CN114807270B - Burdock root polysaccharide prepared by fermenting rhizopus nigricans, and production process and application thereof - Google Patents
Burdock root polysaccharide prepared by fermenting rhizopus nigricans, and production process and application thereof Download PDFInfo
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- CN114807270B CN114807270B CN202210209313.2A CN202210209313A CN114807270B CN 114807270 B CN114807270 B CN 114807270B CN 202210209313 A CN202210209313 A CN 202210209313A CN 114807270 B CN114807270 B CN 114807270B
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- burdock root
- rhizopus nigricans
- burdock
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention relates to burdock root polysaccharide prepared by fermenting rhizopus nigricans, and a production process and application thereof. The weight average molecular weight of the burdock root polysaccharide is 5 multiplied by 10 4 ～1×10 6 Da, the mol content of galactose and galacturonic acid in the burdock root polysaccharide is more than 50 percent. The invention also provides a production process for preparing burdock root polysaccharide by fermenting rhizopus nigricans. The invention utilizes the vigorous fecundity of the rhizopus nigricans, the rich biological enzyme system and the stronger biological conversion capability to ferment the burdock root and the burdock root water extraction residues and then separate and purify the burdock root polysaccharide RB3 component, and the research results of in vitro tumor cell experiments and in vivo tumor-bearing mouse experiments show that the burdock root polysaccharide RB3 has more obvious effect of regulating immunity and resisting tumors, can be applied to the fields of health care foods and medical medicaments, and provides scientific support for the development and application of rhizopus nigricans and burdock resources.
Description
Technical Field
The invention relates to burdock root polysaccharide prepared by fermenting rhizopus nigricans, and a production process and application thereof, belonging to the technical field of medicines and health-care foods.
Background
Cancer is the second most common lethal factor worldwide. According to the world cancer report data of the world health organization IARC, the number of new cancer in China in 2020 is 457 ten thousand, which accounts for 23.7% of the total number of new cancer and 30% of cancer death cases. Among the major cancer types in China, the most common cancer types are lung cancer, breast cancer, gastric cancer, colorectal cancer, liver cancer, esophageal cancer and the like. In addition, malignancy morbidity remains at about 3.9% increase per year, and mortality remains at 2.5% increase per year. Chinese cancer treatment regimens are more limited than those in developed countries such as the United states, and on the one hand, traditional chemotherapeutic drug treatment remains an important tumor treatment modality. However, the traditional chemotherapy has poor selectivity and great toxic and side effects, and can strongly stimulate the digestive system, easily cause damage to liver and kidney functions, and even destroy the hematopoietic function of bone marrow and the immune function of human body. The biological polysaccharide has the advantages of easy acquisition, safety, effectiveness, small toxic and side effects, and can be combined with chemotherapeutics, so that the biological polysaccharide is always a hotspot for innovative research and development of antitumor drugs.
Burdock (Arctium Lappa l.) is a food and drug homologous biennial plant resource of the asteraceae family, and is widely planted in countries and regions such as China, east asia, europe, south america, etc. In addition to their edible value as vegetables, burdock roots and extracts thereof also show various potential in medicine, including anti-inflammation and antioxidant, gastrointestinal function protection, regulation of glycolipid metabolism, improvement of intestinal flora, and immunoregulatory antitumor activity. Wherein burdock polysaccharide is the main active ingredient of burdock root, and is fructooligosaccharide with the molecular weight of about 2100 Da. At present, the primary processed product of burdock root and the extract thereof form industrialized scale, and the health care and medical value of burdock is more and more concerned and welcome by people.
Rhizopus nigricans (Rhizopus nigricans) is a filamentous fungus belonging to the genus rhizopus of the phylum zygomycotina, which is a commonly used microorganism strain in the fermentation industry because of its biocatalysis and bioconversion ability, and has abundant biological enzymes. Research on rhizopus nigricans metabolites has found that extracellular polysaccharide thereof is prominent in enhancing immunity, inducing apoptosis of tumor, etc. since 2013.
Chinese patent document CN103859578A discloses a method for extracting burdock crude polysaccharide and application thereof in tobacco. Cleaning burdock root, slicing, adding water, crushing in a homogenizer, ultrasonic extracting for 1-2 hr, extracting with water at 80-120 deg.c for 1-2 hr, filtering, concentrating the filtrate, adding alcohol to the concentrated solution to reach alcohol concentration over 80%, mixing, standing at 4 deg.c for over 8 hr, centrifuging, filtering and depositing to obtain burdock root polysaccharide. Although the patent discloses a preparation method of burdock polysaccharide, the polysaccharide is extracted only by adopting a physical method, and the obtained burdock polysaccharide has complex components and limited functions.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides burdock root polysaccharide prepared by fermenting rhizopus nigricans, and a production process and application thereof.
The technical scheme of the invention is as follows:
a burdock root polysaccharide prepared by fermenting Rhizopus niveus, wherein the weight average molecular weight of burdock root polysaccharide is 5×10 4 ～1×10 6 Da, the mol content of galactose and galacturonic acid in the burdock root polysaccharide is more than 50 percent.
The invention also provides a production process for preparing burdock root polysaccharide by fermenting rhizopus nigricans, which comprises the following steps:
(1) Inoculating rhizopus nigricans into a potato dextrose agar medium, culturing at 26-30 ℃ for 4-6 days until white hyphae are fully paved and black spores grow, selecting rhizopus nigricans hypha blocks, transferring the rhizopus nigricans hypha blocks into the potato dextrose agar medium, continuously culturing for 2-4 days until a large number of black spores appear, flushing rhizopus nigricans bodies with sterile water, collecting spores, and obtaining rhizopus nigricans spore suspension;
(2) Selecting fresh burdock roots or burdock root water insoluble matters or burdock root water insoluble matter dry powder, adding the burdock roots or burdock root water insoluble matters dry powder into a shake flask of a potato dextrose liquid culture medium, inoculating a rhizopus nigricans spore suspension according to the volume ratio of 0.5-5% after wet sterilization, and then placing the burdock roots or burdock root water insoluble matters dry powder into the shake flask at the temperature of 25-30 ℃ and the shaking rotation speed of 120-200rpm for fermentation culture for 7-15 days to obtain burdock root fermentation liquor;
(3) Filtering and clarifying the burdock root fermentation broth, concentrating the supernatant, precipitating with ethanol, redissolving, deproteinizing, decolorizing, dialyzing, performing anion exchange DEAE fast flow chromatography to obtain crude burdock root polysaccharide, and performing molecular sieve exclusion agarose gel G100 chromatography to obtain purified burdock root polysaccharide.
According to a preferred embodiment of the present invention, in the step (1), the size of the rhizopus nigricans mycelium pellet is 1×1cm.
According to a preferred embodiment of the present invention, in step (1), the density of the Rhizopus nigricans spores in the suspension of Rhizopus nigricans spores is 1X 10 5 ～10 8 And each mL.
According to a preferred embodiment of the invention, in step (2), the suspension of rhizopus nigricans spores is dosed at 2% by volume of potato dextrose broth.
According to the invention, in the step (2), the fresh burdock roots are subjected to cleaning, peeling and chopping treatment, and the weight-volume ratio of the fresh burdock roots to the potato dextrose liquid medium is (1-4): 10, the unit is: g/mL.
According to the invention, in the step (2), the burdock root water insoluble matter is obtained by treating fresh burdock roots by a hot water leaching method, and the weight volume ratio of the burdock root water insoluble matter to a potato dextrose liquid medium is (1-4): 10, the unit is: g/mL.
According to the invention, in the step (2), the burdock root water-insoluble matter dry powder is obtained by drying and crushing burdock root water-insoluble matter at 50-70 ℃, and the weight-volume ratio of the burdock root water-insoluble matter dry powder to potato glucose liquid culture medium is (1-5): 100, the unit is: g/mL.
According to a preferred embodiment of the present invention, in the step (3), the specific method of anion exchange DEAE fast flow chromatography is as follows: and eluting 0.02M tris-hydrochloric acid, 0.02M tris-hydrochloric acid+0.1M sodium chloride solution, 0.02M tris-hydrochloric acid+0.2M sodium chloride solution, 0.02M tris-hydrochloric acid+0.3M sodium chloride solution, 0.02M tris-hydrochloric acid+0.4M sodium chloride solution and 0.02M tris-hydrochloric acid+0.5M sodium chloride solution in sequence from small to large according to the concentration of sodium chloride, and collecting eluent of 0.02M tris-hydrochloric acid+0.3M sodium chloride solution to obtain burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans.
The application of the burdock root polysaccharide in preparing an immunomodulatory anti-tumor drug or food.
According to the invention, the burdock root polysaccharide is combined with 5-fluorouracil, the dosage of the burdock root polysaccharide is 50-150 mg/kg/d, and the dosage of the 5-fluorouracil is 15-25 mg/kg/d.
The invention has the technical characteristics and beneficial effects that:
1. the invention utilizes the vigorous fecundity of the black rhizopus, the rich biological enzyme system and the stronger biological conversion capability, obtains the burdock root polysaccharide RB3 component by separating and purifying the burdock root and the burdock root water extraction residues after fermentation, has excellent immunoregulation anti-tumor activity for the burdock root polysaccharide RB3, has higher tumor inhibition rate for tumors, has NO cytotoxin effect on normal cells, can also enhance the phagocytic activity of macrophages, and induces the secretion of cytokines such as NO, IL-6, TNF-alpha and the like of the macrophages. The invention not only effectively utilizes the biological resources such as burdock, rhizopus nigricans and the like, but also provides scientific support for the development and application of burdock root and rhizopus nigricans in the field of medicine.
2. The invention provides a production process for preparing burdock root polysaccharide by fermenting rhizopus nigricans, which obtains the weight average molecular weight of 5 multiplied by 10 4 ～1×10 6 The composition of the Da burdock root polysaccharide RB3 and the burdock root polysaccharide RB3 mainly comprises galactose and galacturonic acid, the molar content of galactose and galacturonic acid in the burdock root polysaccharide is more than 50%, and the research results of in vitro tumor cell experiments and in vivo tumor-bearing mouse experiments show that the burdock root polysaccharide RB3 has a remarkable immunity regulating and anti-tumor effect, can be applied to the fields of health-care foods and medical medicaments, and provides scientific support for development and application of rhizopus nigricans and burdock resources.
Description of the drawings:
FIG. 1 is a agarose gel DEAE fast flow chart of burdock root polysaccharide (left) extracted from a burdock root water insoluble matter without rhizopus nigricans fermentation and of burdock root polysaccharide RB3 (right) prepared by rhizopus nigricans fermentation according to the present invention.
FIG. 2 is an HPLC chart of the invention for preparing burdock root polysaccharide RB3 by fermentation of Rhizopus nigricans.
FIG. 3 is an infrared spectrum of the burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans.
FIG. 4 is a diagram showing the analysis of polysaccharide methylated sugar alcohol acetyl ester (PMAA) of burdock root polysaccharide RB3 prepared by fermentation of Rhizopus niveus in the present invention.
FIG. 5 is a nuclear magnetic pattern of burdock root polysaccharide RB3 prepared by fermentation of Rhizopus niveus.
In the figure: a is a hydrogen spectrogram, B is a carbon spectrogram, C is an HMBC spectrogram, D is an HSQC spectrogram, and E is a NOESY spectrogram. F is COSY pattern.
FIG. 6 is a graph showing the result of cytotoxicity test of burdock root polysaccharide RB3 prepared by fermentation of Rhizopus nigricans on human normal liver cells LO 2.
FIG. 7 is a graph showing the experimental results of the influence of the burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans on the proliferation of the mouse macrophage RAW 264.7.
FIG. 8 is a graph showing the effect of the fermentation of Rhizopus nigricans to prepare burdock root polysaccharide RB3 on cytokine secretion of mouse macrophage RAW 264.7.
Detailed Description
The invention will be further illustrated below in connection with examples which will enable those skilled in the art to more effectively understand the invention and are not intended to be limiting in any way.
The potato dextrose medium formulation used in the examples below: 200g of potato, 15-20 g of sucrose, 20-30 g of agar and 1000mL of distilled water.
Example 1
A production process for preparing burdock root polysaccharide by fermenting rhizopus nigricans comprises the following steps:
(1) Inoculating Rhizopus nigricans strain into a plate containing potato glucose agar medium (PDA), culturing at 28deg.C for 5 days until white mycelia are spread on the plate and black spores grow, inoculating 1×1cm Rhizopus nigricans mycelia into a culture bottle containing potato glucose agar medium, and culturing for 3 days until a large amount of black spores appearWashing the rhizopus nigricans in the flask with sterile water, collecting spores, and counting with counting plate to obtain spores with density of 1×10 5 ～10 8 individual/mL rhizopus nigricans spore suspension;
(2) Selecting 200g of fresh burdock root after cleaning, peeling and cutting, adding into a 3L shake flask of 1000mL potato glucose liquid medium, sterilizing by wet method, and inoculating spore density of 1×10 according to 2% volume ratio 7 The rhizopus nigricans spore suspension with the volume of one mL is placed in a condition that the temperature is 28 ℃ and the shaking speed is 150rpm for fermentation culture for 12 days, so as to obtain burdock root fermentation liquor;
(3) Filtering and clarifying the burdock root fermentation broth, concentrating the collected supernatant to 1/5 of the original volume, adding 95% ethanol, measuring with an alcohol meter until the final concentration of the ethanol is 75%, and standing at 4 ℃ overnight; adding distilled water with the weight-volume ratio of 5-10 times of the collected alcohol precipitation substances, stirring and dissolving, centrifuging to remove precipitate, and mixing the crude polysaccharide solution with deproteinizing agent (chloroform: n-butanol=3:1) according to the weight ratio of 4:1 into a conical flask, stirring for 30min, pouring into a separating funnel, standing for 30min to separate an organic phase from a water phase, collecting the water phase, and repeating the deproteinizing step until no white substance is separated out between the organic phase and the water phase when standing; decolorizing the deproteinized crude polysaccharide solution by using macroporous resin D301 or D301R chromatographic column until the crude polysaccharide solution is light yellow or colorless; concentrating the decolored crude polysaccharide solution, filling the concentrated crude polysaccharide solution into a dialysis bag with the molecular weight of 5000Da, placing the dialysis bag into distilled water, dialyzing for multiple times, and freeze-drying the solution to obtain a crude polysaccharide dry product;
dissolving crude polysaccharide dry product in distilled water, passing through DEAE fast flow chromatographic column, eluting with 0.02M tris-hydrochloric acid, 0.02M tris-hydrochloric acid+0.1M sodium chloride solution, 0.02M tris-hydrochloric acid+0.2M sodium chloride solution, 0.02M tris-hydrochloric acid+0.3M sodium chloride solution, 0.02M tris-hydrochloric acid+0.4M sodium chloride solution and 0.02M tris-hydrochloric acid+0.5M sodium chloride solution, collecting eluate of 0.02M tris-hydrochloric acid+0.3M sodium chloride solution from small to large according to sodium chloride concentration, collecting single-peak sugar solution, and purifying to obtain crude burdock root polysaccharide RB3.
The polysaccharide is extracted from the burdock root water insoluble matter which is not fermented by rhizopus nigricans by a hot water extraction method, and then the burdock root polysaccharide RB3 prepared by rhizopus nigricans fermentation and the polysaccharide of the burdock root water insoluble matter which is not fermented by rhizopus nigricans in this example are subjected to a DEAE fast flow chromatographic column, and the result is shown in figure 1.
As can be seen from fig. 1, the polysaccharide component extracted from the water insoluble matter of burdock root without rhizopus nigricans fermentation is mainly neutral polysaccharide, and the polysaccharide component extracted from the water insoluble matter of burdock root by rhizopus nigricans fermentation in this embodiment is mainly acidic polysaccharide.
The burdock root polysaccharide RB3 prepared by using the rhizopus nigricans fermentation in this example is prepared into a 5mg/ml solution, the solution is centrifuged at 12000rpm for 10min, the supernatant is filtered by a microporous filter membrane of 0.22 μm, then the sample is transferred into a sample injection small bottle of 1.8ml, high Performance Liquid Chromatography (HPLC) is performed,
the conditions are as follows: chromatographic column: BRT105-104-102 series gel column (8X 300 mm); mobile phase: 0.05M NaCl solution; flow rate: 0.6ml/min, column temperature: 40 ℃; sample injection amount: 20 μl; a detector: the results of the differential detector RI-502 are shown in FIG. 2.
As can be seen from fig. 2, the weight average molecular weight of the burdock root polysaccharide RB3 prepared by fermentation with rhizopus nigrum in this example is 140.0kDa, and the sum of the molar content ratios of galactose and galacturonic acid is 63.3%, and the ratio of galactose (Gal), galacturonic acid (GalA), rhamnose (Rha), arabinose (Ara), glucuronic acid (GlcA) and glucose (Glc) is 31.3:32.2:15.1:14.9:5.7:0.8 molar ratio.
The infrared spectrogram of the burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans in the embodiment is shown in fig. 3.
As can be seen from FIG. 3, RB3 is between 4000 and 400cm -1 There are a large number of characteristic absorption peaks in the range. At 3417.88cm -1 The broad absorption peak in the vicinity is the stretching vibration of the hydroxyl group OH. At 2928.37cm -1 The peak at which corresponds to the stretching vibration of the CH group. 1612.87cm -1 And 1415.02cm -1 The presence of carbonyl CO suggests the possible presence of uronic acid. 1200-1000cm -1 Absorption peaks in the range can be attributed to C-O-C and C-O-H stretching vibrations. In particular at 1070.28cm -1 ,1042.66cm -1 And 896.41cm -1 Characteristic peaks at this point indicate the presence of beta-pyranose-beta-arabinogalactan and/or beta-galactan. At the same time at 823.80cm -1 And 783.99cm -1 The weak absorption peak at this point indicates the presence of sugar units of the alpha configuration.
The analysis of polysaccharide methylated sugar alcohol acetyl ester (PMAA) of Arctium lappa polysaccharide RB3 prepared by fermentation of Rhizopus niveus in this example is shown in FIG. 4.
As can be seen from FIG. 4, the type of linkage of each glycosidic bond in RB3, wherein the ratio of Gal1-4 is the sum of the reductive yield of galactal GalA1-4 and Gal1-4 itself.
The nuclear magnetic spectrum of burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans is shown in fig. 5.
As can be seen from FIG. 5, the signals in the hydrogen spectrum A are mainly concentrated between 3.0 and 5.5 ppm. Delta 3.2-4.0ppm is sugar ring proton signal, and main end group proton peaks delta 5.20, 5.17, 5.07, 5.03, 5.01, 4.96, 4.62, 4.56, 4.47, 4.44, 4.43 and 4.40 are intensively distributed in the region of 4.3-5.5 ppm. Carbon spectrum B analysis is carried out in 13 C NMR(201MHz,D 2 O): the nuclear magnetic carbon spectrum signal is mainly concentrated between 60 and 120 ppm. By observing the carbon spectrum, it can be seen that the main anomeric carbon signal peaks δ 110.59, 104.51 are mainly between δ93 and 105. Whereas the main signal peaks of delta 110.62, 108.88, 108.80, 108.77, 107.12, 105.80, 105.80, 104.48, 103.84, 103.13, 99.53, 98.77 are distributed in the 60-85 ppm region. 174.8ppm is the carbonyl peak of uronic acid and 18.23ppm is the C6 signal peak of rhamnose.
Through the HSQC spectrum D, an isocephalic carbon signal delta 110.56 can be observed, the corresponding isocephalic hydrogen signal in the HSQC spectrum is delta 5.17, and the signal of H1-2 combined with the COSY spectrum F is 5.17/4.13; the signal of H2-3 is 4.13/3.87; the signal of H3-4 is 3.87/3.99; the signal of H4-5a is 3.99/3.76; we can infer that H1, H2, H3, H4, H5a are δ5.17, 4.13, 3.87, 3.99, 3.76, respectively. Corresponding C1-C5 are 110.62, 82.62, 77.97, 85.22, 62.64; thus, this signal is attributed to the glycosidic bond α -L-Araf- (1. Fwdarw. The other anomeric carbon signal is δ104.48, the corresponding anomeric hydrogen signal in HSQC pattern D is δ4.4, the signal of H1-2 is 4.4/3.28 by COSY pattern F, the signal of H1-3 is 3.28/3.46, the signal of H3-4 is 3.46/3.21, the signal of H4-5a is 3.21/3.60, we can infer that H1, H2, H3, H4, H5a are δ4.4, 3.28, 3.46, 3.21, 3.6. The corresponding C1-C5 is 104.48, 74.66, 76.62, 83.4, 77.36, 62.3, respectively, and thus, this signal is attributed to the glycosidic bond β -D-p- (1. Fwdarw. Combining HMBC and NOESY, proceeding attributing all glycosidic bond signals according to a similar law,
RB3 backbone analysis: in NOESY pattern E, the → 2, 4) -alpha-L-Rha- (1 → anomeric hydrogen and the → 4) -alpha-D-GalAp- (1 → H4 have correlation peaks, indicating the presence of → 2, 4) -alpha-L-Rha- (1 → 4) -alpha-D-GalAp- (1 → N. Glycosidic bond → 4) -alpha-D-GalAp- (1 → anomeric hydrogen has related signal peak with H4 of itself; indicating the existence of a linkage mode of → 4) -alpha-D-GalAp- (1 → linkage mode. Glycosidic bond → 4) -alpha-D-GalAp- (1 → isocephalic hydrogen and → 4) -beta-D-Galp- (1 → H4 have relevant signal peaks; indicating the existence of a → 4) -alpha-D-GalAp- (1 → 4) -beta-D-Galp- (1 → linkage mode. 4) -beta-D-Galp- (1- & gt, the anomeric hydrogen and H4 of the beta-D-Galp have related signal peaks; indicating the existence of the linkage mode of → 4) -beta-D-Galp- (1 → linkage mode. 4) -beta-D-Galp- (1- > isocephalic hydrogen and 3, 6) -beta-D-Galp- (1- > H3 have relevant signal peaks; indicating the existence of the linkage mode of → 4) -beta-D-Galp- (1 → 3, 6) -beta-D-Galp- (1 → linkage mode. The → 3, 6) -beta-D-Galp- (1 → isocephalic hydrogen and → 2, 4) -alpha-L-Rha- (1 → H4 have relevant signal peaks; indicating the existence of the linkage mode of → 3, 6) -beta-D-Galp- (1 → 2, 4) -alpha-L-Rha- (1 → link.
RB3 branched analysis: in the HMBC spectrum C, the nuclear magnetism one-dimensional two-dimensional spectrum is combined to attribute the glycosidic bond signal of the RB3 polysaccharide; the glycosidic bond α -L-Araf- (1- > isohead hydrogen has a signal peak associated with the C5 of →5) - α -L-Araf- (1- > indicating the presence of the alpha-L-Araf- (1- > 5) - α -L-Araf- (1- > linkage means the glycosidic bond →5) - α -L-Araf- (1- > isohead hydrogen has a signal peak associated with the C5 of itself, indicating the presence of the →5) - α -L-Araf- (1- > 5) - α -L-Araf- (1- > linkage means the glycosidic bond →5) - α -L-Araf- (1- > isohead hydrogen has a signal peak associated with the C5 of →3,5- α -L-Araf- (1- > C5, indicating the presence of the beta-5) - α -L-Araf- (1- > 3, 5) - α -Araf- (1- > linkage means the isohead hydrogen of the glycosidic bond α -L-Araf- (1- > 3- > 5) - α -Araf- (1- > linkage means the 3, 5) - α -Araf- (1- > 3, -5) - α -Araf- (1- > linkage means the 3, -L-Araf- (1- > 3, -5- > and the signal peak associated with the C5 of the →3-Araf- (1- > linkage means of Gal-L-Araf- (1-,5-, indicating the presence of beta-D-Galp- (1- & gt 6) -beta-D-Galp- (1- & gt 6-C6 having a correlation peak with its 3, 6) -beta-D-Galp- (1- & gt, indicating the presence of 6) -beta-D-Galp- (1- & gt 3, 6) -beta-D-Galp- (1- & gt beta-D-Glcp- (1- & gt carbon having a correlation peak with 4) -beta-D-Glcp- (1- & gt H4 indicating the presence of beta-D-Glcp- (1- & gt 4) -beta-D-Glcp- (1- & gt 4) having a correlation peak with its own H4.
To sum up: the burdock root polysaccharide RB3 mainly comprises monosaccharides, wherein the molar ratio of rhamnose to arabinose to galactose to galacturonic acid is 1:1:2:2, and other glycosidic bond contents are too low to be ignored as a main reference. The main chain of the polysaccharide is mainly formed by → 4) -alpha-L-Rha- (1 → 4) -alpha-D-GalAp- (1 → 4) -beta-D-Galp- (1 → and the branched chains are respectively connected to the main chain through → 2, 4) -alpha-L-Rha- (1 → and → 3, 6) -beta-D-Galp- (1 → O-2, O-6.
Example 2
A production process for preparing burdock root polysaccharide by fermenting rhizopus nigricans comprises the following steps:
(1) Inoculating Rhizopus nigricans strain into a plate containing potato glucose culture medium (PDA), culturing at 28deg.C for 6 days until white mycelia are spread on the plate and black spores grow, inoculating 1×1cm Rhizopus nigricans mycelia block into a culture bottle containing potato glucose agar culture medium, culturing for 4 days until a large number of black spores appear, washing Rhizopus nigricans body in the culture bottle with sterile water, collecting spores, and counting with counting plate to obtain spores with density of 1×10 5 individual/mL rhizopus nigricans spore suspension;
(2) Selecting fresh burdock root, treating with hot water extraction method to obtain burdock root water insoluble matter, adding 250g burdock root water insoluble matter into 3L triangular flask of 1000mL potato glucose liquid culture medium, and performing wet processAfter sterilization, the spore density is 1 multiplied by 10 according to the volume ratio of 5 percent V/V 5 The rhizopus nigricans spore suspension with the volume of one mL is placed in a condition that the temperature is 28 ℃ and the shaking speed is 150rpm for 15 days of fermentation culture, and burdock root fermentation liquor is obtained; (3) Filtering and clarifying the burdock root fermentation broth, concentrating the supernatant to 1/4 of the original volume, adding 95% ethanol, measuring with an alcohol meter until the final concentration of the ethanol is 75%, and standing at 4 ℃ overnight; adding distilled water with the weight-volume ratio of 5-10 times of the collected alcohol precipitation substances, stirring and dissolving, centrifuging, and removing precipitate to obtain a tan crude polysaccharide solution; the crude polysaccharide aqueous solution was combined with deproteinizing agent (chloroform: n-butanol=3:1) according to 3:1 into a conical flask, stirring for 30min, pouring into a separating funnel, standing for 30min to separate an organic phase from a water phase, collecting the water phase, and repeating the deproteinizing step until no white substance is separated out between the organic phase and the water phase when standing; decolorizing the deproteinized crude polysaccharide solution by using macroporous resin D301 or D301R chromatographic column until the crude polysaccharide solution is light yellow or colorless; concentrating the decolored crude polysaccharide solution, filling the concentrated crude polysaccharide solution into a dialysis bag with the molecular weight of 5000Da, placing the dialysis bag into distilled water, dialyzing for multiple times, and freeze-drying the solution to obtain a crude polysaccharide dry product;
dissolving crude polysaccharide in distilled water, passing through DEAE fast flow chromatographic column, eluting with 0.02M tris-hydrochloric acid, 0.02M tris-hydrochloric acid+0.2M sodium chloride solution and 0.02M tris-hydrochloric acid+0.4M sodium chloride solution sequentially from small to large according to sodium chloride concentration, collecting eluate of 0.02M tris-hydrochloric acid+0.4M sodium chloride solution to obtain crude burdock root polysaccharide, and subjecting to molecular sieve exclusion agarose gel G100 chromatography to obtain purified burdock root polysaccharide RB3.
Wherein, the process of obtaining the burdock root water insoluble matter after the hot water leaching method treatment is specifically as follows: selecting fresh burdock root, inactivating enzyme in boiling water bath for 5-10min, peeling burdock root, slicing, adding deionized water at a mass ratio of 1:10, setting the leaching condition at 80deg.C for 60min, leaching for 3 times, mixing the leaching solutions, filtering and suction filtering, centrifuging the filtrate, and collecting precipitate to obtain burdock root water insoluble substance.
The weight average molecular weight of the burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans is 149.5kDa, the sum of the molar content ratio of galactose and galacturonic acid is 50.5%, and the burdock root polysaccharide RB3 mainly consists of galactose, galacturonic acid, rhamnose, arabinose, glucuronic acid, glucose, mannose, xylose and fructose in a molar ratio of 26.7:23.8:17.7:16.3:3.9:1.5:6.0:2.4:1.7.
Example 3
A production process for preparing burdock root polysaccharide by fermenting rhizopus nigricans comprises the following steps:
(1) Inoculating Rhizopus nigricans strain into a plate containing potato glucose culture medium (PDA), culturing at 28deg.C for 4 days until white mycelia are spread on the plate and black spores grow, inoculating 1×1cm Rhizopus nigricans mycelia block into a culture bottle containing potato glucose agar culture medium, culturing for 2 days until a large amount of black spores appear, washing Rhizopus nigricans body in the culture bottle with sterile water, collecting spores, and counting with counting plate to obtain spores with density of 1×10 8 individual/mL rhizopus nigricans spore suspension;
(2) Selecting fresh burdock root, treating by hot water leaching to obtain burdock root water insoluble matter, drying the burdock root water insoluble matter at 60 ℃ and pulverizing to obtain burdock root water insoluble matter dry powder, adding 50g burdock root water insoluble matter dry powder into a 3L triangular flask of 1000mL potato glucose liquid medium, sterilizing by wet method, and inoculating spores with the density of 1X 10 according to the volume ratio of 3%V/V 8 The rhizopus nigricans spore suspension with the volume of one mL is placed in a condition that the temperature is 28 ℃ and the shaking speed is 150rpm for 7 days of fermentation culture, so as to obtain burdock root fermentation liquor;
(3) Filtering and clarifying the burdock root fermentation broth, concentrating the supernatant to 1/4 of the original volume, adding 95% ethanol, measuring with an alcohol meter until the final concentration of the ethanol is 75%, and standing at 4 ℃ overnight; adding distilled water with the weight-volume ratio of 5-10 times of the collected alcohol precipitation substances, stirring and dissolving, centrifuging, and removing precipitate to obtain a tan crude polysaccharide solution; the crude polysaccharide aqueous solution was combined with deproteinizing agent (chloroform: n-butanol=3:1) according to 3:1 into a conical flask, stirring for 30min, pouring into a separating funnel, standing for 30min to separate an organic phase from a water phase, collecting the water phase, and repeating the deproteinizing step until no white substance is separated out between the organic phase and the water phase when standing; decolorizing the deproteinized crude polysaccharide solution by using macroporous resin D301 or D301R chromatographic column until the crude polysaccharide solution is light yellow or colorless; concentrating the decolored crude polysaccharide solution, filling the concentrated crude polysaccharide solution into a dialysis bag with the molecular weight of 5000Da, placing the dialysis bag into distilled water, dialyzing for multiple times, and freeze-drying the solution to obtain a crude polysaccharide dry product;
dissolving crude polysaccharide in distilled water, passing through DEAE fast flow chromatographic column, eluting with 0.02M tris-hydrochloric acid, 0.02M tris-hydrochloric acid+0.2M sodium chloride solution and 0.02M tris-hydrochloric acid+0.4M sodium chloride solution sequentially from small to large according to sodium chloride solution concentration, collecting eluate of 0.02M tris-hydrochloric acid+0.4M sodium chloride solution to obtain crude burdock root polysaccharide, and subjecting to molecular sieve exclusion agarose gel G100 chromatography to obtain purified burdock root polysaccharide RB3.
The weight average molecular weight of the burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans is 128.1kDa, galactose and galacturonic acid are used as main materials, the sum of the molar content ratio is 54.7%, and the burdock root polysaccharide RB is mainly composed of galactose, galacturonic acid, rhamnose, arabinose, glucuronic acid, mannose and xylose in a molar ratio of 29.8:24.9:19.3:13.5:4.2:7.1:1.2.
Example 4 cell assay
1. Cytotoxicity test of burdock root polysaccharide RB3 prepared in example 1 on human normal liver cell LO2
The specific method comprises the following steps:
human normal hepatocytes LO2 were cultured with RPMI1640 basal medium+10% fetal bovine serum+1% diabody, and placed in a constant temperature incubator containing 5% carbon dioxide, and cultured at 37deg.C. After digestion of the cells, the cells were then treated at 5X 10 3 The well is spread in a 96-well plate, RB3 is dissolved in RPMI160 culture solution, filtered and sterilized, cells are treated by RB3 with different concentration gradients of 0-1500 mug/mL, after 24 hours, the culture solution is removed, CCK-8 reagent is added, after 1 hour of reaction at 37 ℃, absorbance at 540nm is detected by an enzyme-labeled instrument.
As shown in FIG. 6, the burdock root polysaccharide RB3 with the concentration of 100-1500 mug/mL has no obvious inhibition effect on the survival rate of LO2, and the burdock root polysaccharide RB3 has no cytotoxic effect on normal cells.
2. The specific method for performing the immunocompetence experiment on the mouse macrophage RAW264.7 by burdock root polysaccharide RB3 prepared in example 1 is as follows:
mouse macrophage RAW264.7 is cultivated by RPMI1640 basic culture solution, 10 percent of fetal bovine serum and 1 percent of double antibody, and the mice are placed in a constant temperature incubator containing 5 percent of carbon dioxide for cultivation at 37 ℃. After digestion of the cells, the cells were then treated at 5X 10 3 The well is spread in a 96-well plate, RB3 is dissolved in RPMI160 culture solution and filtered for sterilization, the cells are treated by RB3 with different concentration gradients of 0-900 mug/mL, after 24 hours, the culture solution is sucked out, CCK-8 reagent is added, after 1 hour of reaction at 37 ℃, absorbance at 540nm is detected by an enzyme-labeled instrument to detect the influence of RB3 on macrophage proliferation. In addition, the culture medium was collected and centrifuged at 5000 Xg, and the amounts of cytokines such as NO, IL-beta, IL-6, TNF-alpha, etc., were measured.
As shown in FIG. 7, compared with the control group, the concentration of the burdock root polysaccharide RB3 of 50-900 mug/mL can obviously promote the proliferation of macrophages and is in direct proportion to the concentration of polysaccharide, and the proliferation rate can reach 135% at most.
As shown in FIG. 8, compared with the control group, the concentration of burdock root polysaccharide RB3 of 50-900 mug/mL can also enhance the phagocytic activity of macrophages, and induce the secretion of cytokines such as NO, IL-beta, IL-6, TNF-alpha and the like of the macrophages.
3. Proliferation experiment of Burdock root polysaccharide RB3 prepared in example 1 on tumor cells
The specific method comprises the following steps: human colorectal cancer cells HCT-116, human breast cancer cells MDA-MB-231, human liver cancer cells A549 and human glioma cells U87 are cultured in RPMI1640 basal medium plus 10% fetal bovine serum plus 1% double antibody for 24 hours. The burdock root polysaccharide RB3 is dissolved in RPMI160 culture solution, filtered and sterilized, and treated by prepared culture solution containing RB3 with different concentrations for 48 hours, and the survival rate of cancer cells is detected by CCK-8 reagent.
Experimental results show that the burdock root polysaccharide RB3 has a good inhibition effect on proliferation of the above tumor cells, and has obvious dose dependency, and the IC50 doses of the burdock root polysaccharide RB3 on the above tumor cells are 753, 521, 243 and 855 mug/mL in sequence.
EXAMPLE 5 in vivo tumor-bearing mouse Experimental results
The right armpit of Balb/c female mice was injected with an appropriate amount of S180 cells to establish an S180 tumor-bearing mouse model. The burdock root polysaccharide RB3 prepared in example 1 was used, and a burdock root polysaccharide RB3 low dose group (50 mg/kg/d), a medium dose group (100 mg/kg/d), a high dose group (150 mg/kg/d), a 5-fluorouracil group (20 mg/kg/d), and a 5-fluorouracil (20 mg/kg/d) +medium dose (100 mg/kg/d) combination group were set. The administration mode is that the gastric lavage is 0.2ml/d, and the negative control group 1-2 is burdock root polysaccharide (500 mg/kg/d) extracted by modeling the equal dosage of distilled water and hot water of the mice gastric lavage.
After the mice are selected for about 8 weeks and the common feed is fed adaptively for 1 week, the right armpit is injected with a proper amount of S180 cells, the modeling is successful about one week after inoculation, and then the administration is continued for 14 days. The last day overnight fast without water, the following day after weighing, the mice were sacrificed by cervical scission, tumor was peeled off and weighed, and the results are shown in table 1.
TABLE 1
As can be seen from Table 1, the tumor inhibition rates of the low dose group (50 mg/kg/d), the medium dose group (100 mg/kg/d) and the high dose group (150 mg/kg/d) and the 5-fluorouracil group (20 mg/kg/d) of burdock root polysaccharide RB3 were 34%, 60%, 81% and 86%, respectively, by comparison with the average tumor volume of the negative control group. Wherein the difference between the medium dose group and the high dose group is statistically significant (p < 0.05). In addition, the 5-fluorouracil and the medium dose combination have better tumor inhibiting effect than the single 5-fluorouracil, and have better recovery effect on the weight, immune tissues and the like of mice. But only the hot water extracted burdock root polysaccharide has no obvious in vivo anti-tumor activity.
Claims (9)
1. A burdock root polysaccharide prepared by fermenting rhizopus nigricans is characterized in that the weight average molecular weight of the burdock root polysaccharide is 5 multiplied by 10 4 ～1×10 6 Da, wherein the molar content of galactose and galacturonic acid in the burdock root polysaccharide is more than 50%;
the production process for preparing burdock root polysaccharide by fermenting rhizopus nigricans comprises the following steps:
(1) Inoculating rhizopus nigricans into a potato dextrose agar medium, culturing at 26-30 ℃ for 4-6 days until white hyphae are fully paved and black spores grow, selecting rhizopus nigricans hypha blocks, transferring the rhizopus nigricans hypha blocks into the potato dextrose agar medium, continuously culturing for 2-4 days until a large number of black spores appear, flushing rhizopus nigricans bodies with sterile water, collecting spores, and obtaining rhizopus nigricans spore suspension;
(2) Selecting fresh burdock roots or burdock root water insoluble matters or burdock root water insoluble matter dry powder, adding the burdock roots or burdock root water insoluble matters dry powder into a shake flask of a potato dextrose liquid culture medium, inoculating a rhizopus nigricans spore suspension according to the volume ratio of 0.5-5% after wet sterilization, and then placing the burdock roots or burdock root water insoluble matters dry powder into the shake flask at the temperature of 25-30 ℃ and the shaking rotation speed of 120-200rpm for fermentation culture for 7-15 days to obtain burdock root fermentation liquor;
(3) Filtering and clarifying the burdock root fermentation broth, concentrating the supernatant, precipitating with ethanol, redissolving, deproteinizing, decolorizing, dialyzing, performing anion exchange DEAE fast flow chromatography to obtain crude burdock root polysaccharide, and performing molecular sieve exclusion agarose gel G100 chromatography to obtain purified burdock root polysaccharide.
2. The process according to claim 1, wherein in step (1), the size of the rhizopus nigricans mycelium pellet is 1 x 1cm; the density of the rhizopus nigricans spores in the rhizopus nigricans spore suspension is 1 multiplied by 10 5 ～10 8 And each mL.
3. The process of claim 1, wherein in step (2) the suspension of rhizopus nigricans spores is introduced in an amount of 2% of the volume of potato dextrose broth.
4. The production process according to claim 1, wherein in the step (2), the fresh burdock root is subjected to washing, peeling and chopping treatment, and the weight-volume ratio of the fresh burdock root to the potato dextrose liquid medium is (1-4): 10, the unit is: g/mL.
5. The production process according to claim 1, wherein in the step (2), the burdock root water-insoluble matter is obtained by treating fresh burdock root by a hot water leaching method, and the weight-volume ratio of the burdock root water-insoluble matter to potato dextrose liquid medium is (1-4): 10, the unit is: g/mL.
6. The production process according to claim 1, wherein in the step (2), the burdock root water-insoluble matter dry powder is obtained by drying and crushing burdock root water-insoluble matter at 50-70 ℃, and the weight-volume ratio of the burdock root water-insoluble matter dry powder to potato dextrose liquid medium is (1-5): 100, the unit is: g/mL.
7. The process according to claim 1, wherein in step (3), the anion exchange DEAE fast flow chromatography is performed as follows: and eluting 0.02M tris-hydrochloric acid, 0.02M tris-hydrochloric acid+0.1M sodium chloride solution, 0.02M tris-hydrochloric acid+0.2M sodium chloride solution, 0.02M tris-hydrochloric acid+0.3M sodium chloride solution, 0.02M tris-hydrochloric acid+0.4M sodium chloride solution and 0.02M tris-hydrochloric acid+0.5M sodium chloride solution in sequence from small to large according to the concentration of sodium chloride, and collecting eluent of 0.02M tris-hydrochloric acid+0.3M sodium chloride solution to obtain burdock root polysaccharide RB3 prepared by fermenting rhizopus nigricans.
8. The use of burdock root polysaccharide according to claim 1 in the preparation of an immunomodulatory anti-tumor drug; the tumor is colorectal cancer, breast cancer, liver cancer, glioma or ascites tumor.
9. The use according to claim 8, wherein the burdock root polysaccharide is used in combination with 5-fluorouracil in an amount of 50 to 150mg/kg/d and 5-fluorouracil in an amount of 15 to 25mg/kg/d.
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