreae is 30g / 70kg / d, which is equivalent to 3.9g / kg / d in mice.

[0174] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A use of an effective part of a fermented broth extract of Phytolacca americana fruit in the preparation of an anti-tumor drug, characterized in that: The effective fraction is a petroleum ether extraction component, and the petroleum ether extraction component is used to treat glioma. The preparation method of the petroleum ether extraction component comprises the following steps: The concentrated solution of the Phytolacca oleracea fermentation broth was added with 8 times the volume of 75% methanol and ultrasonically extracted for 3 times, each time for 40 min. After the methanol was recovered by rotary evaporator, the extracts were combined to obtain the total methanol extract of Phytolacca oleracea. Petroleum ether with the same volume as the total methanol extract was added to the separatory funnel, and the extraction was continued for 3 times. The upper layers were combined to recover the petroleum ether to obtain the petroleum ether extract component of Phytolacca oleracea. The preparation method of the pokeweed fruit fermentation liquid comprises the following steps: (1) Preparation of fermented yeast liquid: Take ripe and fresh pokeweed fruit, crush it, ferment it, and stir it continuously. Add appropriate amount of inorganic salts and white sugar to adjust the culture conditions. Ferment it fully to obtain fermented yeast liquid. (2) Preparation of the first fermentation liquid: fresh and ripe pokeweed fruit is washed, drained, and dried in a cool place until 60% to 70% dry. The fruit is placed in a non-iron fermentation container, 2-3% fermentation yeast solution is added, and the mixture is mixed evenly with the pokeweed fruit. The mixture is fermented aerobically at 20-30°C for 30-60 days. An appropriate amount of white sugar and organic acid is added to adjust the sugar content and acidity. The fermentation liquid is separated to obtain the first fermentation liquid. (3) Preparation of the second fermentation liquid: Place the first fermentation liquid in a non-iron container, place it in a cold place at 5-25°C, seal it and keep it away from light, and ferment it again under anaerobic conditions for more than 30 days to obtain the second fermentation liquid, i.e., the Phytolacca fruit fermentation liquid. In the preparation of the fermentation liquid, 0.1% to 0.5% of ammonium phosphate and 15% to 25% of crystallized white sugar are added to adjust the culture conditions; in the preparation of the first fermentation liquid, 15% to 25% of white sugar is added to adjust the sugar content, and 0.5% to 2.0% of citric acid is added to adjust the acidity. The pokeweed fermented liquid contains Penicillium sublateritium , Golubevia pallescens , Aspergillus penicillioides , Aspergillus parvulus , Meyerozyma guilliermondii, Candida intermedia, Lodderomyces elongisporus and Aspergillus sydowii A mixture of eight bacterial strains, wherein the abundance of each bacterial strain in the fermentation liquid is at least 100 times greater than that of the fresh fruit before fermentation.

2. The use according to claim 1, characterized in that: The dosage form of the drug is selected from: tablets, capsules, oral liquid or granules.

Images