CN112190603A

CN112190603A - Phytolacca acinosa fruit fermentation liquor and preparation method and application thereof

Info

Publication number: CN112190603A
Application number: CN202011079683.6A
Authority: CN
Inventors: 刘文龙; 王秋红; 李彪; 张志宏; 林晗; 朱金莲
Original assignee: Hengyang Wen Long Wild Fruits And Vegetables Planting Professional Farmers Cooperatives
Current assignee: Hengyang Wen Long Wild Fruits And Vegetables Planting Professional Farmers Cooperatives
Priority date: 2020-10-10
Filing date: 2020-10-10
Publication date: 2021-01-08

Abstract

The invention relates to a pokeberry fruit fermentation liquid and a preparation method thereof, the pokeberry fruit fermentation liquid is prepared by adopting a mixed strain fermentation method, the method takes mature pokeberry fresh fruit as a raw material, takes Penicillium sublateritium, Golubeville pallens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozyma guilliermondii, Candida intermedia, Loddermomyces elongii and Aspergillus sydowii 8 mixed strains as fermentation strains, and the pokeberry fruit fermentation liquid is prepared by secondary fermentation at 18-32 ℃. The preparation method has definite fermentation strain and controllable process. The prepared fermentation liquor has clear chemical components and obvious anticancer activity.

Description

Phytolacca acinosa fruit fermentation liquor and preparation method and application thereof

Technical Field

The invention belongs to the fields of biological medicines, foods and health-care products, and particularly relates to pokeberry fruit fermentation liquor, a preparation method thereof and application thereof in preparing medicines, foods and health-care products for treating cancers.

Background

Phytolaccae acinosa Radix is a traditional Chinese medicine, is taken as a medicine by roots of years, is bitter and cold, enters lung, spleen, kidney and large intestine channels, has the effects of expelling water and reducing swelling, detoxifying and resolving masses and inducing diuresis, is externally used for detoxifying and resolving masses, is used for treating symptoms such as edema, turgor, carbuncle and swelling, and is also used for treating diseases such as liver ascites type tumor, edema in modern clinic. Phytolacca acinosa is originally recorded in Shen nong Ben Cao Jing (Shen nong's herbal Jing), listed as the first-generation product, and recorded as "Phytolacca acinosa, pungent and mild flavor, mainly manifested as water swelling and tumor arthralgia, removing carbuncle and swelling, and killing ghost essence". The Chinese generation recorded in famous medical records that the pokeberry root has sour flavor and treats pathogenic qi in the chest, edema, paralysis, abdominal fullness, five internal organs and water and qi dispersing. The jin Dynasty Ku hong (elbow posterior prescription) records: for swelling, swollen and swollen, the surface is flooded, radix Phytolaccae is cooked to be thoroughly cooked (peeled and thinly cut), mutton is added for one jin, and salted with scallion, so that the treatment is performed randomly, and the treatment is suitable for treating swelling and healing after eating. Also can be pounded into pokeberry and mixed with rice to be steamed into cake for eating. The Chinese herbal medicine has the following characteristics: the prescription is not used much, but edema is treated, roots are cut, and the mixed carps are boiled to be taken as soup. Taoist-jia is used in powder form, and decocted … … is used as a miraculous drug. The flower is called Yao Hua, especially Lian. The chapter willow, seed, root, seedling and stem in the book Ji ban Ben Cao can be steamed and eaten.

The Chinese pharmacopoeia stipulates that the original plant source of the pokeberry has 2 varieties, namely pokeberry Phytolacca acinosa Roxb and Phytolacca americana L, but the toxicity of the two varieties is different obviously. According to the herbal examination and modern research, the toxicity of pokeberry is low, so that pokeberry is used as a food material, while the toxicity of serendipity is severe. In long-term mixed use, the traditional Chinese medicine generally considers that the traditional Chinese medicine is a toxic traditional Chinese medicine and can cause symptoms such as vomit, headache, diarrhea and the like, so the traditional Chinese medicine is rarely used or is limited to use clinically. However, it is commonly used in national medicine, such as Yi nationality, Mongolian nationality, Tujia nationality and Miao nationality. South China recorded Lei Gong Pao Zhi Lun recorded that "Shang Lu Hua Bai, which the celestial being collected as preserved fruit after more years, can also drink wine. Tang Dynasty Sun Simiao (thousand gold wing prescription) the root seedling can be eaten with purple color, the one with better taste is more superior than white, and the herb is clean, thoroughly steamed and nontoxic. Then, the use of phytolacca esculenta diet therapy is available. To the Song Dynasty "Taiping Shenghui Fang" (Taiping Shenghui Fang): "improving eyesight and benefiting hearing" is more clear to think that it has tonifying action. Shang Lu Xiren also breeds vegetables in the compendium of materia Medica of Ming Dynasty Li Shizhen, white root and purple are taken as tillering seeds for ridge cultivation or seeds. The seedlings can be washed and steamed or cooked well with ash, so they are suitable for people who eat Dansha and Rushi.

The pokeberry is a perennial plant, the infructescence of the pokeberry is terminal or lateral and umbrella-shaped, the annual yield of a single mature fresh fruit is 5-15 kg, the pokeberry can be continuously grown for many years, the yield is extremely high, the resources are rich, but the fresh fruit of the pokeberry is not effectively developed and utilized all the time. The method is characterized in that fresh Phytolacca acinosa Roxb, which is low in toxicity, is used as a raw material, and the fresh fruit contains a large amount of pectin polysaccharide, protein, trace elements and the like besides chemical components of saponins, flavonoids, anthraquinones, phenolic acids and sterols which are consistent with those in roots, so that sufficient nutrient substances can be provided for fermentation, and in addition, the fresh fruit contains rich natural symbiotic strains, so that a condition guarantee is provided for fermentation of the fresh fruit. The fermented fresh fruit fermentation liquor has the effects of removing water and swelling, and resolving hard mass and relieving pain, and is used for treating cancer, AIDS, arteriosclerosis, skin diseases and the like in China folk, and the curative effect is obvious.

Disclosure of Invention

The invention provides a pokeberry fruit fermentation liquid and a preparation method thereof, and separates and identifies mixed fermentation strains and therapeutic active ingredients in the fermentation liquid, thereby obtaining a fermentation process which is controllable, has definite fermentation products, can be used for treating serious and difficult diseases such as cancer, AIDS, arteriosclerosis, skin diseases and the like, and provides a new solution for clinic.

In a first aspect, the invention provides a method for preparing a fermentation liquid of fresh pokeberry fruit, which comprises the following steps:

(1) preparation of fermentation mother liquor: crushing and naturally fermenting mature and fresh pokeberry fruit, continuously stirring, adding a proper amount of inorganic salt and white granulated sugar to adjust culture conditions, and fully fermenting to obtain fermentation mother liquor;

(2) preparation of a first fermentation broth: taking fresh and mature pokeberry fruit, cleaning, draining, drying in the shade until the pokeberry fruit becomes dry, placing in a non-iron fermentation container, adding 2-3% of fermentation mother liquor, uniformly mixing with the pokeberry fruit, carrying out aerobic fermentation for 30-60 days at 20-30 ℃, adding a proper amount of white granulated sugar and organic acid to adjust sugar degree and acidity, and separating fermentation liquor to obtain first fermentation liquor;

(3) preparation of a second fermentation liquid: and placing the primary fermentation liquor in a non-iron container, placing the container in a cold place at 5-25 ℃, sealing in the dark, standing, and fully fermenting for more than 30 days under an anaerobic condition to obtain secondary fermentation liquor, namely the pokeberry fruit fermentation liquor.

In one embodiment, ammonium phosphate accounting for 0.1-0.5% of the weight of fresh fruits and crystallized white granulated sugar accounting for 15% -25% of the weight of the fresh fruits are added to prepare the fermentation mother liquor to adjust the culture conditions; in the preparation of the first fermentation liquor, white granulated sugar which accounts for 15 to 25 percent of the weight of the fresh fruits is added to adjust the sugar degree, and citric acid which accounts for 0.5 to 2.0 percent of the weight of the fresh fruits is added to adjust the acidity.

The fermentation broth obtained by the method for preparing the pokeweed fruit fermentation broth, wherein the fermentation flora of the pokeweed fruit fermentation broth is mainly characterized by eight strains of Penicillium sublaterium, Golubepiallescens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozyma guilliermondii, Candida intermedia, Lodderomyces elengiensis and Aspergillus sydowii.

In a second aspect, the present invention provides a fresh pokeweed fruit fermentation broth obtained according to the above-mentioned preparation method, comprising a mixed species of eight species of bacteria selected from the group consisting of Penicillium sublaterium, Golubevia pallesens, Aspergillus penicillioides, Aspergillus parvus, Meyerozyma guilliermondii, Candida intermedia, loddermyces elongisporus, and Aspergillus sywii, wherein the abundance of each species in the fermentation broth is increased by at least 100-fold or more compared to the fresh fruit before fermentation.

In a third aspect, the present invention provides a pokeweed fruit fermentation broth containing, as active ingredients, anthraquinones and glycosides thereof including compounds of formulae (a) to (F) and pokeweed polysaccharides consisting of arabinose (Ara), rhamnose (Rha), galacturonic acid (GalUA), mannose (Man), glucose (Glc) and galactose (Gal) in a molar ratio of 12: 6: 1: 20: 30: 36;

the fresh pokeweed fruit fermentation broth is prepared from fresh pokeweed fruit by taking mixed bacteria of eight bacteria including Penicillium sublateritium, Golubeva pallesens, Aspergillus penicillioides, Aspergillus parvus, Meyerozymea guilliermondii, Candida intermedia, Lodderomyces elongii and Aspergillus sydowii as fermentation strains.

In a fourth aspect, the present invention provides a fermentation broth of fresh pokeweed fruit, the broth being prepared from a mixture of eight species of pokeweed fruit, including Penicillium sublateritium, Golubeva pallesens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozyma guilliermondii, Candida intermedia, Loddermyces elongii and Aspergillus sydowii.

The fermentation process is a complex biochemical reaction process taking specific strains as cores, so certain environmental conditions are required. In order to further illustrate the present invention, the fermentation conditions of the method for preparing the pokeweed fruit fermentation liquid are characterized in that, in addition to adopting a proper fermentation strain: mature and fresh pokeberry is adopted, and the pokeberry fresh fruit cannot be mildewed or rotten; the first fermentation day is 30-60 days, and the fermentation temperature is 20-30 ℃; the second fermentation day is more than 30 days, the sugar degree, acidity and inorganic salt content of the fermentation liquor, and the temperature, humidity and dissolved oxygen of the environment can be properly adjusted to ensure that the fermentation of the strains characterized by 8 strains can be smoothly carried out, and the screened proper humidity is 70-80% of relative humidity and dissolved oxygen.

On the basis, however, the method is not limited to adding other new strains for promoting and ensuring the smooth proceeding of the directional fermentation, and one or a plurality of other strains are added, so that the fermentation results of dominant strains cannot be obviously different, and the method falls into the invention.

In addition, any one of Penicillium sublateritium, Golubeevia pallesens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozyma guilliermondii, Candida intermedia, Lodderomyces elongisporus and Aspergillus sydowii and/or a mixed strain of any combination of two or more of them is used as the fermentation broth prepared by the fermentation strain, and it also falls into the present invention.

In a fifth aspect, the present invention provides a use of the aforementioned fresh pokeberry fruit fermentation broth in the preparation of a medicament for treating a disease selected from cancer, aids, arteriosclerosis and skin diseases.

In a sixth aspect, the invention provides an application of the fermented pokeberry fruit liquid in preparing a medicament for treating cancer, which is characterized in that the cancer is selected from liver cancer, lung cancer, pancreatic cancer, cervical cancer, breast cancer, ovarian cancer, endometrial cancer and prostate cancer, and is preferably liver cancer.

Further, the medicine can be prepared into oral liquid preparations or solid preparations, and the solid preparations are selected from tablets, capsules and granules.

In a seventh aspect, the present invention provides a use of the fermented liquid of fresh pokeberry fruit in the preparation of non-therapeutic food, drink or health food.

In an eighth aspect, the present invention provides a non-therapeutic food or drink comprising the fermented liquid of fresh pokeweed fruit described above.

In a ninth aspect, the usage of the pokeberry fruit fermentation liquid of the invention: the pokeberry root fermentation liquor can be directly prepared into an oral liquid preparation; or extracting, purifying, drying, and making into extract, dried product or solid, and adding pharmaceutically acceptable excipient or adjuvant into the extract, dried product or solid to make into other medicinal preparations, health products, foods, etc.

Drawings

FIG. 1 comparison of species level heat maps of fresh fruit juice (PAR-1), fermentation mother liquor, and first and second fermentation broth (PAR-2) strains

FIG. 2 structural formula of small molecule component in pokeberry fermentation liquid

FIG. 3 the effect of fermentation broth of Phytolacca acinosa on the tumor weight of H22 hepatoma bearing mice

Advantageous effects

The fresh pokeberry fruit fermentation liquid prepared by the method has definite fermentation strain, controllable fermentation process, stable quality of the fermentation liquid, definite pharmacological active ingredients and obvious treatment effect on cancer.

Detailed Description

EXAMPLE 1 preparation of fermented Phytolacca acinosa fruit liquid

Preparation of fermentation mother liquor: crushing fresh and mature Phytolacca acinosa fruits, naturally fermenting, continuously stirring, adding ammonium phosphate accounting for 0.1-0.5% of the weight of the fresh fruits and crystallized white granulated sugar accounting for 16% of the weight of the fresh fruits, adjusting culture conditions, and performing aerobic fermentation to obtain fermentation mother liquor.

Preparing a first fermentation liquid: 200kg of fresh mature pokeberry is taken, cleaned, drained and dried in the shade until the six seven fruits become dry for later use. Putting the clean pokeberry fruit into a PVC plastic barrel, and adding fermentation mother liquor accounting for 2-3% of the fresh fruit mass. Adding white granulated sugar accounting for 15-25% of the weight of the fresh fruits for sugar degree adjustment, and adding citric acid accounting for 0.5-2% of the weight of the fresh fruits for acidity adjustment. Carrying out aerobic fermentation for 30-60 days at 20-30 ℃, and separating or squeezing to separate fermentation liquor to obtain the fermented liquid.

Preparing a second fermentation liquid: and placing the first fermentation liquid in a PVC plastic barrel, standing, settling, keeping out of the sun, storing, performing anaerobic fermentation, performing full fermentation for more than 30 days to obtain a second fermentation, and separating to obtain supernatant fermentation liquid, namely the pokeberry fruit fermentation liquid.

Example 2 identification of Phytolacca acinosa Zymobacter

The identification of the fermentation strain of the invention comprises the following steps:

sampling: respectively taking 3ml of each of the fresh fruit sample and the pokeberry fruit fermentation liquid sample in the example 1 by using a freezing tube, directly putting the samples into liquid nitrogen for quick freezing for 1h, then labeling the freezing tube with a sealing bag, subpackaging, and putting the samples into a refrigerator at the temperature of-80 ℃ for storage.

2. Extracting genome DNA, wherein the extraction step comprises:

1) subjecting the sample to homogenization and lysis: adding 0.25-0.5 g of sample and 0.6mL of Buffer SOL into 2mL of beads Tube, and carrying out vortex for 5-10 min at the highest speed on a vortex instrument; add 60. mu.L Buffer SDS to the sample and vortex for 15 s.

2) Further lysis of the bacteria: placing the sample into a 70 ℃ water bath kettle, keeping the temperature for 10min, and centrifuging for a short time to collect liquid drops on the tube wall.

3) Add 200. mu.L Buffer PS and 150. mu.L adsorbent Solution to the lysate, vortex and mix well for 20s, stand at room temperature for 5min, occasionally invert 2-3 times during this period, centrifuge for 5min at 13 kg.

4) Transferring the supernatant to a 2mL centrifuge tube, adding equal volume of Buffer GDP, and reversing and mixing to prepare a mixed solution. And (3) loading the DNA column into a collecting pipe, transferring half of the mixed solution in the centrifugal pipe to the DNA column, centrifuging for 30-60 s for 13kg, pouring out the effluent, transferring the remaining mixed solution to the DNA column, repeating the centrifuging operation, pouring out the effluent, and loading the DNA column into a collecting pipe.

5) Adding 400 mu L of Buffer GDP to the DNA column, centrifuging for 30-60 s at 13kg, pouring the effluent, and loading the DNA column into a collection pipe.

6) Then 600. mu.L of Buffer GDP was added to the DNA column, 13kg was centrifuged for 30 to 60 seconds, and the effluent was discarded and the DNA column was collected in a collection tube.

7) And 6, repeating the step.

8) The DNA column is placed in a 1.5mL centrifuge tube, 50 μ L of AE preheated to 70 ℃ Buffer is added to the center of the membrane of the column, the column is placed for 3-5 min, and 13kg is centrifuged for 1 min. The DNA binding column was discarded and the DNA was stored in a refrigerator at 2-8 ℃.

9) The primer sequence is as follows:

GATGAAGAACGYAGYRAATCCTCCGCTTATTGATATGC, PCR amplification of the target region is performed, and the amplified product is recovered and quantified.

3. 16S, ITS sequencing of DNA, library expansion, database search: library quantification and sequencing the second round of amplification products were purified using AMPure XP Beads, quantified using an ABI StepOnePelus Real-Time PCR System (Life Technologies, USA of origin), and sequenced on a Pooling machine according to the PE250 model of Hiseq 2500.

4. Sequencing results

According to the abundance value of the strains, the main fermentation strains in the fermentation liquor of the fresh pokeweed fruits after fermentation are finally determined to be eight strains of Penicillium sublateritium, Golubeva pallesens, Aspergillus penicillioides, Aspergillus parvul, Meyerozyma guilliermondii, Candida intermedia, Lodderomyces elengisporus and Aspergillus sydowii. Wherein Penicillium sublaterium is 1 species of Penicillium; golubeva pallesens, Meyerozyma guilliermondii, Candida intermedia, Lodderomyces elongisporus are yeasts; aspergillus penicilloides, Aspergillus parvus and Aspergillus sydowii are Aspergillus species. The heat map of strain level of fresh fruit liquid (marked as PAR-1) and fermentation broth (marked as PAR-2) is shown in figure 1, and the abundance of strain level of fresh fruit liquid (PAR-1) and fermentation broth (PAR-2) is shown in Table 1.

TABLE 1 seed-level abundance of fresh fruit juice (PAR-1) and fermentation broth (PAR-2) strains

EXAMPLE 3 preparation of fermented liquid of fresh Phytolacca acinosa fruit

Fermenting with 8 strains

200kg of fresh and mature Phytolacca acinosa fruits are cleaned, water is drained, the Phytolacca acinosa fruits are dried in the shade to be six-seven dry, the dried Phytolacca acinosa fruits are placed in a PVC plastic barrel, eight strains of Penicillium sublaterium, Golubepalascens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozymea guillieri, Candida intermedia, Lodderomyces elongii and Aspergillus sydowii are added, the sugar degree and the acidity of the fermentation liquor are adjusted according to the titer ratio of 3.37:3.31:2.32:2.16:1.52:1.45:1.11:1 in sequence, 2.5kg of white granulated sugar and 0.6kg of citric acid are added, the temperature in the daytime is 25-30 ℃, the temperature is 19-27 ℃, and after 30 days of fermentation at night, the first-time fermentation liquor is prepared by squeezing with gauze.

And placing the obtained first fermentation liquid in a clean PVC plastic barrel, sealing and avoiding light, carrying out anaerobic fermentation, standing, carrying out secondary fermentation for 30 days at the daytime temperature of 15-27 ℃ and the nighttime temperature of 8-20 ℃, fully settling, and removing precipitates to obtain a second fermentation liquid, namely the pokeberry fruit fermentation liquid fermented by 8 strains. The pokeberry fruit fermentation liquor can be directly prepared into an oral liquid preparation and can also be used as a raw material.

EXAMPLE 4 preparation of fermented Phytolacca acinosa fruit liquid

Adopting 8 strains and 2 strains for mixed fermentation

200kg of fresh and mature Phytolacca acinosa fruits are cleaned, drained, dried in the shade to six-seven dry, placed in a PVC plastic barrel, added with Penicillium sublateritium, Golubevillescens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozymea guillieri, Candida intermedia, Lodderomyces elosporus and Aspergillus sydowii, and added with Candida parapsilosis and Malassezia restica according to the titer ratio of 3.37:3.31:2.32:2.16:1.52:1.45:1.11:1:0.5:0.5 in sequence, fermented together with 10 strains, added with 2.5kg of white granulated sugar and 0.6kg of citric acid, fermented at the temperature of 25-30 ℃ for fermentation, and the fermentation broth is separated at the temperature of 30-19 ℃ for the first time, and the fermentation broth is fermented at the temperature of 25-19 ℃ for the first time.

And placing the obtained primary fermentation liquid in a clean PVC plastic barrel, sealing and avoiding light, carrying out anaerobic fermentation, standing, carrying out secondary fermentation for 60 days at the daytime temperature of 15-27 ℃ and the nighttime temperature of 8-20 ℃, fully settling, and removing precipitates to obtain secondary fermentation liquid, namely the pokeberry fruit fermentation liquid.

Example 5 isolation and characterization of active ingredients in fermented fresh Phytolacca acinosa fruit solution

In order to further explain the material composition of the target fermentation liquor, the invention analyzes and identifies the chemical micromolecule components and the polysaccharide macromolecule components of the fermentation liquor obtained by fermenting the pokeberry fruit by adopting 8 mixed strains.

1. Isolation and characterization of active ingredients of small molecule drugs

Taking the pokeberry fruit fermentation liquid prepared in the example 2, freeze-drying, adding a proper amount of ethanol to ensure that the alcohol concentration is 80-85%, standing and alcohol precipitating, repeating the steps once, combining the supernatant, carrying out reduced pressure rotary evaporation and concentration until no alcohol smell exists, adding mass spectrum methanol to dilute the solution to 10ul/ml, and carrying out Q-TOF-MS analysis. The main component analysis of the data is carried out by Marker View software, the data is collected in an anion mode, the compound is screened according to the accurate mass number and the isotope kurtosis ratio by Peak View2.0/Masterview1.0 software, then the cracking rule analysis is carried out by comparing a reference product and a reference product secondary spectrogram in a Natural products HR-MS/MS Spectral Library 1.0software, the component composition of the fermentation liquor is determined, and the result is shown in Table 2.

TABLE 2 MS2 of sample based on ESI-QTOF

The structural formula of each compound is obtained by database retrieval, and the compounds are identified as quinones and their glycosides A-F, and the results are shown in figure 2.

2. Analysis of macromolecular polysaccharide component of pokeberry fermentation liquor

Taking the pokeberry fruit fermentation liquid prepared in the example 2, freezing and drying, adding a small amount of water for redissolving, adding a proper amount of ethanol to ensure that the alcohol concentration is 80%, standing and precipitating with ethanol, and repeating the steps for three times to obtain total polysaccharide precipitate. Further adopting a Sevage method to remove protein, dialyzing with running water for 48h, dialyzing with pure water for 24h to remove small molecules, and freeze-drying to obtain the refined total polysaccharide. And (3) performing monosaccharide composition analysis by adopting complete acid hydrolysis, pre-column derivatization and a GC-MS method. The polysaccharide in the pokeweed fruit fermentation liquor mainly comprises arabinose (Ara), rhamnose (Rha), galacturonic acid (GalUA), mannose (Man), glucose (Glc) and galactose (Gal) according to the molar ratio of 12: 6: 1: 20: 30: 36, wherein the content of galactose and glucose is higher, and the content of mannose, rhamnose and galacturonic acid is lower.

Example 6 study of antitumor Effect of Phytolacca acinosa fruit fermentation broth on H22 tumor-bearing mice

Reagent testing: freeze-drying the pokeberry root fermentation liquor to obtain a freeze-dried product of the fermentation liquor, and re-dissolving a certain amount of pure water into suspension with low (0.75g/kg), medium (1.5g/kg) and high (3g/kg) doses according to the mass of the freeze-dried product for administration. Positive drugs: cyclophosphamide, administered at 0.07 g/kg.

Tumor strain: mouse H22 hepatoma cell line, purchased from Shanghai Rich-Duty Biotech, Inc. Inoculating H22 cell with good growth state into abdominal cavity of mouse, inoculating 0.2ml, continuously passaging in vivo for 3 times, collecting ascites of mouse with obvious abdominal expansion, adding appropriate amount of normal saline, and adjusting cell density to 1 × 10⁷One/ml, as an inoculum for H22 tumor-bearing mice.

Animal and model: SPF grade ICR mice, each half male and female, weight 17.3-30.5g, provided by the laboratory animal center of Heilongjiang university of traditional Chinese medicine, and the qualification certificate number: SCXK Black: 2013-004. The inoculation is carried out under the armpit of the mice, and 50 mice are randomly divided into a pokeweed fruit fermentation liquid low, medium and high dose group, a cyclophosphamide group and a model control group (equal volume of normal saline) according to sex and weight.

The administration method comprises the following steps: the body mass of the mice was recorded daily, once daily for 14 days starting on the third day after the inoculation of the mice, and after the last administration, subcutaneous tumor tissues were dissected and weighed.

The statistical method comprises the following steps: tumor inhibition rate (%) (1-administration tumor weight/model tumor weight) × 100%

Statistical processing is carried out by adopting SPSS16.0, and data are all adopted

Data between groups are shown to be compared by one-way anova.

The results are shown in Table 3.

TABLE 3 Effect of pokeberry fruit fermentation broth on the body weight of H22 tumor-bearing mice

Note: comparison with before administration^**P<0.01; comparison with the Cyclophosphamide group^▲P<0.05

As can be seen from table 3, there was no significant difference in body weight between groups before dosing (P > 0.05); after 14 days of administration, the body weight of each group is respectively compared with that before administration, the body weight of the cyclophosphamide group is obviously reduced (P is less than 0.01), and the pokeberry root fermentation broth has no obvious difference in each group; after 14 days of administration, the pokeberry fruit fermentation liquid groups have obvious difference (P <0.05) compared with the cyclophosphamide group in the pokeberry fruit fermentation liquid low-dose groups.

TABLE 4 Effect of pokeberry root fermentation broth on tumor weight of H22 tumor-bearing mice

Note: comparison with model group^*P<0.05，^**P<0.01

As can be seen from Table 4, after 14 days of administration, the tumor weights of the respective administration groups were significantly reduced as compared with the model group (^*P<0.05；^**P<0.01); compared with the model group, the tumor inhibition rates of the pokeberry root fermentation liquid in the high, medium and low dose groups are 47.45%, 32.66% and 42.88% respectively. The tumor volume of the mice in the administration group is obviously smaller than that of the mice in the model group, and the influence of the pokeberry root fermentation liquid on the tumor weight of the H22 liver cancer tumor-bearing mice is shown in figure 3.

Example 7 preparation of fermented liquid of fresh pokeberry fruit for oral liquid preparation

Directly filling the prepared pokeberry fruit fermentation liquid into a 50ml brown light-proof straight-mouth glass bottle in an oral liquid filling production line to prepare oral liquid, and storing the oral liquid in a light-proof shade place.

EXAMPLE 8 preparation of enteric hard Capsule formulation from fermented fresh Phytolacca acinosa fruit

Adding 400g of medicinal anhydrous calcium hydrogen phosphate powder into 5L of fresh pokeberry fruit fermentation liquid, uniformly mixing, freeze-drying to obtain freeze-dried powder, adding 400g of anhydrous calcium hydrogen phosphate powder, uniformly mixing, sieving with a 80-mesh sieve, and filling into enteric hard capsules to obtain the product. The preparation can properly reduce the stimulation effect on the stomach caused by the fermentation of the pokeberry fruit fermentation liquid with higher content of the fermented ethanol and the like.

EXAMPLE 9 preparation of enteric Soft Capsule preparation from fermented liquid of fresh Phytolacca acinosa fruit

Adding 400g of medicinal anhydrous calcium hydrogen phosphate powder into 5L of fresh pokeberry fruit fermentation liquid, uniformly mixing, freeze-drying to obtain freeze-dried powder, adding a proper amount of medicinal auxiliary materials such as emulsifier, grease and the like, preparing a semisolid filling material, and filling the semisolid filling material into enteric soft capsules to obtain the phytolacca acinosa capsule. The preparation can properly reduce the stimulation effect on the stomach caused by the fermentation of the pokeberry fruit fermentation liquid with higher content of the fermented ethanol and the like.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the raw material of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a pokeberry fresh fruit fermentation liquid is characterized by comprising the following steps:

(1) preparation of fermentation mother liquor: crushing and fermenting mature and fresh pokeberry fruit, continuously stirring, optionally adding a proper amount of inorganic salt and white granulated sugar to adjust culture conditions, and fully fermenting to obtain fermentation mother liquor;

2. The preparation method according to claim 1, characterized in that ammonium phosphate accounting for 0.1-0.5% of the weight of fresh fruits and crystallized white granulated sugar accounting for 15% -25% of the weight of fresh fruits are added in the preparation of the fermentation mother liquor to adjust the culture conditions; in the preparation of the first fermentation liquor, white granulated sugar which accounts for 15 to 25 percent of the weight of the fresh fruits is added to adjust the sugar degree, and citric acid which accounts for 0.5 to 2.0 percent of the weight of the fresh fruits is added to adjust the acidity.

3. A fermented liquid of fresh pokeweed fruit obtained according to the method of claim 1 or 2, characterized in that the fermented liquid of pokeweed fruit contains a mixed species of eight species of bacteria selected from the group consisting of Penicillium sublateritium, Golubevia pallesens, Aspergillus penicilioides, Aspergillus parvulus, Meyerozyma guilliermondii, Candida intermedia, loddermyces elongii and Aspergillus sydowii, wherein the abundance of each species of bacteria in the fermented liquid is increased by at least 100-fold or more compared to the fresh fruit before fermentation.

4. A pokeweed fruit fermentation liquid is characterized in that the fermentation liquid contains anthraquinone and glycosides thereof and pokeweed polysaccharide as active ingredients, wherein the anthraquinone and glycosides thereof comprise compounds of formulas (A) to (F), and the pokeweed polysaccharide consists of arabinose (Ara), rhamnose (Rha), galacturonic acid (GalUA), mannose (Man), glucose (Glc) and galactose (Gal) according to the molar ratio of 12: 6: 1: 20: 30: 36;

5. a fermentation broth according to claim 4, characterized in that it is prepared from fresh Phytolacca acinosa fruit as a fermenting species from a mixture of eight species comprising Penicillium sublateritium, Golubeva pallesens, Aspergillus penicillioides, Aspergillus parvul, Meyerozymea guilliermondii, Candida intermedia, Lodderomyces elongisporus and Aspergillus sydowii.

6. A fermentation broth of fresh pokeweed fruit, wherein the fermentation broth is prepared from the fermentation of fresh pokeweed fruit with a mixture of eight bacteria comprising Penicillium sublateritium, Golubeva pallesens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozymea guilliermondii, Candida intermedia, Loddeplomyces elengiensis and Aspergillus sydowii.

7. Use of a mixture of bacteria comprising eight bacteria of the group consisting of Penicillium sublateritium, Golubeva pallesens, Aspergillus penicillioides, Aspergillus parvulus, Meyerozymea guilliermondii, Candida intermedia, Lodderomyces elongii and Aspergillus sydowii as fermenting species for the preparation of a fermented broth of fresh Phytolacca acinosa.

8. Use of a fermented pokeweed fruit broth according to any one of claims 3-6 for the preparation of a medicament for the treatment of a disease selected from the group consisting of cancer, AIDS, arteriosclerosis and skin disorders.

9. Use of a pokeweed fruit broth according to any one of claims 3-6 for the preparation of a medicament for the treatment of cancer selected from liver cancer, lung cancer, pancreatic cancer, cervical cancer, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, preferably liver cancer.

10. Use of a fermented pokeweed fruit solution according to any one of claims 3-6 in the preparation of a non-therapeutic food or drink or health food.